Secondary literature sources for KISc
The following references were automatically generated.
- Mitchell A
- On track with kinesin.
- Nat Rev Mol Cell Biol. 2001; 2: 86-86
- Nedelec F, Surrey T, Maggs AC
- Dynamic concentration of motors in microtubule arrays.
- Phys Rev Lett. 2001; 86: 3192-5
- Display abstract
We present experimental and theoretical studies of the dynamics of molecular motors in microtubule arrays and asters. By solving a convection-diffusion equation we find that the density profile of motors in a two-dimensional aster is characterized by continuously varying exponents. Simulations are used to verify the assumptions of the continuum model. We observe the concentration profiles of kinesin moving in quasi-two-dimensional artificial asters by fluorescent microscopy and compare with our theoretical results.
- Minehardt TJ, Cooke R, Pate E, Kollman PA
- Molecular dynamics study of the energetic, mechanistic, and structural implications of a closed phosphate tube in ncd.
- Biophys J. 2001; 80: 1151-68
- Display abstract
The switch 1 region of myosin forms a lid over the nucleotide phosphates as part of a structure known as the phosphate-tube. The homologous region in kinesin-family motors is more open, not interacting with the nucleotide. We used molecular dynamics (MD) simulations to examine a possible displacement of switch 1 of the microtubule motor, ncd, from the open conformation to the closed conformation seen in myosin. MD simulations were done of both the open and the closed conformations, with either MgADP or MgATP at the active site. All MD structures were stable at 300 K for 500 ps, implying that the open and closed conformers all represented local minima on a global free energy surface. Free energy calculations indicated that the open structure was energetically favored with MgADP at the active site, suggesting why only the open structure has been captured in crystallographic work. With MgATP, the closed and open structures had roughly equal energies. Simulated annealing MD showed the transformation from the closed phosphate-tube ncd structure to an open configuration. The MD simulations also showed that the coordination of switch 1 to the nucleotide dramatically affected the position of both the bound nucleotide and switch 2 and that a closed phosphate-tube may be necessary for catalysis.
- Kliche W, Fujita-Becker S, Kollmar M, Manstein DJ, Kull FJ
- Structure of a genetically engineered molecular motor.
- EMBO J. 2001; 20: 40-6
- Display abstract
Molecular motors move unidirectionally along polymer tracks, producing movement and force in an ATP-dependent fashion. They achieve this by amplifying small conformational changes in the nucleotide-binding region into force-generating movements of larger protein domains. We present the 2.8 A resolution crystal structure of an artificial actin-based motor. By combining the catalytic domain of myosin II with a 130 A conformational amplifier consisting of repeats 1 and 2 of alpha-actinin, we demonstrate that it is possible to genetically engineer single-polypeptide molecular motors with precisely defined lever arm lengths and specific motile properties. Furthermore, our structure shows the consequences of mutating a conserved salt bridge in the nucleotide-binding region. Disruption of this salt bridge, which is known to severely inhibit ATP hydrolysis activity, appears to interfere with formation of myosin's catalytically active 'closed' conformation. Finally, we describe the structure of alpha-actinin repeats 1 and 2 as being composed of two rigid, triple-helical bundles linked by an uninterrupted alpha-helix. This fold is very similar to the previously described structures of alpha-actinin repeats 2 and 3, and alpha-spectrin repeats 16 and 17.
- Maney T, Wagenbach M, Wordeman L
- Molecular dissection of the microtubule depolymerizing activity of mitotic centromere-associated kinesin.
- J Biol Chem. 2001; 276: 34753-8
- Display abstract
Mitotic centromere-associated kinesin (MCAK) is a microtubule depolymerizer that is consistent with its role in promoting chromosome segregation during mitosis. Here we show that the conserved motor domain of MCAK is necessary but not sufficient for microtubule depolymerization in cells or in vitro. The addition of only 30 amino acids N-terminal to the motor restores depolymerization activity. Furthermore, dimerization studies revealed that the smallest functional MCAK deletion constructs are monomers. These results define a highly conserved domain within MCAK and related (KIN I) kinesins that is critical for depolymerization activity and show that this depolymerization is not dependent on MCAK dimerization.
- Yuan YR, Blecker S, Martsinkevich O, Millen L, Thomas PJ, Hunt JF
- The crystal structure of the MJ0796 ATP-binding cassette. Implications for the structural consequences of ATP hydrolysis in the active site of an ABC transporter.
- J Biol Chem. 2001; 276: 32313-21
- Display abstract
The crystal structure of the MJ0796 ATP-binding cassette, a member of the o228/LolD transporter family, has been determined at 2.7-A resolution with MgADP bound at its active site. Comparing this structure with that of the ATP-bound form of the HisP ATP-binding cassette (Hung, L. W., Wang, I. X., Nikaido, K., Liu, P. Q., Ames, G. F., and Kim, S. H. (1998) Nature 396, 703-707) shows a 5-A withdrawal of a phylogenetically invariant glutamine residue from contact with the gamma-phosphate of ATP in the active site. This glutamine is located in a protein segment that links the rigid F(1)-type ATP-binding core of the enzyme to an ABC transporter-specific alpha-helical subdomain that moves substantially away from the active site in the MgADP-bound structure of MJ0796 compared with the ATP-bound structure of HisP. A similar conformational effect is observed in the MgADP-bound structure of MJ1267 (Karpowich, N., et al. (2001) Structure, in press), establishing the withdrawal of the glutamine and the coupled outward rotation of the alpha-helical subdomain as consistent consequences of gamma-phosphate release from the active site of the transporter. Considering this subdomain movement in the context of a leading model for the physiological dimer of cassettes present in ABC transporters indicates that it produces a modest mechanical change that is likely to play a role in facilitating nucleotide exchange out of the ATPase active site. Finally, it is noteworthy that one of the intersubunit packing interactions in the MJ0796 crystal involves antiparallel beta-type hydrogen bonding interactions between the outermost beta-strands in the two core beta-sheets, leading to their fusion into a single extended beta-sheet, a type of structural interaction that has been proposed to play a role in mediating the aggregation of beta-sheet-containing proteins.
- Kawaguchi K, Ishiwata S
- Nucleotide-dependent single- to double-headed binding of kinesin.
- Science. 2001; 291: 667-9
- Display abstract
The motility of kinesin motors is explained by a "hand-over-hand" model in which two heads of kinesin alternately repeat single-headed and double-headed binding with a microtubule. To investigate the binding mode of kinesin at the key nucleotide states during adenosine 5'-triphosphate (ATP) hydrolysis, we measured the mechanical properties of a single kinesin-microtubule complex by applying an external load with optical tweezers. Both the unbinding force and the elastic modulus in solutions containing AMP-PNP (an ATP analog) were twice the value of those in nucleotide-free solution or in the presence of both AMP-PNP and adenosine 5'-diphosphate. Thus, kinesin binds through two heads in the former and one head in the latter two states, which supports a major prediction of the hand-over-hand model.
- Miki H, Setou M, Kaneshiro K, Hirokawa N
- All kinesin superfamily protein, KIF, genes in mouse and human.
- Proc Natl Acad Sci U S A. 2001; 98: 7004-11
- Display abstract
Intracellular transport is essential for morphogenesis and functioning of the cell. The kinesin superfamily proteins (KIFs) have been shown to transport membranous organelles and protein complexes in a microtubule- and ATP-dependent manner. More than 30 KIFs have been reported in mice. However, the nomenclature of KIFs has not been clearly established, resulting in various designations and redundant names for a single KIF. Here, we report the identification and classification of all KIFs in mouse and human genome transcripts. Previously unidentified murine KIFs were found by a PCR-based search. The identification of all KIFs was confirmed by a database search of the total human genome. As a result, there are a total of 45 KIFs. The nomenclature of all KIFs is presented. To understand the function of KIFs in intracellular transport in a single tissue, we focused on the brain. The expression of 38 KIFs was detected in brain tissue by Northern blotting or PCR using cDNA. The brain, mainly composed of highly differentiated and polarized cells such as neurons and glia, requires a highly complex intracellular transport system as indicated by the increased number of KIFs for their sophisticated functions. It is becoming increasingly clear that the cell uses a number of KIFs and tightly controls the direction, destination, and velocity of transportation of various important functional molecules, including mRNA. This report will set the foundation of KIF and intracellular transport research.
- Hiratsuka Y, Tada T, Oiwa K, Kanayama T, Uyeda TQ
- Controlling the Direction of Kinesin-Driven Microtubule Movements along Microlithographic Tracks.
- Biophys J. 2001; 81: 1555-61
- Display abstract
Motor proteins are able to move protein filaments in vitro. However, useful work cannot be extracted from the existing in vitro systems because filament motions are in random directions on two-dimensional surfaces. We succeeded in restricting kinesin-driven movements of microtubules along linear tracks by using micrometer-scaled grooves lithographically fabricated on glass surfaces. We also accomplished the extraction of unidirectional movement from the bidirectional movements along the linear tracks by adding arrowhead patterns on the tracks. These "rectifiers" enabled us to construct microminiturized circulators in which populations of microtubules rotated in one direction, and to actively transport microtubules between two pools connected by arrowheaded tracks in the fields of micrometer scales.
- Schliwa M, Woehlke G
- Molecular motors. Switching on kinesin.
- Nature. 2001; 411: 424-5
- Goldstein LS
- Kinesin molecular motors: transport pathways, receptors, and human disease.
- Proc Natl Acad Sci U S A. 2001; 98: 6999-7003
- Display abstract
Kinesin molecular motor proteins are responsible for many of the major microtubule-dependent transport pathways in neuronal and non-neuronal cells. Elucidating the transport pathways mediated by kinesins, the identity of the cargoes moved, and the nature of the proteins that link kinesin motors to cargoes are areas of intense investigation. Kinesin-II recently was found to be required for transport in motile and nonmotile cilia and flagella where it is essential for proper left-right determination in mammalian development, sensory function in ciliated neurons, and opsin transport and viability in photoreceptors. Thus, these pathways and proteins may be prominent contributors to several human diseases including ciliary dyskinesias, situs inversus, and retinitis pigmentosa. Kinesin-I is needed to move many different types of cargoes in neuronal axons. Two candidates for receptor proteins that attach kinesin-I to vesicular cargoes were recently found. One candidate, sunday driver, is proposed to both link kinesin-I to an unknown vesicular cargo and to bind and organize the mitogen-activated protein kinase components of a c-Jun N-terminal kinase signaling module. A second candidate, amyloid precursor protein, is proposed to link kinesin-I to a different, also unknown, class of axonal vesicles. The finding of a possible functional interaction between kinesin-I and amyloid precursor protein may implicate kinesin-I based transport in the development of Alzheimer's disease.
- Turner J, Anderson R, Guo J, Beraud C, Fletterick R, Sakowicz R
- Crystal structure of the mitotic spindle kinesin Eg5 reveals a novel conformation of the neck-linker.
- J Biol Chem. 2001; 276: 25496-502
- Display abstract
Success of mitosis depends upon the coordinated and regulated activity of many cellular factors, including kinesin motor proteins, which are required for the assembly and function of the mitotic spindle. Eg5 is a kinesin implicated in the formation of the bipolar spindle and its movement prior to and during anaphase. We have determined the crystal structure of the Eg5 motor domain with ADP-Mg bound. This structure revealed a new intramolecular binding site of the neck-linker. In other kinesins, the neck-linker has been shown to be a critical mechanical element for force generation. The neck-linker of conventional kinesin is believed to undergo an ordered-to-disordered transition as it translocates along a microtubule. The structure of Eg5 showed an ordered neck-linker conformation in a position never observed previously. The docking of the neck-linker relies upon residues conserved only in the Eg5 subfamily of kinesin motors. Based on this new information, we suggest that the neck-linker of Eg5 may undergo an ordered-to-ordered transition during force production. This ratchet-like mechanism is consistent with the biological activity of Eg5.
- Yang Z, Roberts EA, Goldstein LS
- Functional analysis of mouse C-terminal kinesin motor KifC2.
- Mol Cell Biol. 2001; 21: 2463-6
- Display abstract
Proteins of the kinesin superfamily define a class of microtubule-dependent motors that play crucial roles in cell division and intracellular transport. In the mouse, several kinesin motors have been characterized and are suggested to play roles in axonal and/or dendritic transport. One such kinesin is KifC2. Sequence and secondary structure analysis revealed that KifC2 is a member of the C-terminal motor family. Northern and Western blot analyses indicated that KifC2 is specifically expressed in both the central and peripheral nervous systems. The cellular locations of the KifC2 proteins were found to be mainly in neural cell bodies and dendrites but also in axons. To understand the in vivo function of the KifC2 gene, we used homologous recombination in embryonic stem cells to construct knockout mouse strains for the KifC2 gene. Homozygous KifC2 mutants were viable and reproduced normally, and their development was apparently normal. These results suggest that KifC2 is dispensable for normal neural development and behavior in the mouse.
- Schief WR, Howard J
- Conformational changes during kinesin motility.
- Curr Opin Cell Biol. 2001; 13: 19-28
- Display abstract
Nucleotide-dependent movements of the head and neck of kinesin have been visualized by cryoelectron microscopy and have been inferred from single-molecule studies. Key predictions of the hand-over-hand model for dimeric kinesin have been confirmed, and a novel processivity mechanism for the one-headed, kinesin-related motor KIF1A has been discovered.
- Reilein AR, Rogers SL, Tuma MC, Gelfand VI
- Regulation of molecular motor proteins.
- Int Rev Cytol. 2001; 204: 179-238
- Display abstract
Motor proteins in the kinesin, dynein, and myosin superfamilies are tightly regulated to perform multiple functions in the cell requiring force generation. Although motor proteins within families are diverse in sequence and structure, there are general mechanisms by which they are regulated. We first discuss the regulation of the subset of kinesin family members for which such information exists, and then address general mechanisms of kinesin family regulation. We review what is known about the regulation of axonemal and cytoplasmic dyneins. Recent work on cytoplasmic dynein has revealed the existence of multiple isoforms for each dynein chain, making the study of dynein regulation more complicated than previously realized. Finally, we discuss the regulation of myosins known to be involved in membrane trafficking. Myosins and kinesins may be evolutionarily related, and there are common themes of regulation between these two classes of motors.
- Gibbons F, Chauwin JF, Desposito M, Jose JV
- A dynamical model of kinesin-microtubule motility assays.
- Biophys J. 2001; 80: 2515-26
- Display abstract
A two-dimensional stochastic model for the dynamics of microtubules in gliding-assay experiments is presented here, which includes the viscous drag acting on the moving fiber and the interaction with the kinesins. For this purpose, we model kinesin as a spring, and explicitly use parameter values to characterize the model from experimental data. We numerically compute the mean attachment lifetimes of all motors, the total force exerted on the microtubules at all times, the effects of a distribution in the motor speeds, and also the mean velocity of a microtubule in a gliding assay. We find quantitative agreement with the results of J. Howard, A. J. Hudspeth, and R. D. Vale, Nature. 342:154-158. We perform additional numerical analysis of the individual motors, and show how cancellation of the forces exerted by the many motors creates a resultant longitudinal force much smaller than the maximum force that could be exerted by a single motor. We also examine the effects of inhomogeneities in the motor-speeds. Finally, we present a simple theoretical model for microtubules dynamics in gliding assays. We show that the model can be analytically solved in the limit of few motors attached to the microtubule and in the opposite limit of high motor density. We find that the speed of the microtubule goes like the mean speed of the motors in good quantitative agreement with the experimental and numerical results.
- Hays T, Li M
- Kinesin transport: driving kinesin in the neuron.
- Curr Biol. 2001; 11: 1369-1369
- Display abstract
A plethora of cytoplasmic motors contribute to the directed transport of a wide range of cellular organelles and molecules. Recent studies have advanced our understanding of cargo attachment to motor molecules and the regulation of intracellular transport.
- Cross RA
- Molecular motors: Kinesin's string variable.
- Curr Biol. 2001; 11: 1479-1479
- Display abstract
A recent model suggests that the walking action of kinesin is due to a 13 residue 'fundamental engine' called the neck linker domain, which cyclically zips and unzips to the main part of the heads. New experiments confirm one prediction of the model: that crosslinking the neck linker to the head should block motility.
- Quinlan ME, Forkey JN, Goldman YE
- Kinesin-ADP: whole lotta shakin' goin' on.
- Nat Struct Biol. 2001; 8: 478-80
- Bloom GS
- The UNC-104/KIF1 family of kinesins.
- Curr Opin Cell Biol. 2001; 13: 36-40
- Display abstract
Most UNC-104/KIF1 kinesins are monomeric motors that transport membrane-bounded organelles toward the plus ends of microtubules. Recent evidence implies that KIF1A, a synaptic vesicle motor, moves processively. This surprising behavior for a monomeric motor depends upon a lysine-rich loop in KIF1A that binds to the negatively charged carboxyl terminus of tubulin and, in the context of motor processivity, compensates for the lack of a second motor domain on the KIF1A holoenzyme.
- Kikkawa M, Sablin EP, Okada Y, Yajima H, Fletterick RJ, Hirokawa N
- Switch-based mechanism of kinesin motors.
- Nature. 2001; 411: 439-45
- Display abstract
Kinesin motors are specialized enzymes that use hydrolysis of ATP to generate force and movement along their cellular tracks, the microtubules. Although numerous biochemical and biophysical studies have accumulated much data that link microtubule-assisted ATP hydrolysis to kinesin motion, the structural view of kinesin movement remains unclear. This study of the monomeric kinesin motor KIF1A combines X-ray crystallography and cryo-electron microscopy, and allows analysis of force-generating conformational changes at atomic resolution. The motor is revealed in its two functionally critical states-complexed with ADP and with a non-hydrolysable analogue of ATP. The conformational change observed between the ADP-bound and the ATP-like structures of the KIF1A catalytic core is modular, extends to all kinesins and is similar to the conformational change used by myosin motors and G proteins. Docking of the ADP-bound and ATP-like crystallographic models of KIF1A into the corresponding cryo-electron microscopy maps suggests a rationale for the plus-end directional bias associated with the kinesin catalytic core.
- Yang Z, Xia C, Roberts EA, Bush K, Nigam SK, Goldstein LS
- Molecular cloning and functional analysis of mouse C-terminal kinesin motor KifC3.
- Mol Cell Biol. 2001; 21: 765-70
- Display abstract
Proteins of the kinesin superfamily define a class of microtubule-dependent motors that play crucial roles in cell division and intracellular transport. To study the molecular mechanism of intracellular transport involving microtubule-dependent motors, a cDNA encoding a new kinesin-like protein called KifC3 was cloned from a mouse brain cDNA library. Sequence and secondary structure analysis revealed that KifC3 is a member of the C-terminal motor family. In contrast to other mouse C-terminal motors, KifC3 is apparently ubiquitous and may have a general role in intracellular transport. To understand the in vivo function of the KifC3 gene, we used homologous recombination in embryonic stem cells to construct knockout mouse strains for the KifC3 gene. Homozygous mutants of the KifC3 gene are viable, reproduce normally, and apparently develop normally. These results suggest that KifC3 is dispensable for normal development and reproduction in the mouse.
- Surrey T, Nedelec F, Leibler S, Karsenti E
- Physical properties determining self-organization of motors and microtubules.
- Science. 2001; 292: 1167-71
- Display abstract
In eukaryotic cells, microtubules and their associated motor proteins can be organized into various large-scale patterns. Using a simplified experimental system combined with computer simulations, we examined how the concentrations and kinetic parameters of the motors contribute to their collective behavior. We observed self-organization of generic steady-state structures such as asters, vortices, and a network of interconnected poles. We identified parameter combinations that determine the generation of each of these structures. In general, this approach may become useful for correlating the morphogenetic phenomena taking place in a biological system with the biophysical characteristics of its constituents.
- De Marco V, Burkhard P, Le Bot N, Vernos I, Hoenger A
- Analysis of heterodimer formation by Xklp3A/B, a newly cloned kinesin-II from Xenopus laevis.
- EMBO J. 2001; 20: 3370-9
- Display abstract
kinesin-II motor proteins are composed of two different kinesin-like motor proteins and one cargo binding subunit. Here we report the cloning of a new member of the kinesin-II superfamily, Xklp3A from Xenopus laevis, which forms a heterodimeric complex with Xklp3B. The heterodimer formation properties between Xklp3A and B have been tested in vitro using reticulocyte lysate expression and immunoprecipitation. To this end we produced a series of Xklp3A and B constructs of varying length and tested their propensity for heterodimer formation. We could demonstrate that, in contrast to conventional kinesin, the critical domains for heterodimer formation in Xklp3A/B are located at the C-terminal end of the stalk. Neither the neck nor the highly charged stretches after the neck region, which are typical of kinesins-II, are required for heterodimer formation, nor do they prevent homodimer formation. Dimerization is controlled by a cooperative mechanism between the C-terminal coiled-coil segments. Classical trigger sites were not identified. The critical regions for dimerization exhibit a very high degree of sequence conservation among equivalent members of the kinesin-II family.
- Hackney DD, Jiang W
- Assays for kinesin microtubule-stimulated ATPase activity.
- Methods Mol Biol. 2001; 164: 65-71
- DeLuca JG, Newton CN, Himes RH, Jordan MA, Wilson L
- Purification and characterization of native conventional kinesin, HSET, and CENP-E from mitotic hela cells.
- J Biol Chem. 2001; 276: 28014-21
- Display abstract
We have developed a strategy for the purification of native microtubule motor proteins from mitotic HeLa cells and describe here the purification and characterization of human conventional kinesin and two human kinesin-related proteins, HSET and CENP-E. We found that the 120-kDa HeLa cell conventional kinesin is an active motor that induces microtubule gliding at approximately 30 microm/min at room temperature. This active form of HeLa cell kinesin does not contain light chains, although light chains were detected in other fractions. HSET, a member of the C-terminal kinesin subfamily, was also purified in native form for the first time, and the protein migrates as a single band at approximately 75 kDa. The purified HSET is an active motor that induces microtubule gliding at a rate of approximately 5 microm/min, and microtubules glide for an average of 3 microm before ceasing movement. Finally, we purified native CENP-E, a kinesin-related protein that has been implicated in chromosome congression during mitosis, and we found that this form of CENP-E does not induce microtubule gliding but is able to bind to microtubules.
- Yun M, Zhang X, Park CG, Park HW, Endow SA
- A structural pathway for activation of the kinesin motor ATPase.
- EMBO J. 2001; 20: 2611-8
- Display abstract
Molecular motors move along actin or microtubules by rapidly hydrolyzing ATP and undergoing changes in filament-binding affinity with steps of the nucleotide hydrolysis cycle. It is generally accepted that motor binding to its filament greatly increases the rate of ATP hydrolysis, but the structural changes in the motor associated with ATPase activation are not known. To identify the conformational changes underlying motor movement on its filament, we solved the crystal structures of three kinesin mutants that decouple nucleotide and microtubule binding by the motor, and block microtubule-activated, but not basal, ATPase activity. Conformational changes in the structures include a disordered loop and helices in the switch I region and a visible switch II loop, which is disordered in wild-type structures. Switch I moved closer to the bound nucleotide in two mutant structures, perturbing water-mediated interactions with the Mg2+. This could weaken Mg2+ binding and accelerate ADP release to activate the motor ATPASE: The structural changes we observe define a signaling pathway within the motor for ATPase activation that is likely to be essential for motor movement on microtubules.
- Nishiyama M, Muto E, Inoue Y, Yanagida T, Higuchi H
- Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules.
- Nat Cell Biol. 2001; 3: 425-8
- Display abstract
Kinesin is a molecular motor that moves processively by regular 8-nm steps along microtubules. The processivity of this movement is explained by a hand-over-hand model in which the two heads of kinesin work in a coordinated manner. One head remains bound to the microtubule while the other steps from the alphabeta-tubulin dimer behind the attached head to the dimer in front. The overall movement is 8 nm per ATPase cycle. To investigate elementary processes within the 8-nm step, we have developed a new assay that resolves nanometre displacements of single kinesin molecules with microsecond accuracy. Our data show that the 8-nm step can be resolved into fast and slow substeps, each corresponding to a displacement of approximately 4 nm. The substeps are most probably generated by structural changes in one head of kinesin, leading to rectified forward thermal motions of the partner head. It is also possible that the kinesin steps along the 4-nm repeat of tubulin monomers.
- Meech R, Holley M
- Ion-age molecular motors.
- Nat Neurosci. 2001; 4: 771-3
- Touitou I, Lhomond G, Pruliere G
- Boursin, a sea urchin bimC kinesin protein, plays a role in anaphase and cytokinesis.
- J Cell Sci. 2001; 114: 481-91
- Display abstract
We have isolated and characterized Boursin, a kinesin-related protein of the bimC family, from Paracentrotus lividus sea urchin eggs. Boursin is expressed at high levels in eggs and embryos during early cleavage stages. Boursin was found to be associated with different parts of the mitotic spindle from early prophase to telophase. Expression of a form of the protein predicted to act as a dominant negative mutant caused severe defects in cell division and resulted in the formation of embryos with polyploid and multiastral blastomeres. Immunofluorescence analysis indicated that these defects did not arise from failure in either centrosome separation or bipolar spindle formation. Time-lapse observations showed rather that these perturbations in cell division resulted from abnormal anaphase and failure to complete cytokinesis. These phenotypes differ from the phenotype described following perturbation of the function of bimC family members in other organisms. Our study has thus uncovered roles for a bimC kinesin in late stages of cell division.
- Beuron F, Hoenger A
- Structural analysis of the microtubule-kinesin complex by cryo-electron microscopy.
- Methods Mol Biol. 2001; 164: 235-54
- Kozielski F, Svergun D, Zaccai G, Wade RH, Koch MH
- The overall conformation of conventional kinesins studied by small angle X-ray and neutron scattering.
- J Biol Chem. 2001; 276: 1267-75
- Display abstract
The quaternary structures of several monomeric and dimeric kinesin constructs from Homo sapiens and Drosophila melanogaster were analyzed using small angle x-ray and neutron scattering. The experimental scattering curves of these proteins were compared with simulated scattering curves calculated from available crystallographic coordinates. These comparisons indicate that the overall conformations of the solution structures of D. melanogaster and H. sapiens kinesin heavy chain dimers are compatible with the crystal structure of dimeric kinesin from Rattus norvegicus. This suggests that the unusual asymmetric conformation of dimeric kinesin in the microtubule-independent ADP state is likely to be a general feature of the kinesin heavy chain subfamily. An intermediate length Drosophila construct (365 residues) is mostly monomeric at low protein concentration whereas at higher concentrations it is dimeric with a tendency to form higher oligomers.
- Chen YD, Yan B
- Theoretical formalism for bead movement powered by single two-headed motors in a motility assay.
- Biophys Chem. 2001; 91: 79-91
- Display abstract
Kinesins and dyneins are protein motors that can use the free energy of ATP hydrolysis to carry a cargo and move uni-directionally along a microtubule filament. The purpose of this paper is to derive the formalism connecting the ATP-driven translocation reactions of these motors on microtubule filaments and the movement of the bead carried by the motor in a motility assay in which the bead is clamped at an arbitrary constant force. The formalism is thus useful in elucidating the load-dependent kinetic mechanism of the free-energy transduction of the motor using the mechanical data obtained from the motility assay. The formalism is also useful in assessing the effect on the measured motility data of various physical and hydrodynamic parameters of the assay, such as the size of the bead, the viscosity of the medium, the stiffness of the elastic element connecting the motor and the bead, etc. In a previous paper [Biophys. J. 67 (2000) 313] (hereafter referred to as paper I), we have derived the formalism for the case that the motor in the assay has only one head. In this paper we extend the derivation to the case that the motor is two-headed. The formalism is derived based on a simple two-state hand-over-hand model for the movement of the motor on microtubule, but can be easily extended to more complicated kinetic models. Effects of various hydrodynamic parameters on the velocity of the bead are studied with numerical calculations of the model. The difference between the formalism presented in this paper and the widely used "chemical" formalism, in which the movement of the kinesin and the bead is described by pure chemical reactions, is discussed.
- Verhey KJ, Rapoport TA
- Kinesin carries the signal.
- Trends Biochem Sci. 2001; 26: 545-50
- Display abstract
Conventional kinesin has long been known to be a molecular motor that transports vesicular cargo, but only recently have we begun to understand how it functions in cells. Regulation of kinesin involves self-inhibition in which a head-to-tail interaction prevents microtubule binding. Although the mechanism of motor activation remains to be clarified, recent progress with respect to cargo binding might provide a clue. Kinesin binds directly to the JIPs (JNK-interacting proteins), identified previously as scaffolding proteins in the JNK (c-Jun NH(2)-terminal kinase) signaling pathway. The JIPs can allow kinesin to transport many different cargoes and to concentrate and respond to signaling pathways at certain sites within the cell. The use of scaffolding proteins could be a general mechanism by which molecular motors link to their cargoes.
- Nedelec F, Surrey T
- Assaying spatial organization of microtubules by kinesin motors.
- Methods Mol Biol. 2001; 164: 213-22
- Kahana J
- Yet another function for kinesin.
- Trends Cell Biol. 2001; 11: 13-13
- Reddy AS
- Molecular motors and their functions in plants.
- Int Rev Cytol. 2001; 204: 97-178
- Display abstract
Molecular motors that hydrolyze ATP and use the derived energy to generate force are involved in a variety of diverse cellular functions. Genetic, biochemical, and cellular localization data have implicated motors in a variety of functions such as vesicle and organelle transport, cytoskeleton dynamics, morphogenesis, polarized growth, cell movements, spindle formation, chromosome movement, nuclear fusion, and signal transduction. In non-plant systems three families of molecular motors (kinesins, dyneins, and myosins) have been well characterized. These motors use microtubules (in the case of kinesines and dyneins) or actin filaments (in the case of myosins) as tracks to transport cargo materials intracellularly. During the last decade tremendous progress has been made in understanding the structure and function of various motors in animals. These studies are yielding interesting insights into the functions of molecular motors and the origin of different families of motors. Furthermore, the paradigm that motors bind cargo and move along cytoskeletal tracks does not explain the functions of some of the motors. Relatively little is known about the molecular motors and their roles in plants. In recent years, by using biochemical, cell biological, molecular, and genetic approaches a few molecular motors have been isolated and characterized from plants. These studies indicate that some of the motors in plants have novel features and regulatory mechanisms. The role of molecular motors in plant cell division, cell expansion, cytoplasmic streaming, cell-to-cell communication, membrane trafficking, and morphogenesis is beginning to be understood. Analyses of the Arabidopsis genome sequence database (51% of genome) with conserved motor domains of kinesin and myosin families indicates the presence of a large number (about 40) of molecular motors and the functions of many of these motors remain to be discovered. It is likely that many more motors with novel regulatory mechanisms that perform plant-specific functions are yet to be discovered. Although the identification of motors in plants, especially in Arabidopsis, is progressing at a rapid pace because of the ongoing plant genome sequencing projects, only a few plant motors have been characterized in any detail. Elucidation of function and regulation of this multitude of motors in a given species is going to be a challenging and exciting area of research in plant cell biology. Structural features of some plant motors suggest calcium, through calmodulin, is likely to play a key role in regulating the function of both microtubule- and actin-based motors in plants.
- Schalley CA, Beizai K, Vogtle F
- On the way to rotaxane-based molecular motors: studies in molecular mobility and topological chirality.
- Acc Chem Res. 2001; 34: 465-76
- Display abstract
ATP synthase represents a machine at the molecular level which couples the rotation of an axle in a wheel with the endergonic production of ATP, the general source of chemical energy in the cell. The natural system prototypically bears all features of a macroscopic motor: a rotor within a stator held by a membrane and fueled by a difference in chemical potential in the form of a proton gradient combined with a machine for ATP production. The assembly of axle and wheel to a rotor device reminds one very much of a rotaxane. In this Account, we discuss some important features of motors and their (potential) realization in simpler artificial model systems, that is, the molecular mobility of mechanically bound molecules, the importance of chirality for unidirectional motion, the sources of energy for driving the rotation, and the potential of using membranes and surfaces for ordering a large number of devices to achieve macroscopic effects.
- Morfini G, Tsai MY, Szebenyi G, Brady ST
- Approaches to study interactions between kinesin motors and membranes.
- Methods Mol Biol. 2001; 164: 147-62
- Reddy AS, Day IS
- Kinesins in the Arabidopsis genome: A comparative analysis among eukaryotes.
- BMC Genomics. 2001; 2: 2-2
- Display abstract
BACKGROUND: Kinesins constitute a superfamily of microtubule motor proteins that are found in eukaryotic organisms. Members of the kinesin superfamily perform many diverse cellular functions such as transport of vesicles and organelles, spindle formation and elongation, chromosome segregation, microtubule dynamics and morphogenesis. Only a few kinesins have been characterized in plants including Arabidopsis thaliana. Because of the diverse cellular functions in which kinesins are involved, the number, types and characteristics of kinesins present in the Arabidopsis genome would provide valuable information for many researchers. RESULTS: Here we have analyzed the recently completed Arabidopsis genome sequence and identified sixty-one kinesin genes in the Arabidopsis genome. Among the five completed eukaryotic genomes the Arabidopsis genome has the highest percentage of kinesin genes. Further analyses of the kinesin gene products have resulted in identification of several interesting domains in Arabidopsis kinesins that provide clues in understanding their functions. A phylogenetic analysis of all Arabidopsis kinesin motor domain sequences with 113 motor domain sequences from other organisms has revealed that Arabidopsis has seven of the nine recognized subfamilies of kinesins whereas some kinesins do not fall into any known family. CONCLUSION: There are groups of Arabidopsis kinesins that are not present in yeast, Caenorhabditis elegans and Drosophila melanogaster that may, therefore, represent new subfamilies specific to plants. The domains present in different kinesins may provide clues about their functions in cellular processes. The comparative analysis presented here provides a framework for future functional studies with Arabidopsis kinesins.
- Sosa H, Peterman EJ, Moerner WE, Goldstein LS
- ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy.
- Nat Struct Biol. 2001; 8: 540-4
- Display abstract
Kinesin is an ATP-driven molecular motor protein that moves processively along microtubules. Despite considerable research, the detailed mechanism of kinesin motion remains elusive. We applied an enhanced suite of single- and multiple-molecule fluorescence polarization microscopy assays to report the orientation and mobility of kinesin molecules bound to microtubules as a function of nucleotide state. In the presence of analogs of ATP, ADP-Pi or in the absence of nucleotide, the kinesin head maintains a rigid orientation. In the presence of ADP, the motor domain of kinesin, still bound to the microtubule, adopts a previously undescribed, highly mobile state. This state may be general to the chemomechanical cycle of motor proteins; in the case of kinesin, the transition from a highly mobile to a rigid state after ADP release may contribute to the generation of the 8 nm step.
- Kamimoto T, Zama T, Aoki R, Muro Y, Hagiwara M
- Identification of a Novel Kinesin-related Protein, KRMP1, as a Target for Mitotic Peptidyl-prolyl Isomerase Pin1.
- J Biol Chem. 2001; 276: 37520-37528
- Display abstract
Mitosis utilizes a number of kinesin-related proteins (KRPs). Here we report the identification of a novel KRP termed KRMP1, which has a deduced 1780-amino acid sequence composed of ternary domains. The amino-terminal head domain is most similar to the kinesin motor domain of the MKLP-1 subfamily and has an intrinsic ATPase activity that is diminished by substituting the consensus Lys-168 with Arg. The central stalk domain is predicted to form a long alpha-helical coiled-coil, and can interact with each other in vivo. An in vivo labeling experiment revealed that KRMP1 is phosphorylated, and we also found that the region within the tail domain containing Thr-1604 as the cdc2 kinase phosphorylation site differs from the bimC box conserved in the bimC subfamily of KRPs. Immunofluorescence analysis showed that endogenous KRMP1 was localized predominantly to the cytoplasm during interphase and dispersed throughout the cell during mitosis. Consistent with this finding, overexpressed KRMP1 was detected in a complicated nuclear or cytoplasmic pattern reflecting multiple nuclear localization/export signals. Furthermore, KRMP1 interacted with the mitotic peptidyl-prolyl isomerase Pin1 in vivo, and an in vitro interaction was detected between the tail domain of KRMP1 and the WW domain of Pin1. Overexpression of KRMP1 caused COS-7 cells to arrest at G(2)-M, and co-expression of Pin1 reversed this effect, indicating their physiological interaction. Together, our results suggest that KRMP1 is a mitotic target regulated by Pin1 and vice versa.
- Fox RF, Choi MH
- Rectified Brownian motion and kinesin motion along microtubules.
- Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics. 2001; 63: 51901-51901
- Display abstract
The mechanism of rectified Brownian movement is used to analyze measured data for kinesin motion along microtubules. A key component of the mechanism is the diffusive movement of the microtubule binding heads of kinesin during the adenosine triphosphate (ATP) cycle. The first-passage time distribution for this step is analyzed in detail and is shown to be responsible for observed load-velocity profiles. The ATPase activity of the kinesin heads is that of a nucleotide switch and not that of a direct chemomechanical energy converter. Experimental data acquisition, rate constants, and alternative explanations are discussed. The mechanism described in this paper is fundamental to the nanobiology of intracellular processes.
- Yang Z, Roberts EA, Goldstein LS
- Functional analysis of mouse kinesin motor Kif3C.
- Mol Cell Biol. 2001; 21: 5306-11
- Display abstract
Members of the kinesin II family are thought to play essential roles in many types of intracellular transport. One distinguishing feature of kinesin II is that it generally contains two different motor subunits from the Kif3 family. Three Kif3 family members (Kif3A, Kif3B, and Kif3C) have been identified and characterized in mice. Intracellular localization and biochemical studies previously suggested that Kif3C is an anterograde motor involved in anterograde axonal transport. To understand the in vivo function of the Kif3C gene, we used homologous recombination in embryonic stem cells to construct two different knockout mouse strains for the Kif3C gene. Both homozygous Kif3C mutants are viable, reproduce normally, and apparently develop normally. These results suggest that Kif3C is dispensable for normal neural development and behavior in the mouse.
- Szilagyi AN, Ghosh M, Garman E, Vas M
- A 1.8 A resolution structure of pig muscle 3-phosphoglycerate kinase with bound MgADP and 3-phosphoglycerate in open conformation: new insight into the role of the nucleotide in domain closure.
- J Mol Biol. 2001; 306: 499-511
- Display abstract
3-phosphoglycerate kinase (PGK) is a typical kinase with two structural domains. The domains each bind one of the two substrates, 3-phosphoglycerate (3-PG) and MgATP. For the phospho-transfer reaction to take place the substrates must be brought closer by a hinge-bending domain closure. Open and closed structures of the enzyme with different relative domain positions have been determined from different species, but a comprehensive description of this conformational transition is yet to be attained. Crystals of pig muscle PGK in complex with MgADP and 3-phosphoglycerate were grown under the conditions which have previously resulted in crystals of the closed, catalytically competent conformation of Trypanosoma brucei PGK. The X-ray structure of the pig muscle ternary complex was determined at 1.8 A and the model was refined to R=20.8% and Rfree=24.1%. Contrary to expectation, however, it represents an essentially open conformation compared to that of T. brucei PGK. In addition, the beta-phosphate group of ADP is mobile in the new structure, in contrast to its well-defined position in T. brucei PGK. An extensive comparison of the ternary complexes from these remote species has been carried out in order to establish general differences between the two conformations and is reported here. A second pair of the open and closed structures was also compared. These analyses have made it possible to define several characteristic changes which accompany the structural transition, in addition to those identified previously: (1) the operation of a hinge at beta-strand L in the inter-domain region which greatly affects the relative domain positions; (2) the rearrangement and movement of helix 8, regulated through the interactions with the nucleotide phosphate; and (3) the existence of another hinge between helix 14 and the rest of the C-terminal part of the chain, which allows fine adjustment of the N-domain position.The main hinge at beta-strand L acts in concert with the C-terminal hinge at helix 7 described previously. Simultaneous interactions of the nucleotide phosphate groups with the loop that precedes helix 8, beta-strand J and the N terminus of helix 13 are required for propagation of the nucleotide effect towards the beta-strand L molecular hinge. A detailed description of the role of nucleotide binding in the hinge operation is presented.
- Mogilner A, Fisher AJ, Baskin RJ
- Structural changes in the neck linker of kinesin explain the load dependence of the motor's mechanical cycle.
- J Theor Biol. 2001; 211: 143-57
- Display abstract
The two-headed motor protein kinesin hydrolyzes ATP and moves on microtubule tracks towards the plus end. The motor develops speeds and forces of the order of hundreds of nanometers per second and piconewtons, respectively. Recently, the dependence of the velocity, the dissociation rate and the displacement variance on the load and the ATP concentration were measured in vitro for individual kinesin molecules (Coppin et al., 1997; Visscher et al., 1999) over a wide range of forces. The structural changes in the kinesin motor that drive motility were discovered by Rice et al. (1999). Here we present a phenomenological model for force generation in kinesin based on the bi-stable, nucleotide-dependent behavior of the neck linker. We demonstrate that the model explains the mechanical, kinetic and statistical (experimental) data of Coppin et al. (1997). We also discuss the relationship between the model results and experimental data of Visscher et al. (1999). Copyright 2001 Academic Press.
- deCastro MJ, Fondecave RM, Clarke LA, Schmidt CF, Stewart RJ
- Working strokes by single molecules of the kinesin-related microtubule motor ncd.
- Nat Cell Biol. 2000; 2: 724-9
- Display abstract
The ncd protein is a dimeric, ATP-powered motor that belongs to the kinesin family of microtubule motor proteins. Here we resolve single mechanochemical cycles of recombinant, dimeric, full-length ncd, using optical-tweezers-based instrumentation and a three-bead, suspended-microtubule assay. Under conditions of limiting ATP, isolated and transient microtubule-binding events exhibit exponentially distributed and ATP-concentration-dependent lifetimes. These events do not involve consecutive steps along the microtubule, quantitatively confirming that ncd is non-processive. At low loads, a single motor molecule produces ATP-triggered working strokes of about 9 nm, which occur at the ends of binding events.
- Hildebrandt ER, Hoyt MA
- Mitotic motors in Saccharomyces cerevisiae.
- Biochim Biophys Acta. 2000; 1496: 99-116
- Display abstract
The budding yeast Saccharomyces cerevisiae provides a unique opportunity for study of the microtubule-based motor proteins that participate in mitotic spindle function. The genome of Saccharomyces encodes a relatively small and genetically tractable set of microtubule-based motor proteins. The single cytoplasmic dynein and five of the six kinesin-related proteins encoded have been implicated in mitotic spindle function. Each motor protein is unique in amino acid sequence. On account of functional overlap, no single motor is uniquely required for cell viability, however. The ability to create and analyze multiple mutants has allowed experimental dissection of the roles performed by each mitotic motor. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle (kinesin-related Cin8p, Kip1p, Kip3p and Kar3p). Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell (dynein and kinesin-related Kip2p, Kip3p and Kar3p). The six motors apparently contribute three fundamental activities to spindle function: motility, microtubule cross-linking and regulation of microtubule dynamics.
- Astumian RD
- The role of thermal activation in motion and force generation by molecular motors.
- Philos Trans R Soc Lond B Biol Sci. 2000; 355: 511-22
- Display abstract
The currently accepted mechanism for ATP-driven motion of kinesin is called the hand-over-hand model, where some chemical transition during the ATP hydrolysis cycle stretches a spring, and motion and force production result from the subsequent relaxation. It is essential in this mechanism for the moving head of kinesin to dissociate, while the other head remains firmly attached to the microtubule. Here we propose an alternative Brownian motor model where the action of ATP modulates the interaction potential between kinesin and the microtubule rather than a spring internal to the kinesin molecule alone. In this model neither head need dissociate (which predicts that under some circumstances a single-headed kinesin can display processive motion) and the transitions by which the motor moves are best described as thermally activated steps. This model is consistent with a wide range of experimental data on the force-velocity curves, the one ATP to one-step stoichiometry observed at small load, and the stochastic properties of the stepping.
- Lipowsky R, Harms T
- Molecular motors and nonuniform ratchets.
- Eur Biophys J. 2000; 29: 542-8
- Display abstract
Dimeric kinesin presumably moves in a "hand-over-hand" fashion via alternating steps of its two heads, which can cooperate in various ways. This motion is discussed in the framework of nonuniform ratchet models in which the molecular motor is described by M internal states and undergoes transitions at K spatial locations within the period of the molecular force potentials. Two subclasses of models with (M, K)=(3, 2) and (M, K)=(2, 2) are studied which correspond to weakly and strongly cooperative heads, respectively. Both subclasses lead to the same universal relationship between the motor velocity and the unbinding rate constant of the motor heads which is reminiscent of, but distinct from, Michaelis-Menten kinetics.
- Seeberger C, Mandelkow E, Meyer B
- Conformational preferences of a synthetic 30mer peptide from the interface between the neck and stalk regions of kinesin.
- Biochemistry. 2000; 39: 12558-67
- Display abstract
The conformation of a synthetic peptide, consisting of 30 amino acids spanning the neck and hinge regions of rat brain kinesin, was investigated by NMR spectroscopy. The peptide extends from K357 to D386 and has the sequence KSVIQHLEVELNRWRNGEAVPEDEQISAKD. A total of 82 distance range constraints and 23 dihedral angle constraints could be obtained from NOESY and E.COSY spectra, respectively. These were used to calculate 500 structures by applying the REDAC algorithm of the software package DYANA. The first half of the peptide matched the helical structure of the neck determined from an X-ray crystal structure of kinesin. This part normally dimerizes into a coiled-coil by virtue of a leucine zipper interaction, but it is alpha-helical even in the monomeric state. The second half (not visible in the X-ray structure because of disorder) contains locally defined structure elements (extended chain, helical loop) connected by flexible joints. This is consistent with the "hinge" function postulated for this domain which is important for kinesin's motility and orientation.
- Hoenger A et al.
- Surface topography of microtubule walls decorated with monomeric and dimeric kinesin constructs.
- Biol Chem. 2000; 381: 1001-11
- Display abstract
The surface topography of opened-up microtubule walls (sheets) decorated with monomeric and dimeric kinesin motor domains was investigated by freeze-drying and unidirectional metal shadowing. Electron microscopy of surface-shadowed specimens produces images with a high signal/noise ratio, which enable a direct observation of surface features below 2 nm detail. Here we investigate the inner and outer surface of microtubules and tubulin sheets with and without decoration by kinesin motor domains. Tubulin sheets are flattened walls of microtubules, keeping lateral protofilament contacts intact. Surface shadowing reveals the following features: (i) when the microtubule outside is exposed the surface relief is dominated by the bound motor domains. Monomeric motor constructs generate a strong 8 nm periodicity, corresponding to the binding of one motor domain per alpha-beta-tubulin heterodimer. This surface periodicity largely disappears when dimeric kinesin motor domains are used for decoration, even though it is still visible in negatively stained or frozen hydrated specimens. This could be explained by disorder in the binding of the second (loosely tethered) kinesin head, and/or disorder in the coiled-coil tail. (ii) Both surfaces of undecorated sheets or microtubules, as well as the inner surface of decorated sheets, reveal a strong 4 nm repeat (due to the periodicity of tubulin monomers) and a weak 8 nm repeat (due to slight differences between alpha- and beta-tubulin). The differences between alpha- and beta-tubulin on the inner surface are stronger than expected from cryo-electron microscopy of unstained microtubules, indicating the existence of tubulin subdomain-specific surface properties that reflect the surface corrugation and hence metal deposition during evaporation. The 16 nm periodicity visible in some negatively stained specimens (caused by the pairing of cooperatively bound kinesin dimers) is not detected by surface shadowing.
- Kanai Y, Okada Y, Tanaka Y, Harada A, Terada S, Hirokawa N
- KIF5C, a novel neuronal kinesin enriched in motor neurons.
- J Neurosci. 2000; 20: 6374-84
- Display abstract
Kinesin superfamily proteins (KIFs) are the molecular motors conveying cargos along microtubules. KIF5s, the heavy chains of conventional kinesin (KHC), are originally identified members of KIFs, and neuronal KIF5A and ubiquitous KIF5B have been identified so far. In the present work, we cloned a novel member of KIF5, KIF5C, and generated specific antibodies against three KIF5s to investigate their distribution and functions. KIF5A showed pan-neuronal distribution in the nervous system. KIF5B showed a glial cell distribution pattern in general; however, interestingly, its expression was strongly upregulated in axon-elongating neurons, such as olfactory primary neurons and mossy fibers. KIF5C was also a neuronal KIF5 like KIF5A but was highly expressed in lower motor neurons in 2-week-old or older mice, suggesting its important roles in the maintenance of motor neurons rather than in their formation, such as axonal elongation. Because a large part of KIF5s in adult motor neurons were expected to be KIF5C, we generated mice lacking the kif5C gene to investigate the functions of KIF5C in neurons in living animals. The mutant mice showed smaller brain size but were viable and did not show gross changes in the nervous system. Closer examinations revealed the relative loss of motor neurons to sensory neurons. Because three KIF5s showed high similarity in the amino acid sequence, could rescue the KIF5B mutant cells, and could form heterodimers, we think that there are functional redundancy among the three KIF5s and that KIF5A and KIF5B prevented the KIF5C null mice from the severe phenotype.
- Bohm KJ, Stracke R, Unger E
- Speeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parameters.
- Cell Biol Int. 2000; 24: 335-41
- Display abstract
So far, there has been a discrepancy between the velocities of kinesin-dependent microtubule motility measured in vitro and within cells. By changing ATP, Mg(2+), and kinesin concentrations, pH and ionic strength, we tried to find conditions that favour microtubule gliding across kinesin-covered glass surfaces. For porcine brain kinesin, we found that raising the molar Mg(2+)/ATP ratio can substantially elevate gliding velocity. Gliding became also faster after temperature elevation or lowering the number of kinesin molecules bound to the glass surface. The highest mean gliding velocity (1.8 microm/s+/-0.09 microm/s), approaching velocities measured for anterograde transport in vivo, was achieved by combination of favourable factors (2.5 m m ATP, 12.5 m m Mg(2+), 37 degrees C, 450 kinesin molecules/microm(2)).
- Vale RD, Case R, Sablin E, Hart C, Fletterick R
- Searching for kinesin's mechanical amplifier.
- Philos Trans R Soc Lond B Biol Sci. 2000; 355: 449-57
- Display abstract
Kinesin, a microtubule-based motor, and myosin, an actin-based motor, share a similar core structure, indicating that they arose from a common ancestor. However, kinesin lacks the long lever-arm domain that is believed to drive the myosin power stroke. Here, we present evidence that a much smaller region of ca. 10-40 amino acids serves as a mechanical element for kinesin motor proteins. These 'neck regions' are class conserved and have distinct structures in plus-end and minus-end-directed kinesin motors. Mutagenesis studies also indicate that the neck regions are involved in coupling ATP hydrolysis and energy into directional motion along the microtubule. We suggest that the kinesin necks drive motion by undergoing a conformational change in which they detach and re-dock onto the catalytic core during the ATPase cycle. Thus, kinesin and myosin have evolved unique mechanical elements that amplify small, nucleotide-dependent conformational changes that occur in their similar catalytic cores.
- Cross RA, Crevel I, Carter NJ, Alonso MC, Hirose K, Amos LA
- The conformational cycle of kinesin.
- Philos Trans R Soc Lond B Biol Sci. 2000; 355: 459-64
- Display abstract
The stepping mechanism of kinesin can be thought of as a programme of conformational changes. We briefly review protein chemical, electron microscopic and transient kinetic evidence for conformational changes, and working from this evidence, outline a model for the mechanism. In the model, both kinesin heads initially trap Mg x ADP. Microtubule binding releases ADP from one head only (the trailing head). Subsequent ATP binding and hydrolysis by the trailing head progressively accelerate attachment of the leading head, by positioning it closer to its next site. Once attached, the leading head releases its ADP and exerts a sustained pull on the trailing head. The rate of closure of the molecular gate which traps ADP on the trailing head governs its detachment rate. A speculative but crucial coordinating feature is that this rate is strain sensitive, slowing down under negative strain and accelerating under positive strain.
- Woehlke G, Schliwa M
- Directional motility of kinesin motor proteins.
- Biochim Biophys Acta. 2000; 1496: 117-27
- Display abstract
Kinesin motor proteins are molecules capable of moving along microtubules. They share homology in the so-called core motor domain which acts as a microtubule-dependent ATPase. The surprising finding that different members of the superfamily move in opposite directions along microtubules despite their close similarity has stimulated intensive research on the determinants of motor directionality. This article reviews recent biophysical, biochemical, structural and mutagenic studies that contributed to the elucidation of the mechanisms that cause directional motion of kinesin motor proteins.
- Xing J, Wriggers W, Jefferson GM, Stein R, Cheung HC, Rosenfeld SS
- Kinesin has three nucleotide-dependent conformations. Implications for strain-dependent release.
- J Biol Chem. 2000; 275: 35413-23
- Display abstract
Although crystallographic information is available on several nucleotide-induced states in myosin, little is known about the corresponding structural changes in kinesin, since a crystallographic model is only available for the kinesin:ADP complex. This makes it difficult to characterize at a molecular level the structural changes that occur in this motor through the course of its ATPase cycle. In this study, we report on the production of a series of single tryptophan mutants of a monomeric human kinesin motor domain, which demonstrate nucleotide-dependent changes in microtubule affinity that are similar to wild type. We have used these mutations to measure intramolecular distances in both strong and weak binding states, using fluorescence resonance energy transfer. This work provides direct evidence that movement of the switch II loop and helix are essential to mediate communication between the catalytic and microtubule binding sites, evidence that is supported as well by molecular modeling. Kinetic studies of fluorescent nucleotide binding to these mutants are consistent with these distance changes, and demonstrate as well that binding of ADP produces two structural transitions, neither of which are identical to that produced by the binding of ATP. This study provides a basis for understanding current structural models of the kinesin mechanochemical cycle.
- Okada Y, Hirokawa N
- Mechanism of the single-headed processivity: diffusional anchoring between the K-loop of kinesin and the C terminus of tubulin.
- Proc Natl Acad Sci U S A. 2000; 97: 640-5
- Display abstract
A motor-domain construct of KIF1A, a single-headed kinesin superfamily protein, was demonstrated to take more than 600 steps before detaching from a microtubule. However, its molecular mechanism remained unclear. Here we demonstrate the nucleotide-dependent binding between the lysine-rich, highly positively charged loop 12 of the KIF1A motor domain (K-loop) and the glutamate-rich, highly negatively charged C-terminal region of tubulin (E-hook). This binding did not contribute in the strong binding state but only in the weak binding state. This binding was demonstrated to be essential for the single-headed processivity by functioning as the anchor for the one-dimensional simple Brownian movement in the weak binding state. This Brownian movement will allow the small KIF1A motor domain to span the distance between the binding sites on microtubule and also will give the diffusive nature to the movement of single KIF1A molecules. These observations quantitatively fitted well to the predictions made from our Brownian motor model on the mechanism of the single-headed processive movement.
- Signor D, Rose LS, Scholey JM
- Analysis of the roles of kinesin and dynein motors in microtubule-based transport in the Caenorhabditis elegans nervous system.
- Methods. 2000; 22: 317-25
- Display abstract
The heteromeric kinesins constitute a subfamily of kinesin-related motor complexes that function in several distinct intracellular transport events. The founding member of this subfamily, heterotrimeric kinesin II, has been purified and characterized from early sea urchin embryos, where it was shown using antibody perturbation to be required for the synthesis of motile cilia, presumably by driving the anterograde transport of raft complexes. To further characterize heteromeric kinesin transport pathways, and to attempt to identify cargo molecules, we are using the model organism Caenorhabditis elegans to exploit its well-characterized nervous system and simple genetics. Here we describe methods for large-scale nematode growth and partial purification of kinesin-related holoenzymes from C. elegans, and an in vivo transport assay that allows the direct labeling and visualization of motor complexes and putative cargo molecules moving in living C. elegans neurons. This transport assay is being used to characterize the in vivo transport properties of motor enzymes in living cells, and to exploit a number of existing mutations in C. elegans that may represent constituents of heteromeric kinesin-driven transport pathways, for example, the retrograde intraflagellar transport motor CHE-3 dynein, as well as cargo molecules and/or regulatory molecules.
- Sharp DJ, Rogers GC, Scholey JM
- Roles of motor proteins in building microtubule-based structures: a basic principle of cellular design.
- Biochim Biophys Acta. 2000; 1496: 128-41
- Display abstract
Eukaryotic cells must build a complex infrastructure of microtubules (MTs) and associated proteins to carry out a variety of functions. A growing body of evidence indicates that a major function of MT-associated motor proteins is to assemble and maintain this infrastructure. In this context, we examine the mechanisms utilized by motors to construct the arrays of MTs and associated proteins contained within the mitotic spindle, neuronal processes, and ciliary axonemes. We focus on the capacity of motors to drive the 'sliding filament mechanism' that is involved in the construction and maintenance of spindles, axons and dendrites, and on a type of particle transport called 'intraflagellar transport' which contributes to the assembly and maintenance of axonemes.
- Barroso C, Chan J, Allan V, Doonan J, Hussey P, Lloyd C
- Two kinesin-related proteins associated with the cold-stable cytoskeleton of carrot cells: characterization of a novel kinesin, DcKRP120-2.
- Plant J. 2000; 24: 859-68
- Display abstract
We have previously described the biochemical isolation of 65 kDa and 120 kDa microtubule-associated proteins from carrot cytoskeletons. The 65 kDa MAPs have subsequently been shown to be structural MAPs that reconstitute 30 nm cross-bridges of the kind that maintain cortical microtubules in parallel groups. By exploiting its avid binding to microtubules, we have now devised a method for isolating MAP120 from protoplast extracts, and shown that it has properties of a kinesin-related protein. MAP120 segregates with the cold stable pool of microtubules in carrot cytoskeletons, whilst the 65 kDa MAPs are also associated with the cold-sensitive microtubules. On gradient gels, MAP120 resolves as two kinesin-like bands. We report the isolation of a carrot cDNA, DcKRP120-2, corresponding to a novel kinesin of the BimC class known to move to the plus ends of microtubules. Antibodies raised against specific expressed sequences recognize the upper band, while the lower band is recognized by antibodies to the tobacco kinesin-related protein, TKRP125. We have also isolated a partial genomic carrot DNA, DcKRP120-1, homologous to the motor region of tobacco TKRP125. Immunofluorescence of the two proteins produces different staining patterns. Anti-TKRP125 labels the cortical microtubules and the pre-prophase band, but anti-DcKRP120-2 does so only weakly. Both clearly stain the spindle and the phragmoplast, but in a proportion of cells anti-DcKRP120-2 strongly decorates the phragmoplast mid-line where the plus ends of the microtubules overlap. We discuss the potential roles of these proteins during the microtubule cycle.
- Yabe JT, Jung C, Chan WK, Shea TB
- Phospho-dependent association of neurofilament proteins with kinesin in situ.
- Cell Motil Cytoskeleton. 2000; 45: 249-62
- Display abstract
Recent studies demonstrate co-localization of kinesin with neurofilament (NF) subunits in culture and suggest that kinesin participates in NF subunit distribution. We sought to determine whether kinesin was also associated with NF subunits in situ. Axonal transport of NF subunits in mouse optic nerve was perturbed by the microtubule (MT)-depolymerizing drug vinblastine, indicating that NF transport was dependent upon MT dynamics. Kinesin co-precipitated during immunoprecipitation of NF subunits from optic nerve. The association of NFs and kinesin was regulated by NF phosphorylation, since (1) NF subunits bearing developmentally delayed phospho-epitopes did not co-purify in a microtubule motor preparation from CNS while less phosphorylated forms did; (2) subunits bearing these phospho-epitopes were selectively not co-precipitated with kinesin; and (3) phosphorylation under cell-free conditions diminished the association of NF subunits with kinesin. The nature and extent of this association was further examined by intravitreal injection of (35)S-methionine and monitoring NF subunit transport along optic axons. As previously described by several laboratories, the wave of NF subunits underwent a progressive broadening during continued transport. The front, but not the trail, of this broadening wave of NF subunits was co-precipitated with kinesin, indicating that (1) the fastest-moving NFs were associated with kinesin, and (2) that dissociation from kinesin may foster trailing of NF subunits during continued transport. These data suggest that kinesin participates in NF axonal transport either by directly translocating NFs and/or by linking NFs to transporting MTs. Both Triton-soluble as well as cytoskeleton-associated NF subunits were co-precipitated with kinesin; these data are considered in terms of the form(s) in which NF subunits undergo axonal transport.
- Wade RH, Kozielski F
- Structural links to kinesin directionality and movement.
- Nat Struct Biol. 2000; 7: 456-60
- Display abstract
The kinesin motor proteins generate directional movement along microtubules and are involved in many vital processes, including cell division, in eukaryotes. The kinesin superfamily is characterized by a conserved motor domain of approximately 320 residues. Dimeric constructs of N and C class kinesins, with the motor domains at opposite ends of the heavy chain, move towards microtubule plus and minus ends, respectively. Their crystal structures differ mainly in the region linking the motor domain core to the alpha-helical coiled coil dimerization domain. Chimeric kinesins show that regions outside of the motor domain core determine the direction of movement and mutations in the linker region have a strong effect on motility. Recent work on chimeras and mutants is discussed in a structural context giving insights to possible molecular mechanisms of kinesin directionality and motility.
- Cai G et al.
- Identification and characterization of a novel microtubule-based motor associated with membranous organelles in tobacco pollen tubes.
- Plant Cell. 2000; 12: 1719-36
- Display abstract
Pollen tube growth depends on the differential distribution of organelles and vesicles along the tube. The role of microtubules in organelle movement is uncertain, mainly because information at the molecular level is limited. In an effort to understand the molecular basis of microtubule-based movement, we isolated from tobacco pollen tubes polypeptides that cosediment with microtubules in an ATP-dependent manner. Major polypeptides released from microtubules by ATP (ATP-MAPs) had molecular masses of 90, 80, and 41 kD. Several findings indicate that the 90-kD ATP-MAP is a kinesin-related motor: binding of the polypeptide to microtubules was enhanced by the nonhydrolyzable ATP analog AMP-PNP; the 90-kD polypeptide reacted specifically with a peptide antibody directed against a highly conserved region in the motor domain of the kinesin superfamily; purified 90-kD ATP-MAP induced microtubules to glide in motility assays in vitro; and the 90-kD ATP-MAP cofractionated with microtubule-activated ATPase activity. Immunolocalization studies indicated that the 90-kD ATP-MAP binds to organelles associated with microtubules in the cortical region of the pollen tube. These findings suggest that the 90-kD ATP-MAP is a kinesin-related microtubule motor that moves organelles in the cortex of growing pollen tubes.
- Amos LA
- Kinesin sticks its neck out.
- Nat Cell Biol. 2000; 2: 156-156
- Bowman AB et al.
- Kinesin-dependent axonal transport is mediated by the sunday driver (SYD) protein.
- Cell. 2000; 103: 583-94
- Display abstract
A broadly conserved membrane-associated protein required for the functional interaction of kinesin-I with axonal cargo was identified. Mutations in sunday driver (syd) and the axonal transport motor kinesin-I cause similar phenotypes in Drosophila, including aberrant accumulations of axonal cargoes. GFP-tagged mammalian SYD localizes to tubulovesicular structures that costain for kinesin-I and a marker of the secretory pathway. Coimmunoprecipitation analysis indicates that mouse SYD forms a complex with kinesin-I in vivo. Yeast two-hybrid analysis and in vitro interaction studies reveal that SYD directly binds kinesin-I via the tetratricopeptide repeat (TPR) domain of kinesin light chain (KLC) with K(d) congruent with 200 nM. We propose that SYD mediates the axonal transport of at least one class of vesicles by interacting directly with KLC.
- Gilbert SP, Mackey AT
- Kinetics: a tool to study molecular motors.
- Methods. 2000; 22: 337-54
- Display abstract
Molecular motors are enzymes that couple the energy from nucleoside triphosphate hydrolysis to movement along a filament lattice. The three cytoskeletal motor superfamilies include myosin, dynein, and kinesin. However, in the last decade it has become apparent that the nucleic acid-based enzymes (DNA and RNA polymerases as well as the DNA helicases) share a number of mechanistic features in common with the microtubule and actin motors despite the fact that their cellular functions are so different. This review addresses the mechanistic approaches that have been used to study molecular motors. We discuss the basic biochemical techniques used to characterize a protein preparation, including active site determination and steady-state kinetics. In addition, we present the transient-state kinetic approaches used to define a mechanochemical cycle. We attempt to integrate the information obtained from kinetic studies within the context of motility results to provide a better understanding of the contribution of each approach for dissecting unidirectional force generation.
- Tomishige M, Vale RD
- Controlling kinesin by reversible disulfide cross-linking. Identifying the motility-producing conformational change.
- J Cell Biol. 2000; 151: 1081-92
- Display abstract
Conventional kinesin, a dimeric molecular motor, uses ATP-dependent conformational changes to move unidirectionally along a row of tubulin subunits on a microtubule. Two models have been advanced for the major structural change underlying kinesin motility: the first involves an unzippering/zippering of a small peptide (neck linker) from the motor catalytic core and the second proposes an unwinding/rewinding of the adjacent coiled-coil (neck coiled-coil). Here, we have tested these models using disulfide cross-linking of cysteines engineered into recombinant kinesin motors. When the neck linker motion was prevented by cross-linking, kinesin ceased unidirectional movement and only showed brief one-dimensional diffusion along microtubules. Motility fully recovered upon adding reducing agents to reverse the cross-link. When the neck linker motion was partially restrained, single kinesin motors showed biased diffusion towards the microtubule plus end but could not move effectively against a load imposed by an optical trap. Thus, partial movement of the neck linker suffices for directionality but not for normal processivity or force generation. In contrast, preventing neck coiled-coil unwinding by disulfide cross-linking had relatively little effect on motor activity, although the average run length of single kinesin molecules decreased by 30-50%. These studies indicate that conformational changes in the neck linker, not in the neck coiled-coil, drive processive movement by the kinesin motor.
- Thorn KS, Ubersax JA, Vale RD
- Engineering the processive run length of the kinesin motor.
- J Cell Biol. 2000; 151: 1093-100
- Display abstract
Conventional kinesin is a highly processive molecular motor that takes several hundred steps per encounter with a microtubule. Processive motility is believed to result from the coordinated, hand-over-hand motion of the two heads of the kinesin dimer, but the specific factors that determine kinesin's run length (distance traveled per microtubule encounter) are not known. Here, we show that the neck coiled-coil, a structure adjacent to the motor domain, plays an important role in governing the run length. By adding positive charge to the neck coiled-coil, we have created ultra-processive kinesin mutants that have fourfold longer run lengths than the wild-type motor, but that have normal ATPase activity and motor velocity. Conversely, adding negative charge on the neck coiled-coil decreases the run length. The gain in processivity can be suppressed by either proteolytic cleavage of tubulin's negatively charged COOH terminus or by high salt concentrations. Therefore, modulation of processivity by the neck coiled-coil appears to involve an electrostatic tethering interaction with the COOH terminus of tubulin. The ability to readily increase kinesin processivity by mutation, taken together with the strong sequence conservation of the neck coiled-coil, suggests that evolutionary pressures may limit kinesin's run length to optimize its in vivo function.
- Taylor EW, Borisy GG
- Kinesin processivity.
- J Cell Biol. 2000; 151: 279-279
- Terada S, Hirokawa N
- Moving on to the cargo problem of microtubule-dependent motors in neurons.
- Curr Opin Neurobiol. 2000; 10: 566-73
- Display abstract
Vigorous investigation has finally begun to shed light on the cargo problem of the microtubule-dependent motors, kinesin and dynein superfamily proteins. Biochemical observations have suggested that the potential cargoes of certain populations of motor proteins seem to be in vesicle-form, each vesicle possessing specific functional marker molecules. In addition to the close relationship between microtubule-dependent motors and cargoes in vesicle-form, kinesin has also been highlighted as an apparent driving force for another cargo in non-vesicle-form, cytoplasmic protein. On the basis of new biophysical and cell-biological evidence, the controversy over the movement of cytoplasmic cargoes has entered a new phase.
- Hoenger A et al.
- A new look at the microtubule binding patterns of dimeric kinesins.
- J Mol Biol. 2000; 297: 1087-103
- Display abstract
The interactions of monomeric and dimeric kinesin and ncd constructs with microtubules have been investigated using cryo-electron microscopy (cryo-EM) and several biochemical methods. There is a good consensus on the structure of dimeric ncd when bound to a tubulin dimer showing one head attached directly to tubulin, and the second head tethered to the first. However, the 3D maps of dimeric kinesin motor domains are still quite controversial and leave room for different interpretations. Here we reinvestigated the microtubule binding patterns of dimeric kinesins by cryo-EM and digital 3D reconstruction under different nucleotide conditions and different motor:tubulin ratios, and determined the molecular mass of motor-tubulin complexes by STEM. Both methods revealed complementary results. We found that the ratio of bound kinesin motor-heads to alphabeta-tubulin dimers was never reaching above 1.5 irrespective of the initial mixing ratios. It appears that each kinesin dimer occupies two microtubule-binding sites, provided that there is a free one nearby. Thus the appearances of different image reconstructions can be explained by non-specific excess binding of motor heads. Consequently, the use of different apparent density distributions for docking the X-ray structures onto the microtubule surface leads to different and mutually exclusive models. We propose that in conditions of stoichiometric binding the two heads of a kinesin dimer separate and bind to different tubulin subunits. This is in contrast to ncd where the two heads remain tightly attached on the microtubule surface. Using dimeric kinesin molecules crosslinked in their neck domain we also found that they stabilize protofilaments axially, but not laterally, which is a strong indication that the two heads of the dimers bind along one protofilament, rather than laterally bridging two protofilaments. A molecular walking model based on these results summarizes our conclusions and illustrates the implications of symmetry for such models.
- Chui KK et al.
- Roles of two homotetrameric kinesins in sea urchin embryonic cell division.
- J Biol Chem. 2000; 275: 38005-11
- Display abstract
To improve our understanding of the roles of microtubule cross-linking motors in mitosis, we analyzed two sea urchin embryonic kinesin-related proteins. It is striking to note that both of these proteins behave as homotetramers, but one behaves as a more compact molecule than the other. These observations suggest that these two presumptive motors could cross-link microtubules into bundles with different spacing. Both motors localize to mitotic spindles, and antibody microinjection experiments suggest that they have mitotic functions. Thus, one of these kinesin-related proteins may cross-link spindle microtubules into loose bundles that are "tightened" by the other.
- Bohm KJ, Stracke R, Baum M, Zieren M, Unger E
- Effect of temperature on kinesin-driven microtubule gliding and kinesin ATPase activity.
- FEBS Lett. 2000; 466: 59-62
- Display abstract
DeCuevas et al. [J. Cell Biol. 116 (1992) 957-965] demonstrated by circular dichroism spectroscopy for the kinesin stalk fragment that shifting temperature from 25 to 30 degrees C caused a conformational transition. To gain insight into functional consequences of such a transition, we studied the temperature dependence of a full-length kinesin by measuring both the velocity of microtubule gliding across kinesin-coated surfaces and microtubule-promoted kinesin ATPase activity in solution. The corresponding Arrhenius plots revealed distinct breaks at 27 degrees C, corroborating the temperature-dependent conformational transition for a motility-competent full-length kinesin. Microtubules were found to glide up to 45 degrees C; at higher temperatures, kinesin was irreversibly damaged.
- Brendza KM, Sontag CA, Saxton WM, Gilbert SP
- A kinesin mutation that uncouples motor domains and desensitizes the gamma-phosphate sensor.
- J Biol Chem. 2000; 275: 22187-95
- Display abstract
Conventional kinesin is a processive, microtubule-based motor protein that drives movements of membranous organelles in neurons. Amino acid Thr(291) of Drosophila kinesin heavy chain is identical in all superfamily members and is located in alpha-helix 5 on the microtubule-binding surface of the catalytic motor domain. Substitution of methionine at Thr(291) results in complete loss of function in vivo. In vitro, the T291M mutation disrupts the ATPase cross-bridge cycle of a kinesin motor/neck construct, K401-4 (Brendza, K. M., Rose, D. J., Gilbert, S. P., and Saxton, W. M. (1999) J. Biol. Chem. 274, 31506-31514). The pre-steady-state kinetic analysis presented here shows that ATP binding is weakened significantly, and the rate of ATP hydrolysis is increased. The mutant motor also fails to distinguish ATP from ADP, suggesting that the contacts important for sensing the gamma-phosphate have been altered. The results indicate that there is a signaling defect between the motor domains of the T291M dimer. The ATPase cycles of the two motor domains appear to become kinetically uncoupled, causing them to work more independently rather than in the strict, coordinated fashion that is typical of kinesin.
- Amos LA
- Focusing-in on microtubules.
- Curr Opin Struct Biol. 2000; 10: 236-41
- Display abstract
A good approximation of the atomic structure of a microtubule has been derived from docking the high-resolution structure of tubulin, solved by electron crystallography, into lower resolution maps of whole microtubules. Some structural interactions with other molecules, including nucleotides, drugs, motor proteins and microtubule-associated proteins, can now be predicted.
- Shimizu T, Thorn KS, Ruby A, Vale RD
- ATPase kinetic characterization and single molecule behavior of mutant human kinesin motors defective in microtubule-based motility.
- Biochemistry. 2000; 39: 5265-73
- Display abstract
Conventional kinesin is a microtubule-based motor protein that is an important model system for understanding mechanochemical transduction. To identify regions of the kinesin protein that participate in microtubule binding and force production, Woehlke et al. [(1997) Cell 90, 207-216] generated 35 alanine mutations in solvent-exposed residues. Here, we have performed presteady-state kinetic and single molecule motility analyses on three of these mutants [Y138A, loop 11 triple (L248A/D249A/E250A), and E311A] that exhibited a similar approximately 3-fold reduction in both microtubule gliding velocity and microtubule-stimulated ATPase activity. All mutants showed normal second-order ATP binding kinetics, indicating correct folding of the active site. The Y138A and loop 11 triple mutants were defective both in nucleotide hydrolysis and in microtubule-stimulated ADP release rates, the latter suggesting a defect in allosteric communication between the microtubule and the active site. A single molecule fluorescence assay further revealed that the loop 11 mutant is defective in initiating processive motion, suggesting that this loop is important for the initial contact between kinesin and the microtubule. Y138A, on the other hand, can bind to the microtubule normally but cannot move processively. For E311A, neither the rate of nucleotide hydrolysis nor ADP release could account for its slower ATPase and gliding velocity, which suggests that either phosphate release or a conformational transition is rate-limiting in this mutant. The single molecule assay showed that E311A has a reduced velocity of movement, but is not defective in processivity. Thus, while these mutants behave similarly in solution ATPase and multiple motor gliding assays, kinetic and single molecule analyses reveal defects in distinct processes in kinesin's mechanochemical cycle.
- Kawaguchi K, Ishiwata S
- Temperature dependence of force, velocity, and processivity of single kinesin molecules.
- Biochem Biophys Res Commun. 2000; 272: 895-9
- Display abstract
Using the bead assay in optical microscopy equipped with optical tweezers, we have examined the effect of temperature on the gliding velocity, force, and processivity of single kinesin molecules interacting with a microtubule between 15 and 35 degrees C. The gliding velocity increased with the Arrhenius activation energy of 50 kJ/mol, consistent with the temperature dependence of the microtubule-dependent ATPase activity. Also, the average run length, i.e., a measure of processivity of kinesin, increased on increasing temperature. On the other hand, the generated force was independent of temperature, 7.34 +/- 0.33 pN (average +/- S.D., n = 70). The gliding velocities decreased almost linearly with an increase in force irrespective of temperature, implying that the efficiency of mechano-chemical energy conversion is maintained constant in this temperature range. Thus, we suggest that the force generation is attributable to the temperature-insensitive nucleotide-binding state(s) and/or conformational change(s) of kinesin-microtubule complex, whereas the gliding velocity is determined by the ATPase rate.
- Woehlke G, Schliwa M
- Walking on two heads: the many talents of kinesin.
- Nat Rev Mol Cell Biol. 2000; 1: 50-8
- Display abstract
The gallop of a race horse and the minute excursions of a cellular vesicle have one thing in common: they are based on the directional movement of proteins termed molecular motors -- many trillions in the case of the horse, just a few in the case of the cell vesicle. These tiny machines take nanometre steps on a millisecond timescale to drive all biological movements. Over the past 15 years new biochemical and biophysical approaches have allowed us to take a giant step forward in understanding the molecular basis of motor mechanics.
- Ng HL, Kopka ML, Dickerson RE
- The structure of a stable intermediate in the A <--> B DNA helix transition.
- Proc Natl Acad Sci U S A. 2000; 97: 2035-9
- Display abstract
The DNA dodecamer CATGGGCCCATG in a crystal structure of resolution 1.3 A has a conformation intermediate between A and B DNA. This trapping of a stable intermediate suggests that the A and B DNA families are not discrete, as previously believed. The structure supports a base-centered rather than a backbone-centered mechanism for the A <--> B transition mediated by guanine tracts. Interconversion between A and B DNA provides another means for regulating protein-DNA recognition.
- Koonce MP
- Dictyostelium, a model organism for microtubule-based transport.
- Protist. 2000; 151: 17-25
- Seiler S, Kirchner J, Horn C, Kallipolitou A, Woehlke G, Schliwa M
- Cargo binding and regulatory sites in the tail of fungal conventional kinesin.
- Nat Cell Biol. 2000; 2: 333-8
- Display abstract
Here, using a quantitative in vivo assay, we map three regions in the carboxy terminus of conventional kinesin that are involved in cargo association, folding and regulation, respectively. Using C-terminal and internal deletions, point mutations, localization studies, and an engineered 'minimal' kinesin, we identify five heptads of a coiled-coil domain in the kinesin tail that are necessary and sufficient for cargo association. Mutational analysis and in vitro ATPase assays highlight a conserved motif in the globular tail that is involved in regulation of the motor domain; a region preceding this motif participates in folding. Although these sites are spatially and functionally distinct, they probably cooperate during activation of the motor for cargo transport.
- Endow SA, Higuchi H
- A mutant of the motor protein kinesin that moves in both directions on microtubules.
- Nature. 2000; 406: 913-6
- Display abstract
Molecular motors move directionally to either the plus or the minus end of microtubules or actin filaments. Kinesin moves towards microtubule plus ends, whereas the kinesin-related Ncd motor moves to the minus ends. The 'neck'--the region between the stalk and motor domain--is required for Ncd to move to microtubule minus ends, but the mechanism underlying directional motor movement is not understood. Here we show that a single amino-acid change in the Ncd neck causes the motor to reverse directions and move with wild-type velocities towards the plus or minus end; thus, the neck is functional but directionality is defective. Mutation of a motor-core residue that touches the neck residue in crystal structures also results in movement in both directions, indicating that directed movement to the minus end requires interactions of the neck and motor core. Low-density laser-trap assays show that a conformational change or working stroke of the Ncd motor is directional and biased towards the minus end, whereas that of the neck mutant occurs in either direction. We conclude that the directional bias of the working stroke is dependent on neck/motor core interactions. Absence of these interactions removes directional constraints and permits movement in either direction.
- Hirose K et al.
- Structural comparison of dimeric Eg5, Neurospora kinesin (Nkin) and Ncd head-Nkin neck chimera with conventional kinesin.
- EMBO J. 2000; 19: 5308-14
- Display abstract
Cryo-electron microscopy and 3D image reconstruction of microtubules saturated with kinesin dimers has shown one head bound to tubulin, the other free. The free head of rat kinesin sits on the top right of the bound head (with the microtubule oriented plus-end upwards) in the presence of 5'-adenylylimido-diphosphate (AMPPNP) and on the top left in nucleotide-free solutions. To understand the relevance of this movement, we investigated other dimeric plus-end-directed motors: Neurospora kinesin (Nkin); Eg5, a slow non-processive kinesin; and a chimera of Ncd heads attached to Nkin necks. In the AMPPNP (ATP-like) state, all dimers have the free head to the top right. In the absence of nucleotide, the free head of an Nkin dimer appears to occupy alternative positions to either side of the bound head. Despite having the Nkin neck, the free head of the chimera was only seen to the top right of the bound head. Eg5 also has the free head mostly to the top right. We suggest that processive movement may require kinesins to move their heads in alternative ways.
- Cross RA
- Motor proteins. Directing direction.
- Nature. 2000; 406: 839-40
- Sablin EP
- Kinesins and microtubules: their structures and motor mechanisms.
- Curr Opin Cell Biol. 2000; 12: 35-41
- Display abstract
Atomic resolution three-dimensional structures of two oppositely directed kinesin motors - conventional kinesin and non-claret disjunctional (ncd) protein - are now available in their functional dimeric form. A detailed model of the microtubule has also been recently obtained by docking the 3.7 A structure of tubulin into a 20 A map of the microtubule. Recent structural studies of kinesin motors and their microtubule tracks are contributing to our current understanding of kinesin motor mechanisms.
- Rogers SL, Gelfand VI
- Membrane trafficking, organelle transport, and the cytoskeleton.
- Curr Opin Cell Biol. 2000; 12: 57-62
- Display abstract
Cytoskeleton-associated motor proteins typically drive organelle movements in eukaryotic cells in a manner that is tightly regulated, both spatially and temporally. In the past year, a novel organelle transport mechanism utilizing actin polymerization was described. Important advances were also made in the assignment of functions to several new motors and in our understanding of how motor proteins are regulated during organelle transport. In addition, insights were gained into how and why organelles are transported cooperatively along the microtubule and actin cytoskeletons, and into the importance of motor-mediated transport in the organization of the cytoskeleton itself.
- Foster KA, Gilbert SP
- Kinetic studies of dimeric Ncd: evidence that Ncd is not processive.
- Biochemistry. 2000; 39: 1784-91
- Display abstract
Ncd is a kinesin-related motor protein which drives movement to the minus-end of microtubules. The kinetics of Ncd were investigated using the dimeric construct MC1 (Leu(209)-Lys(700)) expressed in Escherichia coli strain BL21(DE) as a nonfusion protein [Chandra, R., Salmon, E. D., Erickson, H. P., Lockhart, A., and Endow, S. A. (1993) J. Biol. Chem. 268, 9005-9013]. Acid chemical quench flow methods were used to measure directly the rate of ATP hydrolysis, and stopped-flow kinetic methods were used to determine the kinetics of mantATP binding, mantADP release, dissociation of MC1 from the microtubule, and binding of MC1 to the microtubule. The results define a minimal kinetic mechanism, M.N + ATP M.N.ATP M.N.ADP.P N. ADP.P N.ADP + P M.N.ADP M.N + ADP, where N, M, and P represent Ncd, microtubules, and inorganic phosphate respectively, with k(+1) = 2.3 microM(-1) s(-1), k(+2) =23 s(-1), k(+3) =13 s(-1), k(+5)= 0.7 microM(-)(1) s(-)(1), and k(+6) = 3.7 s(-)(1). Phosphate release (k(+4)) was not measured directly although it is assumed to be fast relative to ADP release because Ncd is purified with ADP tightly bound at the active site. ATP hydrolysis occurs at 23 s(-)(1) prior to Ncd dissociation at 13 s(-)(1). The pathway for ATP-promoted detachment (steps 1-3) of Ncd from the microtubule is comparable to kinesin's. However, there are two major differences between the mechanisms of Ncd and kinesin. In contrast to kinesin, mantADP release for Ncd at 3.7 s(-)(1) is the slowest step in the pathway and is believed to limit steady-state turnover. Additionally, the burst amplitude observed in the pre-steady-state acid quench experiments is stoichiometric, indicating that Ncd, in contrast to kinesin, is not processive for ATP hydrolysis.
- Hirokawa N
- Stirring up development with the heterotrimeric kinesin KIF3.
- Traffic. 2000; 1: 29-34
- Display abstract
KIF3 is a heterotrimeric member of the kinesin superfamily of microtubule associated motors. This functionally diverse family of motor is involved in anterograde transport of membrane bound organelles in neurons and melanosomes, mediates transport between the endoplasmic reticulum and the Golgi, and transports protein complexes within cilia and flagella required for their morphogenesis. Interestingly, a mutation of KIF3, which impairs ciliogenesis in nodal cells, prevents the unidirectional leftward flow (nodal flow) of putative morphogens during embryogenesis, thereby altering the development of left-right asymmetry in mammals.
- Cross RA
- Molecular motors: Kinesin's dynamically dockable neck.
- Curr Biol. 2000; 10: 1246-1246
- Display abstract
Kinesin is a molecular walking machine with two identical motor heads connected to a coiled-coil tail. Details of the coordination mechanism, which causes kinesin to walk directionally, and the tracking mechanism, which guides each detaching head to its next site on the microtubule, are beginning to emerge.
- Chen Yd
- Theoretical formalism for kinesin motility I. Bead movement powered by single one-headed kinesins.
- Biophys J. 2000; 78: 313-21
- Display abstract
The directional movement on a microtubule of a plastic bead connected elastically to a single one-headed kinesin motor is studied theoretically. The kinesin motor can bind and unbind to periodic binding sites on the microtubule and undergo conformational changes while catalyzing the hydrolysis of ATP. An analytic formalism relating the dynamics of the bead and the ATP hydrolysis cycle of the motor is derived so that the calculation of the average velocity of the bead can be easily carried out. The formalism was applied to a simple three-state biochemical model to investigate how the velocity of the bead movement is affected by the external load, the diffusion coefficient of the bead, and the stiffness of the elastic element connecting the bead and the motor. The bead velocity was found to be critically dependent on the diffusion coefficient of the bead and the stiffness of the elastic element. A linear force-velocity relation was found for the model no matter whether the bead velocity was modulated by the diffusion coefficient of the bead or by the externally applied load. The formalism should be useful in modeling the mechanisms of chemimechanical coupling in kinesin motors based on in vitro motility data.
- Pennisi E
- Cell biology. Kinesin movements revealed.
- Science. 2000; 287: 2325-2325
- Stafford P, Brown J, Langford GM
- Interaction of actin- and microtubule-based motors in squid axoplasm probed with antibodies to myosin V and kinesin.
- Biol Bull. 2000; 199: 203-5
- Goldstein LS, Yang Z
- Microtubule-based transport systems in neurons: the roles of kinesins and dyneins.
- Annu Rev Neurosci. 2000; 23: 39-71
- Display abstract
The large size and extreme polarization of neurons is crucial to their ability to communicate at long distances and to form the complex cellular networks of the nervous system. The size, shape, and compartmentalization of these specialized cells must be generated and supported by the cytoskeletal systems of intracellular transport. One of the major systems is the microtubule-based transport system along which kinesin and dynein motor proteins generate force and drive the traffic of many cellular components. This review describes our current understanding of the functions of kinesins and dyneins and how these motor proteins may be harnessed to generate some of the unique properties of neuronal cells.
- Vale RD, Milligan RA
- The way things move: looking under the hood of molecular motor proteins.
- Science. 2000; 288: 88-95
- Display abstract
The microtubule-based kinesin motors and actin-based myosin motors generate motions associated with intracellular trafficking, cell division, and muscle contraction. Early studies suggested that these molecular motors work by very different mechanisms. Recently, however, it has become clear that kinesin and myosin share a common core structure and convert energy from adenosine triphosphate into protein motion using a similar conformational change strategy. Many different types of mechanical amplifiers have evolved that operate in conjunction with the conserved core. This modular design has given rise to a remarkable diversity of kinesin and myosin motors whose motile properties are optimized for performing distinct biological functions.
- Hopkins SC, Vale RD, Kuntz ID
- Inhibitors of kinesin activity from structure-based computer screening.
- Biochemistry. 2000; 39: 2805-14
- Display abstract
Kinesin motor proteins use ATP hydrolysis for transport along microtubules in the cell. We sought to identify small organic ligands to inhibit kinesin's activity. Candidate molecules were identified by computational docking of commercially available compounds using the computer program DOCK. Compounds were docked at either of two sites, and a selection was tested for inhibition of microtubule-stimulated ATPase activity. Twenty-two submillimolar inhibitors were identified. Several inhibitors appeared to be competitive for microtubule binding and not for ATP binding, and three compounds showed 50% inhibition down to single-digit micromolar levels. Most inhibitors grouped into four distinct classes (fluoresceins, phenolphthaleins, anthraquinones, and naphthylene sulfonates). We measured the binding of one inhibitor, rose bengal lactone (RBL), to kinesin (dissociation constant 2.5 &mgr;M) by its increase in steady-state fluorescence anisotropy. The RBL binding site on kinesin was localized by fluorescent resonance energy transfer (FRET) using a donor fluorophore (coumarin) covalently attached at unique, surface-exposed cysteine residues engineered at positions 28, 149, 103, 220, or 330. RBL was found to bind in its original docked site: the pocket cradled by loop 8 and beta-strand 5 in kinesin's three-dimensional structure. These results confirm this region's role in microtubule binding and identify this pocket as a novel binding site for kinesin inhibition.
- Schnitzer MJ, Visscher K, Block SM
- Force production by single kinesin motors.
- Nat Cell Biol. 2000; 2: 718-23
- Display abstract
Motor proteins such as kinesin, myosin and polymerase convert chemical energy into work through a cycle that involves nucleotide hydrolysis. Kinetic rates in the cycle that depend upon load identify transitions at which structural changes, such as power strokes or diffusive motions, are likely to occur. Here we show, by modelling data obtained with a molecular force clamp, that kinesin mechanochemistry can be characterized by a mechanism in which a load-dependent isomerization follows ATP binding. This model quantitatively accounts for velocity data over a wide range of loads and ATP levels, and indicates that movement may be accomplished through two sequential 4-nm substeps. Similar considerations account for kinesin processivity, which is found to obey a load-dependent Michaelis-Menten relationship.
- Hackney DD, Stock MF
- Kinesin's IAK tail domain inhibits initial microtubule-stimulated ADP release.
- Nat Cell Biol. 2000; 2: 257-60
- Display abstract
Kinesin undergoes a global folding conformational change from an extended active conformation at high ionic concentrations to a compact inhibited conformation at physiological ionic concentrations. Here we show that much of the observed ATPase activity of folded kinesin is due to contamination with proteolysis fragments that can still fold, but retain an activated ATPase function. In contrast, kinesin that contains an intact IAK-homology region exhibits pronounced inhibition of its ATPase activity (140-fold in 50 mM KCl) and weak net affinity for microtubules in the presence of ATP, resulting from selective inhibition of the release of ADP upon initial interaction with a microtubule. Subsequent processive cycling is only partially inhibited. Fusion proteins containing residues 883-937 of the kinesin alpha-chain bind tightly to microtubules; exposure of this microtubule-binding site in proteolysed species is probably responsible for their activated ATPase activities at low microtubule concentrations.
- Kikkawa M, Okada Y, Hirokawa N
- 15 A resolution model of the monomeric kinesin motor, KIF1A.
- Cell. 2000; 100: 241-52
- Display abstract
A two-headed structure has been widely believed to be essential for the kinesin molecular motor to move processively on the track, microtubules. However, we have recently demonstrated that a monomeric motor domain construct of KIF1A (C351), a kinesin superfamily protein, moves processively, taking about 700 steps before being detached from microtubules. To elucidate the mechanism of its single-headed processivity, we examined the C351 -MT interaction by mutant analysis and high-resolution cryo-EM. Mutant analysis indicated the importance of a highly positively charged loop, the "K loop," for such processivity. A 15 A resolution structure unambiguously docked with the available atomic models revealed "K loop" as an extra microtubule-binding domain specific to KIF1A, and bound to the C terminus of tubulin. The site-specific cross-linking further confirmed this model.
- Ong LL, Lim AP, Er CP, Kuznetsov SA, Yu H
- Kinectin-kinesin binding domains and their effects on organelle motility.
- J Biol Chem. 2000; 275: 32854-60
- Display abstract
Intracellular organelle motility involves motor proteins that move along microtubules or actin filaments. One of these motor proteins, kinesin, was proposed to bind to kinectin on membrane organelles during movement. Whether kinectin is the kinesin receptor on organelles with a role in organelle motility has been controversial. We have characterized the sites of interaction between human kinectin and conventional kinesin using in vivo and in vitro assays. The kinectin-binding domain on the kinesin tail partially overlaps its head-binding domain and the myosin-Va binding domain. The kinesin-binding domain on kinectin resides near the COOH terminus and enhances the microtubule-stimulated kinesin-ATPase activity, and the overexpression of the kinectin-kinesin binding domains inhibited kinesin-dependent organelle motility in vivo. These data, when combined with other studies, suggest a role for kinectin in organelle motility.
- Smyczynski C, Derancourt J, Chaussepied P
- Regulation of ncd by the oligomeric state of tubulin.
- J Mol Biol. 2000; 295: 325-36
- Display abstract
We have compared the interaction of ncd (non-claret disjunctional), a kinesin related protein, with microtubules and tubulin heterodimer. Ultracentrifugation experiments revealed that the ncd motor domain, residues 335-700 (ncd335), does not induce tubulin polymerization but stabilizes pre-formed microtubules with a maximum effect at a 1:1 ncd335:tubulin ratio. Ncd335 binding to tubulin or microtubules was estimated by following the change in fluorescence polarization of an exogenous dye attached to Cys670 of ncd335. Ncd335 binding to tubulin (containing GTP or GDP-bound) is characterized by a 2:1 stoichiometry, a higher affinity and an increased sensitivity towards salt, ADP, ATP and AMPPNP, as compared with ncd335 binding to microtubules. Maximum ATPases were 0.06-0.08 sec(-1) and 1.8-2.0 sec(-1) for the ncd335-tubulin and ncd335-microtubules complexes, respectively. Only the polymerized complex is fully functional, suggesting the presence of additional contacts between adjacent protofilaments. Moreover, the data reveal that the oligomeric state of microtubules is a potent regulator for the activity of kinesin related proteins.
- Case RB, Rice S, Hart CL, Ly B, Vale RD
- Role of the kinesin neck linker and catalytic core in microtubule-based motility.
- Curr Biol. 2000; 10: 157-60
- Display abstract
Kinesin motor proteins execute a variety of intracellular microtubule-based transport functions [1]. Kinesin motor domains contain a catalytic core, which is conserved throughout the kinesin superfamily, followed by a neck region, which is conserved within subfamilies and has been implicated in controlling the direction of motion along a microtubule [2] [3]. Here, we have used mutational analysis to determine the functions of the catalytic core and the approximately 15 amino acid 'neck linker' (a sequence contained within the neck region) of human conventional kinesin. Replacement of the neck linker with a designed random coil resulted in a 200-500-fold decrease in microtubule velocity, although basal and microtubule-stimulated ATPase rates were within threefold of wild-type levels. The catalytic core of kinesin, without any additional kinesin sequence, displayed microtubule-stimulated ATPase activity, nucleotide-dependent microtubule binding, and very slow plus-end-directed motor activity. On the basis of these results, we propose that the catalytic core is sufficient for allosteric regulation of microtubule binding and ATPase activity and that the kinesin neck linker functions as a mechanical amplifier for motion. Given that the neck linker undergoes a nucleotide-dependent conformational change [4], this region might act in an analogous fashion to the myosin converter, which amplifies small conformational changes in the myosin catalytic core [5,6].
- Gigant B et al.
- The 4 A X-ray structure of a tubulin:stathmin-like domain complex.
- Cell. 2000; 102: 809-16
- Display abstract
Phosphoproteins of the stathmin family interact with the alphabeta tubulin heterodimer (tubulin) and hence interfere with microtubule dynamics. The structure of the complex of GDP-tubulin with the stathmin-like domain of the neural protein RB3 reveals a head-to-tail assembly of two tubulins with a 91-residue RB3 alpha helix in which each copy of an internal duplicated sequence interacts with a different tubulin. As a result of the relative orientations adopted by tubulins and by their alpha and beta subunits, the tubulin:RB3 complex forms a curved structure. The RB3 helix thus most likely prevents incorporation of tubulin into microtubules by holding it in an assembly with a curvature very similar to that of the depolymerization products of microtubules.
- Mackey AT, Gilbert SP
- Moving a microtubule may require two heads: a kinetic investigation of monomeric Ncd.
- Biochemistry. 2000; 39: 1346-55
- Display abstract
Ncd is a minus-end-directed microtubule motor and a member of the kinesin superfamily. The Ncd dimer contains two motor domains, and cooperative interactions between the heads influence the interactions of each respective motor domain with the microtubule. The approach we have taken to understand the cooperativity between the two motor domains is to analyze the ATPase cycle of dimeric MC1 and monomeric MC6. The steps in the ATPase cycle where cooperativity occurs can be identified by comparing the two mechanisms. The rate-limiting step in the MC6 mechanism is ADP release at 3.4 s(-)(1). The observed rate constant for ATP-induced dissociation from the microtubule is 14 s(-)(1). However, the relative amplitude associated with MC6 dissociation is extremely small in comparison to the amplitude associated with dimeric MC1 dissociation kinetics. The amplitude data indicate that monomeric MC6 does not detach from the microtubule during the initial turnovers of ATP, and ATP hydrolysis is uncoupled from movement. The results show that cooperative interactions between the motor domains of the dimer are required for ATP-dependent dissociation; therefore, one function of the partner motor domain may be to weaken the interaction of the adjacent head with the microtubule.
- Endow SA
- Microtubule motors in spindle and chromosome motility.
- Eur J Biochem. 1999; 262: 12-8
- Display abstract
Many of the kinesin microtubule motor proteins discovered during the past 8-9 years have roles in spindle assembly and function or chromosome movement during meiosis or mitosis. The discovery of kinesin motor proteins with a clear involvement in spindle and chromosome motility, together with recent evidence that cytoplasmic dynein plays a role in chromosome distribution, has attracted great interest. The identification of microtubule motors that function in chromosome distribution represents a major advance in understanding the forces that underlie chromosome and spindle movements during cell division.
- Olsen JG, Kadziola A, von Wettstein-Knowles P, Siggaard-Andersen M, Lindquist Y, Larsen S
- The X-ray crystal structure of beta-ketoacyl [acyl carrier protein] synthase I.
- FEBS Lett. 1999; 460: 46-52
- Display abstract
The crystal structure of the fatty acid elongating enzyme beta-ketoacyl [acyl carrier protein] synthase I (KAS I) from Escherichia coli has been determined to 2.3 A resolution by molecular replacement using the recently solved crystal structure of KAS II as a search model. The crystal contains two independent dimers in the asymmetric unit. KAS I assumes the thiolase alpha(beta)alpha(beta)alpha fold. Electrostatic potential distribution reveals an acyl carrier protein docking site and a presumed substrate binding pocket was detected extending the active site. Both subunits contribute to each substrate binding site in the dimer.
- Kapoor TM, Mitchison TJ
- Allele-specific activators and inhibitors for kinesin.
- Proc Natl Acad Sci U S A. 1999; 96: 9106-11
- Display abstract
Members of the kinesin superfamily are force-generating ATPases that drive movement and influence cytoskeleton organization in cells. Often, more than one kinesin is implicated in a cellular process, and many kinesins are proposed to have overlapping functions. By using conventional kinesin as a model system, we have developed an approach to activate or inhibit a specific kinesin allele in the presence of other similar motor proteins. Modified ATP analogs are described that do not activate either conventional kinesin or another superfamily member, Eg5. However, a kinesin allele with Arg-14 in its nucleotide binding pocket mutated to alanine can use a subset of these nucleotide analogs to drive microtubule gliding. Cyclopentyl-ATP is one such analog. Cyclopentyl-adenylylimidodiphosphate, a nonhydrolyzable form of this analog, inhibits the mutant allele in microtubule-gliding assays, but not wild-type kinesin or Eg5. We anticipate that the incorporation of kinesin mutants and allele-specific activators and inhibitors in in vitro assays should clarify the role of individual motor proteins in complex cellular processes.
- Huang JD et al.
- Direct interaction of microtubule- and actin-based transport motors.
- Nature. 1999; 397: 267-70
- Display abstract
The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actin network is proposed to be used for short-range transport, although the mechanism(s) by which this transport is coordinated is poorly understood. For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport. In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination. Here we show that an actin-based vesicle-transport motor, MyoVA, can interact directly with a microtubule-based transport motor, KhcU. As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell. These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules.
- Phelps KK, Walker RA
- N-ethylmaleimide inhibits Ncd motor function by modification of a cysteine in the stalk domain.
- Biochemistry. 1999; 38: 10750-7
- Display abstract
N-Ethylmaleimide (NEM), which reacts readily with exposed sulfhydryl groups, has been shown to inhibit the activity of the microtubule (MT) motors kinesin, Ncd, and dynein. Currently, the mechanism of inhibition is not known for any of these proteins. To investigate the mechanism by which NEM inhibits Ncd, the recombinant Ncd motor-stalk protein MC1 (modified claret 1) was treated with varying concentrations of NEM (0-10 mM) and cosedimentation and ATPase assays were used to assess the effects of modification on MC1 interactions with MTs. In the cosedimentation assay, treatment with =0.1 mM NEM enhanced MC1 binding to MTs in the presence of MgATP but had no effect on MC1 binding to MTs in the presence of MgAMP-PNP. In comparison, treatment with >/=0.5 mM NEM induced aggregation of MC1 and resulted in sedimentation of the motor in the absence of MTs. NEM modification had no effect on the basal ATPase rate but produced a decrease in the MT-stimulated ATPase rate. Labeling of MC1 with [3H]NEM indicated that enhanced MT binding was associated with an average labeling of 1 Cys residue per MC1 polypeptide, while aggregation was associated with an average labeling of 2 Cys residues per MC1 polypeptide. Protein digestion, structural analysis, and mass spectrometry indicate that modification of Cys313 or Cys324 in the stalk domain is correlated with enhanced binding of MC1 to MTs. These results suggest that NEM enhances Ncd binding to MTs by disruption of neck and/or stalk function and demonstrate the importance of this region in motor function.
- Brendza KM, Rose DJ, Gilbert SP, Saxton WM
- Lethal kinesin mutations reveal amino acids important for ATPase activation and structural coupling.
- J Biol Chem. 1999; 274: 31506-14
- Display abstract
To study the relationship between conventional kinesin's structure and function, we identified 13 lethal mutations in the Drosophila kinesin heavy chain motor domain and tested a subset for effects on mechanochemistry. S246F is a moderate mutation that occurs in loop 11 between the ATP- and microtubule-binding sites. While ATP and microtubule binding appear normal, there is a 3-fold decrease in the rate of ATP turnover. This is consistent with the hypothesis that loop 11 provides a structural link that is important for the activation of ATP turnover by microtubule binding. T291M is a severe mutation that occurs in alpha-helix 5 near the center of the microtubule-binding surface. It impairs the microtubule-kinesin interaction and directly effects the ATP-binding pocket, allowing an increase in ATP turnover in the absence of microtubules. The T291M mutation may mimic the structure of a microtubule-bound, partially activated state. E164K is a moderate mutation that occurs at the beta-sheet 5a/loop 8b junction, remote from the ATP pocket. Surprisingly, it causes both tighter ATP-binding and a 2-fold decrease in ATP turnover. We propose that E164 forms an ionic bridge with alpha-helix 5 and speculate that it helps coordinate the alternating site catalysis of dimerized kinesin heavy chain motor domains.
- Seiler S, Plamann M, Schliwa M
- Kinesin and dynein mutants provide novel insights into the roles of vesicle traffic during cell morphogenesis in Neurospora.
- Curr Biol. 1999; 9: 779-85
- Display abstract
BACKGROUND: Kinesin and cytoplasmic dynein are force-generating molecules that move in opposite directions along microtubules. They have been implicated in the directed transport of a wide variety of cellular organelles, but it is unclear whether they have overlapping or largely independent functions. RESULTS: We analyzed organelle transport in kinesin and dynein single mutants, and in a kinesin and dynein double mutant of Neurospora crassa. Remarkably, the simultaneous mutation of kinesin and dynein was not lethal and resulted in an additive phenotype that combined the features of the single mutants. The mutation of kinesin and dynein had opposite effects on the apical and retrograde transport, respectively, of vesicular organelles. In the kinesin mutant, apical movement of submicroscopic, secretory vesicles to the Spitzenkorper - an organelle in the hyphal apex - was defective, whereas the predominantly retrograde movement of microscopic organelles was only slightly reduced. In contrast, the dynein mutant still had a prominent Spitzenkorper, demonstrating that apical transport was intact, but retrograde transport was essentially inhibited completely. A major defect in vacuole formation and dynamics was also evident. In agreement with the observations on apical transport, protein secretion into the medium was markedly inhibited in the kinesin mutant but not in the dynein mutant. CONCLUSIONS: Transport of secretory vesicles is necessary but not sufficient for normal apical extension. A component of retrograde transport, presumably precursors of the vacuole system, is also essential. Our findings provide new information on the role microtubule motors play in cell morphogenesis and suggest that kinesin and cytoplasmic dynein have largely independent functions within separate pathways.
- Hirsch JA, Schubert C, Gurevich VV, Sigler PB
- The 2.8 A crystal structure of visual arrestin: a model for arrestin's regulation.
- Cell. 1999; 97: 257-69
- Display abstract
G protein-coupled signaling is utilized by a wide variety of eukaryotes for communicating information from the extracellular environment. Signal termination is achieved by the action of the arrestins, which bind to activated, phosphorylated G protein-coupled receptors. We describe here crystallographic studies of visual arrestin in its basal conformation. The salient features of the structure are a bipartite molecule with an unusual polar core. This core is stabilized in part by an extended carboxy-terminal tail that locks the molecule into an inactive state. In addition, arrestin is found to be a dimer of two asymmetric molecules, suggesting an intrinsic conformational plasticity. In conjunction with biochemical and mutagenesis data, we propose a molecular mechanism by which arrestin is activated for receptor binding.
- Steinrauf LK, Chiang MY, Shiuan D
- Molecular structure of the amyloid-forming protein kappa I Bre.
- J Biochem (Tokyo). 1999; 125: 422-9
- Display abstract
The molecular structure of the amyloid-forming Bence-Jones protein kappa I Bre has been determined by X-ray crystallography at 2.0 A resolution. The fragment from the kappa chain of immunoprotein contains 107 amino acid residues, and polymerizes in the crystal form into a giant helical spiral, surrounding a cylinder of water 50 A in diameter with a repeat of 77.56 A, containing 12 kappa molecules, plus another 12 molecules from neighboring parallel spirals. The resulting structure has many features which have been found or suggested from studies on the protein fibrils found in amyloid deposits. From the results of the X-ray crystal structure a hypothesis is presented for the structure and formation of the amyloid fibril.
- Desai A, Verma S, Mitchison TJ, Walczak CE
- Kin I kinesins are microtubule-destabilizing enzymes.
- Cell. 1999; 96: 69-78
- Display abstract
Using in vitro assays with purified proteins, we show that XKCM1 and XKIF2, two distinct members of the internal catalytic domain (Kin I) kinesin subfamily, catalytically destabilize microtubules using a novel mechanism. Both XKCM1 and XKIF2 influence microtubule stability by targeting directly to microtubule ends where they induce a destabilizing conformational change. ATP hydrolysis recycles XKCM1/XKIF2 for multiple rounds of action by dissociating a XKCM1/ XKIF2-tubulin dimer complex released upon microtubule depolymerization. These results establish Kin I kinesins as microtubule-destabilizing enzymes, distinguish them mechanistically from kinesin superfamily members that use ATP hydrolysis to translocate along microtubules, and have important implications for the regulation of microtubule dynamics and for the intracellular functions and evolution of the kinesin superfamily.
- Stephen S, Talbot NJ, Stebbings H
- Poly(A) mRNA is attached to insect ovarian microtubules in vivo in a nucleotide-sensitive manner.
- Cell Motil Cytoskeleton. 1999; 43: 159-66
- Display abstract
In ovarioles of hemipteran insects, RNA passes from anteriorly positioned nurse cells to the chain of developing oocytes via extended nutritive tubes. These intercellular connections may reach several millimeters in length. Each nutritive tube is comprised of many thousands of parallel microtubules. We have extracted microtubule bundles from isolated nutritive tubes of Notonecta glauca and, using hybridization techniques, provide evidence of poly(A) mRNA attachment to microtubules in vivo. We also show this attachment to be nucleotide-sensitive, which is typical of a motor protein-mediated interaction. The pattern of nucleotide sensistivity is indicative of a kinesin motor mechanism. We provide evidence that a kinesin is present in the nutritive tube translocation channels and is a component of the mRNA/microtubule bundles isolated and extracted from them. Our findings are consistent with kinesin-driven transport of mRNA along the nutritive tube microtubules.
- Crevel I, Carter N, Schliwa M, Cross R
- Coupled chemical and mechanical reaction steps in a processive Neurospora kinesin.
- EMBO J. 1999; 18: 5863-72
- Display abstract
We show using single molecule optical trapping and transient kinetics that the unusually fast Neurospora kinesin is mechanically processive, and we investigate the coupling between ATP turnover and the mechanical actions of the motor. Beads carrying single two-headed Neurospora kinesin molecules move in discrete 8 nm steps, and stall at approximately 5 pN of retroactive force. Using microtubule-activated release of the fluorescent analogue 2'-(3')-O-(N-methylanthraniloyl) adenosine 5'-diphosphate (mantADP) to report microtubule binding, we found that initially only one of the two motor heads binds, and that the binding of the other requires a nucleotide 'chase'. mantADP was released from the second head at 4 s(-1) by an ADP chase, 5 s(-1) by 5'-adenylylimidodiphosphate (AMPPNP), 27 s(-1) by ATPgammaS and 60 s(-1) by ATP. We infer a coordination mechanism for molecular walking, in which ATP hydrolysis on the trailing head accelerates leading head binding at least 15-fold, and leading head binding then accelerates trailing head unbinding at least 6-fold.
- Muresan V, Lyass A, Schnapp BJ
- The kinesin motor KIF3A is a component of the presynaptic ribbon in vertebrate photoreceptors.
- J Neurosci. 1999; 19: 1027-37
- Display abstract
Kinesin motors are presumed to transport various membrane compartments within neurons, but their specific in vivo functions, cargoes, and expression patterns in the brain are unclear. We have investigated the distribution of KIF3A, a member of the heteromeric family of kinesins, in the vertebrate retina. We find KIF3A at two distinct sites within photoreceptors: at the basal body of the connecting cilium axoneme and at the synaptic ribbon. Immunoelectron microscopy of the photoreceptor ribbon synapse shows KIF3A to be concentrated both at the ribbon matrix and on vesicles docked at the ribbon, a result that is consistent with the presence of both detergent-extractable and resistant KIF3A fractions at these synapses. KIF3A is also present in the inner plexiform layer, again at presynaptic ribbons. These findings suggest that within a single cell, the photoreceptor, one kinesin polypeptide, KIF3A, can serve two distinct functions, one specific for ribbon synapses.
- Muller J, Marx A, Sack S, Song YH, Mandelkow E
- The structure of the nucleotide-binding site of kinesin.
- Biol Chem. 1999; 380: 981-92
- Display abstract
Kinesin is a microtubule-based motor protein responsible for anterograde transport of vesicles and organelles in nerve axons and other cell types. The energy necessary for this transport is derived from the hydrolysis of ATP which is thought to induce conformational changes in the protein. We have solved the X-ray crystal structures of rat brain kinesin in three conditions intended to mimic different nucleotide states: (1) with ADP bound to the nucleotide-binding site, (2) with bound ADP in the presence of AIF(-)4, and (3) with ADP hydrolyzed to AMP by apyrase. In contrast to analogous cases observed in GTP-binding proteins or the muscle motor myosin, the structure of kinesin remained nearly unchanged. This highlights the stability of kinesin's ADP state in the absence of microtubules. Surprisingly, even after hydrolysis of ADP to AMP by apyrase a strong density peak remains at the position of the beta-phosphate which is compatible either with a phosphate or a sulfate from the solvent and appears to stabilize the nucleotide-binding pocket through several hydrogen bonds.
- Pechatnikova E, Taylor EW
- Kinetics processivity and the direction of motion of Ncd.
- Biophys J. 1999; 77: 1003-16
- Display abstract
The kinetic mechanism of the nonclaret disjunctional protein (Ncd) motor was investigated using the dimer termed MC1 (residues 209-700), which has been shown to exhibit negative-end directed motility (Chandra et al., 1993). The kinetic properties are similar to those of the monomeric Ncd motor domain (Pechatnikova and Taylor, 1997). The maximum steady-state ATPase activity of 1.5 s(-1) is half as large as for the monomeric motor. Dissociation constants in the presence of nucleotides showed the same trend but with approximately a two-fold decrease in the values: K(d) values are 1.0 microM for ADP-AlF(4), 1.1 microM for ATPgammaS, 1.5 microM for ATP, 3 microM for ADP, and 10 microM for ADP-vanadate (in 25 mM NaCl, 22 degrees C). The apparent second-order rate constants for the binding of ATP and ADP to the microtubule-motor complex (MtMC1) are 2 microM(-1) s(-1). Based on measurements at high microtubule concentrations the kinetic steps were fitted to the scheme,[see text] where N refers to one head of the dimer and T, D, and P stand for ATP, ADP, and inorganic phosphate. k(1) and k(-4) are the first-order rate constants of the transition induced by the binding of mant ATP and mant ADP respectively. ADP release is the main rate-limiting step in the MtMC1 mechanism. The binding of the MC1-mant ADP complex to microtubules released less than half of the mant ADP (alternating site reactivity). The second mant ADP is only released by the binding of nucleotides that dissociate the MtMC1 complex (ATP and ADP but not AMPPNP). The apparent rate constant for dissociation of the second mant ADP is four times smaller than the first and much smaller than the rate of dissociation of MtMC1 by ATP or ADP. These results are explained by a model in which MC1.ADP is first dissociated from the microtubule by ATP, followed by rebinding to the microtubule by the ADP-containing head. Ncd may follow a different reaction pathway than does kinesin, but the differences in rate constants do not explain the opposite direction of motion. The kinetic evidence and the high ratio of motile velocity to ATPase support a nonprocessive, low duty cycle mechanism for the Ncd motor.
- Astumian RD, Derenyi I
- A chemically reversible Brownian motor: application to kinesin and Ncd.
- Biophys J. 1999; 77: 993-1002
- Display abstract
Kinesin and nonclaret disjunctional protein (ncd) are two microtubule-based molecular motors that use energy from ATP hydrolysis to drive motion in opposite directions. They are structurally very similar and bind with similar orientations on microtubule. What is the origin of the different directionality? Is it some subtle feature of the structure of the motor domains, not apparent in x-ray diffraction studies, or possibly some difference near the neck regions far from the microtubule binding site? Perhaps because the motors function as dimers, the explanation involves differences in the strength of the interaction between the two motor monomers themselves. Here we present another possibility, based on a Brownian ratchet, in which the direction of motion of the motor is controlled by the chemical mechanism of ATP hydrolysis and is an inherent property of a single head. In contrast to conventional power stroke models, dissociation of the individual heads is not obligatory in the chemomechanical cycle, and the steps during which motion and force generation occurs are best described as one-dimensional thermally activated transitions that take place while both heads are attached to the microtubule. We show that our model is consistent with experiments on kinesin in which the velocity is measured as a function of external force and with the observed stiochiometry of one ATP/8-nm step at low load. Further, the model provides a way of understanding recent experiments on the ATP dependence of the variance (randomness) of the distance moved in a given time.
- Kirchner J, Woehlke G, Schliwa M
- Universal and unique features of kinesin motors: insights from a comparison of fungal and animal conventional kinesins.
- Biol Chem. 1999; 380: 915-21
- Display abstract
Kinesins are microtubule motors that use the energy derived from the hydrolysis of ATP to move unidirectionally along microtubules. The founding member of this still growing superfamily is conventional kinesin, a dimeric motor that moves processively towards the plus end of microtubules. Within the family of conventional kinesins, two groups can be distinguished to date, one derived from animal species, and one originating from filamentous fungi. So far no conventional kinesin has been reported from plant cells. Fungal and animal conventional kinesins differ in several respects, both in terms of their primary sequence and their physiological properties. Thus all fungal conventional kinesins move at velocities that are 4-5 times higher than those of animal conventional kinesins, and all of them appear to lack associated light chains. Both groups of motors are characterized by a number of group-specific sequence features which are considered here with respect to their functional importance. Animal and fungal conventional kinesins also share a number of sequence characteristics which point to common principles of motor function. The overall domain organization is remarkably similar. A C-terminal sequence motif common to all kinesins, which constitutes the only region of high homology outside the motor domain, suggests common principles of cargo association in both groups of motors. Consideration of the differences of, and similarities between, fungal and animal kinesins offers novel possibilities for experimentation (e. g., by constructing chimeras) that can be expected to contribute to our understanding of motor function.
- Rice S et al.
- A structural change in the kinesin motor protein that drives motility.
- Nature. 1999; 402: 778-84
- Display abstract
Kinesin motors power many motile processes by converting ATP energy into unidirectional motion along microtubules. The force-generating and enzymatic properties of conventional kinesin have been extensively studied; however, the structural basis of movement is unknown. Here we have detected and visualized a large conformational change of an approximately 15-amino-acid region (the neck linker) in kinesin using electron paramagnetic resonance, fluorescence resonance energy transfer, pre-steady state kinetics and cryo-electron microscopy. This region becomes immobilized and extended towards the microtubule 'plus' end when kinesin binds microtubules and ATP, and reverts to a more mobile conformation when gamma-phosphate is released after nucleotide hydrolysis. This conformational change explains both the direction of kinesin motion and processive movement by the kinesin dimer.
- Sakowicz R, Farlow S, Goldstein LS
- Cloning and expression of kinesins from the thermophilic fungus Thermomyces lanuginosus.
- Protein Sci. 1999; 8: 2705-10
- Display abstract
The motor domain regions of three novel members of the kinesin superfamily TLKIF1, TLKIFC, and TLBIMC were identified in a thermophilic fungus Thermomyces lanuginosus. Based on sequence similarity, they were classified as members of the known kinesin families Unc104/KIF1, KAR3, and BIMC. TLKIF1 was subsequently expressed in Escherichia coli. The expression level was high, and the protein was mostly soluble, easy to purify, and enzymatically active. TLKIF1 is a monomeric kinesin motor, which in a gliding motility assay displays a robust plus-directed microtubule movement up to 2 microm/s. The discovery of TLKIF1 also demonstrates that a family of kinesin motors not previously found in fungi may in fact be used in this group of organisms.
- Visscher K, Schnitzer MJ, Block SM
- Single kinesin molecules studied with a molecular force clamp.
- Nature. 1999; 400: 184-9
- Display abstract
Kinesin is a two-headed, ATP-driven motor protein that moves processively along microtubules in discrete steps of 8 nm, probably by advancing each of its heads alternately in sequence. Molecular details of how the chemical energy stored in ATP is coupled to mechanical displacement remain obscure. To shed light on this question, a force clamp was constructed, based on a feedback-driven optical trap capable of maintaining constant loads on single kinesin motors. The instrument provides unprecedented resolution of molecular motion and permits mechanochemical studies under controlled external loads. Analysis of records of kinesin motion under variable ATP concentrations and loads revealed several new features. First, kinesin stepping appears to be tightly coupled to ATP hydrolysis over a wide range of forces, with a single hydrolysis per 8-nm mechanical advance. Second, the kinesin stall force depends on the ATP concentration. Third, increased loads reduce the maximum velocity as expected, but also raise the apparent Michaelis-Menten constant. The kinesin cycle therefore contains at least one load-dependent transition affecting the rate at which ATP molecules bind and subsequently commit to hydrolysis. It is likely that at least one other load-dependent rate exists, affecting turnover number. Together, these findings will necessitate revisions to our understanding of how kinesin motors function.
- Kuleva NV
- [The current concepts on the mechanism of energy transformation by molecular motors of differing natures]
- Zh Evol Biokhim Fiziol. 1999; 35: 78-85
- Cross RA
- Molecular motors: Walking talking heads.
- Curr Biol. 1999; 9: 8546-8546
- Display abstract
Small tension signals that pass between the two linked heads of kinesin allow the motor protein to coordinate its walking action. Two new studies suggest that certain members of the two other major families of motor proteins, the myosins and dyneins, can do the same thing.
- Karabay A, Walker RA
- Identification of microtubule binding sites in the Ncd tail domain.
- Biochemistry. 1999; 38: 1838-49
- Display abstract
Nonclaret disjunctional (Ncd) is a minus end-directed, C-terminal motor protein that is required for spindle assembly and maintenance during meiosis and early mitosis in Drosophila oocytes and early embryos. Ncd has an ATP-independent MT binding site in the N-terminal tail domain, and an ATP-dependent MT binding site in the C-terminal motor domain. The ability of Ncd to cross-link MTs through the action of these binding sites may be important for Ncd function in vivo. To identify the region(s) responsible for ATP-independent MT interactions of Ncd, 12 cDNAs coding various regions of Ncd tail domain were expressed in E. coli as C-terminal fusions to thioredoxin (Trx). Ncd tail fusion proteins (TrxNT) were purified by ion exchange (S-Sepharose) and/or Talon metal affinity chromatography. Purified TrxNT and NT proteins were analyzed in microtubule (MT) cosedimentation and bundling assays to identify which tail proteins were able to bind and bundle MTs. Based on the results of these experiments, all TrxNT and NT proteins that showed MT binding activity also bundled MTs, and there are two ATP-independent MT interaction sites in the tail region: one within amino acids 83-100 that exhibits conformation-independent, high-affinity MT binding activity; and another within amino acids 115-187 that exhibits conformation-dependent, lower affinity MT binding activity. It is possible that both of these MT interacting sites combine in the native protein to form a single MT binding site that allows the Ncd tail to bind cargo MTs in vivo.
- Hirose K, Amos LA
- Three-dimensional structure of motor molecules.
- Cell Mol Life Sci. 1999; 56: 184-99
- Display abstract
Images, calculated from electron micrographs, show the three-dimensional structures of microtubules and tubulin sheets decorated stoichiometrically with motor protein molecules. Dimeric motor domains (heads) of kinesin and ncd, the kinesin-related protein that moves in the reverse direction, each appeared to bind to tubulin in the same way, by one of their two heads. The second heads show an interesting difference in position that seems to be related to the directions of movement of the two motors. X-ray crystallographic results showing the structures of kinesin and ncd to be very similar at atomic resolution, and homologous also to myosin, suggest that the two motor families may use mechanisms that have much in common. Nevertheless, myosins and kinesins differ kinetically. Also, whereas conformational changes in the myosin catalytic domain are amplified by a long lever arm that connects it to the stalk domain, kinesin and ncd do not appear to possess a structure with a similar function but may rely on biased diffusion in order to move along microtubules.
- Martin MA, Hurd DD, Saxton WM
- Kinesins in the nervous system.
- Cell Mol Life Sci. 1999; 56: 200-16
- Display abstract
Both the development and the maintenance of neurons require a great deal of active cytoplasmic transport. Much of this transport is driven by microtubule motor proteins. Membranous organelles and other macromolecular assemblies bind motor proteins that then use cycles of adenosine 5'-triphosphate hydrolysis to move these 'cargoes' along microtubules. Different sets of cargoes are transported to distinct locations in the cell. The resulting differential distribution of materials almost certainly plays an important part in generating polarized neuronal morphologies and in maintaining their vectorial signalling activities. A number of different microtubule motor proteins function in neurons; presumably they are specialized for accomplishing different transport tasks. Questions about specific motor functions and the functional relationships between different motors present a great challenge. The answers will provide a much deeper understanding of fundamental transport mechanisms, as well as how these mechanisms are used to generate and sustain cellular asymmetries.
- Cole DG
- Kinesin-II, the heteromeric kinesin.
- Cell Mol Life Sci. 1999; 56: 217-26
- Display abstract
The kinesins constitute a large family of motor proteins which are responsible for the distribution of numerous organelles, vesicles and macromolecular complexes throughout the cell. One class of these molecular motors, kinesin-II, is unique in that these proteins are typically found as heterotrimeric complexes containing two different, though related, kinesin-like motor subunits, and a single nonmotor subunit. The heteromeric nature of these kinesins appears to have resulted in a class of combinatorial kinesins which can 'mix and match' different motor subunits. Another novel feature of these motors is that the activities of several kinesin-II representatives are essential in the assembly of motile and nonmotile cilia, a role not attributed to any other kinesin. This review presents a brief overview of the structure and biological functions of kinesin-II, the heteromeric kinesin.
- Oliver TN, Berg JS, Cheney RE
- Tails of unconventional myosins.
- Cell Mol Life Sci. 1999; 56: 243-57
- Display abstract
In addition to the conventional myosins (class II) required for processes such as muscle contraction and cytokinesis, the myosin superfamily of actin-based motor proteins includes at least 14 'unconventional' classes. These unconventional myosins are defined by myosin-like head (motor) domains attached to class-specific tail domains that differ greatly from those of myosin-II. The unconventional myosins account for almost two-thirds of the 28 or more myosin genes currently believed to be expressed in humans and 80-90% of the approximately 10 or more myosin genes expressed in a typical nonmuscle cell. Although these members of the myosin superfamily have not been as intensively investigated as the conventional myosins, unconventional myosins are known or believed to power many forms of actin-based motility and organelle trafficking. The presence of signaling domains such as kinase domains, SH3 domains, PH domains or GTPase-activating domains in the tails of unconventional myosins indicates that these proteins can also be components of signal transduction pathways. Since several classes of the myosin superfamily have been found only in lower eukaryotes or plants (VIII, XI, XIII and XIV), in this review we will focus on the structures and properties of the unconventional myosins found in multicellular animals (excluding classes I and V, which have been reviewed elsewhere recently). Special attention will be focused on the three classes of unconventional myosins that can cause deafness in mouse or humans when mutated. In addition, we discuss the discovery of a pair of intriguing domains, the Myosin Tail Homology 4 (MyTH4) and FERM (band 4.1, Ezrin, Radixin, Moesin) domains, that are present in the tails of otherwise very different myosins as well as a plant kinesin-like protein. Recent progress in the identification of novel unconventional myosins will also be summarized.
- Heck MM
- Dr. Dolittle and the making of the mitotic spindle.
- Bioessays. 1999; 21: 985-90
- Display abstract
The intrinsic polarity of microtubules within cells is exploited each time cells divide. Kinesins, microtubule-associated motor proteins, are required to execute the dramatic events of mitosis: bipolar spindle assembly, metaphase chromosome alignment, anaphase chromosome segregation, and separation of spindle poles prior to cytokinesis. Surprisingly, kinesin-related proteins have been found to move in either "plus-ward" or "minus-ward" directions along microtubules. Evidence from genetic analyses of simple eukaryotes and in vitro activity assays supports the notion that certain subfamilies of kinesin-related proteins provide antagonistic activities necessary to balance mitotic forces. A recent study by Sharp et al.((1)) sheds further light on the subject by exploiting the genetics and cytology of the fruit fly embryo.
- Lunin VI et al.
- [Intracellular endonuclease from Serratia marcescens. I. Spatial structure of the protein and crystalline state at a resolution of 1.7 A]
- Mol Biol (Mosk). 1999; 33: 214-22
- Knight AE, Molloy JE
- Coupling ATP hydrolysis to mechanical work.
- Nat Cell Biol. 1999; 1: 879-879
- Sack S, Kull FJ, Mandelkow E
- Motor proteins of the kinesin family. Structures, variations, and nucleotide binding sites.
- Eur J Biochem. 1999; 262: 1-11
- Display abstract
Microtubule-dependent motors of the kinesin family convert the energy from ATP hydrolysis into mechanical work in order to transport vesicles and organelles along microtubules. The motor domains of several kinesins have been solved by X-ray diffraction, but the conformational changes associated with force development remain unknown. Here we describe conformational properties of kinesin that might be related to the mechanism of action. First, we have evaluated the conformational variability among all known kinesin structures and find they are concentrated in six areas, most of which are functionally important either in microtubule binding or in linking the core motor to the stalk. Secondly, we show that there is an important difference between kinesins when compared with myosins or GTPases (with which kinesin motor domains bear structural and catalytic similarities); in the diphosphate-state (with bound ADP), all kinesins show a 'tight' nucleotide-binding pocket, comparable with myosin or GTPases in the triphosphate state, whose nucleotide-binding pockets become open, or 'loose', following nucleotide hydrolysis. Thus, kinesin-ADP appears to be in a tense state, resembling that observed in myosin-ATP or p21ras-GTP.
- Pollock N, de Hostos EL, Turck CW, Vale RD
- Reconstitution of membrane transport powered by a novel dimeric kinesin motor of the Unc104/KIF1A family purified from Dictyostelium.
- J Cell Biol. 1999; 147: 493-506
- Display abstract
Motor-powered movement along microtubule tracks is important for membrane organization and trafficking. However, the molecular basis for membrane transport is poorly understood, in part because of the difficulty in reconstituting this process from purified components. Using video microscopic observation of organelle transport in vitro as an assay, we have purified two polypeptides (245 and 170 kD) from Dictyostelium extracts that independently reconstitute plus-end-directed membrane movement at in vivo velocities. Both polypeptides were found to be kinesin motors, and the 245-kD protein (DdUnc104) is a close relative of Caenorhabditis elegans Unc104 and mouse KIF1A, neuron-specific motors that deliver synaptic vesicle precursors to nerve terminals. A knockout of the DdUnc104 gene produces a pronounced defect in organelle transport in vivo and in the reconstituted assay. Interestingly, DdUnc104 functions as a dimeric motor, in contrast to other members of this kinesin subfamily, which are monomeric.
- Kozielski F, De Bonis S, Burmeister WP, Cohen-Addad C, Wade RH
- The crystal structure of the minus-end-directed microtubule motor protein ncd reveals variable dimer conformations.
- Structure Fold Des. 1999; 7: 1407-16
- Display abstract
BACKGROUND: The kinesin superfamily of microtubule-associated motor proteins are important for intracellular transport and for cell division in eukaryotes. Conventional kinesins have the motor domain at the N terminus of the heavy chain and move towards the plus end of microtubules. The ncd protein is necessary for chromosome segregation in meiosis. It belongs to a subfamily of kinesins that have the motor domain at the C terminus and move towards the minus end of microtubules. RESULTS: The crystal structure of dimeric ncd has been obtained at 2.9 A resolution from crystals with the C222(1) space group, with two independent dimers per asymmetric unit. The motor domains in these dimers are not related by crystallographic symmetry and the two ncd dimers have significantly different conformations. An alpha-helical coiled coil connects, and interacts with, the motor domains. CONCLUSIONS: The ncd protein has a very compact structure, largely due to extended interactions of the coiled coil with the head domains. Despite this, we find that the overall conformation of the ncd dimer can be rotated by as much as 10 degrees away from that of the twofold-symmetric archetypal ncd. The crystal structures of conventional kinesin and of ncd suggest a structural rationale for the reversal of the direction of movement in chimeric kinesins.
- Endow SA
- Determinants of molecular motor directionality.
- Nat Cell Biol. 1999; 1: 1637-1637
- Display abstract
Work over the past two years has led to a breakthrough in our understanding of the molecular basis of the directionality of the kinesin motor proteins. This breakthrough has come first from the reversal of directionality of the kinesin-related motor Ncd, followed closely by the reversal of kinesin's directionality and the finding that the Ncd 'neck' can convert Ncd or kinesin, which are intrinsically plus-end-directed microtubule motors, into a minus-end motor. These findings raise several outstanding questions, foremost, how does the neck function in motor directionality?
- Hartman JJ, Vale RD
- Microtubule disassembly by ATP-dependent oligomerization of the AAA enzyme katanin.
- Science. 1999; 286: 782-5
- Display abstract
Katanin, a member of the AAA adenosine triphosphatase (ATPase) superfamily, uses nucleotide hydrolysis energy to sever and disassemble microtubules. Many AAA enzymes disassemble stable protein-protein complexes, but their mechanisms are not well understood. A fluorescence resonance energy transfer assay demonstrated that the p60 subunit of katanin oligomerized in an adenosine triphosphate (ATP)- and microtubule-dependent manner. Oligomerization increased the affinity of katanin for microtubules and stimulated its ATPase activity. After hydrolysis of ATP, microtubule-bound katanin oligomers disassembled microtubules and then dissociated into free katanin monomers. Coupling a nucleotide-dependent oligomerization cycle to the disassembly of a target protein complex may be a general feature of ATP-hydrolyzing AAA domains.
- Han Y, Sablin EP, Nogales E, Fletterick RJ, Downing KH
- Visualizing a new binding site of ncd-motor domain on tubulin.
- J Struct Biol. 1999; 128: 26-33
- Display abstract
Ncd is a microtubule minus-end directed motor of the kinesin superfamily. Previously it has been shown that ncd and kinesin motor domains share the same major binding site on microtubules. Here we report a three-dimensional EM reconstruction of negatively stained two-dimensional Zn-induced tubulin crystal sheets (Zn-sheets) decorated with the ncd motor domain at a resolution of 16 A. This work has revealed a second specific binding site for the ncd motor domain. The motor binding site on the tubulin Zn-sheets spans both alpha and beta tubulin subunits. This binding site is located at a position different from the previously identified ncd binding site on microtubules and may play a role in motor function.
- Sheetz MP
- Motor and cargo interactions.
- Eur J Biochem. 1999; 262: 19-25
- Display abstract
The movements of intracellular cargo along microtubules within cells are often saltatory or of short duration. Further, calculations of the fraction of membrane vesicles that are moving at any period, indicate that active motor complexes are rare. From observations of normal vesicle traffic in cells, there appears to be position-dependent activation of motors and a balance of traffic in the inward and outward directions. In-vitro binding of motors to cargo is observed under many conditions but motility is not. Multi-component complexes appear to be involved in producing active organelle movements by a graded activation system that is highly localized in the cell. The basis of the activation of motility of the organelle motor complexes is still unknown but phosphorylation has been implicated in many systems. In the case of the motor-binding protein, kinectin, it has been linked to active organelle movements powered by conventional kinesin. From the coiled-coil structure of kinectin and the coiled-coil tail of kinesin, it is postulated that a coiled-coil assembly is responsible for the binding interaction. Many other cargoes are transported but the control of transport will be customized for each function, such as axonemal rafts or cytoskeletal complexes. Each function will have to be analyzed separately and motor activity will need to be integrated into the specific aspects of the function.
- Iwatani S, Iwane AH, Higuchi H, Ishii Y, Yanagida T
- Mechanical and chemical properties of cysteine-modified kinesin molecules.
- Biochemistry. 1999; 38: 10318-23
- Display abstract
To probe the structural changes within kinesin molecules, we made the mutants of motor domains of two-headed kinesin (4-411 aa) in which either all the five cysteines or all except Cys45 were mutated. A residual cysteine (Cys45) of the kinesin mutant was labeled with an environment-sensitive fluorescent probe, acrylodan. ATPase activity, mechanical properties, and fluorescence intensity of the mutants were measured. Upon acrylodan-labeled kinesin binding to microtubules in the presence of 1 mM AMPPNP, the peak intensity was enhanced by 3.4-fold, indicating the structural change of the kinesin head by the binding. Substitution of cysteines decreased both the maximum microtubule-activated ATPase and the sliding velocity to the same extent. However, the maximum force and the step size were not affected; the force produced by a single molecule was 6-6.5 pN, and a step size due to the hydrolysis of one ATP molecule by kinesin molecules was about 10 nm for all kinesins. This step size was close to a unitary step size of 8 nm. Thus, the mechanical events of kinesin are tightly coupled with the chemical events.
- Cross R, Scholey J
- Kinesin: the tail unfolds.
- Nat Cell Biol. 1999; 1: 11921-11921
- Friedman DS, Vale RD
- Single-molecule analysis of kinesin motility reveals regulation by the cargo-binding tail domain.
- Nat Cell Biol. 1999; 1: 293-7
- Display abstract
Conventional kinesin transports membranes along microtubules in vivo, but the majority of cellular kinesin is unattached to cargo. The motility of non-cargo-bound, soluble kinesin may be repressed by an interaction between the amino-terminal motor and carboxy-terminal cargo-binding tail domains, but neither bead nor microtubule-gliding assays have shown such inhibition. Here we use a single-molecule assay that measures the motility of kinesin unattached to a surface. We show that full-length kinesin binds microtubules and moves about ten times less frequently and exhibits discontinuous motion compared with a truncated kinesin lacking a tail. Mutation of either the stalk hinge or neck coiled-coil domain activates motility of full-length kinesin, indicating that these regions are important for tail-mediated repression. Our results suggest that the motility of soluble kinesin in the cell is inhibited and that the motor becomes activated by cargo binding.
- Rahman A, Kamal A, Roberts EA, Goldstein LS
- Defective kinesin heavy chain behavior in mouse kinesin light chain mutants.
- J Cell Biol. 1999; 146: 1277-88
- Display abstract
Conventional kinesin, kinesin-I, is a heterotetramer of two kinesin heavy chain (KHC) subunits (KIF5A, KIF5B, or KIF5C) and two kinesin light chain (KLC) subunits. While KHC contains the motor activity, the role of KLC remains unknown. It has been suggested that KLC is involved in either modulation of KHC activity or in cargo binding. Previously, we characterized KLC genes in mouse (Rahman, A., D.S. Friedman, and L.S. Goldstein. 1998. J. Biol. Chem. 273:15395-15403). Of the two characterized gene products, KLC1 was predominant in neuronal tissues, whereas KLC2 showed a more ubiquitous pattern of expression. To define the in vivo role of KLC, we generated KLC1 gene-targeted mice. Removal of functional KLC1 resulted in significantly smaller mutant mice that also exhibited pronounced motor disabilities. Biochemical analyses demonstrated that KLC1 mutant mice have a pool of KIF5A not associated with any known KLC subunit. Immunofluorescence studies of sensory and motor neuron cell bodies in KLC1 mutants revealed that KIF5A colocalized aberrantly with the peripheral cis-Golgi marker giantin in mutant cells. Striking changes and aberrant colocalization were also observed in the intracellular distribution of KIF5B and beta'-COP, a component of COP1 coatomer. Taken together, these data best support models that suggest that KLC1 is essential for proper KHC activation or targeting.
- Service RF
- Borrowing from biology to power the petite.
- Science. 1999; 283: 27-8
- Houdusse A, Kalabokis VN, Himmel D, Szent-Gyorgyi AG, Cohen C
- Atomic structure of scallop myosin subfragment S1 complexed with MgADP: a novel conformation of the myosin head.
- Cell. 1999; 97: 459-70
- Display abstract
The crystal structure of a proteolytic subfragment from scallop striated muscle myosin, complexed with MgADP, has been solved at 2.5 A resolution and reveals an unusual conformation of the myosin head. The converter and the lever arm are in very different positions from those in either the pre-power stroke or near-rigor state structures; moreover, in contrast to these structures, the SH1 helix is seen to be unwound. Here we compare the overall organization of the myosin head in these three states and show how the conformation of three flexible "joints" produces rearrangements of the four major subdomains in the myosin head with different bound nucleotides. We believe that this novel structure represents one of the prehydrolysis ("ATP") states of the contractile cycle in which the myosin heads stay detached from actin.
- Coy DL, Hancock WO, Wagenbach M, Howard J
- Kinesin's tail domain is an inhibitory regulator of the motor domain.
- Nat Cell Biol. 1999; 1: 288-92
- Display abstract
When not bound to cargo, the motor protein kinesin is in an inhibited state that has low microtubule-stimulated ATPase activity. Inhibition serves to minimize the dissipation of ATP and to prevent mislocalization of kinesin in the cell. Here we show that this inhibition is relieved when kinesin binds to an artificial cargo. Inhibition is mediated by kinesin's tail domain: deletion of the tail activates the ATPase without need of cargo binding, and inhibition is re-established by addition of exogenous tall peptide. Both ATPase and motility assays indicate that the tail does not prevent kinesin from binding to microtubules, but rather reduces the motor's stepping rate.
- Coy DL, Wagenbach M, Howard J
- Kinesin takes one 8-nm step for each ATP that it hydrolyzes.
- J Biol Chem. 1999; 274: 3667-71
- Display abstract
Conventional kinesin is a motor protein that moves stepwise along microtubules carrying membrane-bound organelles toward the periphery of cells. The steps are of amplitude 8.1 nm, the distance between adjacent tubulin binding sites, and are powered by the hydrolysis of ATP. We have asked: how many steps does kinesin take for each molecule of ATP that it hydrolyzes? To answer this question, the motility and ATP hydrolysis of recombinant, heterotetrameric and homodimeric conventional Drosophila kinesins adsorbed to 200-nm-diameter casein-coated silica beads were assayed under identical, single-molecule conditions. Division of the speed by the maximum microtubule-activated ATPase rate gave a stoichiometry of 1. 08 +/- 0.09 steps for each ATP hydrolyzed at 1 mM ATP. Therefore, under low loads in which the drag force << 1 pN, coupling between the chemical and mechanical cycles of kinesin is tight, consistent with conventional power stroke models. Our results rule out models that require two or more ATPs/step, such as some thermal ratchet models, or that propose multiple steps powered by single ATPs.
- Chen MC, Detrich HW 3rd
- Kinesin-like microtubule motors in early development.
- Methods Cell Biol. 1999; 59: 227-50
- Goldstein LS, Philp AV
- The road less traveled: emerging principles of kinesin motor utilization.
- Annu Rev Cell Dev Biol. 1999; 15: 141-83
- Display abstract
Proteins of the kinesin superfamily utilize a conserved catalytic motor domain to generate movements in a wide variety of cellular processes. In this review, we discuss the rapid expansion in our understanding of how eukaryotic cells take advantage of these proteins to generate force and movement in diverse functional contexts. We summarize several recent examples revealing that the simplest view of a kinesin motor protein binding to and translocating a cargo along a microtubule track is inadequate. In fact, this paradigm captures only a small subset of the many ways in which cells harness force production of the generation of intracellular movements and functions. We also highlight several situations where the catalytic kinesin motor domain may not be used to generate movement, but instead may be used in other biochemical and functional contexts. Finally, we review some recent ideas about kinesin motor regulation, redundancy, and cargo attachment strategies.
- Nautiyal S, Alber T
- Crystal structure of a designed, thermostable, heterotrimeric coiled coil.
- Protein Sci. 1999; 8: 84-90
- Display abstract
Electrostatic interactions are often critical for determining the specificity of protein-protein complexes. To study the role of electrostatic interactions for assembly of helical bundles, we previously designed a thermostable, heterotrimeric coiled coil, ABC, in which charged residues were employed to drive preferential association of three distinct, 34-residue helices. To investigate the basis for heterotrimer specificity, we have used multiwavelength anomalous diffraction (MAD) analysis to determine the 1.8 A resolution crystal structure of ABC. The structure shows that ABC forms a heterotrimeric coiled coil with the intended arrangement of parallel chains. Over half of the ion pairs engineered to restrict helix associations were apparent in the experimental electron density map. As seen in other trimeric coiled coils, ABC displays acute knobs-into-holes packing and a buried anion coordinated by core polar amino acids. These interactions validate the design strategy and illustrate how packing and polar contacts determine structural uniqueness.
- Hancock WO, Howard J
- Kinesin's processivity results from mechanical and chemical coordination between the ATP hydrolysis cycles of the two motor domains.
- Proc Natl Acad Sci U S A. 1999; 96: 13147-52
- Display abstract
Kinesin is a processive motor protein: A single molecule can walk continuously along a microtubule for several micrometers, taking hundreds of 8-nm steps without dissociating. To elucidate the biochemical and structural basis for processivity, we have engineered a heterodimeric one-headed kinesin and compared its biochemical properties to those of the wild-type two-headed molecule. Our construct retains the functionally important neck and tail domains and supports motility in high-density microtubule gliding assays, though it fails to move at the single-molecule level. We find that the ATPase rate of one-headed kinesin is 3-6 s(-1) and that detachment from the microtubule occurs at a similar rate (3 s(-1)). This establishes that one-headed kinesin usually detaches once per ATP hydrolysis cycle. Furthermore, we identify the rate-limiting step in the one-headed hydrolysis cycle as detachment from the microtubule in the ADP.P(i) state. Because the ATPase and detachment rates are roughly an order of magnitude lower than the corresponding rates for two-headed kinesin, the detachment of one head in the homodimer (in the ADP.P(i) state) must be accelerated by the other head. We hypothesize that this results from internal strain generated when the second head binds. This idea accords with a hand-over-hand model for processivity in which the release of the trailing head is contingent on the binding of the forward head. These new results, together with previously published ones, allow us to propose a pathway that defines the chemical and mechanical cycle for two-headed kinesin.
- Stone DB, Hjelm RP Jr, Mendelson RA
- Solution structures of dimeric kinesin and ncd motors.
- Biochemistry. 1999; 38: 4938-47
- Display abstract
The dimeric structure of the members of the kinesin family of motor proteins determines the individual characteristics of their microtubule-based motility. Crystal structures for ncd and kinesin dimers, which move in opposite directions on microtubules, show possible states of these dimers with ADP bound but give no information about these dimers in solution. Here, low-angle X-ray and neutron scattering were used to investigate their solution structures. Scattering profiles of Drosophila ncd 281-700 (NCD281) and human kinesin 1-420 (hKIN420) were compared with models made from the crystallographically determined structures of NCD281 and rat kinesin 1-379 (rKIN379). From the low-angle region it was found that the radius of gyration (Rg) of NCD281 is 3.60 +/- 0.075 nm, which is in agreement with the crystallography-based model. Scattering by longer ncd constructs (NCD250 and NCD224) is also well fit by the appropriate crystallography-based models. However, the measured Rg of hKIN420, 4.05 +/- 0.075 nm, is significantly smaller than that of the crystallography-based model. In addition, the overall scattering pattern of NCD281 is well fit by the model, but that of hKIN420 is poorly fit. Model calculations indicate that the orientation of the catalytic cores is different from that observed in the rKIN379 crystal structure. Like the crystal structure, the best-fitting models do not show 2-fold symmetry about the neck axis; however, their overall shape more resembles a mushroom than the "T"-like orientation of the catalytic cores found in the crystal structure. The center of mass separations of the catalytic cores in the best-fitting models are 0.7-1 nm smaller than in the crystal structure.
- Volz K
- A test case for structure-based functional assignment: the 1.2 A crystal structure of the yjgF gene product from Escherichia coli.
- Protein Sci. 1999; 8: 2428-37
- Display abstract
The YER057c/YIL051c/YjgF protein family is a set of 24 full-length homologs, each approximately 130 residues in length, and each with no known function or relationship to proteins of known structure. To determine the function of this family, the structure of one member--the YjgF protein from Escherichia coli--was solved and refined at a resolution of 1.2 A. The YjgF molecule is a homotrimer with exact threefold symmetry. Its tertiary and quaternary structures are related to that of Bacillus subtilis chorismate mutase, although their active sites are completely different. The YjgF protein has an active site curiously similar to protein tyrosine phosphatases, including a covalently modified cysteine, but it is unlikely to be functionally related. The lessons learned from this attempt to deduce function from structure may be useful to future projects in structural genomics.
- Hirose K, Lowe J, Alonso M, Cross RA, Amos LA
- Congruent docking of dimeric kinesin and ncd into three-dimensional electron cryomicroscopy maps of microtubule-motor ADP complexes.
- Mol Biol Cell. 1999; 10: 2063-74
- Display abstract
We present a new map showing dimeric kinesin bound to microtubules in the presence of ADP that was obtained by electron cryomicroscopy and image reconstruction. The directly bound monomer (first head) shows a different conformation from one in the more tightly bound empty state. This change in the first head is amplified as a movement of the second (tethered) head, which tilts upward. The atomic coordinates of kinesin.ADP dock into our map so that the tethered head associates with the bound head as in the kinesin dimer structure seen by x-ray crystallography. The new docking orientation avoids problems associated with previous predictions; it puts residues implicated by proteolysis-protection and mutagenesis studies near the microtubule but does not lead to steric interference between the coiled-coil tail and the microtubule surface. The observed conformational changes in the tightly bound states would probably bring some important residues closer to tubulin. As expected from the homology with kinesin, the atomic coordinates of nonclaret disjunctional protein (ncd).ADP dock in the same orientation into the attached head in a map of microtubules decorated with dimeric ncd.ADP. Our results support the idea that the observed direct interaction between the two heads is important at some stages of the mechanism by which kinesin moves processively along microtubules.
- Okada Y, Hirokawa N
- A processive single-headed motor: kinesin superfamily protein KIF1A.
- Science. 1999; 283: 1152-7
- Display abstract
A single kinesin molecule can move "processively" along a microtubule for more than 1 micrometer before detaching from it. The prevailing explanation for this processive movement is the "walking model," which envisions that each of two motor domains (heads) of the kinesin molecule binds coordinately to the microtubule. This implies that each kinesin molecule must have two heads to "walk" and that a single-headed kinesin could not move processively. Here, a motor-domain construct of KIF1A, a single-headed kinesin superfamily protein, was shown to move processively along the microtubule for more than 1 micrometer. The movement along the microtubules was stochastic and fitted a biased Brownian-movement model.
- Rogers GC, Hart CL, Wedaman KP, Scholey JM
- Identification of kinesin-C, a calmodulin-binding carboxy-terminal kinesin in animal (Strongylocentrotus purpuratus) cells.
- J Mol Biol. 1999; 294: 1-8
- Display abstract
Several novel members of the kinesin superfamily, until now identified only in plants, are unique in their ability to bind calmodulin in the presence of Ca(2+). Here, we identify the first such kinesin in an animal system. Sequence analysis of this new motor, called kinesin-C, predicts that it is a large carboxy-terminal kinesin, 1624 amino acid residues in length, with a predicted molecular mass of 181 kDa. Kinesin-C is predicted to contain a kinesin motor domain at its carboxy terminus, linked to a segment of alpha-helical coiled-coil 950 amino acid residues long, ending with an amino-terminal proline-rich tail domain. A putative calmodulin-binding domain resides at the extreme carboxy terminus of the motor polypeptide, and recombinant kinesin-C binds to a calmodulin-affinity column in a Ca(2+)-dependent fashion. The presence of this novel calmodulin-binding motor in sea urchin embryos suggests that it plays a critical role in Ca(2+)-dependent events during early sea urchin development.
- Oster G, Wang H
- ATP synthase: two motors, two fuels.
- Structure Fold Des. 1999; 7: 6772-6772
- Display abstract
FoF1 ATPase is the universal protein responsible for ATP synthesis. The enzyme comprises two reversible rotary motors: Fo is either an ion 'turbine' or an ion pump, and F1 is either a hydrolysis motor or an ATP synthesizer. Recent biophysical and biochemical studies have helped to elucidate the operating principles for both motors.
- Moreau V, Way M
- In vitro approaches to study actin and microtubule dependent cell processes.
- Curr Opin Cell Biol. 1999; 11: 152-8
- Display abstract
Actin and microtubules represent complex polymer systems that play essential roles during many cellular processes including chromosome segregation, cytokinesis and motility. The dynamic nature of actin and microtubules together with their regulation by a myriad of proteins makes their study both fascinating and challenging. Over the past few years there has been an increasing move towards development of in vitro systems to facilitate the elucidation of the molecular basis of actin and microtubule dependent cell processes. This review focuses on some of the recent developments using in vitro assays to dissect the cellular role of the actin and microtubule cytoskeleton.
- Mandelkow E, Hoenger A
- Structures of kinesin and kinesin-microtubule interactions.
- Curr Opin Cell Biol. 1999; 11: 34-44
- Display abstract
Several X-ray crystal structures of kinesin motor domains have recently been solved at high resolution ( approximately 0.2-0.3 nm), in both their monomeric and dimeric states. They show the folding of the polypeptide chain and different arrangements of subunits in the dimer. In addition, cryo-electron microscopy and image reconstruction have revealed microtubules decorated with kinesin at intermediate resolution ( approximately 2 nm), showing the distribution and orientation of kinesin heads on the microtubule surface. The comparison of the X-ray and electron microscopy results yields a model of how monomeric motor domains bind to the microtubule but the binding of dimeric motors, their stoichiometry, or the influence of nucleotides remains a matter of debate.
- Wade RH, Meurer-Grob P, Metoz F, Arnal I
- Organisation and structure of microtubules and microtubule-motor protein complexes.
- Eur Biophys J. 1998; 27: 446-54
- Display abstract
We present a short overview of the current status of work on the organisation and structure of microtubules and of microtubule-motor protein complexes. At present there is great interest in obtaining structural information that can help us to understand the movement of the kinesin family of microtubule associated molecular motors. Using electron cryomicroscopy and image reconstruction methods three dimensional maps of microtubule-motor complexes have been obtained in the presence of different nucleotides. We address a number of principles involved in different aspects of this work.
- Grummt M, Pistor S, Lottspeich F, Schliwa M
- Cloning and functional expression of a 'fast' fungal kinesin.
- FEBS Lett. 1998; 427: 79-84
- Display abstract
Conventional kinesins are molecular motors that move towards the plus end of microtubules. In animal species, they have been shown to be remarkably conserved in terms of both their primary sequence and several physiological properties, including their velocity of movement. Here we report the cloning of Synkin, a homologue of conventional kinesin from the zygomycete fungus Syncephalastrum racemosum [Steinberg, Eur. J. Cell Biol. 73 (1997) 124-131] that is 4-5 times faster than its animal counterparts. Expression in bacteria yields a fully functional motor that moves at the same speed as the native motor isolated from fungal hyphae and has similar hydrodynamic properties. Its sequence is most closely related to that of two other fungal kinesins from Neurospora and Ustilago, and shares several biochemical properties with the Neurospora motor. Fungal kinesins therefore seem to form a conserved subfamily of conventional kinesins distantly related to animal kinesins. They may help to identify sequence features important for determining motor velocity.
- Kozielski F, Arnal I, Wade RH
- A model of the microtubule-kinesin complex based on electron cryomicroscopy and X-ray crystallography.
- Curr Biol. 1998; 8: 191-8
- Display abstract
BACKGROUND: Motor proteins of the kinesin superfamily play an organising role in eukaryotic cells and participate in many crucial phases of the cell cycle by moving along microtubules and thereby changing the position of attached organelles. In their 'standard' form, kinesin motors are elongated heterotetrameric protein complexes composed of two identical heavy chains and two light chains; the central regions of the heavy chains intertwine, forming a coiled coil, with the globular 'heads' of the microtubule-interacting motor domains at one end. In order to understand how kinesin motors interact with and move along microtubules, we have combined electron cryomicroscopy and X-ray crystallographic data to build a model of the complex. RESULTS: Using electron cryomicroscopy and image reconstruction, we have obtained three-dimensional maps of complexes of kinesin motor domain dimers and microtubules. Motor domain dimers interact one to one with tubulin dimers, with one head attached--lying along the microtubule protofilament--and the other unattached--pointing sideways and upwards towards the microtubule plus end. Using currently available crystallographic data, we have built an atomic resolution model of the motor domain dimer, which can be successfully 'docked' into the three-dimensional framework of the maps from electron cryomicroscopy. CONCLUSIONS: Docking the atomic resolution model into the map of the microtubule-kinesin complex with the coiled coil of kinesin pointing away from the microtubule surface shows that the attached and unattached heads have similar relative positions on the microtubule and in the crystal. Three regions of the attached head appear likely to interact with the microtubule.
- Song H, Endow SA
- Decoupling of nucleotide- and microtubule-binding sites in a kinesin mutant.
- Nature. 1998; 396: 587-90
- Display abstract
Molecular motors require ATP to move along microtubules or actin filaments. To understand how molecular motors function, it is crucial to know how binding of the motor to its filamentous track stimulates the hydrolysis of ATP by the motor, enabling it to move along the filament. A mechanism for the enhanced ATP hydrolysis has not been elucidated, but it is generally accepted that conformational changes in the motor proteins occur when they bind to microtubules or actin filaments, facilitating the release of ADP. Here we report that a mutation in the motor domain of the microtubule motor proteins Kar3 and Ncd uncouples nucleotide- and microtubule-binding by the proteins, preventing activation of the motor ATPase by microtubules. Unlike the wild-type motors, the mutants bind tightly to both ADP and microtubules, indicating that interactions between the nucleotide- and microtubule-binding sites are blocked. The region of the motor that includes the mutated amino acid could transmit or undergo a conformational change required to convert the motor ATPase into a microtubule-stimulated state.
- Young EC, Mahtani HK, Gelles J
- One-headed kinesin derivatives move by a nonprocessive, low-duty ratio mechanism unlike that of two-headed kinesin.
- Biochemistry. 1998; 37: 3467-79
- Display abstract
A single molecule of the "two-headed" motor enzyme kinesin can move along a microtubule continuously for many enzymatic turnovers (processive movement), and the velocity produced by one kinesin molecule is the same as that produced by many kinesin molecules (high duty ratio). We studied the microtubule movement driven at 1 mM ATP by biotinated N-terminal fragments of Drosophila kinesin heavy chain attached to streptavidin-coated coverslips at various surface densities. K448-BIO has velocity at a high density of vmax = 750 nm s-1 and is dimeric (hence two-headed); K365-BIO (vmax = 200 nm s-1) and K340-BIO (vmax = 90 nm s-1) are monomeric. Escape of microtubules from the surface was prevented by methylcellulose so that continuous trajectories of microtubules not continuously attached to motor molecules could be recorded by video microscopy. The component of instantaneous velocity parallel to the microtubule axis (v) was analyzed in trajectories with a mean velocity 0.4-0.7 times vmax. In K448-BIO trajectories, the distribution of v was bimodal with peaks near 0 and 750 nm s-1. Temporal autocorrelation analysis of v detected lengthy episodes of high-velocity movement consistent with isolated processive microtubule runs driven at vmax by single K448-BIO dimers. K365-BIO and K340-BIO trajectories had unimodal distributions of v and autocorrelation times much shorter than those for K448-BIO. Therefore the monomeric motors have duty ratio < 55% (i.e., no forward movement is generated for at least 45% of the enzymatic cycle time) or processivity below the detection limit of approximately 300 turnovers even in methylcellulose. Continuous movement at maximal velocity thus requires more than one kinesin head.
- Toyoshima I
- [Molecular mechanisms of axonal transport]
- No To Shinkei. 1998; 50: 691-702
- Albert A et al.
- Crystal structure of aspartate decarboxylase at 2.2 A resolution provides evidence for an ester in protein self-processing.
- Nat Struct Biol. 1998; 5: 289-93
- Display abstract
The structure of L-aspartate-alpha-decarboxylase from E. coli has been determined at 2.2 A resolution. The enzyme is a tetramer with pseudofour-fold rotational symmetry. The subunits are six-stranded beta-barrels capped by small alpha-helices at each end. The active sites are located between adjacent subunits. The electron density provides evidence for catalytic pyruvoyl groups at three active sites and an ester at the fourth. The ester is an intermediate in the autocatalytic self-processing leading to formation of the pyruvoyl group. This unprecedented structure provides novel insights into the general phenomenon of protein processing.
- Ditzel L et al.
- Crystal structure of the thermosome, the archaeal chaperonin and homolog of CCT.
- Cell. 1998; 93: 125-38
- Display abstract
We have determined to 2.6 A resolution the crystal structure of the thermosome, the archaeal group II chaperonin from T. acidophilum. The hexadecameric homolog of the eukaryotic chaperonin CCT/TRiC shows an (alphabeta)4(alphabeta)4 subunit assembly. Domain folds are homologous to GroEL but form a novel type of inter-ring contact. The domain arrangement resembles the GroEL-GroES cis-ring. Parts of the apical domains form a lid creating a closed conformation. The lid substitutes for a GroES-like cochaperonin that is absent in the CCT/TRiC system. The central cavity has a polar surface implicated in protein folding. Binding of the transition state analog Mg-ADP-AIF3 suggests that the closed conformation corresponds to the ATP form.
- Lohman TM, Thorn K, Vale RD
- Staying on track: common features of DNA helicases and microtubule motors.
- Cell. 1998; 93: 9-12
- Thormahlen M et al.
- Interaction of monomeric and dimeric kinesin with microtubules.
- J Mol Biol. 1998; 275: 795-809
- Display abstract
The binding stoichiometry of kinesin to microtubules was determined using several biochemical and biophysical approaches (chemical crosslinking, binding assays, scanning transmission electron microscopy (STEM), image reconstruction, and X-ray scattering). The results show that each tubulin dimer associates with one kinesin head, irrespective of whether kinesin occurs in a monomeric or dimeric form in solution. Moreover, these heads appear to align along the protofilament axis generating a 16 nm periodicity of successive kinesin dimers. This is consistent with a "tightrope" model of movement where the first head of the dimer provides a guiding signal for the following one.
- Chaussepied P, Smyczynski C, Van Dijk J
- [Mechanisms of action and cellular functions of molecular motors]
- C R Seances Soc Biol Fil. 1998; 192: 319-34
- Display abstract
Cytoskeleton based molecular motors support most of the cellular movements and by consequence they are associated with a variety of human disorders. The wide functional diversity of these molecular motors is now explained by the presence of three different families: the myosin, kinesin and dynein families. Although they are functionally distinct, these motors present unexpected structural homologies at the ATP and actin or microtubule binding sites. However, these homologies do not seem sufficient to design a common molecular mechanism which allows these proteins to move along the cytoskeleton.
- Muresan V et al.
- KIF3C and KIF3A form a novel neuronal heteromeric kinesin that associates with membrane vesicles.
- Mol Biol Cell. 1998; 9: 637-52
- Display abstract
We have cloned from rat brain the cDNA encoding an 89,828-Da kinesin-related polypeptide KIF3C that is enriched in brain, retina, and lung. Immunocytochemistry of hippocampal neurons in culture shows that KIF3C is localized to cell bodies, dendrites, and, in lesser amounts, to axons. In subcellular fractionation experiments, KIF3C cofractionates with a distinct population of membrane vesicles. Native KIF3C binds to microtubules in a kinesin-like, nucleotide-dependent manner. KIF3C is most similar to mouse KIF3B and KIF3A, two closely related kinesins that are normally present as a heteromer. In sucrose density gradients, KIF3C sediments at two distinct densities, suggesting that it may be part of two different multimolecular complexes. Immunoprecipitation experiments show that KIF3C is in part associated with KIF3A, but not with KIF3B. Unlike KIF3B, a significant portion of KIF3C is not associated with KIF3A. Consistent with these biochemical properties, the distribution of KIF3C in the CNS has both similarities and differences compared with KIF3A and KIF3B. These results suggest that KIF3C is a vesicle-associated motor that functions both independently and in association with KIF3A.
- Nagar B, Jones RG, Diefenbach RJ, Isenman DE, Rini JM
- X-ray crystal structure of C3d: a C3 fragment and ligand for complement receptor 2.
- Science. 1998; 280: 1277-81
- Display abstract
Activation and covalent attachment of complement component C3 to pathogens is the key step in complement-mediated host defense. Additionally, the antigen-bound C3d fragment interacts with complement receptor 2 (CR2; also known as CD21) on B cells and thereby contributes to the initiation of an acquired humoral response. The x-ray crystal structure of human C3d solved at 2.0 angstroms resolution reveals an alpha-alpha barrel with the residues responsible for thioester formation and covalent attachment at one end and an acidic pocket at the other. The structure supports a model whereby the transition of native C3 to its functionally active state involves the disruption of a complementary domain interface and provides insight into the basis for the interaction between C3d and CR2.
- Ferhat L et al.
- Expression of the mitotic motor protein Eg5 in postmitotic neurons: implications for neuronal development.
- J Neurosci. 1998; 18: 7822-35
- Display abstract
It is well established that the microtubules of the mitotic spindle are organized by a variety of motor proteins, and it appears that the same motors or closely related variants organize microtubules in the postmitotic neuron. Specifically, cytoplasmic dynein and the kinesin-related motor known as CHO1/MKLP1 are used within the mitotic spindle, and recent studies suggest that they are also essential for the establishment of the axonal and dendritic microtubule arrays of the neuron. Other motors are required to tightly regulate microtubule behaviors in the mitotic spindle, and it is attractive to speculate that these motors might also help to regulate microtubule behaviors in the neuron. Here we show that a homolog of the mitotic kinesin-related motor known as Eg5 continues to be expressed in rodent neurons well after their terminal mitotic division. In neurons, Eg5 is directly associated with the microtubule array and is enriched within the distal regions of developing processes. This distal enrichment is transient, and typically lost after a process has been clearly defined as an axon or a dendrite. Strong expression can resume later in development, and if so, the protein concentrates within newly forming sprouts at the distal tips of dendrites. We suggest that Eg5 generates forces that help to regulate microtubule behaviors within the distal tips of developing axons and dendrites.
- Rahman A, Friedman DS, Goldstein LS
- Two kinesin light chain genes in mice. Identification and characterization of the encoded proteins.
- J Biol Chem. 1998; 273: 15395-403
- Display abstract
Native kinesin consists of two light chains and two heavy chains in a 1:1 stoichiometric ratio. To date, only one gene for kinesin light chain has been characterized, while a second gene was identified in a genomic sequencing study but not analyzed biochemically. Here we describe new genes encoding kinesin light chains in mouse. One of these light chains is neuronally enriched, while another shows ubiquitous expression. The presence of multiple kinesin light chain genes in mice is especially interesting, since there are two kinesin heavy chain genes in humans (Niclas, J., Navone, F., Hom-Booher, N., and Vale, R. D. (1994) Neuron 12, 1059-1072). To assess the selectivity of kinesin light chain interaction with the heavy chains, we performed immunoprecipitation experiments. The data suggested that the light chains form homodimers with no specificity in their interaction with the two heavy chains. Immunofluorescence and biochemical subfractionation suggested differences in the subcellular localization of the two kinesin light chain gene products. Although both kinesin light chains are distributed throughout the central and peripheral nervous systems, there is enrichment of one in sciatic nerve axons, while the other shows elevated levels in olfactory bulb glomeruli. These results indicate that the mammalian nervous system contains multiple kinesin light chain gene products with potentially distinct functions.
- Kack H, Sandmark J, Gibson KJ, Schneider G, Lindqvist Y
- Crystal structure of two quaternary complexes of dethiobiotin synthetase, enzyme-MgADP-AlF3-diaminopelargonic acid and enzyme-MgADP-dethiobiotin-phosphate; implications for catalysis.
- Protein Sci. 1998; 7: 2560-6
- Display abstract
The crystal structures of two complexes of dethiobiotin synthetase, enzyme-diaminopelargonic acid-MgADP-AlF3 and enzyme-dethiobiotin-MgADP-Pi, respectively, have been determined to 1.8 A resolution. In dethiobiotin synthetase, AlF3 together with carbamylated diaminopelargonic acid mimics the phosphorylated reaction intermediate rather than the transition state complex for phosphoryl transfer. Observed differences in the binding of substrate, diaminopelargonic acid, and the product, dethiobiotin, suggest considerable displacements of substrate atoms during the ring closure step of the catalytic reaction. In both complexes, two metal ions are observed at the active site, providing evidence for a two-metal mechanism for this enzyme.
- Tuma MC, Zill A, Le Bot N, Vernos I, Gelfand V
- Heterotrimeric kinesin II is the microtubule motor protein responsible for pigment dispersion in Xenopus melanophores.
- J Cell Biol. 1998; 143: 1547-58
- Display abstract
Melanophores move pigment organelles (melanosomes) from the cell center to the periphery and vice-versa. These bidirectional movements require cytoplasmic microtubules and microfilaments and depend on the function of microtubule motors and a myosin. Earlier we found that melanosomes purified from Xenopus melanophores contain the plus end microtubule motor kinesin II, indicating that it may be involved in dispersion (Rogers, S.L., I.S. Tint, P.C. Fanapour, and V.I. Gelfand. 1997. Proc. Natl. Acad. Sci. USA. 94: 3720-3725). Here, we generated a dominant-negative construct encoding green fluorescent protein fused to the stalk-tail region of Xenopus kinesin-like protein 3 (Xklp3), the 95-kD motor subunit of Xenopus kinesin II, and introduced it into melanophores. Overexpression of the fusion protein inhibited pigment dispersion but had no effect on aggregation. To control for the specificity of this effect, we studied the kinesin-dependent movement of lysosomes. Neither dispersion of lysosomes in acidic conditions nor their clustering under alkaline conditions was affected by the mutant Xklp3. Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport. We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin. This paper demonstrates that kinesin II moves membrane-bound organelles.
- Sakowicz R et al.
- A marine natural product inhibitor of kinesin motors.
- Science. 1998; 280: 292-5
- Display abstract
Members of the kinesin superfamily of motor proteins are essential for mitotic and meiotic spindle organization, chromosome segregation, organelle and vesicle transport, and many other processes that require microtubule-based transport. A compound, adociasulfate-2, was isolated from a marine sponge, Haliclona (also known as Adocia) species, that inhibited kinesin activity by targeting its motor domain and mimicking the activity of the microtubule. Thus, the kinesin-microtubule interaction site could be a useful target for small molecule modulators, and adociasulfate-2 should serve as an archetype for specific inhibitors of kinesin functions.
- Stewart RJ, Semerjian J, Schmidt CF
- Highly processive motility is not a general feature of the kinesins.
- Eur Biophys J. 1998; 27: 353-60
- Display abstract
Evidence is presented that the kinesin-related ncd protein is not as processive as kinesin. In low surface density motility experiments, a dimeric ncd fusion protein behaved mechanistically more similar to non-processive myosins than to the highly processive kinesin. First, there was a critical microtubule length for motility; only microtubules longer than this critical length moved in low density ncd surfaces, which suggested that multiple ncd proteins must cooperate to move microtubules in the surface assay. Under similar conditions, native kinesin demonstrated no critical microtubule length, consistent with the behavior of a highly processive motor. Second, addition of methylcellulose to decrease microtubule diffusion decreased the critical microtubule length for motility. Also, the rates of microtubule motility were microtubule length dependent in methylcellulose; short microtubules, that interacted with fewer ncd proteins, moved more slowly than long microtubules that interacted with more ncd proteins. In contrast, short microtubules, that interacted with one or a few kinesin proteins, moved on average slightly faster than long microtubules that interacted with multiple kinesins. We conclude that a degree of processivity as high as that of kinesin, where a single dimer can move over distances on the order of one micrometer, may not be a general mechanistic feature of the kinesin superfamily.
- Alonso MC, van Damme J, Vandekerckhove J, Cross RA
- Proteolytic mapping of kinesin/ncd-microtubule interface: nucleotide-dependent conformational changes in the loops L8 and L12.
- EMBO J. 1998; 17: 945-51
- Display abstract
We used a battery of proteases to probe the footprint of microtubules on kinesin and ncd, and to search for nucleotide-induced conformational changes in these two oppositely-directed yet homologous molecular motors. Proteolytic cleavage sites were identified by N-terminal microsequencing and electrospray mass spectrometry, and then mapped onto the recently-determined atomic structures of ncd and kinesin. In both kinesin and ncd, microtubule binding shields a set of cleavage sites within or immediately flanking the loops L12, L8 and L11 and, in ncd, the loop L2. Even in the absence of microtubules, exchange of ADP for AMPPNP in the motor active site drives conformational shifts involving these loops. In ncd, a chymotryptic cleavage at Y622 in L12 is protected in the strong binding AMPPNP conformation, but cleaved in the weak binding ADP conformation. In kinesin, a thermolysin cleavage at L154 in L8 is protected in AMPPNP but cleaved in ADP. We speculate that ATP turnover in the active site governs microtubule binding by cyclically retracting or displaying the loops L8 and L12. Curiously, the retracted state of the loops corresponds to microtubule strong binding. Conceivably, nucleotide-dependent display of loops works as a reversible block on strong binding.
- Mandelkow E, Johnson KA
- The structural and mechanochemical cycle of kinesin.
- Trends Biochem Sci. 1998; 23: 429-33
- Display abstract
Kinesin is a microtubule-based motor protein that pulls vesicles or organelles towards the plus end of microtubules. Structural changes in the protein that drive motility are coupled to ATP binding and hydrolysis. Here, we attempt to integrate recent structural and kinetic results into a picture of the mechanochemical cycle of kinesin.
- Sablin EP et al.
- Direction determination in the minus-end-directed kinesin motor ncd.
- Nature. 1998; 395: 813-6
- Display abstract
Motor proteins of the kinesin superfamily transport intracellular cargo along microtubules. Although different kinesin proteins share 30-50% amino-acid identity in their motor catalytic cores, some move to the plus end of microtubules whereas others travel in the opposite direction. Crystal structures of the catalytic cores of conventional kinesin (a plus-end-directed motor involved in organelle transport) and ncd (a minus-end-directed motor involved in chromosome segregation) are nearly identical; therefore, the structural basis for their opposite directions of movement is unknown. Here we show that the ncd 'neck' made up of 13 class-specific residues next to the superfamily-conserved catalytic core, is essential for minus-end-directed motility, as mutagenesis of these neck residues reverses the direction of ncd motion. By solving the 2.5 A structure of a functional ncd dimer, we show that the ncd neck (a coiled-coil) differs from the corresponding region in the kinesin neck (an interrupted beta-strand), although both necks interact with similar elements in the catalytic cores. The distinct neck architectures also confer different symmetries to the ncd and kinesin dimers and position these motors with appropriate directional bias on the microtubule.
- Harada Y et al.
- Single molecule imaging and nanomanipulation of biomolecules.
- Methods Cell Biol. 1998; 55: 117-28
- Diefenbach RJ, Mackay JP, Armati PJ, Cunningham AL
- The C-terminal region of the stalk domain of ubiquitous human kinesin heavy chain contains the binding site for kinesin light chain.
- Biochemistry. 1998; 37: 16663-70
- Display abstract
The motor protein kinesin is a heterotetramer composed of two heavy chains of approximately 120 kDa and two light chains of approximately 65 kDa protein. Kinesin motor activity is dependent on the presence of ATP and microtubules. The kinesin light chain-binding site in human kinesin heavy chain was determined by reconstituting in vitro a complex of recombinant heavy and light chains. The proteins expressed in bacteria included oligohistidine-tagged fragments of human ubiquitous kinesin heavy chain, spanning most of the stalk and all of the tail domain (amino acids 555-963); and untagged, essentially full-length human kinesin light chain (4-569) along with N-terminal (4-363) and C-terminal (364-569) light chain fragments. Heavy chain fragments were attached to Ni2+-charged beads and incubated with untagged light chain fragments. Analysis of eluted complexes by SDS-PAGE and immunoblotting mapped the light chain-binding site in heavy chain to amino acids 771-813, a region close to the C-terminal end of the heavy chain stalk domain. In addition, only the full-length and N-terminal kinesin light chain fragments bound to this heavy chain region. Within this heavy chain region are four highly conserved contiguous heptad repeats (775-802) which are predicted to form a tight alpha-helical coiled-coil interaction with the heptad repeat-containing N-terminus of the light chain, in particular region 106-152 of human light chain. This predicted hydrophobic, alpha-helical coiled-coil interaction is supported by both circular dichroism spectroscopy of the recombinant kinesin heavy chain fragment 771-963, which displays an alpha-helical content of 70%, and the resistance of the heavy/light chain interaction to high salt (0.5 M).
- Shimizu T, Morii H
- Spectroscopic studies of the ncd motor domain.ADP complex: CD spectrum of ADP induced by binding to the motor domain of ncd.
- Biochemistry. 1998; 37: 16680-5
- Display abstract
Previously, we reported that the nucleotide-free ncd motor domain exhibited a near-UV CD spectrum different from that of the ordinary ncd motor domain.ADP complex [Shimizu and Morii, (1996) J. Biochem. 120, 1176-1181]. In the present study, we exchanged the bound nucleotide ADP with N6-methylADP (MeADP) which has a UV absorption spectrum different from that of ADP. The resultant ncd motor domain. MeADP complex gave a near-UV CD spectrum different from that of the ordinary ncd motor domain with bound ADP. This result indicates that the bound nucleotide contributes to the near-UV CD spectra to a considerable extent although ADP or MeADP free in solution gives a spectrum with negligible peaks and troughs. In addition, the absorption intensity of ADP or MeADP at the peak wavelengths decreased to a considerable extent upon binding to the nucleotide-free ncd motor domain. It is suggested that interaction between adenine moiety and chromophore(s) of the protein contributed to the spectral changes of ADP. A candidate chromophore is Tyr442 which is stacked with the adenine moiety at a distance of 0.43 nm. On the other hand, we detected an intensity decrease of tryptophanyl fluorescence upon binding of a nucleotide to the nucleotide-free ncd motor domain, while at the same time tyrosyl fluorescence increased. The fluorescence changes, as well as the UV absorption change described above, gave similar rates upon addition of a nucleotide to the nucleotide-free ncd motor domain. Therefore, they are likely to originate from the same conformational change of the protein.
- Romberg L, Pierce DW, Vale RD
- Role of the kinesin neck region in processive microtubule-based motility.
- J Cell Biol. 1998; 140: 1407-16
- Display abstract
Kinesin is a dimeric motor protein that can move along a microtubule for several microns without releasing (termed processive movement). The two motor domains of the dimer are thought to move in a coordinated, hand-over-hand manner. A region adjacent to kinesin's motor catalytic domain (the neck) contains a coiled coil that is sufficient for motor dimerization and has been proposed to play an essential role in processive movement. Recent models have suggested that the neck enables head-to-head communication by creating a stiff connection between the two motor domains, but also may unwind during the mechanochemical cycle to allow movement to new tubulin binding sites. To test these ideas, we mutated the neck coiled coil in a 560-amino acid (aa) dimeric kinesin construct fused to green fluorescent protein (GFP), and then assayed processivity using a fluorescence microscope that can visualize single kinesin-GFP molecules moving along a microtubule. Our results show that replacing the kinesin neck coiled coil with a 28-aa residue peptide sequence that forms a highly stable coiled coil does not greatly reduce the processivity of the motor. This result argues against models in which extensive unwinding of the coiled coil is essential for movement. Furthermore, we show that deleting the neck coiled coil decreases processivity 10-fold, but surprisingly does not abolish it. We also demonstrate that processivity is increased by threefold when the neck helix is elongated by seven residues. These results indicate that structural features of the neck coiled coil, although not essential for processivity, can tune the efficiency of single molecule motility.
- Hancock WO, Howard J
- Processivity of the motor protein kinesin requires two heads.
- J Cell Biol. 1998; 140: 1395-405
- Display abstract
A single kinesin molecule can move for hundreds of steps along a microtubule without dissociating. One hypothesis to account for this processive movement is that the binding of kinesin's two heads is coordinated so that at least one head is always bound to the microtubule. To test this hypothesis, the motility of a full-length single-headed kinesin heterodimer was examined in the in vitro microtubule gliding assay. As the surface density of single-headed kinesin was lowered, there was a steep fall both in the rate at which microtubules landed and moved over the surface, and in the distance that microtubules moved, indicating that individual single-headed kinesin motors are not processive and that some four to six single-headed kinesin molecules are necessary and sufficient to move a microtubule continuously. At high ATP concentration, individual single-headed kinesin molecules detached from microtubules very slowly (at a rate less than one per second), 100-fold slower than the detachment during two-headed motility. This slow detachment directly supports a coordinated, hand-over-hand model in which the rapid detachment of one head in the dimer is contingent on the binding of the second head.
- Endow SA, Waligora KW
- Determinants of kinesin motor polarity.
- Science. 1998; 281: 1200-2
- Display abstract
The kinesin motor protein family members move along microtubules with characteristic polarity. Chimeric motors containing the stalk and neck of the minus-end-directed motor, Ncd, fused to the motor domain of plus-end-directed kinesin were analyzed. The Ncd stalk and neck reversed kinesin motor polarity, but mutation of the Ncd neck reverted the chimeric motor to plus-end movement. Thus, residues or regions contributing to motor polarity must be present in both the Ncd neck and the kinesin motor core. The neck-motor junction was critical for Ncd minus-end movement; attachment of the neck to the stalk may also play a role.
- Gulick AM, Song H, Endow SA, Rayment I
- X-ray crystal structure of the yeast Kar3 motor domain complexed with Mg.ADP to 2.3 A resolution.
- Biochemistry. 1998; 37: 1769-76
- Display abstract
The kinesin family of motor proteins, which contain a conserved motor domain of approximately 350 amino acids, generate movement against microtubules. Over 90 members of this family have been identified, including motors that move toward the minus or plus end of microtubules. The Kar3 protein from Saccharomyces cerevisiae is a minus end-directed kinesin family member that is involved in both nuclear fusion, or karyogamy, and mitosis. The Kar3 protein is 729 residues in length with the motor domain located in the C-terminal 347 residues. Recently, the three-dimensional structures of two kinesin family members have been reported. These structures include the motor domains of the plus end-directed kinesin heavy chain [Kull, F. J., et al. (1996) Nature 380, 550-555] and the minus end-directed Ncd [Sablin, E. P., et al. (1996) Nature 380, 555-559]. We now report the structure of the Kar3 protein complexed with Mg.ADP obtained from crystallographic data to 2.3 A. The structure is similar to those of the earlier kinesin family members, but shows differences as well, most notably in the length of helix alpha 4, a helix which is believed to be involved in conformational changes during the hydrolysis cycle.
- Vugmeyster Y, Berliner E, Gelles J
- Release of isolated single kinesin molecules from microtubules.
- Biochemistry. 1998; 37: 747-57
- Display abstract
Previous studies on the motor enzyme kinesin suggesting that the enzyme molecule tightly binds to a microtubule by only one of its two mechanochemical head domains were performed with multiple kinesin molecules on each microtubule, raising the possibility that interactions between adjacent bound molecules may interfere with the binding of the second head. To characterize the microtubule-bound state of isolated single kinesin molecules, we have measured the rates of nucleotide-induced dissociation of the complex between microtubules and bead-labeled single molecules of the dimeric kinesin derivative K448-BIO using novel single-molecule kinetic methods. Complex dissociation by <2 &mgr;M ADP displays an apparent second-order rate constant of 1.2 x 10(4) M-1 s-1. The data suggest that only one of the two heads is bound to the microtubule in the absence of ATP, that binding of a single ADP to the complex is sufficient to induce dissociation, and that even lengthy exposure of kinesin to the microtubule fails to produce significant amounts of a two-head-bound state under the conditions used. The inhibitor adenylyl imidodiphosphate (AMP-PNP) induces stochastic pauses in the movement of bead-labeled enzyme molecules in 1 mM ATP. Exit from pauses occurs at 2 s-1 independent of AMP-PNP concentration. The same rate constant is obtained for dissociation of the transient kinesin-microtubule complexes formed in 1 mM ADP, 0.5 mM AMP-PNP, suggesting that release of a single AMP-PNP molecule from the enzyme is the common rate-limiting step of the two processes. The results are consistent with alternating-sites movement mechanisms in which two-head-bound states do not occur in the enzyme catalytic cycle until after ATP binding.
- Gilbert SP, Moyer ML, Johnson KA
- Alternating site mechanism of the kinesin ATPase.
- Biochemistry. 1998; 37: 792-9
- Display abstract
The processivity of the microtubule-kinesin ATPase has been investigated using stopped-flow kinetic methods to measure the binding of each motor domain of the dimeric kinesin (K401) to the microtubule and the release of the fluorescent ADP analog, 2'(3')-O-(N-methylanthraniloyl)adenosine 5'-diphosphate (mantADP) from the active site of the motor domain. The results show that the release of two molecules of ADP from dimeric kinesin (K401) after the binding of kinesin ADP to the microtubule is a sequential process leading to biphasic kinetics. The maximum rate of release of mantADP from the first motor domain of K401 or monomeric K341 is fast (300 s-1) and independent of added nucleotide. The rate of mantADP release from the second motor domain of K401 is slow in the absence of added nucleotide (0.4 s-1) and reaches a maximum rate of 300 s-1 at saturating concentrations of ATP. High concentrations of ADP stimulate mantADP release from the second head to a maximum rate of 3.8 s-1. The nonhydrolyzable analog AMP-PNP and ATP-gamma S also stimulate ADP release from the second head (maximum rate of 30 s-1), suggesting that ATP hydrolysis is not necessary to stimulate the ADP release. These experiments establish an alternating site mechanism for dimeric kinesin whereby ATP binding to one kinesin active site stimulates the release of ADP from the second site such that the reactions occurring at the active sites of the two monomer units are kept out of phase from each other by interactions between the heads. These results define the steps of the ATPase pathway that lead to the efficient coupling of ATP hydrolysis to force production in a processive reaction whereby force production in forming a tight microtubule complex by one head is coupled to the rate-limiting release of the other head from the microtubule.
- Moyer ML, Gilbert SP, Johnson KA
- Pathway of ATP hydrolysis by monomeric and dimeric kinesin.
- Biochemistry. 1998; 37: 800-13
- Display abstract
The ATPase mechanism for a monomeric Drosophila kinesin construct, K341, was determined by pre-steady-state kinetic methods and compared to dimeric kinesin, K401. We directly measured the kinetics of binding mantATP (a fluorescent ATP analog) to the microtubule K341 complex, the dissociation of K341 from the microtubule, and release of phosphate and ADP from K341. Measurements of phosphate release kinetics at low salt concentration show that K341 hydrolyzes 18 molecules of ATP per kinesin monomer prior to release from the microtubule. At a higher salt concentration the amplitude of the pre-steady-state burst of phosphate release was reduced to 8 molecules per kinesin monomer. The maximum rate of dissociation of K341 from the microtubule following the addition of ATP was 22 s-1. The rate of mantADP release from the M.K341.mantADP complex increased as a function of tubulin concentration with a second-order rate constant of 11 microM-1 s-1 for K341 binding to the microtubule and reached a maximum rate of mantADP release of 303 s-1. ADP release kinetics were also determined by monitoring the binding of mantATP to K341.ADP and K401.ADP after mixing with microtubules. We show that monomeric kinesin remains associated with the microtubule through multiple rounds of ATP hydrolysis. This apparent processivity implies that one of the functions of the cooperative interaction between the two kinesin heads in dimeric kinesin is for the reactions occurring on one kinesin head to facilitate the release of the adjacent head from the microtubule.
- Visscher K, Block SM
- Versatile optical traps with feedback control.
- Methods Enzymol. 1998; 298: 460-89
- Liao G, Gundersen GG
- Kinesin is a candidate for cross-bridging microtubules and intermediate filaments. Selective binding of kinesin to detyrosinated tubulin and vimentin.
- J Biol Chem. 1998; 273: 9797-803
- Display abstract
We showed previously that stable, detyrosinated (Glu) microtubules function to localize vimentin intermediate filaments in fibroblasts (Gurland, G., and Gundersen, G. G. (1995) J. Cell Biol. 131, 1275-1290). To identify candidate proteins that mediate the Glu microtubule-vimentin interaction, we incubated microtubules with microtubule-interacting proteins and saturating levels of antibodies to Glu or tyrosinated (Tyr) tubulin. Antibodies to Glu tubulin prevented the microtubule binding of kinesin obtained from fibroblast or brain extracts more effectively than antibodies to Tyr tubulin. Scatchard plot analysis showed that kinesin heads bound to Glu microtubules with an approximately 2.8-fold higher affinity than to Tyr microtubules. Purified brain kinesin cosedimented with vimentin, but not with neurofilaments, indicating that kinesin specifically associates with vimentin without accessory molecules. Kinesin binding to vimentin was not sensitive to ATP, and kinesin heads failed to bind to vimentin. By SDS-polyacrylamide gel electrophoresis, a kinesin heavy chain of approximately 120 kDa and a light chain of approximately 64 kDa were detected in vimentin/kinesin pellets. The light chain reacted with a general kinesin light chain antibody, but not with two other antibodies that recognize the two known isoforms of kinesin light chain in brain, suggesting that the kinesin involved in binding to vimentin may be a specific one. These results demonstrate a kinesin-based mechanism for the preferential interaction of vimentin with detyrosinated microtubules.
- Thormahlen M, Marx A, Sack S, Mandelkow E
- The coiled-coil helix in the neck of kinesin.
- J Struct Biol. 1998; 122: 30-41
- Display abstract
Kinesin is a microtubule-dependent motor protein. We have recently determined the X-ray structure of monomeric and dimeric kinesin from rat brain. The dimer consists of two motor domains, held together by their alpha-helical neck domains forming a coiled coil. Here we analyze the nature of the interactions in the neck domain (residues 339-370). Overall, the neck helix shows a heptad repeat (abcdefg)n typical of coiled coils, with mostly nonpolar residues in positions a and d. However, the first segment (339-355) contains several nonclassical residues in the a and d positions which tend to weaken the hydrophobic interaction along the common interface. Instead, stabilization is achieved by a hydrophobic "coat" formed by the a and d residues and the long aliphatic moieties of lysines and glutamates, extending away from the coiled-coil core. By contrast, the second segment of the kinesin neck (356-370) shows a classical leucine zipper pattern in which most of the hydrophobic residues are buried at the highly symmetrical dimer interface. The end of the neck reveals the structure of a potential coiled-coil "trigger" sequence.
- Hirokawa N, Noda Y, Okada Y
- Kinesin and dynein superfamily proteins in organelle transport and cell division.
- Curr Opin Cell Biol. 1998; 10: 60-73
- Display abstract
Microtubule-associated motor proteins of the kinesin and dynein superfamilies play important roles in cellular mechanisms such as organelle transport and mitosis. Identification and characterization of new family members (in particular KIFC2, 16 new KIFs, XKlp2 and XKCM1 of the kinesin superfamily, and DHC2 and DHC3 of the dynein superfamily) and further characterization of known family members have improved our understanding of these cellular mechanisms. Sophisticated biophysical and structural analyses of monomeric and dimeric motor proteins have contributed to elucidating the mechanisms behind motor protein motility and polarity.
- Yamashita RA, May GS
- Motoring along the hyphae: molecular motors and the fungal cytoskeleton.
- Curr Opin Cell Biol. 1998; 10: 74-9
- Display abstract
This review discusses molecular motors that use the microfilament and microtubule cytoskeletal systems in filamentous fungi. There has been an explosion in our knowledge of kinesins over the past year, because of the integration of genetic and biochemical data. The recognition of possible interactions between septation genes and cytokinesis has also advanced our understanding of microfilament-based cytoskeletal systems. We review recent findings on microfilament motors, including conventional and unconventional myosins, and the microtubule motors of the kinesin family and cytoplasmic dynein. The roles that these molecules play in hyphal morphogenesis and organelle transport provide an insight into cytoskeletal-based transport systems.
- Lockhart A, Kendrick-Jones J
- Nucleotide-dependent interaction of the N-terminal domain of MukB with microtubules.
- J Struct Biol. 1998; 124: 303-10
- Display abstract
The MukB protein from Escherichia coli has a domain structure that is reminiscent of the eukaryotic motor proteins kinesin and myosin: N-terminal globular domains, a region of coiled-coil, and a specialised C-terminal domain. Sequence alignment of the N-terminal domain of MukB with the kinesin motor domain indicated an approximately 22% sequence identity. These observations raised the possibility that MukB might be a prokaryotic motor protein and, due to the sequence homology shared with kinesin, might bind to microtubules (Mts). We found that a construct encoding the first 342 residues of MukB (Muk342) binds specifically to Mts and shares a number of properties with the motor domain of kinesin. Visualisation of the Muk342 decorated Mt complexes using negative stain electron microscopy indicated that the Muk342 smoothly decorates the outside of Mts. Biochemical data demonstrate that Muk342 decorates Mts with a binding stoichiometry of one Muk342 monomer per tubulin monomer. These findings strongly suggest that MukB has a role in force generation and that it is a prokaryotic homologue of kinesin and myosin.
- Verhey KJ, Lizotte DL, Abramson T, Barenboim L, Schnapp BJ, Rapoport TA
- Light chain-dependent regulation of Kinesin's interaction with microtubules.
- J Cell Biol. 1998; 143: 1053-66
- Display abstract
We have investigated the mechanism by which conventional kinesin is prevented from binding to microtubules (MTs) when not transporting cargo. Kinesin heavy chain (HC) was expressed in COS cells either alone or with kinesin light chain (LC). Immunofluorescence microscopy and MT cosedimentation experiments demonstrate that the binding of HC to MTs is inhibited by coexpression of LC. Association between the chains involves the LC NH2-terminal domain, including the heptad repeats, and requires a region of HC that includes the conserved region of the stalk domain and the NH2 terminus of the tail domain. Inhibition of MT binding requires in addition the COOH-terminal 64 amino acids of HC. Interaction between the tail and the motor domains of HC is supported by sedimentation experiments that indicate that kinesin is in a folded conformation. A pH shift from 7.2 to 6.8 releases inhibition of kinesin without changing its sedimentation behavior. Endogenous kinesin in COS cells also shows pH-sensitive inhibition of MT binding. Taken together, our results provide evidence that a function of LC is to keep kinesin in an inactive ground state by inducing an interaction between the tail and motor domains of HC; activation for cargo transport may be triggered by a small conformational change that releases the inhibition of the motor domain for MT binding.
- Cheng JQ, Jiang W, Hackney DD
- Interaction of mant-adenosine nucleotides and magnesium with kinesin.
- Biochemistry. 1998; 37: 5288-95
- Display abstract
Displacement of the fluorescent substrate analogue methylanthraniloyl ADP (mant-ADP) from kinesin by excess ATP results in a biphasic fluorescent transient. The pH and microtubule dependence of the rates and amplitudes indicates that the two phases are produced by release of bound mant-ADP, with an excess of the 3'-isomer, followed by the subsequent relaxation of the free 2'- and 3'-isomers to their equilibrium distribution. The first phase for release of mant-ADP is accelerated by microtubules and occurs at the same rate as ADP release measured using [32P]ADP. The second phase is subject to base catalysis and occurs at the same rate as the isomerization of isolated 2'- or 3'-mant-ATP over a 100-fold range of rates. The bound mant-ADP isomers undergo isomerization rapidly when bound to kinesin at pH 8.2, whereas mant-ADP isomers interconvert only slowly when bound to myosin. No fluorescence resonance energy transfer occurs between the single tryptophan in the kinesin neck domain and bound mant-ADP, but efficient energy transfer does occur from protein tyrosine groups. The rate of mant-ADP release in the absence of microtubules is minimal (0.005 s-1) at pH 7-8, 2 mM Mg2+, and 25 mM KCl but is accelerated at lower pH (0.04 s-1 at pH 5.5) and either lower or higher [KCl] (0.01 and 0. 06 s-1 at 0 and 800 mM KCl, respectively). The microtubule-stimulated rate of ADP release is accelerated at low pH and inhibited by high concentrations of monovalent salts. Reduction of the free Mg2+ by addition of excess EDTA increases the release of mant-ADP from E.MgADP to 0.03 s-1. This acceleration at low Mg2+ likely represents sequential release of Mg2+ at 0.03 s-1 followed by rapid release of ADP, as the rate of ADP release from Mg-free E.ADP is fast (>0.5 s-1). At high Mg2+, rebinding of Mg2+ to E.ADP forces release of ADP from the E.MgADP complex at 0.005 s-1.
- Grummt M, Woehlke G, Henningsen U, Fuchs S, Schleicher M, Schliwa M
- Importance of a flexible hinge near the motor domain in kinesin-driven motility.
- EMBO J. 1998; 17: 5536-42
- Display abstract
Conventional kinesin is a molecular motor consisting of an N-terminal catalytic motor domain, an extended stalk and a small globular C-terminus. Whereas the structure and function of the catalytic motor domain has been investigated, little is known about the function of domains outside the globular head. A short coiled-coil region adjacent to the motor domain, termed the neck, is known to be important for dimerization and may be required for kinesin processivity. We now provide evidence that a helix-disrupting hinge region (hinge 1) that separates the neck from the first extended coiled-coil of the stalk plays an essential role in basic motor activity. A fast fungal kinesin from Syncephalastrum racemosum was used for these studies. Deletion, substitution by a coiled-coil and truncation of the hinge 1 region all reduce motor speed and uncouple ATP turnover from gliding velocity. Insertion of hinge 1 regions from two conventional kinesins, Nkin and DmKHC, fully restores motor activity, whereas insertion of putative flexible linkers of other proteins does not, suggesting that hinge 1 regions of conventional kinesins can functionally replace each other. We suggest that this region is essential for kinesin movement in its promotion of chemo-mechanical coupling of the two heads and therefore the functional motor domain should be redefined to include not only the catalytic head but also the adjacent neck and hinge 1 domains.
- Hirose K, Cross RA, Amos LA
- Nucleotide-dependent structural changes in dimeric NCD molecules complexed to microtubules.
- J Mol Biol. 1998; 278: 389-400
- Display abstract
Complexes consisting of motor domains of the kinesin-like protein ncd bound to reassembled brain microtubules were visualised using cryoelectron microscopy and helical image reconstruction. Different nucleotide-associated states of a dimeric construct (NDelta295-700) of ncd were analysed to reveal ADP-containing, AMP.PNP-containing and empty (rigor) conformations. In these three states, each thought to mimic a different stage in ATP turnover, the double-headed motors attach to the microtubules by one head only, with the free head tethered in relatively fixed positions. The three structures differ both in the way the attached heads interact with tubulin and in the position of the tethered heads. In the strongly binding rigor and AMP.PNP (ATP-like) states, the attached head makes close contact with both subunits of a tubulin heterodimer. In the weakly bound ADP state, the contact made by the attached head with the monomer closer to the plus end appears to be more loose. Also, in the ATP-like state, the free head tilts nearer to the plus end than in the other two states. The data argue against model mechanisms in which a conformational change in the bound head guides the free head closer to its next binding site; on the contrary, the transition from ADP-filled via rigor to the AMP.PNP (ATP-like) state of the bound head produces a small motion of the free head in the counter-productive direction. However, the observation that the tethered head points towards the minus end, in all three states, is consistent with the idea that the relative arrangement of the heads in a dimer is a major determinant of directionality.
- Wriggers W, Schulten K
- Nucleotide-dependent movements of the kinesin motor domain predicted by simulated annealing.
- Biophys J. 1998; 75: 646-61
- Display abstract
The structure of an ATP-bound kinesin motor domain is predicted and conformational differences relative to the known ADP-bound form of the protein are identified. The differences should be attributed to force-producing ATP hydrolysis. Candidate ATP-kinesin structures were obtained by simulated annealing, by placement of the ATP gamma-phosphate in the crystal structure of ADP-kinesin, and by interatomic distance constraints. The choice of such constraints was based on mutagenesis experiments, which identified Gly-234 as one of the gamma-phosphate sensing residues, as well as on structural comparison of kinesin with the homologous nonclaret disjunctional (ncd) motor and with G-proteins. The prediction of nucleotide-dependent conformational differences reveals an allosteric coupling between the nucleotide pocket and the microtubule binding site of kinesin. Interactions of ATP with Gly-234 and Ser-202 trigger structural changes in the motor domain, the nucleotide acting as an allosteric modifier of kinesin's microtubule-binding state. We suggest that in the presence of ATP kinesin's putative microtubule binding regions L8, L12, L11, alpha4, alpha5, and alpha6 form a face complementary in shape to the microtubule surface; in the presence of ADP, the microtubule binding face adopts a more convex shape relative to the ATP-bound form, reducing kinesin's affinity to the microtubule.
- Hoenger A et al.
- Image reconstructions of microtubules decorated with monomeric and dimeric kinesins: comparison with x-ray structure and implications for motility.
- J Cell Biol. 1998; 141: 419-30
- Display abstract
We have decorated microtubules with monomeric and dimeric kinesin constructs, studied their structure by cryoelectron microscopy and three-dimensional image reconstruction, and compared the results with the x-ray crystal structure of monomeric and dimeric kinesin. A monomeric kinesin construct (rK354, containing only a short neck helix insufficient for coiled-coil formation) decorates microtubules with a stoichiometry of one kinesin head per tubulin subunit (alpha-beta-heterodimer). The orientation of the kinesin head (an anterograde motor) on the microtubule surface is similar to that of ncd (a retrograde motor). A longer kinesin construct (rK379) forms a dimer because of the longer neck helix forming a coiled-coil. Unexpectedly, this construct also decorates the microtubule with a stoichiometry of one head per tubulin subunit, and the orientation is similar to that of the monomeric construct. This means that the interaction with microtubules causes the two heads of a kinesin dimer to separate sufficiently so that they can bind to two different tubulin subunits. This result is in contrast to recent models and can be explained by assuming that the tubulin-kinesin interaction is antagonistic to the coiled-coil interaction within a kinesin dimer.
- Zhang G, Darst SA
- Structure of the Escherichia coli RNA polymerase alpha subunit amino-terminal domain.
- Science. 1998; 281: 262-6
- Display abstract
The 2.5 angstrom resolution x-ray crystal structure of the Escherichia coli RNA polymerase (RNAP) alpha subunit amino-terminal domain (alphaNTD), which is necessary and sufficient to dimerize and assemble the other RNAP subunits into a transcriptionally active enzyme and contains all of the sequence elements conserved among eukaryotic alpha homologs, has been determined. The alphaNTD monomer comprises two distinct, flexibly linked domains, only one of which participates in the dimer interface. In the alphaNTD dimer, a pair of helices from one monomer interact with the cognate helices of the other to form an extensive hydrophobic core. All of the determinants for interactions with the other RNAP subunits lie on one face of the alphaNTD dimer. Sequence alignments, combined with secondary-structure predictions, support proposals that a heterodimer of the eukaryotic RNAP subunits related to Saccharomyces cerevisiae Rpb3 and Rpb11 plays the role of the alphaNTD dimer in prokaryotic RNAP.
- Wu Q, Sandrock TM, Turgeon BG, Yoder OC, Wirsel SG, Aist JR
- A fungal kinesin required for organelle motility, hyphal growth, and morphogenesis.
- Mol Biol Cell. 1998; 9: 89-101
- Display abstract
A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformation-mediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkorper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkorper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubule-based motor protein is essential for normal positioning of the Spitzenkorper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology.
- Gee M, Vallee R
- The role of the dynein stalk in cytoplasmic and flagellar motility.
- Eur Biophys J. 1998; 27: 466-73
- Display abstract
We have recently identified a microtubule binding domain within the motor protein cytoplasmic dynein. This domain is situated at the end of a slender 10-12 nm projection which corresponds to the stalks previously observed extending from the heads of both axonemal and cytoplasmic dyneins. The stalks also correspond to the B-links observed to connect outer arm axonemal dyneins to the B-microtubules in flagella and constitute the microtubule attachment sites during dynein motility. The stalks contrast strikingly with the polymer attachment domains of the kinesins and myosins which are found on the surface of the motor head. The difference in dynein's structural design raises intriguing questions as to how the stalk functions in force production along microtubules. In this article, we attempt to integrate the myriad of biochemical and EM structural data that has been previously collected regarding dynein with recent molecular findings, in an effort to begin to understand the mechanism of dynein motility.
- Marx A, Thormahlen M, Muller J, Sack S, Mandelkow EM, Mandelkow E
- Conformations of kinesin: solution vs. crystal structures and interactions with microtubules.
- Eur Biophys J. 1998; 27: 455-65
- Display abstract
Recently, the molecular structures of monomeric and dimeric kinesin constructs in complex with ADP have been determined by X-ray crystallography (Kull et al. 1996; Kozielski et al. 1997 a; Sack et al. 1997). The "motor" or "head" domains have almost identical conformations in the known crystal structures, yet the kinesin dimer is asymmetric: the orientation of the two heads relative to the coiled-coil formed by their neck regions is different. We used small angle solution scattering of kinesin constructs and microtubules decorated with kinesin in order to find out whether these crystal structures are of relevance for kinesin's structure under natural conditions and for its interaction with microtubules. Our preliminary results indicate that the crystal structures of monomeric and dimeric kinesin are similar to their structures in solution, though in solution the center-of-mass distance between the motor domains of the dimer could be slightly greater. The crystal structure of dimeric kinesin can be interpreted as representing two equivalent conformations. Transitions between these or very similar conformational states may occur in solution. Binding of kinesin to microtubules has conformational effects on both, the kinesin and the microtubule. Solution scattering of kinesin decorated microtubules reveals a peak in intensity that is characteristic for the B-surface lattice and that can be used to monitor the axial repeat of the microtubules under various conditions. In decoration experiments, dimeric kinesin dissociates, at least partly, leading to a stoichiometry of 1:1 (one kinesin head per tubulin dimer; Thormahlen et al. 1998a) in contrast to the stoichiometry of 2:1 reported for dimeric ncd. This discrepancy is possibly due to the effect of steric hindrance between kinesin dimers on adjacent binding sites.
- Foster KA, Correia JJ, Gilbert SP
- Equilibrium binding studies of non-claret disjunctional protein (Ncd) reveal cooperative interactions between the motor domains.
- J Biol Chem. 1998; 273: 35307-18
- Display abstract
Non-claret disjunctional protein (Ncd) is a minus end-directed microtubule motor required for normal spindle assembly and integrity during Drosophila oogenesis. We have pursued equilibrium binding experiments to examine the affinity of Ncd for microtubules in the presence of the ATP nonhydrolyzable analog 5'-adenylyl-beta, gamma-imidodiphosphate (AMP-PNP), ADP, or ADP + Pi using both dimeric (MC1) and monomeric (MC6) Ncd constructs expressed in Escherichia coli. Both MC1 and MC6 sediment with microtubules in the absence of added nucleotide as well as in the presence of either ADP or AMP-PNP. Yet, in the presence of ADP + Pi, there is a decrease in the affinity of both MC1 and MC6 for microtubules. The data for dimeric MC1 show that release of the dimer to the supernatant is sigmoidal with the apparent Kd(Pi) for the two phosphate sites at 23.3 and 1.9 mM, respectively. The results indicate that binding at the first phosphate site enhances binding at the second site, thus cooperatively stimulating release. Stopped-flow kinetics indicate that MgATP promotes dissociation of the Mt.MC1 complex at 14 s-1, yet AMP-PNP has no effect on the Mt.MC1 complex. These results are consistent with a model for the ATPase cycle in which ATP hydrolysis occurs on the microtubule followed by detachment as the Ncd.ADP.Pi intermediate.
- Howard J
- Molecular motors: structural adaptations to cellular functions.
- Nature. 1997; 389: 561-7
- Display abstract
Molecular motors are protein machines whose directed movement along cytoskeletal filaments is driven by ATP hydrolysis. Eukaryotic cells contain motors that help to transport organelles to their correct cellular locations and to establish and alter cellular morphology during cell locomotion and division. The best-studied motors, myosin from skeletal muscle and conventional kinesin from brain, are remarkably similar in structure, yet have very different functions. These differences can be understood in terms of the 'duty ratio', the fraction of the time that a motor is attached to its filament. Differences in duty ratio can explain the diversity of structures, speeds and oligomerization states of members of the large kinesin, myosin and dynein families of motors.
- Harrison RE, Huebner E
- Unipolar microtubule array is directly involved in nurse cell-oocyte transport.
- Cell Motil Cytoskeleton. 1997; 36: 355-62
- Display abstract
The telotrophic ovariole of Rhodnius prolixus is richly endowed with microtubules (MTs). An extensive, stable array of MTs packs the trophic core and trophic cords which link the nurse cell compartments to the growing oocytes. This system is excellent to study MT-based transport as the MTs are believed to play a role in transport of nurse cell-produced mitochondria, ribosomes, and mRNAs to the oocytes. We investigated MT polarity and molecular MT motors in this unidirectional transport system. Hook decoration revealed that the MTs of the trophic core and cords have their plus (+) ends in the tropharium and minus (-) ends in the oocytes. Video differential interference optics (DIC) microscopy showed that vesicle transport was saltatory, ATP-dependent, and had an average velocity of 0.77 micron/sec toward the oocyte. Transport was sensitive to 2 mM N-ethylmaleimide (NEM) and 50 microM vanadate and resistant to 1 mM 5'-adenylylimidodiphosphate (AMP-PNP) and 5 microM vanadate. We report that the unipolar, acetylated trophic cord MTs play a direct role in nurse cell-oocyte transport via a cytoplasmic dynein-like retrograde motor.
- Jiang W, Stock MF, Li X, Hackney DD
- Influence of the kinesin neck domain on dimerization and ATPase kinetics.
- J Biol Chem. 1997; 272: 7626-32
- Display abstract
Motor domains of kinesin were expressed that extend from the N terminus to positions 346, 357, 365, 381, and 405 (designated DKH346-DKH405) to determine if the kinetic differences observed between monomeric DKH340 and dimeric DKH392 (Hackney, D. D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6865-6869) were specific to these constructs or due to their oligomeric state. Sedimentation analysis indicated that DKH346, DKH357, and DKH365 are predominantly monomeric and that DKH381 and DKH405 are predominantly dimeric at 0. 01-0.03 microM, the concentrations used for ATPase assays. In buffer with 25 mM KCl, all have high kcat values of 38-96 s-1 at saturating microtubule (MT) levels. Monomeric DKH346, DKH357, and DKH365 have K0.5(MT) values of 17, 9, and 1.4 microM, respectively, but the K0. 5(MT) values for the dimeric species are significantly lower, with 0. 02 and 0.14 microM for DKH381 and DKH405, respectively. The three new monomers release all of their ADP on association with microtubules, whereas the two new dimers retain approximately half of their ADP, consistent with the half-site reactivity observed previously with dimeric DKH392. Both the kbi(ATPase) (=kcat/K0. 5(MT)) values for stimulation of ATPase by MTs and the kbi(ADP) for stimulation of ADP release by MTs were determined in buffer containing 120 mM potassium acetate. The ratio of these rate constants (kbi(ratio) = kbi(ATPase)/kbi(ADP)) is 60-100 for the dimers, indicating hydrolysis of many ATP molecules per productive encounter with a MT as observed previously for DKH392 (Hackney, D. D. (1995) Nature 377, 448-450). For the monomers, kbi(ratio) values of approximately 4 indicate that they also may hydrolyze more than one ATP molecule per encounter with a MT and that the mechanism of hydrolysis is therefore fundamentally different from that of actomyosin. DKH340 is an exception to this pattern and may undergo uncoupled ATP hydrolysis.
- Yokota E, Mabuchi I
- Interaction of flagellar inner arm dynein isolated from sea urchin sperm with microtubules in the presence of ATP.
- Eur J Cell Biol. 1997; 72: 214-21
- Display abstract
We have isolated C/A dynein, which is considered to be a component of inner arms, from flagellar axonemes of sea urchin sperm (E. Yokota, I. Mabuchi, J. Cell Sci. 107, 345-351 (1994). C/A dynein binds to and bundles the microtubules in the absence of ATP. In contrast to outer arm 21S dynein, C/A dynein is not released from the microtubules in the presence of ATP (E. Yokota, I. Mabuchi, J. Cell Sci. 107, 353-361 (1994)). We further investigated the interaction of C/A dynein with microtubules in the presence of ATP. The turbidity at 350 nm of a mixture of C/A dynein and microtubules increased by the addition of ATP. Both the initial rate and final extent of the turbidity increase were dependent on C/A dynein or ATP concentration and were inhibited by vanadate. ATP hydrolysis by C/A dynein was linear during the time course of the turbidity increase. Negative staining electron microscopy revealed that microtubular bundles which formed in the presence of C/A dynein became thicker and longer after addition of ATP. Furthermore, sliding movements of microtubule(s) in the individual bundles were observed in the presence of ATP. This mode of interaction of C/A dynein with microtubules was distinct from that of flagellar or ciliary dyneins reported so far. These results suggest that C/A dynein, as a component of inner arms, may play a distinct role in the flagellar movement of sea urchin sperm.
- Koonce MP
- Identification of a microtubule-binding domain in a cytoplasmic dynein heavy chain.
- J Biol Chem. 1997; 272: 19714-8
- Display abstract
As a molecular motor, dynein must coordinate ATP hydrolysis with conformational changes that lead to processive interactions with a microtubule and generate force. To understand how these processes occur, we have begun to map functional domains of a dynein heavy chain from Dictyostelium. The carboxyl-terminal 10-kilobase region of the heavy chain encodes a 380-kDa polypeptide that approximates the globular head domain. Attempts to further truncate this region fail to produce polypeptides that either bind microtubules or UV-vanadate cleave, indicating that the entire 10-kilobase fragment is necessary to produce a properly folded functional dynein head. We have further identified a region just downstream from the fourth P-loop that appears to constitute at least part of the microtubule-binding domain (amino acids 3182-3818). When deleted, the resulting head domain polypeptide no longer binds microtubules; when the excised region is expressed in vitro, it cosediments with added tubulin polymer. This microtubule-binding domain falls within an area of the molecule predicted to form extended alpha-helices. At least four discrete sites appear to coordinate activities required to bind the tubulin polymer, indicating that the interaction of dynein with microtubules is complex.
- Kozielski F, Schonbrunn E, Sack S, Muller J, Brady ST, Mandelkow E
- Crystallization and preliminary X-ray analysis of the single-headed and double-headed motor protein kinesin.
- J Struct Biol. 1997; 119: 28-34
- Display abstract
Crystals of the single-headed and double-headed kinesin motor domains of Rattus norvegicus have been grown by vapor diffusion using ammonium sulfate as the precipitant. Both crystal systems belong to the orthorhombic space group P2(1)2(1)2(1). Double-headed kinesin crystallized with unit cell constants of a = 72.2 A, b = 91.9 A, and c = 141.7 A, and so far the best crystals diffracted to a maximum resolution of 2.7 A. Using ammonium sulfate single-headed kinesin crystallized in two different crystal forms with cell constants of a = 73.1 A, b = 73.2 A, c = 84.0 A and a = 73.4 A, b = 74.1 A, c = 74.7 A, respectively. They were found to diffract to 2.1 A resolution. Crystals of monomeric kinesin were also obtained with lithium sulfate as precipitant. They have cell constants of a = 71.6 A, b = 73.7 A, and c = 74.1 A and diffract up to 1.7 A resolution.
- Nedelec FJ, Surrey T, Maggs AC, Leibler S
- Self-organization of microtubules and motors.
- Nature. 1997; 389: 305-8
- Display abstract
Cellular structures are established and maintained through a dynamic interplay between assembly and regulatory processes. Self-organization of molecular components provides a variety of possible spatial structures: the regulatory machinery chooses the most appropriate to express a given cellular function. Here we study the extent and the characteristics of self-organization using microtubules and molecular motors as a model system. These components are known to participate in the formation of many cellular structures, such as the dynamic asters found in mitotic and meiotic spindles. Purified motors and microtubules have previously been observed to form asters in vitro. We have reproduced this result with a simple system consisting solely of multi-headed constructs of the motor protein kinesin and stabilized microtubules. We show that dynamic asters can also be obtained from a homogeneous solution of tubulin and motors. By varying the relative concentrations of the components, we obtain a variety of self-organized structures. Further, by studying this process in a constrained geometry of micro-fabricated glass chambers, we demonstrate that the same final structure can be reached through different assembly 'pathways.
- Song H, Golovkin M, Reddy AS, Endow SA
- In vitro motility of AtKCBP, a calmodulin-binding kinesin protein of Arabidopsis.
- Proc Natl Acad Sci U S A. 1997; 94: 322-7
- Display abstract
AtKCBP is a calcium-dependent calmodulin-binding protein from Arabidopsis that contains a conserved kinesin microtubule motor domain. Calmodulin has been shown previously to bind to heavy chains of the unconventional myosins, where it is required for in vitro motility of brush border myosin I, but AtKCBP is the first kinesin-related heavy chain reported to be capable of binding specifically to calmodulin. Other kinesin proteins have been identified in Arabidopsis, but none of these binds to calmodulin, and none has been demonstrated to be a microtubule motor. We have tested bacterially expressed AtKCBP for the ability to bind microtubules to a glass surface and induce gliding of microtubules across the glass surface. We find that AtKCBP is a microtubule motor protein that moves on microtubules toward the minus ends, with the opposite polarity as kinesin. In the presence of calcium and calmodulin, AtKCBP no longer binds microtubules to the coverslip surface. This contrasts strikingly with the requirement of calmodulin for in vitro motility of brush border myosin I. Calmodulin could regulate AtKCBP binding to microtubules in the cell by inhibiting the binding of the motor to microtubules. The ability to bind to calmodulin provides an evolutionary link between the kinesin and myosin motor proteins, but our results indicate that the mechanisms of interaction and regulation of kinesin and myosin heavy chains by calmodulin are likely to differ significantly.
- Jontes JD, Milligan RA
- Three-dimensional structure of Brush Border Myosin-I at approximately 20 A resolution by electron microscopy and image analysis.
- J Mol Biol. 1997; 266: 331-42
- Display abstract
Brush Border Myosin-I (BBMI) is a single-headed, unconventional myosin found in the microvilli of intestinal epithelial cells where it forms lateral bridges between the core bundle of actin filaments and the plasma membrane of the microvillus. A three-dimensional (3D) reconstruction of BBMI was made from images of negatively stained, two-dimensional (2D) crystals grown on lipid monolayers formed from mixtures of phosphatidylserine and phosphatidylcholine. The resolution of the 3D map extends to approximately 20 A and allows identification of all of the major structural domains of BBMI. The BBMI molecule is composed of three domains: a globular motor domain, a light-chain-binding domain and a lipid-binding domain. In our map, the putative motor domain is connected to an extended density, which we believe to be the light-chain-binding domain. This long, narrow region has three distinct bends, which may delineate the bound calmodulin light chains. Following the last calmodulin there is density which extends for a short distance across the lipid surface and is presumably the carboxy-terminal lipid-binding domain.
- Walczak CE, Verma S, Mitchison TJ
- XCTK2: a kinesin-related protein that promotes mitotic spindle assembly in Xenopus laevis egg extracts.
- J Cell Biol. 1997; 136: 859-70
- Display abstract
We used a peptide antibody to a conserved sequence in the motor domain of kinesins to screen a Xenopus ovary cDNA expression library. Among the clones isolated were two that encoded a protein we named XCTK2 for Xenopus COOH-terminal kinesin 2. XCTK2 contains an NH2-terminal globular domain, a central alpha-helical stalk, and a COOH-terminal motor domain. XCTK2 is similar to CTKs in other organisms and is most homologous to CHO2. Antibodies raised against XCTK2 recognize a 75-kD protein in Xenopus egg extracts that cosediments with microtubules. In Xenopus tissue culture cells, the anti-XCTK2 antibodies stain mitotic spindles as well as a subset of interphase nuclei. To probe the function of XCTK2, we have used an in vitro assay for spindle assembly in Xenopus egg extracts. Addition of antibodies to cytostatic factor-arrested extracts causes a 70% reduction in the percentage of bipolar spindles formed. XCTK2 is not required for maintenance of bipolar spindles, as antibody addition to preformed spindles has no effect. To further evaluate the function of XCTK2, we expressed XCTK2 in insect Sf-9 cells using the baculovirus expression system. When purified (recombinant XCTK2 is added to Xenopus egg extracts at a fivefold excess over endogenous levels) there is a stimulation in both the rate and extent of bipolar spindle formation. XCTK2 exists in a large complex in extracts and can be coimmunoprecipitated with two other proteins from extracts. XCTK2 likely plays an important role in the establishment and structural integrity of mitotic spindles.
- Jiang W, Hackney DD
- Monomeric kinesin head domains hydrolyze multiple ATP molecules before release from a microtubule.
- J Biol Chem. 1997; 272: 5616-21
- Display abstract
Transient kinetic analysis of microtubule-stimulated ATP hydrolysis by the monomeric kinesin motor domain DKH357 was performed to investigate the kinetic pattern of a monomer. Both ATP and ADP produced dissociation of the complex, microtubule (MT).E, of microtubules with DKH357 at a maximum rate of approximately 45 s-1 as determined by decrease in turbidity. The maximum dissociation rate was independent of the KCl concentration between 25 and 200 mM. At subsaturating levels of nucleotide, ATP was more effective than ADP in dissociating DKH357 from MT.E (1.6 and 0.4 &mgr;M-1 s-1 for ATP and ADP, respectively, at 50 mM KCl). Addition of ATP to MT.E results in a burst of product formation with a maximum initial rate of approximately 100 s-1 at saturating levels of ATP. This maximum hydrolysis rate of 100 s-1 is similar to the maximum steady state ATPase rate at saturating microtubules of approximately 70 s-1, and thus hydrolysis is at least partially rate-limiting. When the MT lattice was highly occupied with bound DKH357, the amplitude of the burst was approximately 2 per DKH357 active site (superstoichiometric). The rate constant for the burst transient was approximately 45 s-1, which is the same as the rate for dissociation of DKH357 from the microtubule and this suggests that dissociation and termination of the burst phase are coupled. The size of the burst increased with decreasing initial occupancy of the MT lattice with bound DKH357 and approached the value of approximately 4 ATP molecules predicted by previous steady state measurements (Jiang, W., Stock, M., Li, X., and Hackney, D. D., submitted for publication).
- Steinberg G
- A kinesin-like mechanoenzyme from the zygomycete Syncephalastrum racemosum shares biochemical similarities with conventional kinesin from Neurospora crassa.
- Eur J Cell Biol. 1997; 73: 124-31
- Display abstract
The kinesin superfamily consists of mechanoenzymes that convert chemical energy, stored in nucleoside triphosphates, into movement along microtubules. The founding member of this protein superfamily, the so-called conventional kinesin, was only known from animal sources until the recent description of kinesin from the filamentous fungus Neurospora crassa (G. Steinberg, M. Schliwa, Mol. Biol. Cell 6, 1605-1618 (1995)). To determine whether similar motors with comparable features are common in other filamentous fungi, a kinesin from a zygomycete, Syncephalastrum racemosum, was purified. Here, the isolation and characterization of this motorenzyme is described. The purified protein consisted of a doublet at 112 kDa and 115 kDa with no additional polypeptides. This was consistent with a calculated molecular mass of approximately 240 kDa and suggests that the motor is a dimer with a more globular shape than conventional kinesin from animal sources. In gliding assays the enzyme moved microtubules at 2.5 to 3.4 microns/s and had a nucleotide specificity similar to the Neurospora kinesin motor. Peptide antibodies against conserved regions in the head and the tall domain of conventional kinesins cross-reacted with the Syncephalastrum motor. In vitro, the enzyme was able to drive the microtubule-dependent movement of vesicles isolated from Syncephalastrum racemosum, as well as Neurospora crassa, and Aspergillus nidulans. In summary, the Syncephalastrum motor has many of the unique features in common with the conventional kinesin from the ascomycete Neurospora crassa and probably shares a similar function in living hyphae.
- Kasprzak AA
- [How myosin works]
- Postepy Biochem. 1997; 43: 134-42
- Saito N, Okada Y, Noda Y, Kinoshita Y, Kondo S, Hirokawa N
- KIFC2 is a novel neuron-specific C-terminal type kinesin superfamily motor for dendritic transport of multivesicular body-like organelles.
- Neuron. 1997; 18: 425-38
- Display abstract
We have cloned two novel C-terminal motor domain-type kinesin superfamily motor proteins (KIFCs) from mouse brain by utilizing a KIFC-specific consensus sequence. The first protein was the murine homologue of CHO2 antigen, a member of the kar3-type mitotic motor subfamily, and we designated this protein KIFC1. The other protein, KIFC2 (792 amino acids), is novel, with no significant similarity to known kinesin superfamily proteins (KIFs). KIFC2 was specifically expressed in adult neurons, and was immunofluorescently localized to punctate structures in cell bodies and dendrites, but was not detected in axons. Electron microscopic analysis of the immunoisolated KIFC2-bound organelles revealed that KIFC2 associates with multivesicular body (mvb)-like organelles, suggesting that KIFC2 functions as the motor for the transport of mvb-like organelles in dendrites.
- Hanlon DW, Yang Z, Goldstein LS
- Characterization of KIFC2, a neuronal kinesin superfamily member in mouse.
- Neuron. 1997; 18: 439-51
- Display abstract
Members of the kinesin superfamily of microtubule-associated proteins are involved in a variety of intracellular processes including cell division and organelle transport. In the case of axonal transport, all kinesin superfamily members reported thus far appear to play a role in anterograde transport, while a different type of microtubule motor, dynein, appears to function in retrograde transport. To better understand the role of kinesins in axonal transport, we cloned and characterized KIFC2, a novel kinesin superfamily member from mouse brain. KIFC2 encodes a 792 amino acid protein, which contains the conserved motor domain at the C-terminal end of the protein and is most similar to members of the KAR3 family involved in cell division. However, expression analysis localized KIFC2 mRNA to nonproliferative neuronal cells in the central nervous system, and immunolocalization studies demonstrated that KIFC2 is present in axons and dendrites of neurons in the central and peripheral nervous systems. Immunolocalization and biochemical fractionation studies suggest that KIFC2 localizes with some, but not all, axonally transported organelles. Finally, ligation of mouse peripheral nerves showed that KIFC2 accumulates at the proximal and distal sides of an axonal ligature. Taken together, the data suggest that, unlike other C-terminal motor proteins that appear to be involved in cell division, KIFC2 may play a role in retrograde axonal transport.
- Brokaw CJ
- Are motor enzymes bidirectional?
- Cell Motil Cytoskeleton. 1997; 38: 115-9
- Brokaw CJ
- Mechanical components of motor enzyme function.
- Biophys J. 1997; 73: 938-51
- Display abstract
Motor enzymes use energy from ATP dephosphorylation to generate movement by a mechanical cycle, moving and pushing in one direction while attached to their cytoskeletal substrate, and recovering by moving relative to their substrate to a new attachment site. Mainstream models assert that movement while attached to the substrate results from preexisting strain in the attached motor. The additional underlying ideas can be described in terms of three components for strain amplification: a rotating lever arm, multiple attached states, and elastic compliance. These components determine how energy is recovered during the mechanical cycle and stored in a strained motor. They may coexist in a real motor; the challenge is to determine the contributions of each component. Because these components can generate similar relationships between strain energy and strain, standard measurements of motor function do not discriminate easily between these components. However, important information could be is provided by observations that suggest weak coupling between chemical and mechanical cycles, observations of negative force and movement events in single motor experiments, and the discovery that two motors that move in opposite directions have very similar structures. In models incorporating changes in conformation between attached states, these observations are only explained easily if the conformational changes are tightly coupled to changes in the strength of motor-substrate binding.
- Naber N, Cooke R, Pate E
- Binding of ncd to microtubules induces a conformational change near the junction of the motor domain with the neck.
- Biochemistry. 1997; 36: 9681-9
- Display abstract
We have covalently attached an electron paramagnetic resonance (EPR) spin probe to Cys-670 of the motor domain of ncd (nonclaret disjunctional protein) in order to investigate conformational changes associated with the chemomechanical cycle. Spin-labeling is highly specific and does not affect ncd function as monitored by either the binding affinity to microtubules or the rate of ATP hydrolysis. The EPR spectra can be deconvoluted into two components, one that is highly mobile with respect to the protein and one that is strongly immobilized. In the absence of microtubules, the relative proportions of these two components varied with temperature, showing that the transition between them involves a large change in enthalpy (DeltaH degrees = -75 kJ/mol). This result implies that the two populations represent very different protein conformations. Binding to microtubules results in virtually all probes shifting into the immobilized component, independent of the nucleotide bound. Superposition of the structures of ncd and myosin subfragment 1 reveals that the labeled cysteine is very close to the region which is homologous to the helix containing the two reactive sulfhydryls in myosin and is approximately 10 A from the junction of the motor domain with the remainder of the molecule. We conclude that the binding of ncd to microtubules results in a conformational change in this region which may be involved in the working power stroke.
- Kojima H, Muto E, Higuchi H, Yanagida T
- Mechanics of single kinesin molecules measured by optical trapping nanometry.
- Biophys J. 1997; 73: 2012-22
- Display abstract
We have analyzed the mechanics of individual kinesin molecules by optical trapping nanometry. A kinesin molecule was adsorbed onto a latex bead, which was captured by an optical trap and brought into contact with an axoneme that was bound to a glass surface. The displacement of kinesin during force generation was determined by measuring the position of the beads with nanometer accuracy. As the displacement of kinesin was attenuated because of the compliance of the kinesin-to-bead and kinesin-to-microtubule linkages, the compliance was monitored during force generation and was used to correct the displacement of kinesin. Thus the velocity and the unitary steps could be obtained accurately over a wide force range. The force-velocity curves were linear from 0 to a maximum force at 10 microM and 1 mM ATP, and the maximum force was approximately 7 pN, which is larger by approximately 30% than values previously reported. Kinesin exhibited forward and occasionally backward stepwise displacements with a size of approximately 8 nm. The histograms of step dwell time show a monotonic decrease with time. Model calculations indicate that each kinesin head steps by 16-nm, whereas kinesin molecule steps by 8-nm.
- Lindesmith L, McIlvain JM Jr, Argon Y, Sheetz MP
- Phosphotransferases associated with the regulation of kinesin motor activity.
- J Biol Chem. 1997; 272: 22929-33
- Display abstract
Kinesin, a plus-end-directed microtubule motor protein, functions in concert with accessory factors that have been shown to regulate enzyme activity and may also provide cargo specificity. This report identifies teh 79-kDa kinesin-associated phosphoprotein as a phosphoisoform of kinesin light chain. Increased phosphorylation of this light chain isoform is sufficient to account for the increase in kinesin-mediated microtubule-gliding activity. Additionally, it was found that the degree of phosphorylation of this isoform is regulated by a 100-kDa kinase and 150-kDa type 1 phosphatase. Both the kinesin light chain kinase and phosphatase co-purify with the kinesin heavy chain, suggesting that kinesin exists in a large complex capable of self-regulation.
- Bohm KJ, Steinmetzer P, Daniel A, Baum M, Vater W, Unger E
- Kinesin-driven microtubule motility in the presence of alkaline-earth metal ions: indication for a calcium ion-dependent motility.
- Cell Motil Cytoskeleton. 1997; 37: 226-31
- Display abstract
We studied the effect of alkaline-earth metal ions on the kinesin-driven gliding of microtubules, using a narrow glass chamber enabling the exchange of buffer components without interrupting microscopic observation. Under standard conditions (0.5 mM Mg2+), microtubules were found to glide at a mean velocity of about 0.6 micron/s. Motility was widely ceased after removing Mg2+. Subsequent addition of Ca2+ restored motility (maximal mean gliding velocity measured: 0.26 micron/s at 2.5 mM Ca2+). Also in the presence of Sr2+ or Ba2+ a slow gliding could be observed (0.025 micron/s and 0.014 micron/s, respectively, at 0.5 mM). After removal of Ca2+, Sr2+, or Ba2+ and re-addition of Mg2+, the gliding velocities reached approximately the values determined under standard conditions. Motility was not changed when 0.5 mM Ca2+, Sr2+, or Ba2+ were applied together with Mg2+. Microtubule gliding stopped after substitution of 0.5 mM BeCl2 for Mg2+. When both BeCl2 and Mg2+ were present, the mean gliding velocity was reduced to 0.29 micron/s. In addition, many microtubules were released from the kinesin coated glass surface, indicating that the beryllium salt disorders the binding between kinesin and microtubules. Our results confirm that Mg2+ is the most suitable cofactor for kinesin driven microtubule motility. However, they also demonstrate that brain kinesin can generate motility when Ca2+ was substituted for Mg2+.
- Metoz F, Arnal I, Wade RH
- Tomography without tilt: three-dimensional imaging of microtubule/motor complexes.
- J Struct Biol. 1997; 118: 159-68
- Display abstract
Usually structures such as microtubules are supposed to have surface lattices built from families of continuous helices, giving electron micrographs that can be analyzed by helical diffraction theory with a view to obtaining three-dimensional reconstructions. In the case of microtubules the helical surface lattice may be interrupted by discontinuities, called seams, in which case the usual helical reconstruction approach is no longer applicable. Even so, by virtue of their "superhelical" protofilaments, microtubules are still helical structures and we use this feature to treat a microtubule image as a set of projections equivalent to images obtained in a single axis tilt series. The main thrust of this article is to discuss how to obtain images and image parameters best suited to a tomographic approach to three-dimensional reconstruction. The method is tested by comparing helical and back-projection reconstructions of appropriate microtubules both with and without surface lattice decoration by kinesin family motor proteins. Tomographic reconstruction gives an independent demonstration that in vitro assembled microtubules have a B-type surface lattice. We show that 3-start, 15-protofilament microtubules have a seam, whereas 4-start microtubules have no seam and possess complete helical symmetry. Monomer motor domains attach to the outer ridge of the protofilaments and extend along the protofilament toward the plus end.
- Sosa H, Hoenger A, Milligan RA
- Three different approaches for calculating the three-dimensional structure of microtubules decorated with kinesin motor domains.
- J Struct Biol. 1997; 118: 149-58
- Display abstract
We have used three different electron microscopy approaches to calculate three-dimensional maps of tubulin assemblies decorated with the motor domain of kinesin. The approaches used were: (1) Tilt series reconstruction of negatively stained tubulin sheets. (2) Back-projection reconstruction of microtubules in ice. (3) Helical reconstruction of microtubules in ice. The calculated maps show the overall configuration of the protofilaments and the interactions between the motor and the protofilaments at a resolution of 2-4 nm. The three methods revealed a similar binding configuration of the kinesin motor domain to the protofilament. We also found that seams can be present in potentially helical microtubules, limiting the use of helical reconstruction algorithms. Advantages and disadvantages of each of the three approaches are discussed.
- Coppin CM, Pierce DW, Hsu L, Vale RD
- The load dependence of kinesin's mechanical cycle.
- Proc Natl Acad Sci U S A. 1997; 94: 8539-44
- Display abstract
Kinesin is a dimeric motor protein that transports organelles in a stepwise manner toward the plus-end of microtubules by converting the energy of ATP hydrolysis into mechanical work. External forces can influence the behavior of kinesin, and force-velocity curves have shown that the motor will slow down and eventually stall under opposing loads of approximately 5 pN. Using an in vitro motility assay in conjunction with a high-resolution optical trapping microscope, we have examined the behavior of individual kinesin molecules under two previously unexplored loading regimes: super-stall loads (>5 pN) and forward (plus-end directed) loads. Whereas some theories of kinesin function predict a reversal of directionality under high loads, we found that kinesin does not walk backwards under loads of up to 13 pN, probably because of an irreversible transition in the mechanical cycle. We also found that this cycle can be significantly accelerated by forward loads under a wide range of ATP concentrations. Finally, we noted an increase in kinesin's rate of dissociation from the microtubule with increasing load, which is consistent with a load dependent partitioning between two recently described kinetic pathways: a coordinated-head pathway (which leads to stepping) and an independent-head pathway (which is static).
- Kammerer RA
- Alpha-helical coiled-coil oligomerization domains in extracellular proteins.
- Matrix Biol. 1997; 15: 555-65
- Display abstract
Subunit oligomerization of many proteins is mediated by alpha-helical coiled-coil domains. 3,4-Hydrophobic heptad repeat sequences, the characteristic feature of the coiled-coil protein folding motif, have been found in a wide variety of gene products including cytoskeletal, nuclear, muscle, cell surface, extracellular, plasma, bacterial, and viral proteins. Whereas the majority of coiled-coil structures is represented by intracellular alpha-helical bundles that contain two polypeptide chains, examples of extracellular coiled-coil proteins are fewer in number. Most proteins located in the extracellular space form three-stranded alpha-helical assemblies. Recently, five-stranded coiled coils have been identified in thrombospondins 3 and 4 and in cartilage oligomeric matrix protein, and the formation of a heterotetramer has been observed in in vitro studies with the recombinant asialoglycoprotein receptor oligomerization domain. Coiled-coil domains in laminins and probably also in tenascins and thrombospondins are responsible for the formation of tissue-specific isoforms by selective oligomerization of different polypeptide chains.
- Sosa H et al.
- A model for the microtubule-Ncd motor protein complex obtained by cryo-electron microscopy and image analysis.
- Cell. 1997; 90: 217-24
- Display abstract
Kinesin motors convert chemical energy from ATP hydrolysis into unidirectional movement. To understand how kinesin motors bind to and move along microtubules, we fit the atomic structure of the motor domain of Ncd (a kinesin motor involved in meiosis and mitosis) into three-dimensional density maps of Ncd-microtubule complexes calculated by cryo-electron microscopy and image analysis. The model reveals that Ncd shares an extensive interaction surface with the microtubule, and that a portion of the binding site involves loops that contain conserved residues. In the Ncd dimer, the microtubule-bound motor domain makes intimate contact with its partner head, which is dissociated from the microtubule. This head-head interaction may be important in positioning the dissociated head to take a step to the next binding site on the microtubule protofilament.
- Woehlke G, Ruby AK, Hart CL, Ly B, Hom-Booher N, Vale RD
- Microtubule interaction site of the kinesin motor.
- Cell. 1997; 90: 207-16
- Display abstract
Kinesin and myosin are motor proteins that share a common structural core and bind to microtubules and actin filaments, respectively. While the actomyosin interface has been well studied, the location of the microtubule-binding site on kinesin has not been identified. Using alanine-scanning mutagenesis, we have found that microtubule-interacting kinesin residues are located in three loops that cluster in a patch on the motor surface. The critical residues are primarily positively charged, which is consistent with a primarily electrostatic interaction with the negatively charged tubulin molecule. The core of the microtubule-binding interface resides in a highly conserved loop and helix (L12/alpha5) that corresponds topologically to the major actin-binding domain of myosin. Thus, kinesin and myosin have developed distinct polymer-binding domains in a similar region with respect to their common catalytic cores.
- Tao Y, Strelkov SV, Mesyanzhinov VV, Rossmann MG
- Structure of bacteriophage T4 fibritin: a segmented coiled coil and the role of the C-terminal domain.
- Structure. 1997; 5: 789-98
- Display abstract
BACKGROUND: Oligomeric coiled-coil motifs are found in numerous protein structures; among them is fibritin, a structural protein of bacteriophage T4, which belongs to a class of chaperones that catalyze a specific phage-assembly process. Fibritin promotes the assembly of the long tail fibers and their subsequent attachment to the tail baseplate; it is also a sensing device that controls the retraction of the long tail fibers in adverse environments and, thus, prevents infection. The structure of fibritin had been predicted from sequence and biochemical analyses to be mainly a triple-helical coiled coil. The determination of its structure at atomic resolution was expected to give insights into the assembly process and biological function of fibritin, and the properties of modified coiled-coil structures in general. RESULTS: The three-dimensional structure of fibritin E, a deletion mutant of wild-type fibritin, was determined to 2.2 A resolution by X-ray crystallography. Three identical subunits of 119 amino acid residues form a trimeric parallel coiled-coil domain and a small globular C-terminal domain about a crystallographic threefold axis. The coiled-coil domain is divided into three segments that are separated by insertion loops. The C-terminal domain, which consists of 30 residues from each subunit, contains a beta-propeller-like structure with a hydrophobic interior. CONCLUSIONS: The residues within the C-terminal domain make extensive hydrophobic and some polar intersubunit interactions. This is consistent with the C-terminal domain being important for the correct assembly of fibritin, as shown earlier by mutational studies. Tight interactions between the C-terminal residues of adjacent subunits counteract the latent instability that is suggested by the structural properties of the coiled-coil segments. Trimerization is likely to begin with the formation of the C-terminal domain which subsequently initiates the assembly of the coiled coil. The interplay between the stabilizing effect of the C-terminal domain and the labile coiled-coil domain may be essential for the fibritin function and for the correct functioning of many other alpha-fibrous proteins.
- Hirose K, Amos WB, Lockhart A, Cross RA, Amos LA
- Three-dimensional cryoelectron microscopy of 16-protofilament microtubules: structure, polarity, and interaction with motor proteins.
- J Struct Biol. 1997; 118: 140-8
- Display abstract
We present a three-dimensional (3D) map, reconstructed from electron microscope (EM) images of naturally occurring 16-protofilament (PF) microtubules (MTs) in ice. We compare it with the tubulin in six 3D maps of MTs decorated with motor domains, three from frozen MTs decorated with kinesin or ncd in the tightly bound AMP-PNP state, and three from negatively stained MTs decorated with kinesin in different nucleotide states. The comparison confirms that kinesin and ncd bind to identical sites and interact with both monomers of a tubulin dimer. Maps of specimens in negative stain and in ice are similar except that the protein in the top half of a motor domain appears denser in negative stain. The interactions have only a small effect on tubulin structure; the outward appearance is unchanged, but there seems to be a small internal rearrangement. The relative polarity of undecorated and decorated MTs is evident from their 3D structures. This agrees with the absolute polarities indicated by the orientations of motors in decorated specimens and by polar superposition patterns calculated for undecorated MTs. An image of tubulin PFs in zinc-induced sheets has been tentatively oriented by similar criteria.
- Muresan V, Bendala-Tufanisco E, Hollander BA, Besharse JC
- Evidence for kinesin-related proteins associated with the axoneme of retinal photoreceptors.
- Exp Eye Res. 1997; 64: 895-903
- Display abstract
Situated at the junction between inner and outer segment, the connecting cilium of retinal photoreceptors supports regulated transport of molecules that function distally, while restricting diffusion of membrane proteins from one plasmalemmal domain to the other. Both functions are thought to be performed by a group of proteins stably or transiently associated with the axoneme. We have identified two types of unique polypeptides which associated with the axoneme in a nucleotide-dependent manner: they bind to the axonemes in the presence of adenosine monophosphate (AMP)-PNP, and are solubilized in the presence of adenosine triphosphate (ATP). The first group contained glyconjugates, previously shown to be part of the axoneme-plasmalemma cross-linkers at the connecting cilium. The second group cross-reacted with antibodies to two different conserved peptide sequences (called LAGSE and HIPYR) of kinesin-related proteins, and included polypeptides of approximately 85-97 kDa. Immunofluorescence microscopy of whole-mounted axonemes with the two anti-kinesin antibodies showed labeling throughout the axoneme, including the connecting cilium-basal body region. These results suggest that the identified proteins may serve as motor molecules for transport of material to the outer segment via the connecting cilium.
- Seiler S, Nargang FE, Steinberg G, Schliwa M
- Kinesin is essential for cell morphogenesis and polarized secretion in Neurospora crassa.
- EMBO J. 1997; 16: 3025-34
- Display abstract
Kinesin is a force-generating molecule that is thought to translocate organelles along microtubules, but its precise cellular function is still unclear. To determine the role of kinesin in vivo, we have generated a kinesin-deficient strain in the simple cell system Neurospora crassa. Null cells exhibit severe alterations in cell morphogenesis, notably hyphal extension, morphology and branching. Surprisingly, the movement of organelles visualized by video microscopy is hardly affected, but apical hyphae fail to establish a Spitzenkorper, an assemblage of secretory vesicles intimately linked to cell elongation and morphogenesis in Neurospora and other filamentous fungi. As cell morphogenesis depends on polarized secretion, our findings demonstrate that a step in the secretory pathway leading to cell shape determination and cell elongation cannot tolerate a loss of kinesin function. The defect is suggested to affect the transport of small, secretory vesicles to the site involved in protrusive activity, resulting in the uncoordinated insertion of new cell wall material over much of the cell surface. These observations have implications for the presumptive function of kinesin in more complex cell systems.
- Funatsu T et al.
- Imaging and nano-manipulation of single biomolecules.
- Biophys Chem. 1997; 68: 63-72
- Display abstract
We have developed a new technique for imaging single fluorescent dye molecules by refining epifluorescence and total internal reflection fluorescence microscopies. In contrast to previously reported single fluorescent molecule imaging methods, in which specimens were immobilized on an air-dried surface, our method enables video-rate imaging of single molecules in aqueous solution. This approach enabled us to directly image the processive movement of individual fluorescently labeled kinesin molecules along a microtubule. This method was also used to visualize individual ATP turnover reactions of single myosin molecules. The method can be combined with molecular manipulation using an optical trap. A single kinesin molecule attached to a polystyrene bead was brought into contact with a microtubule adsorbed onto the glass surface. The lifetime of bound Cy3-nucleotide in the absence or presence of the microtubule was 10 s or 0.08 s, respectively, showing that ATPase activity of the kinesin is strongly activated by microtubules. As the present system is equipped with a nanometer sensor, elemental steps of a single kinesin molecule can also be measured. By simultaneously measuring the individual ATP turnovers and elementary mechanical events of a single kinesin molecule, we will be able to obtain a clear answer to the fundamental problem of how the mechanical events are coupled to the ATPase reaction.
- Robertson AM, Allan VJ
- Cell cycle regulation of organelle transport.
- Prog Cell Cycle Res. 1997; 3: 59-75
- Display abstract
Microtubule- and actin-based motors play a wide range of vital roles in the organisation and function of cells during both interphase and mitosis, all of which are likely to be under strict control. Here, we describe how one of these roles--the movement of membranes--is regulated through the cell cycle. Organelle movement in many species is greatly reduced in mitosis as compared to interphase, and this change occurs concomitantly with an inhibition of most membrane traffic functions. Data from in vitro studies is shedding light on how microtubule motor regulation may be achieved.
- Inoue Y, Toyoshima YY, Iwane AH, Morimoto S, Higuchi H, Yanagida T
- Movements of truncated kinesin fragments with a short or an artificial flexible neck.
- Proc Natl Acad Sci U S A. 1997; 94: 7275-80
- Display abstract
To investigate the role of the neck domain of kinesin, we used optical trapping nanometry to perform high-resolution measurements of the movements and forces produced by recombinant kinesin fragments in which the neck domains were shortened or replaced by an artificial random coil. Truncated kinesin fragments (K351) that contain a motor domain consisting of approximately 340 aa and a short neck domain consisting of approximately 11 aa showed fast movement (800 nm/s) and 8-nm steps. Such behavior was similar to that of recombinant fragments containing the full-length neck domain (K411) and to that of native kinesin. Kinesin fragments lacking the short neck domain (K340), however, showed very slow movement (<50 nm/s), as previously reported. Joining an artificial 11-aa sequence that was expected to form a flexible random chain to the motor domain (K340-chain) produced normal fast ( approximately 700 nm/s) and stepwise movement. The results suggest that the neck domain does not act as a rigid lever arm to magnify the structural change at the catalytic domain as has been believed for myosin, but it does act as a flexible joint to guarantee the mobility of the motor domain.
- Nakagawa T et al.
- Identification and classification of 16 new kinesin superfamily (KIF) proteins in mouse genome.
- Proc Natl Acad Sci U S A. 1997; 94: 9654-9
- Display abstract
KIF (kinesin superfamily) proteins are microtubule-dependent molecular motors that play important roles in intracellular transport and cell division. The extent to which KIFs are involved in various transporting phenomena, as well as their regulation mechanism, are unknown. The identification of 16 new KIFs in this report doubles the existing number of KIFs known in the mouse. Conserved nucleotide sequences in the motor domain were amplified by PCR using cDNAs of mouse nervous tissue, kidney, and small intestine as templates. The new KIFs were studied with respect to their expression patterns in different tissues, chromosomal location, and molecular evolution. Our results suggest that (i) there is no apparent tendency among related subclasses of KIFs of cosegregation in chromosomal mapping, and (ii) according to their tissue distribution patterns, KIFs can be divided into two classes-i.e., ubiquitous and specific tissue-dominant. Further characterization of KIFs may elucidate unknown fundamental phenomena underlying intracellular transport. Finally, we propose a straightforward nomenclature system for the members of the mouse kinesin superfamily.
- Hoenger A, Milligan RA
- Motor domains of kinesin and ncd interact with microtubule protofilaments with the same binding geometry.
- J Mol Biol. 1997; 265: 553-64
- Display abstract
Kinesin and ncd (non-claret disjunctional) are microtubule associated motor proteins which share several structural features: both motors are dimers; each monomer is composed of a stalk region, a cargo binding domain and a motor domain; the motor domains have approximately 41% sequence identity. Despite these similarities the two motors have strikingly different movement properties: kinesin is a plus-end directed molecular motor, while ncd is minus-end directed. Here we compare the structure and the microtubule-binding properties of these oppositely directed molecular motors. We determined the three-dimensional structure of tubulin sheets decorated with the motor domains of either kinesin or ncd to a resolution of < 20 A by negative stain electron microscopy and tilt series reconstruction. Comparisons with a control structure of tubulin alone revealed that in both cases the motor domain binds to the outer crest of a single protofilament making contacts with both alpha and beta tubulin. Despite their opposite directionality, the geometry of attachment of the motor domain to the protofilament in the presence of AMP-PNP is very similar for both motors. These data rule out models for directionality which have the motors binding in an opposite orientation to the microtubules. Binding of the ncd as well as the kinesin motor domain appears to induce conformational changes in tubulin. This observation suggests an active role of tubulin in motor movement and/or in the determination of directionality.
- Goodson HV, Valetti C, Kreis TE
- Motors and membrane traffic.
- Curr Opin Cell Biol. 1997; 9: 18-28
- Display abstract
The cytoskeleton is essential for the proper function of many components of secretory and endocytic pathways, and the importance both of microtubule motors (kinesins and dyneins) and of actin motors (myosins) in these pathways is becoming apparent. Recent experiments have improved our understanding of which members of these motor protein families are involved in membrane traffic. Multiple motors are probably involved in the control of the morphology and dynamics of many membranes, and intriguing hints about how these motors are coordinated are appearing.
- Amos LA, Hirose K
- The structure of microtubule-motor complexes.
- Curr Opin Cell Biol. 1997; 9: 4-11
- Display abstract
New images, calculated from electron micrographs, show the three-dimensional structures of microtubules and tubulin sheets decorated stoichiometrically with globular motor protein domains (heads). Single heads of kinesin and ncd, the kinesin-related protein that moves in the reverse direction to kinesin, bind in the same way to the same site on tubulin. Dimeric kinesin and dimeric ncd show an interesting difference in the positions of their second heads.
- Bernstein SI, Milligan RA
- Fine tuning a molecular motor: the location of alternative domains in the Drosophila myosin head.
- J Mol Biol. 1997; 271: 1-6
- Display abstract
Myosin isoform sequence variation is likely critical for generating differences in contraction velocity and force production exhibited by the various skeletal muscles in an animal. To examine how myosin heavy chain (MHC) isoform diversity could affect physiological function, we studied the locations of structural differences in the motor domains of muscle MHCs from Drosophila melanogaster. Drosophila has only one muscle Mhc gene. Isoform variation is achieved by alternative splicing of a limited number of exons, clearly delineating the domains of MHC that are critical for muscle-specific functions. There are four alternative regions that contribute to the motor domain of Drosophila myosin. We used the X-ray structure of chicken skeletal S1 as a framework to examine the locations of these four regions. One lies near the ATP-binding pocket in a position where amino acid changes might be expected to modulate entry or exit of the nucleotide. Interestingly, the other three are clustered at the distal end of the molecule, surrounding the reactive cysteine SH1 and the pivot point about which the light chain-containing region swings. These observations underscore the importance of this region, distant from the site of ATP entry and the actin binding interface, as a part of the molecule where modulation of function can be achieved.
- Morii H, Takenawa T, Arisaka F, Shimizu T
- Identification of kinesin neck region as a stable alpha-helical coiled coil and its thermodynamic characterization.
- Biochemistry. 1997; 36: 1933-42
- Display abstract
The kinesin heavy chain consists of an N-terminal globular domain, referred to as the motor domain, a rod-like middle region, and a C-terminal domain. In this study, the human kinesin neck region, the region adjacent to the motor domain which promotes dimerization, has been investigated. First, we predicted coiled-coil regions including the neck region by our newly devised statistical method. The sequence (335-372) was predominated by a unique heptad amphipathy. A comparison of the bacterially expressed human kinesin heavy chain fragments, K349 (1-349), a monomeric motor domain, and K379 (1-379), a dimer, by circular dichroism (CD) spectroscopy showed that K379 had more alpha-helical content. Chemically synthesized peptides, (332-349), (350-379), and (332-369), gave CD spectra with an alpha-helix-rich pattern, but the spectra varied depending on the peptide concentration. Analysis of the molar ellipticity at 222 nm indicated that those peptides were in monomer-dimer equilibria, and the dissociation isotherms established dissociation constants of 9.6 mM. 60 microM, and 62 nM for the above peptides, respectively. Sedimentation equilibrium measurements verified that the peptide (332-369) existed as a dimeric form. These results strongly suggest that the sequence from 332 to 369 of the neck region forms an alpha-helical coiled coil. The differential peptide of K349 and K379, (350-379), did not show sufficient ability to make K379 dimeric. It is likely that the region (350-379) forms a stable alpha-helical coiled coil only together with the (332-349) region. Fluorescence energy transfer studies of [Cys363]-(332-369) labeled with a fluorescence donor and an acceptor revealed that the peptide formed a parallel coiled coil. This coiled coil was thermodynamically stable against urea and thermal denaturation, and peptide exchange of the coiled coil was undetectable, or extremely slow, at neutral pH. The dissociation free energy was estimated to be 57.7 kJ mol-1 at a peptide concentration of 22 microM. These results indicate that the neck region of kinesin forms a stable coiled coil which may be important for the motility of dimeric kinesin.
- Stenoien DL, Brady ST
- Immunochemical analysis of kinesin light chain function.
- Mol Biol Cell. 1997; 8: 675-89
- Display abstract
The kinesin heterotetramer consists of two heavy and two light chains. Kinesin light chains have been proposed to act in binding motor protein to cargo, but evidence for this has been indirect. A library of monoclonal antibodies directed against conserved epitopes throughout the kinesin light chain sequence were used to map light chain functional architecture and to assess physiological functions of these domains. Immunocytochemistry with all antibodies showed a punctate pattern that was detergent soluble. A monoclonal antibody (KLC-All) made against a highly conserved epitope in the tandem repeat domain of light chains inhibited fast axonal transport in isolated axoplasm by decreasing both the number and velocity of vesicles moving, whereas an antibody against a conserved amino terminus epitope had no effect. KLC-All was equally effective at inhibiting both anterograde and retrograde transport. Neither antibody inhibited microtubule-binding or ATPase activity in vitro. KLC-All was unique among antibodies tested in releasing kinesin from purified membrane vesicles, suggesting a mechanism of action for inhibition of axonal transport. These results provide further evidence that conventional kinesin is a motor for fast axonal transport and demonstrate that kinesin light chains play an important role in kinesin interaction with membranes.
- Higuchi H, Muto E, Inoue Y, Yanagida T
- Kinetics of force generation by single kinesin molecules activated by laser photolysis of caged ATP.
- Proc Natl Acad Sci U S A. 1997; 94: 4395-400
- Display abstract
To relate transients of force by single kinesin molecules with the elementary steps of the ATPase cycle, we measured the time to force generation by kinesin after photorelease of ATP from caged ATP. Kinesin-coated beads were trapped by an infrared laser and brought onto microtubules fixed to a coverslip. Tension was applied to a kinesin-microtubule rigor complex using the optical trap, and ATP was released by flash photolysis of caged ATP with a UV laser. Kinesin started to generate force and move stepwise with a step size of 8 nm at average times of 31, 45, and 79 ms after photorelease of 450, 90, and 18 microM ATP, respectively. The kinetics of force generation were consistent with a two-step reaction: ATP binding, with an apparent second-order rate constant of 0.7 microM-1.s-1, followed by force generation at 45 s-1 per kinesin molecule. The transient rate of force generation was close to the rate of the ATPase cycle in solution, suggesting that the rate-limiting step of ATPase cycle is involved with the force generation.
- Tucker C, Goldstein LS
- Probing the kinesin-microtubule interaction.
- J Biol Chem. 1997; 272: 9481-8
- Display abstract
Kinesin is a mechanoenzyme that couples adenosine triphosphate hydrolysis to the generation of force and movement along microtubules. To gain insight into the interactions of kinesin and microtubules, cross-linking, mapping, and proteolysis experiments were executed. The motor domain of kinesin was consistently cross-linked to both alpha- and beta-tubulin subunits. Initial mapping of the cross-linked kinesin suggested that amino acids within the N- and C-terminal cyanogen bromide fragments of the motor domain formed cross-links to both alpha- and beta-tubulin subunits. Mapping of the cross-linked tubulin suggested that cross-linking to kinesin motors occurred within the negatively charged, C-terminal cyanogen bromide fragments of alpha- and beta-tubulin subunits. Treatment of microtubules with subtilisin, a protease that cleaves C-terminal fragments from alpha- and beta-tubulin, reduced their ability to be cross-linked to kinesin motors supporting the idea that C-terminal sequences of alpha- and beta-tubulin may interact with kinesin motors. Finally, of three synthetic peptides, a peptide consisting of the last 12 C-terminal amino acids of beta-tubulin competitively interfered with the microtubule-stimulated adenosine triphosphatase activity of the kinesin motor, further suggesting that C-terminal sequences of beta-tubulin may be involved in kinesin binding.
- Kaverina IN, Minin AA, Gyoeva FK, Vasiliev JM
- Kinesin-associated transport is involved in the regulation of cell adhesion.
- Cell Biol Int. 1997; 21: 229-36
- Display abstract
It has been recently shown that deploymerization of microtubules induces the elongation of focal contacts at the leading edge. On the other hand, cell shape and pseudopodial activity were found to depend on the microtubule-based motor kinesin. In this paper, we examine whether kinesin is involved in controlling the dynamics of adhesive structures at the cell surface. Microinjection of an antiblocking kinesin activity in vitro causes focal contact elongation similar to the effect of microtubule-depolymerizing drugs. Thus, the role of microtubules in cell adhesion lies in the supporting kinesin-based transport to the adhesion sites.
- Tripet B, Vale RD, Hodges RS
- Demonstration of coiled-coil interactions within the kinesin neck region using synthetic peptides. Implications for motor activity.
- J Biol Chem. 1997; 272: 8946-56
- Display abstract
Kinesin is a dimeric motor protein that can move for several micrometers along a microtubule without dissociating. The two kinesin motor domains are thought to move processively by operating in a hand-over-hand manner, although the mechanism of such cooperativity is unknown. Recently, a approximately 50-amino acid region adjacent to the globular motor domain (termed the neck) has been shown to be sufficient for conferring dimerization and processive movement. Based upon its amino acid sequence, the neck is proposed to dimerize through a coiled-coil interaction. To determine the accuracy of this prediction and to investigate the possible function of the neck region in motor activity, we have prepared a series of synthetic peptides corresponding to different regions of the human kinesin neck (residues 316-383) and analyzed each peptide for its respective secondary structure content and stability. Results of our study show that a peptide containing residues 330-369 displays all of the characteristics of a stable, two-stranded alpha-helical coiled-coil. On the other hand, the NH2-terminal segment of the neck (residues approximately 316-330) has the capacity to adopt a beta-sheet secondary structure. The COOH-terminal residues of the neck region (residues 370-383) are not alpha-helical, nor do they contribute significantly to the overall stability of the coiled-coil, suggesting that these residues mark the beginning of a hinge located between the neck and the extended alpha-helical coiled coil stalk domain. Interestingly, the two central heptads of the coiled-coil segment in the neck contain conserved, "non-ideal" residues located within the hydrophobic core, which we show destabilize the coiled-coil interaction. These residues may enable a portion of the coiled-coil to unwind during the mechanochemical cycle, and we present a model in which such a phenomenon plays an important role in kinesin motility.
- Ma YZ, Taylor EW
- Kinetic mechanism of a monomeric kinesin construct.
- J Biol Chem. 1997; 272: 717-23
- Display abstract
The kinetic mechanism is analyzed for a monomeric human kinesin construct K332. In the absence of microtubules, the rate constants of the ATPase cycle are very similar to dimeric human kinesin K379 and whole kinesin from bovine brain. The microtubule-activated ATPase is 60 s(-1) at 20 degrees C; Km(Mt) is 5 microM; dissociation constants in the presence of ATP and ADP are 9 microM and 16 microM, respectively. The values of dissociation constants are 5 times larger than for K379. Binding of K332 to microtubules increased the rate of the hydrolysis step from 7 s(-1) to greater than 200 s(-1) and the 2'-(3')-O-(N-methylanthraniloyl) (mant) ADP dissociation step from 0.02 s(-1) to greater than 100 s(-1). At higher ionic strength, more than one ATP is hydrolyzed before dissociation of MtK332 (small processivity). Data are fitted to the kinetic scheme. [equation: see text] Approximate values of rate constants are k1 = 500 s(-1), k2 > or = 200 s(-1), k3k4/(k3 + k4) = 100 s(-1), k(dis) = 80+/-10 s(-1). Two experiments to measure k4 gave 110 s(-1) from the maximum rate of dissociation of mant ADP for reaction of K x ADP with microtubules and 300 s(-1) from extrapolation to zero concentration of rate of binding of mant ADP to MtK. It is proposed that mant ADP dissociation is a two-step process. In the simple scheme, k4 is the effective rate of the two-step release of ADP, k4 = 150 s(-1) to 200 s(-1), and k3 = 150 s(-1) to 200 s(-1) to account for the steady state rate.
- Ma YZ, Taylor EW
- Interacting head mechanism of microtubule-kinesin ATPase.
- J Biol Chem. 1997; 272: 724-30
- Display abstract
Kinetic and equilibrium properties are compared for a monomeric kinesin construct (K332) and a dimeric construct (K379). MtK379 has a low affinity (5 x 10(4) M(-1)) and a high affinity (5 x 10(6) M(-1)) binding site for mant ADP while MtK332 has a single low affinity site (5 x 10(4) M(-1)). Rate constants of dissociation of mant ADP are <1 s(-1) for the high affinity site and 75-100 s(-1) for the low affinity site for MtK379. For MtK332, the effective rate constant is 200-300 s(-1). It is proposed that the two heads of the dimer are different through the interaction with the microtubule, a strongly bound head with low affinity for 2'-(3')-O-(N-methylanthraniloyl) adenosine 5'-diphosphate (mant ADP), similar to the single strongly bound head of the monomer and a weakly bound or detached head with high affinity for mant ADP. Rate of binding of mant ADP gave an "S"-shaped dependence on concentration for MtK379 and a hyperbolic dependence for MtK332. Binding of K379 x mant ADP dimer to microtubules releases only one mant ADP at a rate of 50 s(-1). The second strongly bound mant ADP is released by binding of nucleotides to the other head. Rates are 100 s(-1) for ATP, 30 s(-1) for AMPPNP or ATPgammaS, and 2 s(-1) for ADP. The rate of binding of mant ATP to MtK379 showed an "S"-shaped concentration dependence and limiting rate at zero concentration is <1 s(-1) while MtK332 gave a hyperbolic dependence and limiting rate of 100 s(-1). The limiting rate is determined by the rate of dissociation of mant ADP in the hydrolysis cycle. The evidence is consistent with an interacting site model in which binding of ATP to one head is required for release of ADP from the other head in the hydrolysis cycle. This model, in which the cycles are maintained partly out of phase, is an extension of the alternating site model of Hackney (Hackney, D. D. (1994) Proc. Nat. Acad. Sci. U.S.A. 91, 6865-6869). It provides a basis for a processive mechanism.
- Crevel IM, Lockhart A, Cross RA
- Kinetic evidence for low chemical processivity in ncd and Eg5.
- J Mol Biol. 1997; 273: 160-70
- Display abstract
The kinesin molecular motor "walks" processively along microtubules, touching down with alternate motor domains and transiently bridging between sites spaced 8 nm apart axially. To allow bridging, the coiled coil tail of kinesin would need to unzip a region immediately adjacent to the heads, and the tail region sequence at this point indeed contains potentially destabilising interruptions in the regular hydrophobic heptad repeat. We noticed that such interruptions are substantially absent from the coiled coil tails of Eg5, a slow kinesin homologue, and ncd, a reverse-directed homologue, and we wondered if this precluded their processivity. We measured the temperature dependence of kcat/K50% MTs, an index of the chemical processivity of a motor, the number of ATPs split per productive diffusional encounter of motor with microtubule. We found two-headed ncd (GSTMC5) and two-headed Eg5 (E437GST) constructs to be slightly if at all processive in solution over the range 4 degrees C to 30 degrees C. By contrast, two-headed kinesin constructs K401 and K430 were processive, and became substantially more so with increasing temperature. Arrhenius plots for the solution ATPase were linear for all three motors. Arrhenius plots for MT gliding assays were linear and essentially parallel for E437GST and GSTMC5 (Ea = 61 and 63 kJ mol-1) but for K430 the plot was biphasic, with a break at 17 degrees C, corresponding to a 30% reduction in Ea from 84 to 57 kJ mol-1. The data indicate that ncd and Eg5 are only slightly if at all processive, and suggest that this may be related to structural differences in their coiled coil neck regions.
- Stebbings H
- Direct evidence for the nature of the binding of mitochondria to microtubules in ovarian nutritive tubes of an hemipteran insect.
- Cell Tissue Res. 1997; 289: 333-7
- Display abstract
Interactions between mitochondria and microtubules have been investigated in the nutritive tubes that link the nurse cells to the oocytes in ovarioles of the hemipteran insect, Notonecta. The nutritive tubes comprise large numbers of microtubules, which can be dissected manually from ovarioles. This approach, which retains the intrinsic components of the system, has allowed the in vivo interactions between the microtubules and mitochondria, which are also present in the nutritive tubes, to be studied directly. Static binding occurred between mitochondria and microtubules, and investigations of its nucleotide and salt-sensitivities have indicated its microtubule-associated protein (MAP)-dependency.
- Henningsen U, Schliwa M
- Reversal in the direction of movement of a molecular motor.
- Nature. 1997; 389: 93-6
- Display abstract
Kinesin and non-claret disjunctional (ncd) are molecular motors of the kinesin superfamily that move in opposite directions along microtubules. The molecular basis underlying the direction of movement is unclear, although it is thought to be an intrinsic property of the motor domain, a conserved region about 330 amino acids in length. The motor domain is found at the amino terminus in conventional kinesins, but at the carboxy terminus in ncd. Here we report on a chimaera composed of the motor domain of the minus-end-directed kinesin of Neurospora crassa. The bacterially expressed fusion protein was tested in motility assays using polarity-marked microtubules. Surprisingly, the chimaera moved towards the plus end, demonstrating that the polarity of force generation of the ncd motor domain has been reversed. This finding indicates that the domain organization, particularly the position of the motor domain, is of fundamental importance for the polarity of force production. It also demonstrates that the direction of microtubule movement is not controlled solely by the motor domain.
- Schnitzer MJ, Block SM
- Kinesin hydrolyses one ATP per 8-nm step.
- Nature. 1997; 388: 386-90
- Display abstract
Kinesin is a two-headed, ATP-dependent motor protein that moves along microtubules in discrete steps of 8 nm. In vitro, single molecules produce processive movement; motors typically take approximately 100 steps before releasing from a microtubule. A central question relates to mechanochemical coupling in this enzyme: how many molecules of ATP are consumed per step? For the actomyosin system, experimental approaches to this issue have generated considerable controversy. Here we take advantage of the processivity of kinesin to determine the coupling ratio without recourse to direct measurements of ATPase activity, which are subject to large experimental uncertainties. Beads carrying single molecules of kinesin moving on microtubules were tracked with high spatial and temporal resolution by interferometry. Statistical analysis of the intervals between steps at limiting ATP, and studies of fluctuations in motor speed as a function of ATP concentration, allow the coupling ratio to be determined. At near-zero load, kinesin molecules hydrolyse a single ATP molecule per 8-nm advance. This finding excludes various one-to-many and many-to-one coupling schemes, analogous to those advanced for myosin, and places severe constraints on models for movement.
- Suzuki Y, Shimizu T, Morii H, Tanokura M
- Hydrolysis of AMPPNP by the motor domain of ncd, a kinesin-related protein.
- FEBS Lett. 1997; 409: 29-32
- Display abstract
AMPPNP was found to be hydrolyzed by the motor domain of ncd (the product of a Drosophila gene, non-claret disjunctional), a kinesin-related protein. This hydrolysis could be monitored by 31P NMR spectroscopy and by an assay of phosphate, one of the products of the hydrolysis. The rate was approximately 0.00004 s(-1), 1% of the ATP turnover rate. The AMPPNP turnover was not stimulated by microtubules. Kinesin motor domain also turned over AMPPNP but at a somewhat lower rate. Although the turnover was slow, the present finding may present an important caveat, since AMPPNP has been widely used for investigations of kinesin and kinesin-related proteins as a non-hydrolyzable ATP analogue.
- Schnapp BJ
- Retroactive motors.
- Neuron. 1997; 18: 523-6
- Hua W, Young EC, Fleming ML, Gelles J
- Coupling of kinesin steps to ATP hydrolysis.
- Nature. 1997; 388: 390-3
- Display abstract
A key goal in the study of the function of ATP-driven motor enzymes is to quantify the movement produced from consumption of one ATP molecule. Discrete displacements of the processive motor kinesin along a microtubule have been reported as 5 and/or 8 nm. However, analysis of nanometre-scale movements is hindered by superimposed brownian motion. Moreover, because kinesin is processive and turns over stochastically, some observed displacements must arise from summation of smaller movements that are too closely spaced in time to be resolved. To address both of these problems, we used light microscopy instrumentation with low positional drift (< 39 pms[-1]) to observe single molecules of a kinesin derivative moving slowly (approximately 2.5nm s[-1]) at very low (150nM) ATP concentration, so that ATP-induced displacements were widely spaced in time. This allowed increased time-averaging to suppress brownian noise (without application of external force), permitting objective measurement of the distribution of all observed displacement sizes. The distribution was analysed with a statistics-based method which explicitly takes into account the occurrence of unresolved movements, and determines both the underlying step size and the coupling of steps to ATP hydrolytic events. Our data support a fundamental enzymatic cycle for kinesin in which hydrolysis of a single ATP molecule is coupled to a step distance of the microtubule protofilament lattice spacing of 8.12 nm. Step distances other than 8nm are excluded, as is the coupling of each step to two or more consecutive ATP hydrolysis reactions with similar rates, or the coupling of two 8-nm steps to a single hydrolysis. The measured ratio of ATP consumption rate to stepping rate is invariant over a wide range of ATP concentration, suggesting that the 1 ATP to 8nm coupling inferred from behaviour at low ATP can be generalized to high ATP.
- Pechatnikova E, Taylor EW
- Kinetic mechanism of monomeric non-claret disjunctional protein (Ncd) ATPase.
- J Biol Chem. 1997; 272: 30735-40
- Display abstract
The non-claret disjunctional protein (Ncd) is a kinesin-related microtubule motor that moves toward the negative end of microtubules. The kinetic mechanism of the monomer motor domain, residues 335-700, satisfied a simple scheme for the binding of 2'-3'-O-(N-methylanthraniloyl) (MANT) ATP, the hydrolysis step, and the binding and release of MANT ADP, where T, D, and Pi refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively, and MtN is the complex of an Ncd motor domain with a microtubule site. Rate constants k1 and k-4 are the rates of a first order step, an isomerization induced by nucleotide binding. The apparent second order rate constants for the binding steps are 1.5 x 10(6) M-1 s-1 for MANT ATP and 3.5 x 10(6) M-1 s-1 for MANT ADP (conditions, 50 mM NaCl, pH 6.9, 21 degrees C). The rate constant of the hydrolysis step (k2) was obtained from quench flow measurements of the phosphate burst phase corrected for the contribution of the rate of product release to the transient rate constant. The rate of phosphate dissociation was not measured; the value was assigned to account for a steady state rate of 3 s-1. The MtN complex is dissociated by ATP at a rate of 10 s-1 based on light scattering measurements. Dissociation constants of Ncd-nucleotide complexes from microtubules increased in the order adenosine 5'-O-(thiotriphosphate) (ATPgammaS) < ADP-AlF4 < ATP < ADP < ADP-vanadate. Comparison of the properties of Ncd with a monomeric kinesin K332 (Ma and Taylor (1997) J. Biol. Chem. 272, 717-723) showed a close similarity, except that the rate constants for the hydrolysis and ADP release steps and the steady state rate are approximately 15-20 times smaller for Ncd. There are two differences that may affect the reaction pathway. The rate of dissociation of MtN by ATP is comparable to the rate of the hydrolysis step, and N.T may dissociate in the cycle, whereas for kinesin, dissociation occurs after hydrolysis. The rate of dissociation of MtN by ADP is larger than the rate of ADP release from MtN.D, whereas for the microtubule-kinesin complex, the rate of dissociation by ADP is smaller than the rate of ADP release. The monomeric Mt.Ncd complex is not processive.
- Cross RA
- Reversing the kinesin ratchet--a diverting tail.
- Nature. 1997; 389: 15-6
- Case RB, Pierce DW, Hom-Booher N, Hart CL, Vale RD
- The directional preference of kinesin motors is specified by an element outside of the motor catalytic domain.
- Cell. 1997; 90: 959-66
- Display abstract
Members of the kinesin superfamily share a similar motor catalytic domain yet move either toward the plus end (e.g., conventional kinesin) or the minus end (e.g., Ncd) of microtubules. The structural features that determine the polarity of movement have remained enigmatic. Here, we show that kinesin's catalytic domain (316 residues) in a dimeric construct (560 residues) can be replaced with the catalytic domain of Ncd and that the resultant motor moves in the kinesin direction. We also demonstrate that this chimera does not move processively over many tubulin subunits, which is similar to Ncd but differs from the highly processive motion of conventional kinesin. These findings reveal that the catalytic domain contributes to motor processivity but does not control the polarity of movement. We propose that a region adjacent to the catalytic domain serves as a mechanical transducer that determines directionality.
- Amos LA, Cross RA
- Structure and dynamics of molecular motors.
- Curr Opin Struct Biol. 1997; 7: 239-46
- Display abstract
The structures of the oppositely directed microtubule motors kinesin and ncd have been solved to atomic resolution. The two structures are very similar and are also homologous to myosin. Myosins and kinesins differ kinetically but, tantalizingly, cryoelectron microscopy has recently revealed that both structures may tilt during ADP release. Such evidence suggests that the two motor families use common structural mechanisms.
- Imafuku Y, Toyoshima YY, Tawada K
- Fluctuation in the microtubule sliding movement driven by kinesin in vitro.
- Biophys J. 1996; 70: 878-86
- Display abstract
We studied the fluctuation in the translational sliding movement of microtubules driven by kinesin in a motility assay in vitro. By calculating the mean-square displacement deviation from the average as a function of time, we obtained motional diffusion coefficients for microtubules and analyzed the dependence of the coefficients on microtubule length. Our analyses suggest that 1) the motional diffusion coefficient consists of the sum of two terms, one that is proportional to the inverse of the microtubule length (as the longitudinal diffusion coefficient of a filament in Brownian movement is) and another that is independent of the length, and 2) the length-dependent term decreases with increasing kinesin concentration. This latter term almost vanishes within the length range we studied at high kinesin concentrations. From the length-dependence relationship, we evaluated the friction coefficient for sliding microtubules. This value is much larger than the solvent friction and thus consistent with protein friction. The length independence of the motional diffusion coefficient observed at sufficiently high kinesin concentrations indicates the presence of correlation in the sliding movement fluctuation. This places significant constraint on the possible mechanisms of the sliding movement generation by kinesin motors in vitro.
- Thaler CD, Haimo LT
- Microtubules and microtubule motors: mechanisms of regulation.
- Int Rev Cytol. 1996; 164: 269-327
- Display abstract
Microtubule-based motility is precisely regulated, and the targets of regulation may be the motor proteins, the microtubules, or both components of this intricately controlled system. Regulation of microtubule behavior can be mediated by cell cycle-dependent changes in centrosomal microtubule nucleating ability and by cell-specific, microtubule-associated proteins (MAPs). Changes in microtubule organization and dynamics have been correlated with changes in phosphorylation. Regulation of motor proteins may be required both to initiate movement and to dictate its direction. Axonemal and cytoplasmic dyneins as well as kinesin can be phosphorylated and this modification may affect the motor activities of these enzymes or their ability to interact with organelles. A more complete understanding of how motors can be modulated by phosphorylation, either of the motor proteins or of other associated substrates, will be necessary in order to understand how bidirectional transport is regulated.
- Offer G, Knight P
- The structure of the head-tail junction of the myosin molecule.
- J Mol Biol. 1996; 256: 407-16
- Display abstract
An atomic model of the junction between the two heads and tail of a myosin molecule has been created by attaching a scallop regulatory domain to the end of each of the two alpha-helical strands of a model of the scallop alpha-helical coiled coil. The C-terminal alpha-helix of the heavy chain of each regulatory domain was superposed over the corresponding sequence in the coiled coil. In the structure created, the two heads lie alongside one another with their bases in contact but remarkably without steric clash. The principal interactions between the two heads are between the regulatory light chains and there are also head-tail interactions between each regulatory light chain and its heavy chain partner in the coiled coil. The invariant proline residues cause the heavy chains to flare to form the fork. The direction of the turn at the WQW sequence within the regulatory domain causes the long alpha-helix of the heavy chains within the head to continue the sense of the supercoil. With the bases of heads interacting, motion of the heads could still occur by a flexing of the coiled coil close to the heads and by a flexing and twisting of the long alpha-helices in the head. The model accounts for some of the conserved sequence features in myosins from different sources and provides a structural basis for understanding the head-head interactions in regulated myosin. Using the C alpha atoms of subfragment 1 we have also constructed a model with two complete heads. The clockwise curvature of the heads when the model is viewed end-on towards the tail accounts for the most common appearance of myosin molecules in electron micrographs. These models are predicated on the assumption that the entire heptad sequence of the heavy chains forms a coiled coil. Previous evidence from electron micrographs of myosin molecules that this was not the case can be explained by the foreshortening of the tail close to the heads.
- Howard J
- The movement of kinesin along microtubules.
- Annu Rev Physiol. 1996; 58: 703-29
- Display abstract
The molecular motor kinesin is a homodimer containing two heads-globular domains each of which has an ATP- and a microtubule-binding site. It is argued by analogy to other proteins with coiled-coil dimerization domains that the kinesin dimer has an approximate axis of rotational symmetry. The path kinesin follows along the surface of the microtubule is parallel to the protofilaments, and the steps are likely separated by 8 nm, the length of the tubulin dimer. Micromechanical recordings from single kinesin molecules indicate that one motor can exert a force as great as 5 pN. The efficiency of kinesin probably is in the order of 50%, considering the free energy available from ATP hydrolysis. Structural, mechanical, and biochemical experiments suggest that in order not to let go of a microtubule, the two heads of kinesin might move in a coordinated manner, perhaps undergoing a rotary motion.
- Hackney DD
- The kinetic cycles of myosin, kinesin, and dynein.
- Annu Rev Physiol. 1996; 58: 731-50
- Display abstract
The ATPase cycles of the molecular motors myosin, kinesin, and dynein are reviewed, with emphasis on their similarities and differences. Myosin generates motility along actin filaments and functions in muscle contraction, organelle movement, and cytokinesis. Dynein and kinesin produce movement along microtubules. All the motors exhibit burst kinetics with rate-limiting product release. Binding of the products-complex to the filament accelerates product release and completes the ATPase cycle. Kinesin is able to generate processive movement, and the possibilities for how this could be generated by coupling to ATP hydrolysis are discussed.
- Moore JD, Song H, Endow SA
- A point mutation in the microtubule binding region of the Ncd motor protein reduces motor velocity.
- EMBO J. 1996; 15: 3306-14
- Display abstract
Non-claret disjunctional (Ncd) is a kinesin-related microtubule motor protein in Drosophila that functions in meiotic spindle assembly in oocytes and spindle pole maintenance in early embryos. The partial loss-of-function mutant ncdD retains mitotic, but not meiotic, function. The predicted NcdD mutant protein contains a V556-->F mutation in the putative microtubule binding region of the Ncd motor domain. Here we report an analysis of the properties of recombinant Ncd and NcdD proteins. A GST-NcdD fusion protein translocated microtubules approximately 10-fold more slowly than the corresponding wild-type protein in gliding assays. The maximum microtubule-stimulated ATPase activity of an NcdD motor domain protein was reduced approximately 3-fold and an approximately 3-fold greater concentration of microtubules was required for half-maximal stimulation of ATPase activity, compared with the corresponding wild-type protein. The Km for ATP and basal rate of ATP turnover were, in contrast, similar for the NcdD mutant and wild-type Ncd motor domain proteins. Pelleting assays demonstrated that the binding of the mutant NcdD motor protein to microtubules was reduced in the absence of nucleotide, relative to wild-type. The reduced velocity of NcdD translocation on microtubules is therefore correlated with reductions in microtubule-stimulated ATPase activity and affinity of the mutant motor for microtubules. The characteristics of the NcdD motor explain its meiotic loss of function, and are consistent with partial motor activity of Ncd being sufficient for its mitotic, but not its meiotic, role.
- Sickles DW, Brady ST, Testino A, Friedman MA, Wrenn RW
- Direct effect of the neurotoxicant acrylamide on kinesin-based microtubule motility.
- J Neurosci Res. 1996; 46: 7-17
- Display abstract
Acrylamide (ACR) is an environmental toxicant and prototypic tool for studying mechanisms of peripheral neuropathies. Reductions in fast anterograde axonal transport (faAXT) are thought to be a critical step leading to axonal degeneration. Kinesin and microtubules (MT) were evaluated as molecular sites of action using an in vitro MT motility assay. The number of locomoting MT which lifted from a bed of kinesin (MT detachments or MTD), increased from 7% in controls to 80, 89, and 100% following preincubation of kinesin (37 degrees C, 20 min) with 0.1, 0.5, or 1.0 mM ACR, respectively; rates were variably reduced by as much as 20%. Similar alterations were observed with N-ethylmaleimide. A non-neurotoxic analogue, propionamide (1mM), had no effect on either parameter. Preincubation of taxol-stabilized MT with ACR produced a dose-dependent increase in MTD but no changes in rate. We conclude that kinesin and MT are covalently modified by ACR resulting in reduced affinity for each other. The greater sensitivity of kinesin indicates that a primary cause of transient, ACR-induced reductions in faAXT is covalent modification of kinesin. Such reductions in faAXT may be sufficient to produce axonal degeneration. Further, ACR may prove useful as a pharmacological tool to decipher the complex mechanics of kinesin-MT interactions.
- Hackney DD
- Myosin and kinesin: mother and child reunited.
- Chem Biol. 1996; 3: 525-8
- Display abstract
The recent solution of the crystal structure of the kinesin motor domain reveals striking similarities to the core region of the myosin motor domain, implying a strong evolutionary relationship between these two motors. However, a complete understanding of the way that motility is generated will require additional structural information, which may explain how the two motors have adapted to their fundamentally different linear substrates, F-actin and microtubules.
- Vallee RB, Sheetz MP
- Targeting of motor proteins.
- Science. 1996; 271: 1539-44
- Display abstract
Microtubules are responsible for chromosome segregation and the movement and reorganization of membranous organelles. Many aspects of microtubule-based motility can be attributed to the action of motor proteins, producing force directed toward either end of microtubules. How these proteins are targeted to the appropriate organellar sites within the cell, however, has remained a mystery. Recent work has begun to define the targeting mechanism for two well-studied motor proteins, kinesin and cytoplasmic dynein.
- Derenyi I, Vicsek T
- The kinesin walk: a dynamic model with elastically coupled heads.
- Proc Natl Acad Sci U S A. 1996; 93: 6775-9
- Display abstract
Recently individual two-headed kinesin molecules have been studied in in vitro motility assays revealing a number of their peculiar transport properties. In this paper we propose a simple and robust model for the kinesin stepping process with elastically coupled Brownian heads that show all of these properties. The analytic and numerical treatment of our model results in a very good fit to the experimental data and practically has no free parameters. Changing the values of the parameters in the restricted range allowed by the related experimental estimates has almost no effect on the shape of the curves and results mainly in a variation of the zero load velocity that can be directly fitted to the measured data. In addition, the model is consistent with the measured pathway of the kinesin ATPase.
- Ruppel KM, Spudich JA
- Structure-function analysis of the motor domain of myosin.
- Annu Rev Cell Dev Biol. 1996; 12: 543-73
- Display abstract
Motor proteins perform a wide variety of functions in all eukaryotic cells. Recent advances in the structural and mutagenic analysis of the myosin motor has led to insights into how these motors transduce chemical energy into mechanical work. This review focuses on the analysis of the effects of myosin mutations from a variety of organisms on the in vivo and in vitro properties of this ubiquitous motor and illustrates the positions of these mutations on the high-resolution three-dimensional structure of the myosin motor domain.
- Song H, Endow SA
- Binding sites on microtubules of kinesin motors of the same or opposite polarity.
- Biochemistry. 1996; 35: 11203-9
- Display abstract
The kinesin motor proteins translocate toward either the plus or minus end of microtubules (MTs). Competitive microtubule binding assays were carried out with monomeric motor domains of the minus-end-directed nonclaret disjunctional (Ncd) and Kar3 and the plus-end-directed kinesin heavy chain (KHC) to determine whether motors of the same or opposite polarity compete for binding sites on MTs and to test the idea that motor polarity is determined by differences in binding sites on MTs of the motors. The stoichiometries of binding were approximately 1 motor:1 tubulin heterodimer for all three motors. Ncd and Kar3, both minus-end motors, severely inhibited the binding of one another to MTs, as predicted theoretically for binding of the two motors to the same site on MTs, indicating that the binding sites on MTs of Ncd and Kar3 are the same or overlap extensively. Motors of opposite polarity, KHC and Ncd or KHC and Kar3, showed partial or complete inhibition of binding to MTs under different experimental protocols. The differences in binding behavior could be due to experimental conditions or be inherent in the nature of motor binding to MTs. Alternatively, differences in KHC and Ncd or Kar3 binding sites on MTs may exist such that the motors bind to partially overlapping but nonidentical sites on MTs. These differences in binding sites may be related to the opposite polarity of translocation on MTs of the motors.
- Astumian RD, Bier M
- Mechanochemical coupling of the motion of molecular motors to ATP hydrolysis.
- Biophys J. 1996; 70: 637-53
- Display abstract
The typical biochemical paradigm for coupling between hydrolysis of ATP and the performance of chemical or mechanical work involves a well-defined sequence of events (a kinetic mechanism) with a fixed stoichiometry between the number of ATP molecules hydrolyzed and the turnover of the output reaction. Recent experiments show, however, that such a deterministic picture of coupling may not be adequate to explain observed behavior of molecular motor proteins in the presence of applied forces. Here we present a general model in which the binding of ATP and release of ADP serve to modulate the binding energy of a motor protein as it travels along a biopolymer backbone. The mechanism is loosely coupled--the average number of ATPs hydrolyzed to cause a single step from one binding site to the next depends strongly on the magnitude of an applied force and on the effective viscous drag force. The statistical mechanical perspective described here offers insight into how local anisotrophy along the "track" for a molecular motor, combined with an energy-releasing chemical reaction to provide a source of nonequilibrium fluctuations, can lead to macroscopic motion.
- Rosenfeld SS, Correia JJ, Xing J, Rener B, Cheung HC
- Structural studies of kinesin-nucleotide intermediates.
- J Biol Chem. 1996; 271: 30212-21
- Display abstract
We have investigated the structural changes that occur in the molecular motor kinesin during its ATPase cycle, utilizing two bacterially expressed constructs. The structure of both constructs has been examined as a function of the nature of the nucleotide intermediate occupying the active site by means of sedimentation velocity, sedimentation equilibrium, fluorescence solute quenching, fluorescence anisotropy decay, and limited proteolysis. While the molecular weight of monomeric and dimeric human kinesin constructs, as measured by sedimentation velocity and sedimentation equilibrium, and the tryptic cleavage pattern are unaffected by the nucleotide intermediate occupying the active site, significant changes in the rotational correlation time of fluorescently labeled kinesin-nucleotide intermediates can be detected. These results suggest that kinesin contains an internal "hinge" whose flexibility varies through the course of the ATPase cycle. In prehydrolytic, "strong" binding states, this hinge is relatively rigid, while in posthydrolytic, "weak" binding states, it is more flexible. Our results, in conjunction with anisotropy decay studies of myosin, suggest that these two molecular motors may share a common structural feature; viz. weak binding states are characterized by segmental flexibility, which is lost upon assumption of a strong binding conformation.
- Hirokawa N
- The molecular mechanism of organelle transport along microtubules: the identification and characterization of KIFs (kinesin superfamily proteins).
- Cell Struct Funct. 1996; 21: 357-67
- Display abstract
In the cells various kinds of organelles are transported and distributed to their proper destinations in the cell. Organelle transports are very important for cellular morphogenesis and functions, with the conveying and targeting of essential materials to their correct destination being conducted, often at considerable velocities. Recently we have identified at least 10 new microtubule-associated motor proteins named as KIFs (kinesin superfamily proteins). Their characterization reveals that each member can convey a specific organelle or cargo, although there is some redundancy. It has also become clear that there are distinct subclasses of KIFs that form monomeric, heterodimeric and homodimeric motors. Molecular cell biological approaches combining multidisciplinary methods such as new electron microscopy, biochemistry, immunocytochemistry, biophysics, molecular biology and molecular genetics have revealed precise mechanisms of organelle transports in the cells by KIFs.
- Crevel IM, Lockhart A, Cross RA
- Weak and strong states of kinesin and ncd.
- J Mol Biol. 1996; 257: 66-76
- Display abstract
Kinesin superfamily molecular motors step along microtubules (MTs) via a cycle of conformational changes which is coupled to ATP turnover. To probe the coupling mechanism, we titrated the effects of various nucleotides on MT binding by two superfamily members; MT plus-end-directed kinesin and MT minus-end-directed non claret disjunctional (ncd). For both motors, the nucleotide-free state induced by apyrase was the strongest binding (K(kin)d approximately 0.003 micro M, K(ncd)d approximately 0.24 micro M), whilst the ADp state was the weakest binding (K(kin)d approximately 11.32 micro M, K(ncd)d approximately 12.02 micro M). In ATP, the motor. ADP state dominates and the binding is accordingly ADP-like, but in the presence of the slowly hydrolysed analogue adenosine 5'-O-(3-thiotriphosphate) there is a shift towards tighter binding (K(kin)d approximately 4.23 micro M, K(ncd)d approximately 2.34 micro M), consistent with a tight-binding motor. ATP-like state being enriched. In the presence of non-hydrolysable analogue beta,gamma-imidoadenosine 5'-triphosphate the binding is still tighter (K(kin)d approximately <0.27 micro M, K(ncd)d approximately 0.21 micro M), close to the values obtained with apyrase. For both kinesin and ncd, ADP has the unique quality that it traps the motor in a weak binding state. MT tight binding catalyses escape from this state, changing the active site conformation such that both ADP release and ADP binding are accelerated. The data are consistent with a simple two-state scheme in which both kinesis and ncd switch from weak to strong binding via ADP release, and back again via ADP trapping. In a two-state model, the transition from weak to strong binding is force-generating.
- Steinberg G, Schliwa M
- Characterization of the biophysical and motility properties of kinesin from the fungus Neurospora crassa.
- J Biol Chem. 1996; 271: 7516-21
- Display abstract
Neurospora kinesin (Nkin) is a distant relative of the family of conventional kinesins, members of which have been identified in various animal species. As in its animal counterparts, Nkin most likely is an organelle motor. Because it is a functional homologue of the kinesin heavy chain of higher eukaryotes, its biophysical and motility properties were compared with those of other conventional kinesins. Purified Nkin behaves as a homodimeric complex composed of two subunits of a 105-kDa polypeptide. Based on its hydrodynamic properties (Stokes radius and sedimentation coefficient), Nkin is an elongated molecule, although it is more compact than its animal counterparts. A detailed comparison of the motility properties of Nkin with those of animal conventional kinesins reveals similarities and some intriguing differences. Nkin is less effective than other kinesins in the use of natural nucleoside triphosphates but responds to a selection of ATP analogues in a similar fashion as mammalian kinesin. Even in the presence of saturating concentrations of ATP, Nkin is significantly more sensitive to ADP or tripolyphosphate than other kinesins. Both the ATP-driven microtubule gliding activity and the microtubule-stimulated ATPase activity of Nkin obey Michaelis-Menten kinetics. Surprisingly, however, the Km values for both these activities are approximately an order of magnitude higher than those of other kinesins. Whether the low affinity for ATP suggested by these high Km values is related to the high rate of motility remains to be determined.
- Vale RD, Funatsu T, Pierce DW, Romberg L, Harada Y, Yanagida T
- Direct observation of single kinesin molecules moving along microtubules.
- Nature. 1996; 380: 451-3
- Display abstract
Kinesin is a two-headed motor protein that powers organelle transport along microtubules. Many ATP molecules are hydrolysed by kinesin for each diffusional encounter with the microtubule. Here we report the development of a new assay in which the processive movement of individual fluorescently labelled kinesin molecules along a microtubule can be visualized directly; this observation is achieved by low-background total internal reflection fluorescence microscopy in the absence of attachment of the motor to a cargo (for example, an organelle or bead). The average distance travelled after a binding encounter with a microtubule is 600 nm, which reflects a approximately 1% probability of detachment per mechanical cycle. Surprisingly, processive movement could still be observed at salt concentrations as high as 0.3 M NaCl. Truncated kinesin molecules having only a single motor domain do not show detectable processive movement, which is consistent with a model in which kinesin's two force-generating heads operate by a hand-over-hand mechanism.
- Pidoux AL, LeDizet M, Cande WZ
- Fission yeast pkl1 is a kinesin-related protein involved in mitotic spindle function.
- Mol Biol Cell. 1996; 7: 1639-55
- Display abstract
We have used anti-peptide antibodies raised against highly conserved regions of the kinesin motor domain to identify kinesin-related proteins in the fission yeast Schizosaccharomyces pombe. Here we report the identification of a new kinesin-related protein, which we have named pkl1. Sequence homology and domain organization place pkl1 in the Kar3/ncd subfamily of kinesin-related proteins. Bacterially expressed pkl1 fusion proteins display microtubule-stimulated ATPase activity, nucleotide-sensitive binding, and bundling of microtubules. Immunofluorescence studies with affinity-purified antibodies indicate that the pkl1 protein localizes to the nucleus and the mitotic spindle. Pkl1 null mutants are viable but have increased sensitivity to microtubule-disrupting drugs. Disruption of pkl1+ suppresses mutations in another kinesin-related protein, cut7, which is known to act in the spindle. Overexpression of pkl1 to very high levels causes a similar phenotype to that seen in cut7 mutants: V-shaped and star-shaped microtubule structures are observed, which we interpret to be spindles with unseparated spindle poles. These observations suggest that pkl1 and cut7 provide opposing forces in the spindle. We propose that pkl1 functions as a microtubule-dependent motor that is involved in microtubule organization in the mitotic spindle.
- Moyer ML, Gilbert SP, Johnson KA
- Purification and characterization of two monomeric kinesin constructs.
- Biochemistry. 1996; 35: 6321-9
- Display abstract
Steady-state and pre-steady-state kinetic methods were used to analyze two shorter Drosophila kinesin constructs (K341 and K366) in comparison to K401. K341, K366, and K401 represent the kinesin motor domains containing the N-terminal 341, 366, or 401 amino acids, respectively. K401 is dimeric (Kd = 37 +/- 17 nM) whereas both K366 and K341 are monomeric [Correia et al. (1995) Biochemistry 34, 4898-4907]. Like native kinesin and K401, K341 and K366 demonstrate low ATPase activity in the absence of microtubules (0.03 and 0.01 s-1, respectively), and ADP release is rate-limiting during steady-state turnover. Microtubules activate the steady-state ATPase to 84 s-1 for K341 (K(m),ATP = 100 microM; K0.5,MT = 3.2 microM tubulin) and 64 s-1 for K366 (K(m),ATP = 65 microM; K0.5,MT = 2.5 microM tubulin) in comparison to K401 at 20 s-1 (K(m)ATP = 60 microM; K0.5,MT = 1 microM tubulin). The rapid quench experiments for all three constructs show a burst of product formation during the first turnover, indicating the rate-limiting step for the microtubule-activated ATPase occurs after ATP hydrolysis. The interaction of K341 and K366 with the microtubule was analyzed by electron microscopy. The results show that K341 and K366, like K401, bind to the microtubule with an 8 nm axial periodicity. However, the addition of K366 to microtubules resulted in significant aggregation of microtubules. The pre-steady-state kinetic results show that K341 retains the kinetic and structural properties necessary to compare directly the kinetic properties of monomeric and dimeric kinesins, although the microtubule-activated ATPase is significantly faster for the monomeric constructs, suggesting possible interactions in the dimer which inhibit ATP turnover as part of the coupling to force production.
- Turner D, Chang C, Fang K, Cuomo P, Murphy D
- Kinesin movement on glutaraldehyde-fixed microtubules.
- Anal Biochem. 1996; 242: 20-5
- Display abstract
Glutaraldehyde-cross-linked microtubules were investigated as substrates for kinesin motility. Microtubules, formed in vitro from chicken brain tubulin, were stabilized with Taxol and chemically fixed with glutaraldehyde. The degree of tubulin monomer cross-linking as a function of time and glutaraldehyde concentration was characterized using polyacrylamide gel electrophoresis. Atomic force microscopy of fixed microtubules indicated that the cross-linking is sufficient to stabilize the gross structure of the microtubules against air drying or a distilled water challenge. Kinesin movement on immobilized, fixed microtubules was determined using a kinesin-coated bead motility assay observed with differential interference contrast microscopy. Within measurement error, kinesin bead movement velocities were independent of the degree of microtubule cross-linking. Binding affinity, however, decreased with increased cross-linking. Although air- and water-challenged microtubules did not support kinesin motility, a dilute suspension of glutaraldehyde-fixed microtubules in buffer supported kinesin motility for at least 2 days without any substantial degradation of activity. Fixed microtubules may be useful for several applications, including affinity purification of microtubule-associated proteins and motility measurements under extreme conditions of temperature and other variables.
- Arnal I, Metoz F, DeBonis S, Wade RH
- Three-dimensional structure of functional motor proteins on microtubules.
- Curr Biol. 1996; 6: 1265-70
- Display abstract
BACKGROUND: Kinesins are a superfamily of motor proteins that use ATP hydrolysis to fuel movement along microtubules and participate in many crucial phases of the eukaryotic cell cycle. Usually these motors are heterotetramers of two heavy and two light chains, and have globular motor domains on the two heavy chains. Most kinesins move towards the microtubule 'plus end', but some, such as ncd (nonclaret disjunctional protein), move in the opposite direction. Heavy chain dimers produced by overexpression are viable motors. RESULTS: In order to establish whether the opposite directionality of kinesin and ncd dimers is related to notable conformational differences, we have used electron cryo-microscopy and three-dimensional reconstruction methods to investigate the structure of kinesin and ncd dimers attached to microtubules in the presence of AMP-PNP (5'-adenylylimidodiphosphate), a nonhydrolyzable ATP analogue. Three-dimensional maps of the motor-microtubule complexes show the motors to have one unattached, and one attached head per tubulin dimer. The polarity of the reconstructions was determined for each individual microtubule. Attachment occurs on the crest of a protofilament at the end of the tubulin dimer that points towards the plus end of the microtubule. The attached head extends over the next tubulin molecule along the protofilament. The unattached heads of kinesin and ncd have distinctly different conformations. CONCLUSIONS: The attached heads of kinesin and ncd appear to be similar and to interact with the same region of the plus end-oriented tubulin subunits. The free heads, however, are quite different, which suggests that directionality could be determined by differences in the dimer conformations. Work is in progress to obtain three-dimensional maps in the presence of different nucleotides with the aim of understanding how these motors move along microtubules.
- Yamazaki H, Nakata T, Okada Y, Hirokawa N
- Cloning and characterization of KAP3: a novel kinesin superfamily-associated protein of KIF3A/3B.
- Proc Natl Acad Sci U S A. 1996; 93: 8443-8
- Display abstract
We previously reported that KIF3A and KIF3B form a heterodimer that functions as a microtubule-based fast anterograde translocator of membranous organelles. We have also shown that this KIF3A/3B forms a complex with other associated polypeptides, named kinesin superfamily-associated protein 3 (KAP3). In the present study, we purified KAP3 protein by immunoprecipitation using anti-KIF3B antibody from mouse testis. Microsequencing was carried out, and we cloned the full-length KAP3 cDNA from a mouse brain cDNA library. Two isoforms of KAP3 exist [KAP3A (793 aa) and KAP3B (772 aa)], generated by alternative splicing in the carboxyl terminus region. Their amino acid sequences have no homology with those of any other known proteins, and prediction of their secondary structure indicated that almost the entire KAP3 molecule is alpha-helical. We produced recombinant KAP3 and KIF3A/3B using a baculovirus-Sf9 expression system. A reconstruction study in Sf9 cells revealed that KAP3 is a globular protein that binds to the tail domain of KIF3A/3B. The immunolocalization pattern of KAP3 was similar to that of KIF3A/3B in nerve cells. In addition, we found that KAP3 does not affect the motor activity of KIF3A/3B. KAP3 was associated with a membrane-bound form of KIF3A/3B in a fractional immunoprecipitation experiment, and since the KIF3 complex was found to bind to membranous organelles in an EM study, KAP3 may regulate membrane binding of the KIF3 complex.
- Duke T, Leibler S
- Motor protein mechanics: a stochastic model with minimal mechanochemical coupling.
- Biophys J. 1996; 71: 1235-47
- Display abstract
A stochastic model for the action of motor proteins such as kinesin is presented. The mechanical components of the enzyme are 1) two identical head domains that bind to discrete sites on a microtubule and that are capable of undergoing a conformational change; and 2) an elastic element that connects each head to the rest of the molecule. We investigate the situation in which the strain dependence of the chemical reaction rates is minimal and the heads have independent biochemical cycles. The enzyme advances stochastically along a filament when one head detaches and diffuses to a new binding site, while the other head remains bound to the microtubule. We also investigate the case in which the chemical cycles of the heads are correlated so that the molecule shifts each head alternately. The predictions of the model are found to be in agreement with experimentally measured force-velocity relationships for kinesin-both when the force is applied externally and when the enzyme is loaded by a viscous drag. For reasonable values of the parameters, this agreement is quantitative. The molecular stepping characteristics observed in recent motility assays are also reproduced. A number of experiments are suggested that would provide a more stringent test of the model and help determine whether this simple picture is an appropriate description of motor proteins or whether models that include strain-dependent reaction rates or more complicated types of cooperation of the two heads need to be considered.
- Mitsui H, Hasezawa S, Nagata T, Takahashi H
- Cell cycle-dependent accumulation of a kinesin-like protein, KatB/C in synchronized tobacco BY-2 cells.
- Plant Mol Biol. 1996; 30: 177-81
- Display abstract
Immunoblot analysis with antibodies prepared against highly purified recombinant truncated kinesin-like proteins, KatB(5-249) and KatC(207-754), encoded by the katB and katC genes of Arabidopsis thaliana revealed the presence of a kinesin-like polypeptide, termed KatB/C, in cultured tobacco BY-2 cells. The KatB/C polypeptide cosedimented with microtubules in the presence of a nonhydrolyzable ATP analogue and was released from microtubules in the presence of ATP, both of which are characteristics of kinesin proteins. The amount of KatB/C polypeptide in synchronous BY-2 cells increased during M phase of the cell cycle. Microtubule-based structures present in cells at M phase, such as the spindle and phragmoplast, may be the site of action of the KatB/C protein.
- Sellers JR
- Kinesin and NCD, two structural cousins of myosin.
- J Muscle Res Cell Motil. 1996; 17: 173-5
- Muresan V, Godek CP, Reese TS, Schnapp BJ
- Plus-end motors override minus-end motors during transport of squid axon vesicles on microtubules.
- J Cell Biol. 1996; 135: 383-97
- Display abstract
Plus- and minus-end vesicle populations from squid axoplasm were isolated from each other by selective extraction of the minus-end vesicle motor followed by 5'-adenylyl imidodiphosphate (AMP-PNP)-induced microtubule affinity purification of the plus-end vesicles. In the presence of cytosol containing both plus- and minus-end motors, the isolated populations moved strictly in opposite directions along microtubules in vitro. Remarkably, when treated with trypsin before incubation with cytosol, purified plus-end vesicles moved exclusively to microtubule minus ends instead of moving in the normal plus-end direction. This reversal in the direction of movement of trypsinized plus-end vesicles, in light of further observation that cytosol promotes primarily minus-end movement of liposomes, suggests that the machinery for cytoplasmic dynein-driven, minus-end vesicle movement can establish a functional interaction with the lipid bilayers of both vesicle populations. The additional finding that kinesin overrides cytoplasmic dynein when both are bound to bead surfaces indicates that the direction of vesicle movement could be regulated simply by the presence or absence of a tightly bound, plus-end kinesin motor; being processive and tightly bound, the kinesin motor would override the activity of cytoplasmic dynein because the latter is weakly bound to vesicles and less processive. In support of this model, it was found that (a) only plus-end vesicles copurified with tightly bound kinesin motors; and (b) both plus- and minus-end vesicles bound cytoplasmic dynein from cytosol.
- Sharp DJ, Kuriyama R, Baas PW
- Expression of a kinesin-related motor protein induces Sf9 cells to form dendrite-like processes with nonuniform microtubule polarity orientation.
- J Neurosci. 1996; 16: 4370-5
- Display abstract
The microtubules (MTs) within neuronal processes are highly organized with regard to their polarity and yet are not attached to any detectable nucleating structure. Axonal MTs are uniformly oriented with their plus ends distal to the cell body, whereas dendritic MTs are of both orientations. Here, we sought to test the capacity of motor-driven MT transport to organize distinct MT patterns during process outgrowth. We focused on CHO1/MKLP1, a kinesin-related protein present in the midzonal region of the mitotic spindle where MTs of opposite orientation overlap. Insect ovarian Sf9 cells induced to express the N-terminal portion of the molecule form MT-rich processes with a morphology similar to that of neuronal dendrites (Kuriyama et al., 1994). Nascent processes contain uniformly plus-end-distal MTs, but these are joined by minus-end-distal MTs as the processes continue to develop. Thus, this CHO1/MKLP1 fragment establishes a nonuniform MT polarity pattern and does so by a similar sequence of events as occurs with the dendrite, the antecedent of which is a short process with a uniform MT polarity orientation. Two lines of evidence suggest that these results are elicited by motor-driven MT transport. First, there is a depletion of MTs from the cell body during process outgrowth. Second, the same polarity pattern is obtained when net MT assembly is suppressed pharmacologically during process formation. Collectively, these findings provide precedent for the idea that motor-driven transport can organize MTs into distinct patterns of polarity orientation during process outgrowth.
- Hall K, Cole D, Yeh Y, Baskin RJ
- Kinesin force generation measured using a centrifuge microscope sperm-gliding motility assay.
- Biophys J. 1996; 71: 3467-76
- Display abstract
To measure force generation and characterize the relationship between force and velocity in kinesin-driven motility we have developed a centrifuge microscope sperm-gliding motility assay. The average (extrapolated) value of maximum isometric force at low kinesin density was 0.90 +/- 0.14 pN. Furthermore, in the experiments at low kinesin density, sperm pulled off before stall at forces between 0.40 and 0.75 pN. To further characterize our kinesin-demembranated sperm assay we estimated maximum isometric force using a laser trap-based assay. At low kinesin density, 4.34 +/- 1.5 pN was the maximum force. Using values of axoneme stiffness available from other studies, we concluded that, in our centrifuge microscope-based assay, a sperm axoneme functions as a lever arm, magnifying the centrifugal force and leading to pull-off before stall. In addition, drag of the distal portion of the axoneme is increased by the centrifugal force (because the axoneme is rotated into closer proximity to the glass surface) and represents an additional force that the kinesin motor must overcome.
- Lockhart A, Cross RA
- Kinetics and motility of the Eg5 microtubule motor.
- Biochemistry. 1996; 35: 2365-73
- Display abstract
We have investigated the kinetic properties of the slow plus end directed microtubule (MT) motor Eg5. The recombinantly expressed fusion protein E437GST, containing residues 12-437 of Eg5 fused to the N-terminus of glutathione S-transferase (GST), is dimeric and motile, translocating MTs at an average speed of 0.063 (+/-0.01) micrometers(-1). The kinetics of ATP turnover by E437GST were investigated using the fluorescent ATP analogue methylanthraniloyl-ATP (mantATP). In the absence of MTs, mantADP release from E437GST is slow (0.006 s(-1) in 50 mM NaCl) and rate-limiting. MTs accelerate this kinetic step approximately 850-fold to a maximal rate of 4.94 s(-1). Under these conditions, the steady-state rate of mantATP turnover was 1.92 s(-1), indicating that MT-activated mantADP release accounts for at least 40% of the total cycle time of the motor and is probably rate-limiting. This step is around 10-fold slower in Eg5 than in kinesin, consistent with it limiting the rate of physical stepping in both Eg5 and kinesin. The dissociation constants of the motor in the presence of various nucleotides were determined using MT pelleting assays. ADP stabilizes the weakest bound state of the motor, while ATP, ATP gamma S, AMPPNP, and apyrase all induce a shift toward tighter binding states. Overall, the data indicate that Eg5 displays strong kinetic homologies with the two other well-characterized MT motors, kinesin and non claret disjunctional, suggesting that all kinesin superfamily motors may share the same basic mechanochemistry.
- Goody RS
- Motor proteins. A two-way structure.
- Nature. 1996; 380: 483-4
- Sablin EP, Kull FJ, Cooke R, Vale RD, Fletterick RJ
- Crystal structure of the motor domain of the kinesin-related motor ncd.
- Nature. 1996; 380: 555-9
- Display abstract
Microtubule-based ATPases of the kinesin superfamily provide the motile force for many animated features of living cells. Kinesin motors differ in their direction of movement along microtubules. Kinesin and ncd, a kinesin-related motor involved in formation and maintenance of mitotic and meiotic spindles, move in opposite directions along microtubules, even though their motor domains are 40% identical in amino-acid sequence. Here we report the crystal structure of the MgADP complex of the Drosophila ncd motor domain determined to 2.5A by X-ray crystallography, and compare it to the kinesin structure. The ncd and kinesin motor domains are remarkably similar in structure, and the locations of conserved surface amino acids suggest these motors share a common microtubule-binding site. Moreover, structural and functional comparisons of ncd, kinesin, myosin and G proteins indicate that these NTPases may have a similar strategy of changing conformation between NTP and NDP states. We propose a general model for converting a common gamma-phosphate-sensing mechanism into opposite polarities of movement for kinesin and ncd.
- Krupka RM
- Force generation, work, and coupling in molecular motors.
- Biophys J. 1996; 70: 1863-71
- Display abstract
A mechanism is proposed for molecular motors in which force is generated by a protein conformational change driven by binding energy (in muscle, that of myosin with actin as well as with ATP, ADP, or Pi). Work, the product of the force generated by one myosin or kinesin molecule (F) and the distance over which it acts (d), is a function of a ratio of dissociation constants before and after the contractile step: F.d < RT ln(KAe/KAc). From published data the ratio is > 2 x 10(4), which can be explained by conversion of a surface complex to an enclosed, or partly enclosed, complex. Although the complex performing the work stroke is in unstrained conformation, the complex after the work stroke is much more stable, owing to binding forces; the latter, however, is destabilized by the load, which thereby opposes the contractile conformational change, countering the force-generating reaction. The connection between the free energy release and work is implicit in the mechanism, inasmuch as coupling, like force generation, depends on conformational changes driven by binding energy (internal rather than external work being involved in coupling). The principles apply whether ATP or an ion gradient drives the system. At high load, in muscle, the mechanism allows for a summation of the forces generated by several myosin molecules.
- Rosenfeld SS, Rener B, Correia JJ, Mayo MS, Cheung HC
- Equilibrium studies of kinesin-nucleotide intermediates.
- J Biol Chem. 1996; 271: 9473-82
- Display abstract
We have examined the energetics of the interactions of two kinesin constructs with nucleotide and microtubules to develop a structural model of kinesin-dependent motility. Dimerization of the constructs was found to reduce the maximum rate of the microtubule-activated kinesin ATPase 5-fold. Beryllium fluoride and aluminum fluoride also reduce this rate, and they increase the affinity of kinesin for microtubules. By contrast, inorganic phosphate reduces the affinity of a dimeric kinesin construct for microtubules. These findings are consistent with a model in which the kinesin head can assume one of two conformations, "strong" or "weak" binding, determined by the nature of the nucleotide that occupies the active site. Data for dimeric kinesin are consistent with a model in which kinesin.ATP binds to the microtubule in a strong state with positive cooperativity; hydrolysis of ATP to ADP+P(i) leads to dissociation of one of the attached heads and converts the second, attached head to a weak state; and dissociation of phosphate allows the second head to reattach. These results also argue that a large free energy change is associated with formation of kinesin.ADP.P(i) and that this step is the major pathway for dissociation of kinesin from the microtubule.
- Gittes F, Meyhofer E, Baek S, Howard J
- Directional loading of the kinesin motor molecule as it buckles a microtubule.
- Biophys J. 1996; 70: 418-29
- Display abstract
Single kinesin motor molecules were observed to buckle the microtubules along which they moved in a modified in vitro gliding assay. In this assay a central portion of the microtubule was clamped to the glass substrate via biotin-streptavidin bonds, while the plus end of the microtubule was free to interact with motors adsorbed at low density to the substrate. A statistical analysis of the length of microtubules buckled by single motors showed a decreasing probability of buckling for loads greater than 4-6 pN parallel to the filament. This is consistent with kinesin stalling forces found in other experiments. A detailed analysis of some buckling events allowed us to estimate both the magnitude and direction of the loading force as it developed a perpendicular component tending to pull the motor away from the microtubule. We also estimated the motor speed as a function of this changing vector force. The kinesin motors consistently reached unexpectedly high speeds as the force became nonparallel to the direction of motor movement. Our results suggest that a perpendicular component of load does not hinder the kinesin motor, but on the contrary causes the motor to move faster against a given parallel load. Because the perpendicular force component speeds up the motor but does no net work, perpendicular force acts as a mechanical catalyst for the reaction. A simple explanation is that there is a spatial motion of the kinesin molecule during its cycle that is rate-limiting under load; mechanical catalysis results if this motion is oriented away from the surface of the microtubule.
- Lin SX, Pfister KK, Collins CA
- Comparison of the intracellular distribution of cytoplasmic dynein and kinesin in cultured cells: motor protein location does not reliably predict function.
- Cell Motil Cytoskeleton. 1996; 34: 299-312
- Display abstract
While immunolocalization methods have been used as a reasonable means to judge where a given molecule may be active in the cellular milieu, the correlation between distribution and function for proteins involved in intracellular transport may not be clear cut. To address the question of specificity and reproducibility of immunolocalization of microtubule-based motor proteins, we have co-localized cytoplasmic dynein and kinesin by immunofluorescence microscopy using two specific antibodies for each motor molecule. The results indicate that cytoplasmic dynein and kinesin appear to co-localize on a small subset of vesicles, but largely reside or accumulate on morphologically distinct organelles. In addition, anti-kinesin antibodies differing in their epitope specificity label different cellular compartments. To address the question of whether the distribution of motor molecules is representative of organelles that are undergoing active transport, we have altered the activity of vesicle trafficking pathways in fibroblasts using several different methods, including cytoplasmic acidification and disruption of cellular compartments with brefeldin A, nocodazole and okadaic acid. Analysis of the distribution of cytoplasmic dynein and kinesin under these conditions indicates that immunolocalization data alone are not reliable indicators of sites of likely function for these microtubule-based motors.
- Barton NR, Goldstein LS
- Going mobile: microtubule motors and chromosome segregation.
- Proc Natl Acad Sci U S A. 1996; 93: 1735-42
- Display abstract
Proper chromosome segregation in eukaryotes depends upon the mitotic and meiotic spindles, which assemble at the time of cell division and then disassemble upon its completion. These spindles are composed in large part of microtubules, which either generate force by controlled polymerization and depolymerization or transduce force generated by molecular microtubule motors. In this review, we discuss recent insights into chromosome segregation mechanisms gained from the analyses of force generation during meiosis and mitosis. These analyses have demonstrated that members of the kinesin superfamily and the dynein family are essential in all organisms for proper chromosome and spindle behavior. It is also apparent that forces generated by microtubule polymerization and depolymerization are capable of generating forces sufficient for chromosome movement in vitro; whether they do so in vivo is as yet unclear. An important realization that has emerged is that some spindle activities can be accomplished by more than one motor so that functional redundancy is evident. In addition, some meiotic or mitotic movements apparently occur through the cooperative action of independent semiredundant processes. Finally, the molecular characterization of kinesin-related proteins has revealed that variations both in primary sequence and in associations with other proteins can produce motor complexes that may use a variety of mechanisms to transduce force in association with microtubules. Much remains to be learned about the regulation of these activities and the coordination of opposing and cooperative events involved in chromosome segregation; this set of problems represents one of the most important future frontiers of research.
- Coppin CM, Finer JT, Spudich JA, Vale RD
- Detection of sub-8-nm movements of kinesin by high-resolution optical-trap microscopy.
- Proc Natl Acad Sci U S A. 1996; 93: 1913-7
- Display abstract
Kinesin is a molecular motor that transports organelles along microtubules. This enzyme has two identical 7-nm-long motor domains, which it uses to move between consecutive tubulin binding sites spaced 8 nm apart along a microtubular protofilament. The molecular mechanism of this movement, which remains to be elucidated, may be common to all families of motor proteins. In this study, a high-resolution optical-trap microscope was used to measure directly the magnitude of abrupt displacements produced by a single kinesin molecule transporting a microscopic bead. The distribution of magnitudes reveals that kinesin not only undergoes discrete 8-nm movements, in agreement with previous work [Svoboda, K., Schmidt, C. F., Schnapp, B. J. & Block, S.M. (1993) Nature (London) 365, 721-727], but also frequently exhibits smaller movements of about 5 nm. A possible explanation for these unexpected smaller movements is that kinesin's movement from one dimer to the next along a protofilament involves at least two distinct events in the mechanical cycle.
- Sosa H, Milligan RA
- Three-dimensional structure of ncd-decorated microtubules obtained by a back-projection method.
- J Mol Biol. 1996; 260: 743-55
- Display abstract
We have used cryo-electron microscopy and image analysis to obtain the three-dimensional (3D) structure of 11, 12, 14 and 15 protofilament microtubules decorated with the motor domain of ncd. To obtain the 3D maps, we developed a back-projection method that does not require a helical arrangement of the tubulin heterodimers. This method allows the calculation of 3D maps even when lattice discontinuities (seams) are present. The maps show that the microtubules we studied conform to a B-type lattice with one or more seams. In the presence of 5'-adenylim-idodiphosphate (AMP-PNP), the motor domain of ncd binds to the microtubule protofilament crest interacting with only one protofilament. Viewing the structures along the microtubule axis shows that the ncd motor domain and the tubulin are titled in opposite directions. We determined that a clockwise tilt of the tubulin subunits corresponds to a view from the minus end towards the plus end of the microtubule.
- Shimizu T, Morii H
- Comparison of ncd and kinesin motor domains by circular dichroism spectroscopy.
- J Biochem (Tokyo). 1996; 120: 1176-81
- Display abstract
ncd is a microtubule motor protein from Drosophila, having a 40 kDa domain homologous to the kinesin motor domain. In the present study, we investigated the circular dichroism (CD) spectra of the ncd motor domain in comparison with those of the kinesin motor domain. Although the two are about 40% identical in amino acid sequence, and recent X-ray crystallographic studies [Sablin, Kull, Cooke, Vale, and Fletterick (1996) Nature 380, 555-559; Kull, Sablin, Lau, Fletterick, and Vale (1996) Nature 380, 550-555] indicate that their core structures are nearly identical, the far UV CD spectra of ncd and kinesin motor domains, both being monomeric, were considerably different from each other, suggesting a significant difference in the secondary, especially loop structures. The motor domain of ncd, like that of kinesin, contains tightly associating ADP even after purification. We removed ADP from the ncd motor domain by gel filtration in the presence of EDTA and high salt. The resultant protein, however, was likely to be in an inactive state, since it bound ATP slowly. The far UV CD spectrum of the ncd motor domain devoid of ADP was nearly identical to that of the ncd motor domain with bound ADP. This indicated that the removal of ADP did not affect the backbone structure in the presence of high salt. On the other hand, the near UV CD spectrum of the ADP-free ncd motor domain differed from that of the ncd motor domain. ADP complex, one possibility being that the local conformation was changed upon removal of bound ADP. The near UV CD spectra of kinesin motor domain also showed a difference between the ADP-bound form and the nucleotide-free form, although the difference was much smaller.
- Sperry AO, Zhao LP
- Kinesin-related proteins in the mammalian testes: candidate motors for meiosis and morphogenesis.
- Mol Biol Cell. 1996; 7: 289-305
- Display abstract
The kinesin superfamily of molecular motors comprises proteins that participate in a wide variety of motile events within the cell. Members of this family share a highly homologous head domain responsible for force generation attached to a divergent tail domain thought to couple the motor domain to its target cargo. Many kinesin-related proteins (KRPs) participate in spindle morphogenesis and chromosome movement in cell division. Genetic analysis of mitotic KRPs in yeast and Drosophila, as well as biochemical experiments in other species, have suggested models for the function of KRPs in cell division, including both mitosis and meiosis. Although many mitotic KRPs have been identified, the relationship between mitotic motors and meiotic function is not clearly understood. We have used sequence similarity between mitotic KRPs to identify candidates for meiotic and/or mitotic motors in a vertebrate. We have identified a group of kinesin-related proteins from rat testes (termed here testes KRP1 through KRP6) that includes new members of the bimC and KIF2 subfamilies as well as proteins that may define new kinesin subfamilies. Five of the six testes KRPs identified are expressed primarily in testes. Three of these are expressed in a region of the seminiferous epithelia (SE) rich in meiotically active cells. Further characterization of one of these KRPs, KRP2, showed it to be a promising candidate for a motor in meiosis: it is localized to a meiotically active region of the SE and is homologous to motor proteins associated with the mitotic apparatus. Testes-specific genes provide the necessary probes to investigate whether the motor proteins that function in mammalian meiosis overlap with those of mitosis and whether motor proteins exist with functions unique to meiosis. Our search for meiotic motors in a vertebrate testes has successfully identified proteins with properties consistent with those of meiotic motors in addition to uncovering proteins that may function in other unique motile events of the SE.
- Marcussen M, Larsen PJ
- Cell cycle-dependent regulation of cellular ATP concentration, and depolymerization of the interphase microtubular network induced by elevated cellular ATP concentration in whole fibroblasts.
- Cell Motil Cytoskeleton. 1996; 35: 94-9
- Display abstract
In the present work, evidence is presented indicating that an increased cellular ATP concentration during mitosis may, in conjunction with other factors [Verde et al., 1990: Nature 343:233-238; Andersen et al., 1994: J Cell Biol. 127:1289-1299], induce depolymerization of the interphase microtubular network in cultured fibroblasts. It is shown here that the cellular ATP concentration varies through the cell cycle, reaching a peak at G2M- and minimum at late G1/early S-phase. Furthermore, we have found, using indirect immunofluorescent staining with an antitubulin antibody, that depolymerization of the interphase microtubular network may be induced by increasing the intracellular ATP concentration in cultured fibroblasts from 2.2 mM to 4.1 mM. This may be obtained through addition of adenosine and P1 to the growth medium. Our results indicate that this effect of adenosine and Pi is not mediated via adenosine receptors, but through an elevated cellular ATP concentration. ATP is suggested to act through a concentration-dependent effect on the exchangeable GTP site on tubulin, and not through the action of protein kinases or microtubule-associated proteins.
- Waters JC, Salmon ED
- Cytoskeleton: a catastrophic kinesin.
- Curr Biol. 1996; 6: 361-3
- Display abstract
The 'plus' ends of microtubules exhibit dynamic instability, switching stochastically from growth to shortening phases. The first endogenous regulator of such 'catastrophes' has been identified, and is a kinesin-related microtubule motor protein.
- Simmons R
- Molecular motors: single-molecule mechanics.
- Curr Biol. 1996; 6: 392-4
- Display abstract
Novel techniques are revealing the movements and forces associated with single interactions of motor proteins, such as myosin and kinesin, and also of processive enzymes, such as RNA polymerase.
- Malashkevich VN, Kammerer RA, Efimov VP, Schulthess T, Engel J
- The crystal structure of a five-stranded coiled coil in COMP: a prototype ion channel?
- Science. 1996; 274: 761-5
- Display abstract
Oligomerization by the formation of alpha-helical bundles is common in many proteins. The crystal structure of a parallel pentameric coiled coil, constituting the oligomerization domain in the cartilage oligomeric matrix protein (COMP), was determined at 2.05 angstroms resolution. The same structure probably occurs in two other extracellular matrix proteins, thrombospondins 3 and 4. Complementary hydrophobic interactions and conserved disulfide bridges between the alpha helices result in a thermostable structure with unusual properties. The long hydrophobic axial pore is filled with water molecules but can also accommodate small apolar groups. An "ion trap" is formed inside the pore by a ring of conserved glutamines, which binds chloride and probably other monatomic anions. The oligomerization domain of COMP has marked similarities with proposed models of the pentameric transmembrane ion channels in phospholamban and the acetylcholine receptor.
- Insinna EM, Zaborski P, Tuszynski J
- Electrodynamics of microtubular motors: the building blocks of a new model.
- Biosystems. 1996; 39: 187-226
- Display abstract
Microtubules are ubiquitous components of the cytoskeleton. They participate in many motility processes ranging from intracellular transport or chromosome movement during mitosis to ciliary and flagellar beating. The biophysical mechanism inherent in the generation and control of movement in all these motility phenomena has not yet been entirely elucidated. The authors propose a new model based on a charge transfer mechanism capable of shedding a new light on the molecular foundations of all motility processes. Electron transfer along the microtubular lattice is responsible for activation and control of all microtubule-associated ATPases (i.e. force generating enzymes). Microtubules are thus shown to be the basic motors of cell dynamics. The model is first applied to intracellular transport and ciliary and flagellar beating. Through two additional examples, the authors show the heuristic capabilities of the suggested hypothesis. The application of charge transfer control to the Protozoan Euglena gracilis leads to a plausible model capable of accounting for its phototactic response mechanism. Furthermore, the model allows a new interpretation of the electrophysiological response in vertebrate photoreceptors.
- Hirose K, Lockhart A, Cross RA, Amos LA
- Three-dimensional cryoelectron microscopy of dimeric kinesin and ncd motor domains on microtubules.
- Proc Natl Acad Sci U S A. 1996; 93: 9539-44
- Display abstract
Kinesin and ncd motor proteins are homologous in sequence yet move in opposite directions along microtubules. We have previously shown that monomeric kinesin and ncd bind in the same orientation on equivalent sites relative to the ends of tubulin sheets of known polarity. We now report cryoelectron microscope images of 16-protofilament microtubules decorated with both single- and double-headed kinesin and double-headed ncd. Three-dimensional density maps and difference maps show that, in adenosine 5'-[beta,gamma-imido]triphosphate, both dimeric motors bind tightly to microtubules via one head, leaving the other free, though apparently in a fixed position. The attached heads of dimers bind to tubulin in the same way as single kinesin heads. The second heads are connected to the tops of the first but, whereas the second kinesin head is closely associated with the first, pairs of ncd heads are splayed apart. There is also a distinct difference in orientation: the second kinesin head is tilted toward the microtubule plus end, while the second head of ncd points toward the minus end.
- Kashina AS, Baskin RJ, Cole DG, Wedaman KP, Saxton WM, Scholey JM
- A bipolar kinesin.
- Nature. 1996; 379: 270-2
- Display abstract
Chromosome segregation during mitosis depends on the action of the mitotic spindle, a self-organizing, bipolar protein machine which uses microtubules (MTs) and their associated motors. Members of the BimC subfamily of kinesin-related MT-motor proteins are believed to be essential for the formation and functioning of a normal bipolar spindle. Here we report that KRP130, a homotetrameric BimC-related kinesin purified from Drosophila melanogaster embryos, has an unusual ultrastructure. It consists of four kinesin-related polypeptides assembled into a bipolar aggregate with motor domains at opposite ends, analogous to a miniature myosin filament. Such a bipolar 'minifilament' could crosslink spindle MTs and slide them relative to one another. We do not know of any other MT motors that have a bipolar structure.
- Subramanya HS, Bird LE, Brannigan JA, Wigley DB
- Crystal structure of a DExx box DNA helicase.
- Nature. 1996; 384: 379-83
- Display abstract
There are a wide variety of helicases that unwind helical DNA and RNA substrates. The twelve helicases that have been identified in Escherichia coli play a role in almost all cellular processes involving nucleic acids. We have solved the crystal structure of a monomeric form of a DNA helicase from Bacillus stearothermophilus, alone and in a complex with ADP, at 2.5 and 2.9 A resolution, respectively. The enzyme comprises two domains with a deep cleft running between them. The ATP-binding site, which is situated at the bottom of this cleft, is formed by motifs that are conserved across the superfamily of related helicases. Unexpected structural homology with the DNA recombination protein, RecA, suggests how ATP binding and hydrolysis may drive conformational changes of the enzyme during catalysis, and implies that there is a common mechanism for all helicases.
- Sakowicz R, Goldstein LS
- The muscle in kinesin.
- Nat Struct Biol. 1996; 3: 404-7
- Jancsik V, Filliol D, Rendon A
- Tau proteins bind to kinesin and modulate its activation by microtubules.
- Neurobiology (Bp). 1996; 4: 417-29
- Display abstract
Microtubule-associated tau proteins are likely candidates to interfere with axonal transport of membranous organelles. We studied that tau proteins influenced the enzyme activity of kinesin, known to drive anterograd transport along microtubules. An in vitro reconstituted system was applied; microtubules were assembled from purified tubulin with or without tau proteins. Both types of reconstituted microtubules stimulated MgATPase activity of purified kinesin in a concentration dependent, saturable manner. The extent of maximal stimulation by tau-coated microtubules was lower than that of microtubules without tau proteins. Analysis of kinetic data, on the other hand, suggests that tau-coated microtubules apparently bind kinesin with higher affinity then microtubules not associated with tau proteins. Tau proteins, similarly to tubulin dimers, seem to bind to the heavy chain of kinesin. These data support the notion that tau proteins could act as regulators of kinesin-driven processes.
- Gelles J, Berliner E, Young EC, Mahtani HK, Perez-Ramirez B, Anderson K
- Structural and functional features of one- and two-headed biotinated kinesin derivatives.
- Biophys J. 1995; 68: 276281282-276281282
- Display abstract
The oligomeric structure was determined for four recombinant kinesin derivatives containing N-terminal fragments of the kinesin alpha-subunit. Some of the proteins were dimeric (two-headed) molecules with mechanochemical properties similar to those of intact kinesin. Comparison of the primary and quaternary structures of the derivatives with those of intact kinesin suggests that structures distinct from the long alpha-helical coiled-coil rod domain contribute to subunit self-association. Three of the proteins contain a single engineered site for post-translational biotination in vivo; this facilitates analysis of motility in experiments in which the proteins are specifically bound to streptavidin-conjugated microscopic plastic beads. One of the derivatives is monomeric (one-headed); like the two-headed derivatives, it is functional in the motility assay and is a microtubule-dependent ATPase. Unlike intact kinesin and the two-headed derivatives, the one-headed enzyme fails to track microtubule protofilaments. This confirms a prediction of proposed "hand-over-hand" mechanisms of kinesin movement. The ability of molecules with a one-headed solution structure to generate movement is consistent with a translocation-generating conformational change internal to the kinesin head. A simple set of coupling rules can be used to formulate consistent mechano-chemical mechanisms that explain movement by both one- and two-headed kinesin molecules.
- Endow SA
- Determinants of motor polarity in the kinesin proteins.
- Biophys J. 1995; 68: 271274-271274
- Display abstract
Many of the proteins that are members of the kinesin family of microtubule motor proteins are plus-end motors; however, a few of the kinesin proteins have now been found to be minus-end microtubule motors. Overall structural features of the proteins can be used to identify further kinesins that are likely to be minus-end motors. Structural or biochemical differences that may serve as the basis of the "reversed" polarity of a unique subset of the kinesin proteins are discussed.
- Coppin CM, Finer JT, Spudich JA, Vale RD
- Measurement of the isometric force exerted by a single kinesin molecule.
- Biophys J. 1995; 68: 242244-242244
- Astumian RD, Bier M
- Mechanochemical coupling of molecular motors to ATP hydrolysis.
- Biophys J. 1995; 68: 219-219
- Kuo SC, Ramanathan K, Sorg B
- Single kinesin molecules stressed with optical tweezers.
- Biophys J. 1995; 68: 74-74
- Display abstract
Using the optical tweezers to pull on microtubules, we have stretched and twisted single kinesin molecules adsorbed to glass surfaces. Preliminary measurements suggest that the mechanical system is very compliant, with an apparent stretch of 120 nm with < 2 pN of force. Although measurements of the series compliance of the bead-microtubule structure are still in progress, the kinesin attachment site does not slip with stretch. However, under torsional stress, kinesin appears to slip. With torques < 2 pN-microns approximately 1 Hz in 2 mM AMP-PNP, there is no apparent limit to the number of revolutions that the microtubule can rotate around the kinesin attachment site (n = 44). Preliminary data from other nucleotide conditions are similar. Although there are rare instances of torsional elasticity where the attachment site unwinds, the restoring forces are not constant with angular position, also indicating slippage. Mechanisms of mechanochemical transduction must account for linear force generation in the presence of angular "slippage."
- Svoboda K, Mitra PP, Block SM
- Fluctuation analysis of kinesin movement.
- Biophys J. 1995; 68: 69-69
- Tawada K, Imafuku Y, Toyoshima YY
- A single-track fluctuation analysis in the sliding movement by protein motors in vitro.
- Biophys J. 1995; 68: 68-68
- Eden D, Luu BQ, Zapata DJ, Sablin EP, Kull FJ
- Solution structure of two molecular motor domains: nonclaret disjunctional and kinesin.
- Biophys J. 1995; 68: 596465-596465
- Display abstract
The effects of selected ligands on the structure of the truncated heavy-chain chemomechanical motor domains of Drosophila ncd and human kinesin were compared using the technique of transient electric birefringence. The 366-amino acid C-terminal motor domain of Drosophila nonclaret disjunctional, ncd(335-700), and the 349-amino acid N-terminal motor domain of human kinesin, kinesin(349), were studied at 4 degrees C in neutral buffers with ionic strength of 100 mM to form complexes with either MgADP or MgADP.Vi. The rotational diffusion time adjusted to 20 degrees C and water, tau 20,W, for ncd(335-700).MgADP is 32.8 ns, and for ncd(335-700).MgADP.Vi is 34.8 ns, suggesting prolate ellipsoids with dimensions 9.40 x 3.77 nm and 9.73 x 3.70 nm, respectively. The specific Kerr constant, Ksp, of ncd is -1.65 x 10(-12) cm2V-2 for the MgADP complex and -1.15 x 10(-12) cm2V-2 for the MgADP.Vi complex. The large negative Ksp for a prolate protein suggests an unusual charge distribution with two long surfaces with opposite charge. The tau 20,W for kinesin(349).MgADP is longer than the corresponding ncd motor and shows a decrease with increased electric field. The kinesin(349).MgADP.Vi complex has a longer tau 20,W. The Ksp for kinesin(349) is 0.36 x 10(-12) cm2V-2 for each complex.
- Wang Z, Khan S, Sheetz MP
- Single cytoplasmic dynein molecule movements: characterization and comparison with kinesin.
- Biophys J. 1995; 69: 2011-23
- Display abstract
Cytoplasmic dynein is a major microtubule motor for minus-end directed movements including retrograde axonal transport. To better understand the mechanism by which cytoplasmic dynein converts ATP energy into motility, we have analyzed the nanometer-level displacements of latex beads coated with low numbers of cytoplasmic dynein molecules. Cytoplasmic dynein-coated beads exhibited greater lateral movements among microtubule protofilaments (ave. 5.1 times/microns of displacement) compared with kinesin (ave. 0.9 times/micron). In addition, dynein moved rearward up to 100 nm over several hundred milliseconds, often in correlation with off-axis movements from one protofilament to another. We suggest that single molecules of cytoplasmic dynein move the beads because 1) there is a linear dependence of bead motility on dynein/bead ratio, 2) the binding of beads to microtubules studied by laser tweezers is best fit by a first-order Poisson, and 3) the run length histogram of dynein beads follows a first-order decay. At the cellular level, the greater disorder of cytoplasmic dynein movements may facilitate transport by decreasing the duration of collisions between kinesin and cytoplasmic dynein-powered vesicles.
- Walker RA
- ncd and kinesin motor domains interact with both alpha- and beta-tubulin.
- Proc Natl Acad Sci U S A. 1995; 92: 5960-4
- Display abstract
Motor domains of the Drosophila minus-end-directed microtubule (MT) motor protein ncd, were found to saturate microtubule binding sites at a stoichiometry of approximately one motor domain per tubulin dimer. To determine the tubulin subunit(s) involved in binding to ncd, mixtures of ncd motor domain and MTs were treated with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide) (EDC). EDC treatment generated covalently cross-linked products of ncd and alpha-tubulin and of ncd and beta-tubulin, indicating that the ncd motor domain interacts with both alpha- and beta-tubulin. When the Drosophila kinesin motor domain protein was substituted for the ncd motor domain, cross-linked products of kinesin and alpha-tubulin and of kinesin and beta-tubulin were produced. EDC treatment of mixtures of ncd motor domain and unassembled tubulin dimers or of kinesin motor domain and unassembled tubulin dimers produced the same motor-tubulin products generated in the presence of MTs. These results indicate that kinesin family motors of opposite polarity interact with both tubulin monomers and support a model in which some portion of each protein's motor domain overlaps adjacent alpha- and beta-tubulin subunits.
- Naber N, Matuska M, Sablin EP, Pate E, Cooke R
- A novel adenosine triphosphate analog with a heavy atom to target the nucleotide binding site of proteins.
- Protein Sci. 1995; 4: 1824-31
- Display abstract
We have synthesized 2'-deoxy-2'-iodoadenosine-5'-triphosphate (2'-IATP), a heavy-atom analog of adenosine-5'-triphosphate. This compound was made for X-ray structural studies to target the nucleotide site of ATP binding proteins. It was diffused successfully into crystals of the microtubule-based motor proteins ncd (non-claret disjunctional protein from Drosophila melanogaster) and kinesin. With ncd, the nucleotide binding site was 70% occupied and the crystals were able to diffract X-rays to 2.5 A. The iodo-analog provided a useful isomorphous derivative with overall phasing power 1.89 in the range of 25.0-2.5 A. With kinesin, 2'-IATP co-crystallized with the protein. The crystals diffracted to at least 2.8 A with a phasing power of 1.73 in the range of 20.0-5.0 A. The analog was also found to be a substrate for all of the enzymes tested, including creatine kinase, pyruvate kinase, hexokinase, and myosin, with values of Km and Vmax that were within a factor of 10 of those for ATP. The analog supported muscle contraction, relaxing fibers, and producing active tension with values not statistically different from those obtained with ATP. These results all suggest that this analog should be useful for providing a heavy-atom derivative for crystals of enzymes that bind ATP.
- Correia JJ, Gilbert SP, Moyer ML, Johnson KA
- Sedimentation studies on the kinesin motor domain constructs K401, K366, and K341.
- Biochemistry. 1995; 34: 4898-907
- Display abstract
Bacterial expressed kinesin motor domains hydrolyze ATP and promote microtubule-dependent motility. It has routinely been assumed that motor domain preparations are monomeric on the basis of the presumption that dimerization is mediated by the stalk region. However, experimental verification of the oligomeric state of the kinesin construct is required to interpret the results from single-molecule motility assays as well as presteady-state kinetic experiments. We have measured directly the state of assembly of three conventional kinesin motor domain constructs-K401, K366, and K341, comprising the N-terminal 401, 366, and 341 amino acids, respectively, of the Drosophila kinesin heavy chain-by sedimentation velocity and sedimentation equilibrium methods in an analytical ultracentrifuge. K401 (MW of ADP complex, 45,532) is a predominantly a dimer with a sedimentation coefficient, s020,w, of 5.06 S, but it is able to self-associate by means of a 1-2-4 mechanism into higher oligomers. Molecular weight measurements establish the dissociation constant for dimerization at 37 +/- 17 nM in the presence of ATP. The dissociation constant in the presence of ADP is 35 +/- 26 nM and in the presence of AMPPNP is 42 +/- 28 nM. The construct K366 (MW of ADP complex, 41,404) is a monomer (measured MW, 41,768 +/- 1219) at concentrations below 4 microM K366, with a sedimentation coefficient, s020,w, of 3.25 S. At higher concentrations, there is evidence for a weak association of K366 to a 1-2-4-8 model with a slight preference for octamer formation. The smallest construct, K341 (MW of ADP complex, 38,274), is a monomer (measured MW, 38,191 +/- 734) up to at least 10 microM total K341 concentration with a sedimentation coefficient, s020,w, of 2.9 S. Thus, the dimerization domain either is between amino acid residues 367 and 401 or is strongly affected by the removal of this region. Higher oligomers of K401 form by a mechanism involving dimers of dimers, and suggest that native kinesin may also undergo self-association. These results have important implications for the interpretation of ATP-dependent motility assays.
- Langford GM
- Actin- and microtubule-dependent organelle motors: interrelationships between the two motility systems.
- Curr Opin Cell Biol. 1995; 7: 82-8
- Display abstract
Membranous organelles move on both actin filaments and microtubules. Evidence for the presence of myosin and microtubule-based motors on the same organelle has raised the question of the interrelationship between the two motility systems. Functional studies of unconventional myosins are beginning to provide insights into this question and the important roles these motors play in membrane trafficking and organelle movement.
- Nakata T, Hirokawa N
- Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport.
- J Cell Biol. 1995; 131: 1039-53
- Display abstract
In the study of motor proteins, the molecular mechanism of mechanochemical coupling, as well as the cellular role of these proteins, is an important issue. To assess these questions we introduced cDNA of wild-type and site-directed mutant kinesin heavy chains into fibroblasts, and analyzed the behavior of the recombinant proteins and the mechanisms involved in organelle transports. Overexpression of wild-type kinesin significantly promoted elongation of cellular processes. Wild-type kinesin accumulated at the tips of the long processes, whereas the kinesin mutants, which contained either a T93N- or T93I mutation in the ATP-binding motif, tightly bound to microtubules in the center of the cells. These mutant kinesins could bind to microtubules in vitro, but could not dissociate from them even in the presence of ATP, and did not support microtubule motility in vitro, thereby indicating rigor-type mutations. Retrograde transport from the Golgi apparatus to the endoplasmic reticulum, as well as lysosome dispersion, was shown to be a microtubule-dependent, plus-end-directed movement. The latter was selectively blocked in the rigor-mutant cells, although the microtubule minus-end-directed motion of lysosomes was not affected. We found the point mutations that make kinesin motor in strong binding state with microtubules in vitro and showed that this mutant causes a dominant effect that selectively blocks anterograde lysosome membrane transports in vivo.
- Hirose K, Lockhart A, Cross RA, Amos LA
- Nucleotide-dependent angular change in kinesin motor domain bound to tubulin.
- Nature. 1995; 376: 277-9
- Display abstract
Kinesin is a 'motor' molecule, consisting of two head domains, an alpha-helical coiled coil rod, and a tail part that binds to its cargo. When expressed in a bacterial system, the head domain is functional, and can bind to microtubules with the stoichiometry of one head per tubulin dimer. Kinesin moves along microtubules by means of a cyclic process of nucleotide binding, hydrolysis and product release. We have used negative-stain electron microscopy and image analysis to study the structures of microtubules and tubulin sheets decorated with the motor domain (head) of kinesin in three states: in the presence of an unhydrolysable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP); without nucleotides; and with adenosine 5'-diphosphate (ADP). A single kinesin head bound to a microtubule has a pear-shaped structure, with the broader end towards the 'plus' end of the microtubule under all conditions; the reverse motor, ncd, is similarly oriented. Three-dimensional maps reveal that kinesin heads have a spike that is assumed to form the attachment to the tail of a complete kinesin molecule. This spike is perpendicular to the microtubule axis in the presence of ADP, but points towards the plus end (approximately 45 degrees) in the presence of AMP-PNP or absence of nucleotides. Our results provide direct evidence for a conformational change of the kinesin motor domain during the ATPase cycle.
- Kikkawa M, Ishikawa T, Wakabayashi T, Hirokawa N
- Three-dimensional structure of the kinesin head-microtubule complex.
- Nature. 1995; 376: 274-7
- Display abstract
Kinesin is a microtubule (MT)-associated 'motor' molecule fundamental to organelle transport. Recently, various kinesin superfamily members (KIFs) have also been identified and suggested as being responsible for the transport of specific organelles. Kinesin is a heterotetramer composed of two heavy chains and two light chains. The heavy chains form two globular heads, a rod and a fan-like tail completed by the light chains. The globular head, which is composed of approximately 340 amino-terminal residues of the heavy chain, includes both ATP-binding and MT-binding domains, and its recombinant protein also has these properties. To improve the understanding of the mechanism of force generation by an MT-based molecular motor, kinesin, we report here the three-dimensional structure of the complex of a recombinant kinesin head and MTs, as revealed by helical reconstruction from cryo-electron micrographs. A kinesin head is a globular teardrop-like structure binding to the ridge of one protofilament of MTs. We have determined the polarity of the structure of the complex of MTs and the kinesin head in relation to MT polarity.
- Fujiwara S, Kull FJ, Sablin EP, Stone DB, Mendelson RA
- The shapes of the motor domains of two oppositely directed microtubule motors, ncd and kinesin: a neutron scattering study.
- Biophys J. 1995; 69: 1563-8
- Display abstract
The shapes of the motor domains of kinesin and ncd, which move in opposite directions along microtubules, have been investigated. Using proteins expressed in Escherichia coli, it was found that at high salt (> 200 mM) Drosophila ncd motor domain (R335-K700) and human kinesin motor domain (M1-E349) were both sufficiently monomeric to allow an accurate determination of their radii of gyration (Rg) and their molecular weights. The measured Rg values of the ncd and kinesin motor domains in D2O were 2.06 +/- 0.06 and 2.05 +/- 0.04 nm, respectively, and the molecular weights were consistent with those computed from the amino acid compositions. Fitting of the scattering curves to approximately 3.5 nm resolution showed that the ncd and kinesin motor domains can be described adequately by triaxial ellipsoids having half-axes of 1.42 +/- 0.38, 2.24 +/- 0.44, and 3.65 +/- 0.22 nm, and half-axes of 1.52 +/- 0.23, 2.00 +/- 0.25, and 3.73 +/- 0.10 nm, respectively. Both motor domains are described adequately as somewhat flattened prolate ellipsoids with a maximum dimension of approximately 7.5 nm. Thus, it appears that the overall shapes of these motor domains are not the major determinants of the directionality of their movement along microtubules.
- Hackney DD
- Motor proteins. Polar explorations.
- Nature. 1995; 376: 215-6
- Schnitzer MJ, Block SM
- Statistical kinetics of processive enzymes.
- Cold Spring Harb Symp Quant Biol. 1995; 60: 793-802
- Lockhart A, Cross RA, McKillop DF
- ADP release is the rate-limiting step of the MT activated ATPase of non-claret disjunctional and kinesin.
- FEBS Lett. 1995; 368: 531-5
- Display abstract
The motor protein non-claret disjunctional (ncd) moves towards the minus ends of microtubules (MTs), whereas its close relative kinesin moves in the opposite direction towards the plus ends of MTs. The mechanisms of movement and directional reversal for these motor proteins are unknown. Here we report the rate constants for MT activated ADP release from a recombinant double-headed ncd protein, GST-MC5, and a recombinant double-headed kinesin protein, K delta 401, measured using the fluorescent nucleotide analogues methylanthranilyol ATP (mantATP) and mantADP. Comparison of the maximal MT activated mantADP release rates for these proteins with their maximal MT activated mantATP turnover rates indicates that ADP release is the rate-limiting step for ATP turnover for both ncd and kinesin. This data supports the view that directional reversal may result from structural rather than chemical kinetic differences in the way the motors interact with MTs.
- Vernon GG, Woolley DM
- The propagation of a zone of activation along groups of flagellar doublet microtubules.
- Exp Cell Res. 1995; 220: 482-94
- Display abstract
The complexity of the 9 + 2 flagellar axoneme has made it difficult to discover the mechanism of bend propagation. We have studied a simplified preparation of mechanically "opened-out" groups of doublet microtubules that adopt a helical, ribbon-like form. From the very long sperm of the quail, ribbons of doublets up to 130 microns long have been obtained. We reactivated them photolytically by releasing ATP from caged ATP, thus observing the reactivation from the beginning. The response to ATP was a reduction in the pitch and diameter of the helix, in what we refer to as an "active zone" (AZ). Ahead of the AZ was a short region of increase in helical pitch and diameter, the pre-AZ. We ascribe these two altered geometries to the development of tension between the doublets, actively (in the AZ) and passively (in the pre-AZ). The AZ/pre-AZ complex established itself at one end of a helix--almost certainly the proximal end--then it propagated toward the other end of the helix at a mean velocity of 15 microns s-1 (using 1 mM caged ATP), maintaining or increasing its length as it traveled. This is the same velocity as that for bend propagation on cylindrical axonemes detached from their basal structures. Successive propagations on the same helix were seen. Thus, active and inactive segments of the same doublet assembly can coexist, even though all parts are exposed to ATP. The motor response is seen to be a localized event that is transmitted metachronally. The propagation of activation is an intrinsic property of structures in the interdoublet gap and does not require constituents of the cytosol other than ATP and Mg2+. Since it occurs in helical ribbons (3 + 0, 4 + 0, etc.), the propagation of activity must be independent of central axonemal structures; furthermore, it cannot be dependent on the integrity of the 9 + 2 cylinder nor on any feedback from large-scale features of the waveform.
- Brady ST, Sperry AO
- Biochemical and functional diversity of microtubule motors in the nervous system.
- Curr Opin Neurobiol. 1995; 5: 551-8
- Display abstract
The fact that multiple microtubule-based motors exist in brain inevitably raises questions about their function. Transcripts for at least seven kinesin superfamily genes and even more dynein heavy chain genes have been detected in brain cDNA libraries. The challenge now is to match their gene products to specific functions in cells of the nervous system. Recent studies have attempted to establish a function for each microtubule motor by using recombinant protein and immunochemical approaches.
- Wordeman L, Mitchison TJ
- Identification and partial characterization of mitotic centromere-associated kinesin, a kinesin-related protein that associates with centromeres during mitosis.
- J Cell Biol. 1995; 128: 95-104
- Display abstract
Using antipeptide antibodies to conserved regions of the kinesin motor domain, we cloned a kinesin-related protein that associates with the centromere region of mitotic chromosomes. We call the protein MCAK, for mitotic centromere-associated kinesin. MCAK appears concentrated on centromeres at prophase and persists until telophase, after which time the localization disperses. It is found throughout the centromere region and between the kinetochore plates of isolated mitotic CHO chromosomes, in contrast to two other kinetochore-associated microtubule motors: cytoplasmic dynein and CENP-E (Yen et al., 1992), which are closer to the outer surface of the kinetochore plates. Sequence analysis shows MCAK to be a kinesin-related protein with the motor domain located in the center of the protein. It is 60-70% similar to kif2, a kinesin-related protein originally cloned from mouse brain with a centrally located motor domain (Aizawa et al., 1992). MCAK protein is present in interphase and mitotic CHO cells and is transcribed as a single 3.4-kb message.
- Lombillo VA, Nislow C, Yen TJ, Gelfand VI, McIntosh JR
- Antibodies to the kinesin motor domain and CENP-E inhibit microtubule depolymerization-dependent motion of chromosomes in vitro.
- J Cell Biol. 1995; 128: 107-15
- Display abstract
Chromosomes can move with the ends of depolymerizing microtubules (MTs) in vitro, even in the absence of nucleotide triphosphates (Coue, M., V. A. Lombillo, and J. R. McIntosh. 1991. J. Cell Biol. 112:1165-1175.) Here, we describe an immunological investigation of the proteins important for this form of motility. Affinity-purified polyclonal antibodies to kinesin exert a severe inhibitory effect on depolymerization-dependent chromosome motion. These antibodies predominantly recognize a polypeptide of M(r) approximately 250 kD on immunoblots of CHO chromosomes and stain kinetochores as well as some vesicles that are in the chromosome preparation. Antibodies to CENP-E, a kinetochore-associated kinesin-like protein, also recognize a 250-kD electrophoretic component, but they stain only the kinetochroe region of isolated chromosomes. Polyclonal antibodies that recognize specific domains of the CENP-E polypeptide affect MT disassembly-dependent chromosome motion in different ways; antibodies to the head or tail portions slow motility threefold, while those raised against the neck region stop motion completely. Analogous antibodies that block conventional, ATP-dependent motility of cytoplasmic dynein (Vaisberg, G., M. P. Koonce, and J. R. McIntosh. 1993. J. Cell Biol. 123:849-858) have no effect on disassembly-dependent chromosome motion, even though they bind to kinetochores. These observations suggest that CENP-E helps couple chromosomes to depolymerizing MTs. A similar coupling activity may allow spindle MTs to remain kinetochore-bound while their lengths change during both prometaphase and anaphase A.
- Kumar J, Yu H, Sheetz MP
- Kinectin, an essential anchor for kinesin-driven vesicle motility.
- Science. 1995; 267: 1834-7
- Display abstract
The membrane anchor for the molecular motor kinesin is a critical site involved in intracellular membrane trafficking. Monoclonal antibodies specific for the cytoplasmic surface of chick brain microsomes were used to define proteins involved in microtubule-dependent transport. One of four antibodies tested inhibited plus-end-directed vesicle motility by approximately 90 percent even as a monovalent Fab fragment and reduced kinesin binding to vesicles. This antibody bound to the cytoplasmic domain of kinectin, an integral membrane protein of the endoplasmic reticulum that binds to kinesin. Thus, kinectin acted as a membrane anchor protein for kinesin-driven vesicle motility.
- Marks DL, LaRusso NF, McNiven MA
- Isolation of the microtubule-vesicle motor kinesin from rat liver: selective inhibition by cholestatic bile acids.
- Gastroenterology. 1995; 108: 824-33
- Display abstract
BACKGROUND/AIMS: Vesicular transport is supported by microtubule-based, force-transducing adenosine triphosphatases (ATPases), such as kinesin, a ubiquitous motor enzyme that has been well studied in neuronal tissues. Although vesicular transport is important for hepatocellular secretory and clearance activities, the role of kinesin in liver function is poorly understood. Furthermore, the effects of bile acids on kinesin are unknown. METHODS: Kinesin was purified from rat liver cytosol by conventional chromatography and microtubule affinity binding and was characterized by immunoblotting with domain-specific kinesin antibodies and amino acid sequencing of tryptic fragments. Kinesin activity was measured with and without bile acids using an in vitro motility assay and ATPase assays. RESULTS: Immunoblot analysis and partial amino acid sequencing of purified kinesin showed that the sequence at the heavy chain of hepatic kinesin is nearly identical to that of brain kinesin. Purified kinesin transported microtubules in vitro with a velocity of approximately 0.5 microns/s; this activity was significantly inhibited by 0.5-1 mmol/L taurochenodeoxycholate but not by tauroursodeoxycholate. At a dose of 1 mmol/L, chenodeoxycholate conjugates, but not ursodeoxycholate or cholate conjugates, directly inhibited the ATPase activities of kinesin and another microtubule motor, cytoplasmic dynein. CONCLUSIONS: Cholestatic concentrations of chenodeoxycholate conjugates directly inhibit the activity of microtubule motors, suggesting a possible mechanism for impairment of vesicular transport in cholestasis.
- Egelman EH
- Motile systems: Tubulin-based motility races ahead.
- Curr Biol. 1995; 5: 1354-6
- Avsiuk AV, Minin AA, Gioeva FK
- [Kinesin, associated with intermediate vimentin filaments, contains a specific light chain]
- Dokl Akad Nauk. 1995; 345: 119-22
- Hirose K, Fan J, Amos LA
- Re-examination of the polarity of microtubules and sheets decorated with kinesin motor domain.
- J Mol Biol. 1995; 251: 329-33
- Display abstract
Electron microscope images of microtubules and tubulin sheets decorated with kinesin head domains have shown the main mass of the kinesin head domain to be superimposed on one subunit of each tubulin dimer. We have polymerized brain tubulin extensions on to the ends of flagellar axonemes under varied conditions, in order to check the polarity of the tubulin-kinesin head complex. Since the polarity of axonemes incubated with normal brain tubulin may be ambiguous, we also tried 50% N-ethylmaleimide-treated tubulin which specifically blocks minus ends. Our conclusion, which conflicts with recently published results, is that the main mass of the kinesin head is associated with the tubulin subunit closer to the plus end of a microtubule.
- Ma YZ, Taylor EW
- Kinetic mechanism of kinesin motor domain.
- Biochemistry. 1995; 34: 13233-41
- Display abstract
The kinetic mechanism of the human kinesin ATPase motor domain K379, expressed in Escherichia coli, was determined by transient and steady-state kinetic studies. The steps in nucleotide binding were measured using the fluorescent substrate analogues, methylanthraniloyl ATP (mant-ATP) and mant-ADP. Both nucleotides gave a two-step fluorescence signal, an increase followed by a decrease, which indicates that two isomerizations are induced by nucleotide binding. The ATPase mechanism is fitted by a six-step reaction: [formula: see text] where, T, D, and P refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively; K(T) and K(D) are states in rapid equilibrium with the free nucleotide. A set of kinetic constants for 20 degrees C 50 mM NaCl is K1 = 2 x 10(4) M-1, k2 = 200 s-1, k3 = 9 s-1, k5 = 0.01 s-1, and K6 = 2 x 10(-5) M. Values of K1 and K6 are estimates for mant-ATP and mant-ADP, respectively. ADP dissociation is the rate-limiting step. The rate constant for a decrease in fluorescence for the transitions from the high fluorescence K.T state to the low fluorescence K.D state is equal to k3, the rate constant of the hydrolysis step measured by quench flow experiments. The decrease could occur in step 3 or step 4 if k4 > k3.(ABSTRACT TRUNCATED AT 250 WORDS)
- Hyman AA
- Microtubule dynamics. Kinetochores get a grip.
- Curr Biol. 1995; 5: 483-4
- Display abstract
Analysis of the interactions between purified motor proteins or isolated chromosomes and shrinking microtubules has shed light on the mechanism of chromosome segregation at mitosis.
- Julicher F, Prost J
- Cooperative molecular motors.
- PHYSICAL REVIEW LETTERS. 1995; 75: 2618-2621
- Young EC, Berliner E, Mahtani HK, Perez-Ramirez B, Gelles J
- Subunit interactions in dimeric kinesin heavy chain derivatives that lack the kinesin rod.
- J Biol Chem. 1995; 270: 3926-31
- Display abstract
The N-terminal residues of the two heavy chains of the motor enzyme kinesin form two globular "heads"; the heads are attached to a "rod" domain which is a two-stranded alpha-helical coiled-coil. Interaction between the heads is thought to be important to kinesin function. The rod may not be necessary for head-head interactions because a heavy chain N-terminal fragment containing only residues from the head and adjacent region forms dimers (Huang, T.-G., Suhan, J., and Hackney, D. D. (1994) J. Biol. Chem. 269, 16502-16507). However, the nature and stability of the subunit-subunit interactions in such derivatives are unclear. To examine the physical properties of heavy chain interaction in and near the head domains, we characterized the self-association behavior of two dimeric kinesin derivatives predicted (Lupas, A., van Dyke, M., and Stock, J. (1991) Science 252, 1162-1164) to lack the rod. Derivative K448-BIO contains the 448 N-terminal residues of Drosophila kinesin heavy chain fused at the C terminus to a 2-residue linker and a C-terminal fragment from Escherichia coli biotin carboxyl carrier protein; derivative K448-L is the same except that it lacks the biotin carboxyl carrier protein fragment. Both derivatives expressed in insect cells display microtubule-stimulated ATPase activity; K448-BIO also displays microtubule motility. Equilibrium sedimentation and gel filtration indicate that purified K448-BIO and K448-L at 0.02-0.4 mg/ml form homogeneous solutions of homodimers with no detectable formation of monomers or higher order oligomers. Derivative self-association is non-covalent but extremely stable with an association constant > or = 2 x 10(8) M-1. Stable subunit-subunit association induced by structures in and near the kinesin heads may be necessary for full mechanochemical function.
- Steinberg G, Schliwa M
- The Neurospora organelle motor: a distant relative of conventional kinesin with unconventional properties.
- Mol Biol Cell. 1995; 6: 1605-18
- Display abstract
The "conventional" kinesins comprise a conserved family of molecular motors for organelle transport that have been identified in various animal species. Organelle motors from other phyla have not yet been analyzed at the molecular level. Here we report the identification, biochemical and immunological characterization, and molecular cloning of a cytoplasmic motor in a "lower" eukaryote, the Ascomycete fungus Neurospora crassa. This motor, termed Nkin (for Neurospora kinesin), exhibits several unique structural and functional features, including a high rate of microtubule transport, a lack of copurifying light chains, a second P-loop motif, and an overall sequence organization reminiscent of a kinesin-like protein. However, a greater than average sequence homology in the motor domain and the presence of a highly conserved region in the C-terminus identify Nkin as a distant relative of the family of conventional kinesins. A molecular phylogenetic analysis suggests Nkin to have diverged early in the evolution of this family of motors. The discovery of Nkin may help identify domains important for specific biological functions in conventional kinesins.
- Lombillo VA, Stewart RJ, McIntosh JR
- Minus-end-directed motion of kinesin-coated microspheres driven by microtubule depolymerization.
- Nature. 1995; 373: 161-4
- Display abstract
Dynamic changes in microtubule (MT) length have long been thought to contribute to intracellular motility. Both the polymerization and depolymerization of tubulin have been shown to do work in vitro, but the biochemical complexity of objects moved, such as chromosomes, has complicated the identification of proteins that couple MT dynamics with motility. Work with MTs grown from and tethered to pellicles of lysed Tetrahymena has shown that disassembly-dependent movement of chromosomes in vitro can be inhibited with antibodies against the motor domain of kinesin. To study proteins that can function in disassembly-dependent motion, we have refined this motility assay, replacing chromosomes with protein-coated latex microspheres. We report here the ability of several enzymes, including kinesin, to support in vitro motility of latex microspheres on disassembling MTs (Fig. 1a). The polarity of kinesin's motor activity can be reversed by MT disassembly and interactions between a motor and a MT end can either slow or speed the rate of tubulin depolymerization.
- Yamazaki H, Nakata T, Okada Y, Hirokawa N
- KIF3A/B: a heterodimeric kinesin superfamily protein that works as a microtubule plus end-directed motor for membrane organelle transport.
- J Cell Biol. 1995; 130: 1387-99
- Display abstract
We cloned a new member of the murine brain kinesin superfamily, KIF3B, and found that its amino acid sequence is highly homologous but not identical to KIF3A, which we previously cloned and named KIF3 (47% identical). KIF3B is localized in various organ tissues and developing neurons of mice and accumulates with anterogradely moving membranous organelles after ligation of nerve axons. Immunoprecipitation assay of the brain revealed that KIF3B forms a complex with KIF3A and three other high molecular weight (approximately 100 kD)-associated polypeptides, called the kinesin superfamily-associated protein 3 (KAP3). In vitro reconstruction using baculovirus expression systems showed that KIF3A and KIF3B directly bind with each other in the absence of KAP3. The recombinant KIF3A/B complex (approximately 50-nm rod with two globular heads and a single globular tail) demonstrated plus end-directed microtubule sliding activity in vitro. In addition, we showed that KIF3B itself has motor activity in vitro, by making a complex of wild-type KIF3B and a chimeric motor protein (KIF3B head and KIF3A rod tail). Subcellular fractionation of mouse brain homogenates showed a considerable amount of the native KIF3 complex to be associated with membrane fractions other than synaptic vesicles. Immunoprecipitation by anti-KIF3B antibody-conjugated beads and its electron microscopic study also revealed that KIF3 is associated with membranous organelles. Moreover, we found that the composition of KAP3 is different in the brain and testis. Our findings suggest that KIF3B forms a heterodimer with KIF3A and functions as a new microtubule-based anterograde translocator for membranous organelles, and that KAP3 may determine functional diversity of the KIF3 complex in various kinds of cells in vivo.
- Ferro KL, Collins CA
- Microtubule-independent phospholipid stimulation of cytoplasmic dynein ATPase activity.
- J Biol Chem. 1995; 270: 4492-6
- Display abstract
In this study we report that phospholipid vesicles activate ATP hydrolysis by cytoplasmic dynein but not kinesin, consistent with reported differences in the organelle/vesicle binding of these motor proteins. Dynein activation by phospholipids was comparable with that seen in the presence of microtubules but was not sensitive to moderate salt concentrations and was independent of the net charge of the phospholipid, suggesting that the means of interaction between dynein and the lipid vesicle was not strictly ionic in nature. Based on this result, previous data that show that the interaction between dynein and vesicles is not ATP sensitive, and the concentration dependence observed for lipid activation of cytoplasmic dynein, it is likely that the binding interaction between dynein and liposomes is a stable one. In contrast to a previous report, microtubules increased the hydrolysis rate of all naturally occurring nucleotides tested, whereas only ATPase activity was stimulated by phospholipids. As ATP is the physiologically relevant substrate and is the only nucleotide to promote motility, the activation of only the ATPase by phospholipids may represent a means of discriminating between coupled and uncoupled nucleotide hydrolysis in vitro.
- Peskin CS, Oster GF
- Force production by depolymerizing microtubules: load-velocity curves and run-pause statistics.
- Biophys J. 1995; 69: 2268-76
- Display abstract
Experiments indicate that depolymerization of microtubules generates sufficient force to produce the minus-end-directed transport of chromosomes during mitosis (Koshland et al., 1988). In vitro, analogous transport of kinesin-coated microspheres exhibits a paradoxical effect. Minus-end-directed transport of the microspheres driven by depolymerization is enhanced by the presence of ATP, which fuels the motor action of kinesin driving the microspheres in the opposite direction, toward the plus end of the microtubule. Here we present a mathematical model to explain this behavior. We postulate that a microsphere at the plus end of the microtubule facilitates depolymerization and hence enhances minus-end-directed transport. The force-velocity curve of the model is derived; it has the peculiar feature that velocity is maximal at some positive load (opposing the motion) rather than at zero load. The model is used to simulate the stochastic process of microsphere-facilitated depolymerization-driven transport. Simulated trajectories at low load show distinctive runs and pauses, the statistics of which are calculated from the model. The statistics of the process provide sufficient information to determine all of the model's parameters.
- Lockhart A, Crevel IM, Cross RA
- Kinesin and ncd bind through a single head to microtubules and compete for a shared MT binding site.
- J Mol Biol. 1995; 249: 763-71
- Display abstract
Kinesin and non claret disjunctional are closely related molecular motors that move in opposite directions along microtubules. We have used recombinant single-headed and double-headed constructs of both rat kinesin heavy chain and non claret disjunctional to investigate the interactions of these motor proteins with microtubules. At saturation the stoichiometry of binding for non claret disjunctional and kinesin to microtubules is one molecule (single or double-headed) per tubulin heterodimer. In the absence of added nucleotide, addition of increasing amounts of one motor results in the competitive displacement of the other motor from the microtubules. This effect is apparent also in the presence of the nucleotide analogue 5'-adenylimidodiphosphate, which tightens the binding of both kinesin and non claret disjunctional. Competition for binding sites occurs also under conditions of steady-state ATP turnover. We conclude that despite their opposite directionality, kinesin and non claret disjunctional compete for overlapping binding sites on the MT surface. Since the binding of the second head of a double-headed motor is sterically blocked, the data imply also that both kinesin and non claret disjunctional may translocate via a processive (alternating heads) mechanism with a minimum step size of approximately 8 nm.
- Meyhofer E, Howard J
- The force generated by a single kinesin molecule against an elastic load.
- Proc Natl Acad Sci U S A. 1995; 92: 574-8
- Display abstract
To probe the mechanism by which the motor protein kinesin moves along microtubules, we have developed a highly sensitive technique for measuring the force exerted by a single motor molecule. In this technique, one end of a microtubule is attached to the tip of a flexible glass fiber of calibrated stiffness. The other end of the microtubule makes contact with a surface sparsely coated with kinesin. By imaging the tip of the glass fiber on a photodiode detector, displacement of the microtubule by kinesin through as little as 1 nm can be detected and forces as small as 1 pN resolved. Using this force-fiber apparatus we have characterized the mechanical output of this molecular motor. The speed at which a molecule of kinesin moved along the surface of a microtubule decreased linearly as the elastic force was increased. The force required to stop a single kinesin molecule was 5.4 +/- 1.0 pN (mean +/- SD; n = 16), independent of the stiffness of the fiber, the damping from the fluid, and whether the ATP concentration was high or low.
- Elluru RG, Bloom GS, Brady ST
- Fast axonal transport of kinesin in the rat visual system: functionality of kinesin heavy chain isoforms.
- Mol Biol Cell. 1995; 6: 21-40
- Display abstract
The mechanochemical ATPase kinesin is thought to move membrane-bounded organelles along microtubules in fast axonal transport. However, fast transport includes several classes of organelles moving at rates that differ by an order of magnitude. Further, the fact that cytoplasmic forms of kinesin exist suggests that kinesins might move cytoplasmic structures such as the cytoskeleton. To define cellular roles for kinesin, the axonal transport of kinesin was characterized. Retinal proteins were pulse-labeled, and movement of radiolabeled kinesin through optic nerve and tract into the terminals was monitored by immunoprecipitation. Heavy and light chains of kinesin appeared in nerve and tract at times consistent with fast transport. Little or no kinesin moved with slow axonal transport indicating that effectively all axonal kinesin is associated with membranous organelles. Both kinesin heavy chain molecular weight variants of 130,000 and 124,000 M(r) (KHC-A and KHC-B) moved in fast anterograde transport, but KHC-A moved at 5-6 times the rate of KHC-B. KHC-A cotransported with the synaptic vesicle marker synaptophysin, while a portion of KHC-B cotransported with the mitochondrial marker hexokinase. These results suggest that KHC-A is enriched on small tubulovesicular structures like synaptic vesicles and that at least one form of KHC-B is predominantly on mitochondria. Biochemical specialization may target kinesins to appropriate organelles and facilitate differential regulation of transport.
- Ma YZ, Taylor EW
- Mechanism of microtubule kinesin ATPase.
- Biochemistry. 1995; 34: 13242-51
- Display abstract
A six-step mechanism is derived for the activation of kinesin K379 ATPase by microtubules. The data are fitted by the kinetic scheme [Formula see text] where T, D, and P refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively; MtK refers to the complex of a K379 unit with the microtubule binding site. The initial binding and release steps, 1 and 6, are treated as rapid equilibria: k2 = 200 s-1, k3 = 100 s-1, k5 = 35-40 s-1, maximum steady-state rate = 25 s-1 (50 mM NaCl, 20 degrees C). k2 was obtained from the maximum rate of fluorescence enhancement with mant-ATP as substrate, k3 was obtained from the hydrolysis transient phase for ATP or mant-ATP, and k5 was obtained from the rate of decrease in fluorescence of mant-ADP in the reaction [Formula see text]. A large excess of ATP was present with the Mt to block rebinding of mant-ADP. The rate was measured as a function of microtubule concentration and extrapolated to give the maximum rate k5. The same method was used to obtain k5 for ADP by mixing K.ADP with microtubules plus excess mant-ATP. The enhancement of fluorescence for the binding of mant-ATP is followed by a decrease in fluorescence with a rate constant of 35-40 s-1. Since the decrease must occur after hydrolysis, it may be correlated with a step or steps leading to the low fluorescence MtK.D state. In the kinetic scheme, steps 4 and 5 both contribute to determining the maximum turnover rate. At higher ionic strengths or lower protein concentrations, the MtK complex is dissociated by ATP. The maximum rate is 12 +/- 2 s-1 in 50 mM NaCl; consequently, hydrolysis occurs before dissociation. The dissociation constant of MtK in the presence of ADP is twice as large as the dissociation constant in the presence of ATP and four times larger than the KM for microtubule activation. The proposed kinetic scheme, which treats the K379 units of a dimer as independent, provides a satisfactory description of the transient and steady-state properties of the system with the possible exception of results at very low substrate concentrations.
- Wade RH, Horowitz R, Milligan RA
- Toward understanding the structure and interactions of microtubules and motor proteins.
- Proteins. 1995; 23: 502-9
- Display abstract
To obtain an overall three-dimensional picture of the interaction between microtubules and the motor proteins of the kinesin family it will be necessary to take account of both atomic resolution structures obtained by X-ray crystallography and medium resolution reconstructions obtained by electron cryomicroscopy. We examine the problems associated with obtaining the required structural information from electron micrographs of vitreous ice-embedded microtubules decorated with motor domains. We find that the minus-end directed motor, ncd, decorates microtubules with an 80 A periodicity as for kinesin. Our theoretical analysis and experiments with ncd illustrate the difficulty in determining unambiguously the surface lattice organization by diffraction analysis of micrographs. 3D reconstructions of decorated microtubules are required to accurately locate the motor domains. Helical diffraction theory is not usually applicable because microtubules are cylindrical structures that rarely have complete helical symmetry. We propose using a back-projection method based on the long pitch helices formed by individual protofilaments. Model reconstructions show that this approach is feasible.
- Gilbert SP, Webb MR, Brune M, Johnson KA
- Pathway of processive ATP hydrolysis by kinesin.
- Nature. 1995; 373: 671-6
- Display abstract
Direct measurement of the kinetics of kinesin dissociation from microtubules, the release of phosphate and ADP from kinesin, and rebinding of kinesin to the microtubule have defined the mechanism for the kinesin ATPase cycle. The processivity of ATP hydrolysis is ten molecules per site at low salt concentration but is reduced to one ATP per site at higher salt concentration. Kinesin dissociates from the microtubule after ATP hydrolysis. This step is rate-limiting. The subsequent rebinding of kinesin-ADP to the microtubule is fast, so kinesin spends only a small fraction of its duty cycle in the dissociated state. These results provide an explanation for the motility differences between skeletal myosin and kinesin.
- Schnapp BJ
- Molecular motors. Two heads are better than one.
- Nature. 1995; 373: 655-6
- Shimizu T, Sablin E, Vale RD, Fletterick R, Pechatnikova E, Taylor EW
- Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain.
- Biochemistry. 1995; 34: 13259-66
- Display abstract
ncd is a kinesin-related motor protein from Drosophila that moves in the opposite direction along microtubules to kinesin. To learn more about the ncd mechanism, ncd motor domain (R335-K700) was expressed in Escherichia coli and its enzymatic characteristics were studied. The ncd motor domain was purified from the cell lysate by S-Sepharose chromatography, and trace amounts of contaminants were removed by passing through a MonoQ column. The yield was 20 mg from a 500 mL culture of E. coli. The purified ncd motor domain exhibited an unusual UV spectrum with a broad peak around 272-275 nm, which was at least partly due to the bound nucleotide. Upon incubation with radioactive ATP, 3H at adenine but not 32P at gamma-phosphate was retained by the protein on gel filtration, indicating it bound ADP but not ATP. Thus, like kinesin, nucleotide binding to the ncd motor domain is tight, although there is an equilibrium between the protein and free nucleotide. We also used a fluorescent ATP analogue, mantATP, for the kinetic study of ncd motor domain. MantATP was turned over by ncd motor domain slowly in the absence of microtubules, but microtubules activated the turnover to a similar extent to that of ATP. Upon incubation with ncd motor domain, the fluorescent intensity of mantATP increased at 0.005 s-1, which is likely to reflect the release of endogenous ADP and incorporation of mantATP into the protein. The fluorescence intensity of the ncd motor domain having bound mantADP, likewise, decreased upon mixing with ATP, representing the mantADP release.(ABSTRACT TRUNCATED AT 250 WORDS)
- Berliner E, Young EC, Anderson K, Mahtani HK, Gelles J
- Failure of a single-headed kinesin to track parallel to microtubule protofilaments.
- Nature. 1995; 373: 718-21
- Display abstract
Kinesin, a two-headed motor enzyme molecule, hydrolyses ATP to direct organelle transport along microtubules. As it moves along a microtubule, kinesin remains associated with, or 'tracks', microtubule protofilaments. We have prepared truncated kinesin derivatives that contain either two mechanochemical head domains or only a single head. Unlike intact kinesin and the two-headed derivatives, the one-headed enzyme frequently fails to track protofilaments, suggesting that it detaches from microtubules during movement. In this way, the one-headed kinesin derivative is similar to the motor enzyme myosin, which frequently detaches from the actin filament during movement. For myosin (which has two heads), the consequence of this detachment is that single molecules do not appear to drive continuous movement along the filament. Our observations suggest that the ability of single two-headed kinesin molecules to drive continuous movement results from a 'hand-over-hand' mechanism in which one head remains bound to the microtubule while the other detaches and moves forwards.
- Kozminski KG, Beech PL, Rosenbaum JL
- The Chlamydomonas kinesin-like protein FLA10 is involved in motility associated with the flagellar membrane.
- J Cell Biol. 1995; 131: 1517-27
- Display abstract
The Chlamydomonas FLA10 gene was shown to encode a flagellar kinesin-like protein (Walther, Z., M. Vashishtha, and J.L. Hall. 1994. J. Cell Biol. 126:175-188). By using a temperature-sensitive allele of FLA10, we have determined that the FLA10 protein is necessary for both the bidirectional movement of polystyrene beads on the flagellar membrane and intraflagellar transport (IFT), the bidirectional movement of granule-like particles beneath the flagellar membrane (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. (USA). 90:5519-5523). In addition, we have correlated the presence and position of the IFT particles visualized by light microscopy with that of the electron dense complexes (rafts) observed beneath the flagellar membrane by electron microscopy. A role for FLA10 in submembranous or flagellar surface motility is also strongly supported by the immunolocalization of FLA10 to the region between the axonemal outer doublet microtubules and the flagellar membrane.
- Noda Y, Sato-Yoshitake R, Kondo S, Nangaku M, Hirokawa N
- KIF2 is a new microtubule-based anterograde motor that transports membranous organelles distinct from those carried by kinesin heavy chain or KIF3A/B.
- J Cell Biol. 1995; 129: 157-67
- Display abstract
Kinesin is known as a representative cytoskeletal motor protein that is engaged in cell division and axonal transport. In addition to the mutant assay, recent advances using the PCR cloning technique have elucidated the existence of many kinds of kinesin-related proteins in yeast, Drosophila, and mice. We previously cloned five different members of kinesin superfamily proteins (KIFs) in mouse brain (Aizawa, H., Y. Sekine, R. Takemura, Z. Zhang, M. Nangaku, and N. Hirokawa. 1992. J. Cell Biol. 119:1287-1296) and demonstrated that one of them, KIF3A, is an anterograde motor (Kondo, S., R. Sato-Yashitake, Y. Noda, H. Aizawa, T. Nakata, Y. Matsuura, and N. Hirokawa. J. Cell Biol. 1994. 125:1095-1107). We have now characterized another axonal transport motor, KIF2. Different from other KIFs, KIF2 is a central type motor, since its motor domain is located in the center of the molecule. Recombinant KIF2 exists as a dimer with a bigger head and plus-end directionally moves microtubules at a velocity of 0.47 +/- 0.11 microns/s, which is two thirds that of kinesin's. Immunocytological examination showed that native KIF2 is abundant in developing axons and that it accumulates in the proximal region of the ligated nerves after a 20-h ligation. Soluble KIF2 exists without a light chain, and KIF2's associated-vesicles, immunoprecipitated by anti-KIF2 antibody, are different from those carried by existing motors such as kinesin and KIF3A. They are also distinct from synaptic vesicles, although KIF2 is accumulated in so-called synaptic vesicle fractions and embryonal growth cone particles. Our results strongly suggest that KIF2 functions as a new anterograde motor, being specialized for a particular group of membranous organelles involved in fast axonal transport.
- Hackney DD
- Highly processive microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains.
- Nature. 1995; 377: 448-50
- Display abstract
Studies of immobilized kinesin have shown that a single dimeric molecule can maintain contact with and drive sliding of a microtubule. In solution, however, native kinesin binds microtubules too weakly and hydrolyses ATP too slowly to produce the high sliding velocities seen in motility assay. This apparent inhibition in solution appears to be caused by the binding of kinesin's tail domains to its motor (head) domains in a folded conformation. DKH392, a construct containing two heads but no tails, has been shown to display both tight binding to microtubules and high ATPase rates. Furthermore, it retains one molecule of ADP per dimer when bound to microtubules, which could facilitate a 'hand-over-hand' mechanism for processive motion. Here we show that DKH392 hydrolyses more than 100 ATP molecules per diffusional encounter with a microtubule, even in the high-salt conditions encountered physiologically. This provides direct evidence that kinesin's activity is highly processive, with the motor remaining attached to a microtubule through many cycles of ATP hydrolysis.
- Ray S, Wolf SG, Howard J, Downing KH
- Kinesin does not support the motility of zinc-macrotubes.
- Cell Motil Cytoskeleton. 1995; 30: 146-52
- Display abstract
Moving along a microtubule, kinesin follows a course parallel to the protofilaments; but it is not known whether kinesin binds exclusively on a single protofilament. The presence of zinc during tubulin polymerization induces sheets where neighboring protofilaments are antiparallel. If kinesin could support the motility of these zinc-sheets, then the binding site for a kinesin molecule would be limited to a single protofilament. Kamimura and Mandelkow [1992: J. Cell Biol. 118:865-75] reported that kinesin moves along zinc-sheets. We found that zinc-sheets grown under their conditions often had a microtubule-like structure along one edge. We confirmed the possibility that the motility observed by Kamimura and Mandelkow [1992: J. Cell Biol. 118:865-75] is attributed to the microtubule-like structure rather than the zinc-sheet. To resolve the question of whether kinesin can recognize an antiparallel protofilament lattice, we investigated the kinesin-mediated motility of zinc-macrotubes. At higher free zinc concentrations, zinc-sheets roll up as macrotubes, free of edges. In the presence of 10 microM taxol and 100 nM free Zn2+ at pH 6.8, the samples were shown by electron microscopy to contain only macrotubes. Under these buffer conditions, kinesin could bind strongly to axonemal doublets in the presence of AMP-PNP, and generate motility in the presence of ATP, but kinesin did not bind to nor move the macrotubes. This shows that kinesin cannot bind efficiently to nor move on the anti-parallel lattice; it is possible (though not necessary) that the groove between two parallel protofilaments is required for kinesin's motility.
- Cross RA
- On the hand-over-hand footsteps of kinesin heads.
- J Muscle Res Cell Motil. 1995; 16: 91-4
- Jiang MY, Sheetz MP
- Cargo-activated ATPase activity of kinesin.
- Biophys J. 1995; 68: 283284285-283284285
- Display abstract
We have measured the ATPase activity of squid optic lobe kinesin bound to polystyrene beads in the presence of microtubules. We find that there is a substantial increase (> 10-fold) in the microtubule-activated ATPase activity for bead-bound kinesin over free kinesin. We tentatively attribute such cargo-activated ATPase activity to the presence of a self-inhibited form of kinesin in solution, which becomes activated when bound to a bead in the presence of alpha-casein. Further experiments are underway to unravel this phenomenon and, in addition, to associate the traveling distance of beads with the observed ATPase rate to determine the average number of ATP consumed per kinesin-bead per micron of travel along microtubule.
- Sekine Y et al.
- A novel microtubule-based motor protein (KIF4) for organelle transports, whose expression is regulated developmentally.
- J Cell Biol. 1994; 127: 187-201
- Display abstract
To understand the mechanisms of transport for organelles in the axon, we isolated and sequenced the cDNA encoding KIF4 from murine brain, and characterized the molecule biochemically and immunocytochemically. Complete amino acid sequence analysis of KIF4 and ultrastructural studies of KIF4 molecules expressed in Sf9 cells revealed that the protein contains 1,231 amino acid residues (M(r) 139,550) and that the molecule (116-nm rod with globular heads and tail) consists of three domains: an NH2-terminal globular motor domain, a central alpha-helical stalk domain and a COOH-terminal tail domain. KIF4 protein has the property of nucleotide-dependent binding to microtubules, microtubule-activated ATPase activity, and microtubule plus-end-directed motility. Northern blot analysis and in situ hybridization demonstrated that KIF4 is strongly expressed in juvenile tissues including differentiated young neurons, while its expression is decreased considerably in adult mice except in spleen. Immunocytochemical studies revealed that KIF4 colocalized with membranous organelles both in growth cones of differentiated neurons and in the cytoplasm of cultured fibroblasts. During mitotic phase of cell cycle, KIF4 appears to colocalize with membranous organelles in the mitotic spindle. Hence we conclude that KIF4 is a novel microtubule-associated anterograde motor protein for membranous organelles, the expression of which is regulated developmentally.
- Magnasco MO
- Molecular combustion motors.
- PHYSICAL REVIEW LETTERS. 1994; 72: 2656-2659
- Kondo S et al.
- KIF3A is a new microtubule-based anterograde motor in the nerve axon.
- J Cell Biol. 1994; 125: 1095-107
- Display abstract
Neurons are highly polarized cells composed of dendrites, cell bodies, and long axons. Because of the lack of protein synthesis machinery in axons, materials required in axons and synapses have to be transported down the axons after synthesis in the cell body. Fast anterograde transport conveys different kinds of membranous organelles such as mitochondria and precursors of synaptic vesicles and axonal membranes, while organelles such as endosomes and autophagic prelysosomal organelles are conveyed retrogradely. Although kinesin and dynein have been identified as good candidates for microtubule-based anterograde and retrograde transporters, respectively, the existence of other motors for performing these complex axonal transports seems quite likely. Here we characterized a new member of the kinesin super-family, KIF3A (50-nm rod with globular head and tail), and found that it is localized in neurons, associated with membrane organelle fractions, and accumulates with anterogradely moving membrane organelles after ligation of peripheral nerves. Furthermore, native KIF3A (a complex of 80/85 KIF3A heavy chain and a 95-kD polypeptide) revealed microtubule gliding activity and baculovirus-expressed KIF3A heavy chain demonstrated microtubule plus end-directed (anterograde) motility in vitro. These findings strongly suggest that KIF3A is a new motor protein for the anterograde fast axonal transport.
- Hackney DD
- The rate-limiting step in microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains occurs while bound to the microtubule.
- J Biol Chem. 1994; 269: 16508-11
- Display abstract
DKH392 is a construct which contains the first 392 amino acids of the alpha-subunit of Drosophila kinesin and is dimeric in solution (Huang, T.-G., Suhan, J., and Hackney, D. D. (1994) J. Biol. Chem. 269, 16502-16507). The ATPase rate of DKH392 was 0.005 s-1 in the absence of MTs. One ADP bound tightly to each subunit and the release of this ADP was the rate-limiting step in ATP hydrolysis. Microtubules accelerated the rate of ADP release and increased the rate of steady state ATP hydrolysis by almost 10,000-fold (kcat = approximately 45 s-1). The KMT0.5,ATPase value for saturation of the stimulation of the ATPase reaction by microtubules was 50 nM at 8 nM DKH392, but decreased at lower concentrations of DKH392. Physical binding of DKH392 to microtubules in the presence of 1 mM MgATP paralleled saturation of the stimulation of the ATPase activity by microtubules indicating that the rate-limiting step in microtubule-stimulated ATP hydrolysis occurs while DKH392 is bound to the microtubule. These results suggest that microtubule-stimulated ATP hydrolysis by DKH392 may be processive with the hydrolysis of multiple ATP molecules during each diffusional encounter of DKH392 with a microtubule.
- Bloom GS, Endow SA
- Motor proteins. 1: kinesins.
- Protein Profile. 1994; 1: 1059-116
- Sakai H
- [Trends in the study on microtubules]
- Tanpakushitsu Kakusan Koso. 1994; 39: 10-20
- Houliston E, Le Guellec R, Kress M, Philippe M, Le Guellec K
- The kinesin-related protein Eg5 associates with both interphase and spindle microtubules during Xenopus early development.
- Dev Biol. 1994; 164: 147-59
- Display abstract
We have examined the changing abundance and distribution of the kinesin-related protein Eg5 during oogenesis and early development in Xenopus laevis. Antibodies raised against proteins synthesized from parts of a novel Eg5 gene expressed in eggs were used for Western blotting and immunofluorescence. Eg5 protein was highly enriched in oocytes and eggs compared with other adult tissues. It accumulated during the latter stages of oogenesis and increased a further threefold during oocyte maturation. Its level then gradually declined during early development. In oocytes, eggs, and early embryos, Eg5 protein could be detected throughout the cytoplasm and in subcortical aggregates. Eg5 staining was found concentrated in meiotic and mitotic spindles, mainly toward the poles. Some Eg5 staining colocalized with microtubules in interphase cells, including the aligned subcortical microtubules in fertilized eggs implicated in the cortical rotation that specifies the dorsoventral axis. Interphase association of Eg5 with microtubules during early development was confirmed by copelleting the protein with microtubules from egg homogenates. In tadpoles and tissue culture cells, Eg5 colocalized with spindle microtubules throughout mitosis but not with interphase microtubules. These results suggest that the Eg5 microtubule motor may function in meiosis, mitosis, and interphase during early development.
- Sutoh K
- [Protein motors; structural basis of their functions]
- Tanpakushitsu Kakusan Koso. 1994; 39: 1190-201
- Jellali A et al.
- Structural and biochemical properties of kinesin heavy chain associated with rat brain mitochondria.
- Cell Motil Cytoskeleton. 1994; 28: 79-93
- Display abstract
Kinesin, a mechanochemical enzyme that translocates membranous organelles, was initially identified and purified from soluble extracts from vertebrate brains. However, immunocytochemical and morphological approaches have demonstrated that kinesin could be associated to intracellular membranous organelles. We used an antibody raised against the head portion of the Drosophila kinesin heavy chain to reveal the presence of this protein in membranous organelles from rat brain. By using differential centrifugation and immunoblotting we observed a 116 kDa protein that crossreacts with this antibody in microsomes, synaptic vesicles, and mitochondria. This protein could be extracted from mitochondria with low salt concentrations or ATP. The 116 kDa solubilized protein has been identified as conventional kinesin based on limited sequence analysis. We also show that a polyclonal antibody raised against mitochondria-associated kinesin recognizes soluble bovine brain kinesin. The soluble and mitochondrial membrane-associated kinesins show a different isoform pattern. These results are consistent with the idea that kinesin exists as multiple isoforms that might be differentially distributed within the cell. In addition digitonin fractionation of mitochondria combined with KI extraction revealed that kinesin is a peripheral protein, preferentially located in a cholesterol-free outer membrane domain; this domain has the features of contact points between the mitochondrial outer and inner membranes. The significance of these observations on the functional regulation of the mitochondria-associated kinesin is discussed.
- Hackney DD
- Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis.
- Proc Natl Acad Sci U S A. 1994; 91: 6865-9
- Display abstract
The N-terminal 392 amino acids of the Drosophila kinesin alpha subunit (designated DKH392) form a dimer in solution that releases only one of its two tightly bound ADP molecules on association with a microtubule, whereas a shorter monomeric construct (designated DKH340) releases > or = 95% of its one bound ADP on association with a microtubule. This half-site reactivity of dimeric DKH392 is observed over a wide range of ratios of DKH392 to microtubules and steady-state ATPase rates, indicating that it is characteristic of the mechanism of microtubule-stimulated ATP hydrolysis and not the result of a fortuitous balance of rate constants. When [alpha-32P]ATP is included in the medium, incorporation of 32P label into the pool of ADP that is bound to the complex of DKH392 and microtubules occurs rapidly enough for the bound ADP to be an intermediate on the main pathway of ATP hydrolysis. These and other results are consistent with the half-site reactivity being a consequence of the tethering of dimeric DKH392 to the microtubule through one head domain, which is attached in a rigor-like manner without bound nucleotide, whereas the other head is not attached to the microtubule and still contains a tightly bound ADP. An intermediate of this nature and the tight binding of DKH392 to microtubules in the presence of ATP suggest a mechanism for directed motility in which the head domains of dimeric DKH392 alternate in a sequential manner.
- Lafont F, Burkhardt JK, Simons K
- Involvement of microtubule motors in basolateral and apical transport in kidney cells.
- Nature. 1994; 372: 801-3
- Display abstract
The maintenance of a polarized cell surface requires vectorial transport of vesicles to the apical and the basolateral membrane domains. Transport of newly synthesized apical proteins and trans-cytosis from the basolateral to the apical surface have been demonstrated to depend on microtubules. In contrast, movement of membrane proteins to the basolateral surface has been claimed to occur by diffusion and to be microtubule- and actin-independent. We have re-examined the role of microtubules using a recently developed polarized transport assay in permeabilized Madin-Darby canine kidney cells. Here we report that both apical and basolateral transport is inhibited by nocodazole treatment. Transport to the basolateral surface was inhibited by immunodepletion of cytosolic kinesin. In contrast, apical transport involved both dynein and kinesin. Our data demonstrate that in epithelial cells, microtubule motors are involved in the movement of apical and basolateral vesicles. Moreover, we propose that the differential requirement for microtubule-based motors is related to the microtubule organization.
- Feiguin F, Ferreira A, Kosik KS, Caceres A
- Kinesin-mediated organelle translocation revealed by specific cellular manipulations.
- J Cell Biol. 1994; 127: 1021-39
- Display abstract
The distribution of membrane-bound organelles was studied in cultured hippocampal neurons after antisense oligonucleotide suppression of the kinesin-heavy chain (KHC). We observed reduced 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) fluorescent staining in neurites and growth cones. In astrocytes, KHC suppression results in the disappearance of the DiOC6(3)-positive reticular network from the cell periphery, and a parallel accumulation of label within the cell center. On the other hand, mitochondria microtubules and microfilaments display a distribution that closely resembles that observed in control cells. KHC suppression of neurons and astrocytes completely inhibited the Brefeldin A-induced spreading and tubulation of the Golgi-associated structure enriched in mannose-6-phosphate receptors. In addition, KHC suppression prevents the low pH-induced anterograde redistribution of late endocytic structures. Taken collectively, these observations suggest that in living neurons, kinesin mediates the anterograde transport of tubulovesicular structures originated in the central vacuolar system (e.g., the endoplasmic reticulum) and that the regulation of kinesin-membrane interactions may be of key importance for determining the intracellular distribution of selected organelles.
- Svoboda K, Mitra PP, Block SM
- Fluctuation analysis of motor protein movement and single enzyme kinetics.
- Proc Natl Acad Sci U S A. 1994; 91: 11782-6
- Display abstract
We studied fluctuations in the displacement of silica beads driven by single molecules of the motor protein kinesin, moving under low mechanical loads at saturating ATP concentrations. The variance in position was significantly smaller than expected for the case of stepwise movement along a regular lattice of positions with exponentially distributed intervals. The small variance suggests that two or more sequential processes with comparable reaction rates dominate the biochemical cycle. The low value is inconsistent with certain recently proposed thermal ratchet models for motor movement as well as with scenarios where the hydrolysis of a single ATP molecule leads to a cluster of several steps. Fluctuation analysis is a potential powerful tool for studying kinetic behavior whenever the output of a single enzyme can be monitored.
- Song YH, Mandelkow E
- Paracrystalline structure of the stalk domain of the microtubule motor protein kinesin.
- J Struct Biol. 1994; 112: 93-102
- Display abstract
We have studied single molecules and paracrystals of the stalk domain of the microtubule motor protein, kinesin, using circular dichroism, electron microscopy, and optical diffraction. The stalk is a rod-like particle, about 50 nm in length, with about 70% alpha-helical content (lower than tropomyosin and myosin). These data confirm the previous studies of M. De Cuevas, T. Tao, and L.S. B. Goldstein (J. Cell Biol. 116, 957-966, 1992). The particles also show a tendency to self-associate into dimers or higher aggregates, up to paracrystals with a periodic substructure. Four types of paracrystals have been observed, two with short periodicities (8 and 13 nm, types I and II) and two with periodicities comparable with the subunit length (53-63 nm, type III and 38 nm, type IV). Types I and II paracrystals can be interpreted to arise from a polar arrangement of subunits with alternating gaps and overlaps and different staggers between adjacent molecules. Type III and IV paracrystals appear to be formed from sets of antiparallel molecules, forming centrosymmetric patterns. The association properties may be important for functions of the kinesin stalk in microtubule-dependent motility.
- Skoufias DA, Cole DG, Wedaman KP, Scholey JM
- The carboxyl-terminal domain of kinesin heavy chain is important for membrane binding.
- J Biol Chem. 1994; 269: 1477-85
- Display abstract
Sea urchin kinesin is a plus end-directed microtubule-based motor consisting of two heavy chains and two light chains and is proposed to be responsible (a) for the transport of membranous organelles along microtubules in sea urchin mitotic spindles (Wright, B. D., Henson, J. H., Wedaman, K. P., Willy, P. J., Morand, J. N., and Scholey, J. M. (1991) J. Cell Biol. 113, 817-833) and (b) for the radial dispersion of endoplasmic reticulum and endosomal membranes in non-mitotic cultured coelomocytes (Henson, J. H., Nesbitt, D., Wright, B. D., and Scholey, J. M. (1992) J. Cell Sci. 103, 309-320). We report here that sea urchin kinesin is indeed able to bind in a concentration-dependent and saturable manner to microsomal membranes isolated from sea urchin eggs in the presence of MgATP. The kinesin light chains may not be essential for membrane binding since kinesin containing negligible amounts of light chains binds as well as kinesin containing stoichiometric amounts of light chains. Finally, we propose that kinesin binds to membranes with the carboxyl-terminal domain of the heavy chain (amino acid residues 858-1031) since the bacterially expressed and then isolated stalk-tail fragment of kinesin heavy chain, in contrast to the stalk fragment, is able (a) to bind membranes in a concentration-dependent and saturable manner and (b) to compete with native kinesin for membrane binding. Our results support the hypothesis that the carboxyl-terminal domains of the heavy chains attach kinesin molecules to their membranous cargo in mitotic and interphase sea urchin cells.
- Redenbach DM, Richburg JH, Boekelheide K
- Microtubules with altered assembly kinetics have a decreased rate of kinesin-based transport.
- Cell Motil Cytoskeleton. 1994; 27: 79-87
- Display abstract
Microtubules treated with the gamma-diketone 2,5-hexanedione (2,5-HD) have altered assembly behavior characterized by precocious nucleation and rapid elongation. By measuring the rate of microtubule transport, we have examined the potential functional significance of this 2,5-HD-induced microtubule modification. 2,5-HD-treated microtubules were transported at only 70% of the rate of control microtubules in a simple kinesin-based motility assay on glass coverslips using video and computer enhanced differential interference contrast microscopy. Since 2,5-HD is capable of forming both pyrrole adducts and crosslinks with tubulin, the contributions of pyrrole formation and crosslinking to slowed microtubule transport were determined. 3-Acetyl-2,5-hexanedione (AcHD), a pyrrole forming, non-crosslinking congener of 2,5-HD which does not alter microtubule assembly, did not produce slowed microtubule transport as occurs with 2,5-HD. However, glutaraldehyde, a pyrrole-independent crosslinking agent which alters microtubule assembly in the same way as 2,5-HD, slowed microtubule transport. These results indicate that a 2,5-HD-induced microtubule modification, possibly a crosslink-related conformational change, produces both an alteration in the kinetics of assembly and an alteration in the microtubule-motor interaction.
- Huang TG, Hackney DD
- Drosophila kinesin minimal motor domain expressed in Escherichia coli. Purification and kinetic characterization.
- J Biol Chem. 1994; 269: 16493-501
- Display abstract
A truncated motor domain of the alpha subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP. This domain (designated DKH340) extends from the N terminus to amino acid 340. The isolated protein contains a stoichiometric level of tightly bound ADP and has a low basal rate of ATP hydrolysis of 0.029 +/- 0.002 s-1 in the absence of microtubules. The rate of release of bound ADP is 0.026 +/- 0.003 s-1. The approximate equality of the ADP release rate and the steady state ATPase rate indicates that ADP release is the rate-limiting step in ATP hydrolysis in the absence of microtubules. The rate of ATP hydrolysis is stimulated 3000 fold-by addition of microtubules (MT) (kcat = 80 s-1; KMT0.5,ATPase = 160 nM for half-saturation of the ATPase rate by microtubules at saturating ATP levels; KMT0.5ATPase = 43 microns for half-saturation of the ATPase rate by ATP at saturating microtubule levels). Binding of DKH340 to MTs is biphasic in the presence of adenosine 5-(beta-gamma-imido)t-riphosphate. One DKH340 binds tightly per tubulin heterodimer, but greater than one DKH340/tubulin heterodimer can be bound at higher ratios of DKH340/microtubules. In the presence of MgATP, KMT0.5,Binding for physical binding of DKH340 to microtubules is weaker than KMT0.5,ATPase for stimulation of hydrolysis. These results are consistent with a model in which DKH340 cycles on and off the microtubule during hydrolysis of each ATP molecule. For this model, the kcat/KMT0.5,ATPase ratio of 5 x 10(8) M-1 s-1 is at least as large as the bimolecular rate constant for association with microtubules, and this value approaches the diffusion controlled limit. Nucleotide-free DKH340 can be produced by gel filtration in the absence of Mg2+, but it reforms tightly bound ADP slowly in the presence of MgATP (t1/2 > or = 10 min), and thus it is likely to be in a conformational state which is not produced during steady state ATP hydrolysis.
- Lockhart A, Cross RA
- Origins of reversed directionality in the ncd molecular motor.
- EMBO J. 1994; 13: 751-7
- Display abstract
The head or motor domain of the ncd (non-claret disjunctional) molecular motor is 41% identical to that of kinesin, yet moves along microtubules in the opposite direction to kinesin. We show here that despite the reversed directionality of ncd, its kinetics in solution are homologous in key respects to those of kinesin. The rate limiting step, ADP release, occurs at 0.0033 s-1 at 100 mM NaCl and is accelerated approximately 1000-fold when the motor binds to microtubules. Other reaction steps are all very fast (> 0.1 s-1) compared with ADP release, and the motor is consequently paused in the ncd.ADP state until microtubule binding occurs (Kd = 2 microM), at which point ADP release is triggered and the motor locks onto the microtubule in a rigor-like state. These data identify close functional homology between the strong binding states of kinesin and ncd, and in view of this we discuss a possible mechanism for directional reversal, in which the strong binding states of ncd and kinesin are functionally identical, but the weak binding states are biased in opposite directions.
- Svoboda K, Block SM
- Force and velocity measured for single kinesin molecules.
- Cell. 1994; 77: 773-84
- Display abstract
We measured the force-velocity curves of single kinesin molecules attached to silica beads moving in an in vitro motility assay. Optical trapping interferometry was used to track movement with subnanometer precision and to apply calibrated, pN-sized forces to the beads. Velocity decreased linearly with increasing force, and kinesin molecules moved against applied loads of up to 5-6 pN. Comparison of force-velocity curves at limiting and saturating ATP concentrations suggests that the load-dependent diminution in kinesin velocity may be due to a decrease in the net displacement per molecule of ATP hydrolyzed, not simply to a slowing of the ATP turnover rate; kinesin would therefore appear to be a loosely coupled motor.
- Saxton WM
- Isolation and analysis of microtubule motor proteins.
- Methods Cell Biol. 1994; 44: 279-88
- Display abstract
Isolation of microtubule motor proteins is needed both for the discovery of new motors and for characterization of the products of motor-related genes. The sequences of motor-related genes cannot yet be used to predict the mechanochemical properties of the gene products. This was illustrated by the first kinesin-related gene product to be characterized. Protein expressed from the ncd gene moved toward the minus ends of microtubules (Walker et al., 1990; McDonald et al., 1990), while kinesin itself moves toward the plus ends. Until the relationship between mechanochemical function and amino acid sequence is more thoroughly understood, biochemical isolation and characterization of microtubule motor proteins will remain essential. Two approaches for getting useful quantities of microtubule motor proteins have been used: isolation from cytosol as described under Section II above and isolation from bacteria carrying cloned motor protein genes in expression vectors. Bacterial expression of functional microtubule motors has been successful to date in only a few cases (Yang et al., 1990; Walker et al., 1990, McDonald et al., 1990). Additional progress is expected with the expression of cloned genes from viral vectors in cultured eukaryotic cells, but broad success has not yet been reported. Biochemical isolation of motors from their natural cytosol has some distinct advantages. One can have confidence that a given motor will be folded properly and have normal post-translational modifications. In addition, if it exists in vivo as a heteromultimer, a microtubule motor isolated from its native cytosol will carry with it a normal complement of associated proteins. Studies of such associated proteins will be important in learning how motors accomplish their tasks in vivo. Drosophila cytosol should be a rich source of microtubule motors. Drosophila carry at least 11 and perhaps as many as 30 genes that are related to kinesin (Stewart et al., 1991; Endow and Hatsumi, 1991). The work of Tom Hays' lab indicates that Drosophila carry more than nine dynein related genes (Rasmussen et al., 1994). Relatively little effort to isolate the products of these genes from cytosol has been made. The only work that I am aware of has produced a kinesin-like microtubule motor (D.G. Cole, K.B. Sheehan, W.M. Saxton, and J.M. Scholey, in progress) that may be the Drosophila homolog of Xenopus eg5 (Sawin et al., 1992). This isolation was straightforward, and efforts to identify additional motors are almost assured of success.
- Kikkawa M, Ishikawa T, Nakata T, Wakabayashi T, Hirokawa N
- Direct visualization of the microtubule lattice seam both in vitro and in vivo.
- J Cell Biol. 1994; 127: 1965-71
- Display abstract
Microtubules are constructed from alpha- and beta-tubulin heterodimers that are arranged into protofilaments. Most commonly there are 13 or 14 protofilaments. A series of structural investigations using both electron microscopy and x-ray diffraction have indicated that there are two potential lattices (A and B) in which the tubulin subunits can be arranged. Electron microscopy has shown that kinesin heads, which bind only to beta-tubulin, follow a helical path with a 12-nm pitch in which subunits repeat every 8-nm axially, implying a primarily B-type lattice. However, these helical symmetry parameters are not consistent with a closed lattice and imply that there must be a discontinuity or "seam" along the microtubule. We have used quick-freeze deep-etch electron microscopy to obtain the first direct evidence for the presence of this seam in microtubules formed either in vivo or in vitro. In addition to a conventional single seam, we have also rarely found microtubules in which there is more than one seam. Overall our data indicates that microtubules have a predominantly B lattice, but that A lattice bonds between tubulin subunits are found at the seam. The cytoplasmic microtubules in mouse nerve cells also have predominantly B lattice structure and A lattice bonds at the seam. These observations have important implications for the interaction of microtubules with MAPs and with motor proteins, and for example, suggest that kinesin motors may follow a single protofilament track.
- Mercer JA, Albanesi JP, Brady ST
- Molecular motors and cell motility in the brain.
- Brain Pathol. 1994; 4: 167-79
- Display abstract
The advent of video computer-enhanced microscopy has provided a new vision of cell migrations, growth cones, and fast axonal transport in the nervous system. In images obtained in studies of fast transport in isolated axoplasm from the squid giant axon, a virtual torrent of membrane traffic could be seen moving in both directions. Similarly, examination of growth cones and cell migrations in vitro and in vivo revealed properties of cell motility that were previously unsuspected. Evidence has accumulated that many of these activities are driven by a variety of microtubule and microfilament based motors.
- Gilbert SP, Johnson KA
- Pre-steady-state kinetics of the microtubule-kinesin ATPase.
- Biochemistry. 1994; 33: 1951-60
- Display abstract
The pre-steady-state kinetics of the microtubule-kinesin ATPase were investigated by chemical-quench flow methods using the Drosophila kinesin motor domain (K401) expressed in Escherichia coli [Gilbert, S. P., & Johnson, K. A. (1993) Biochemistry 32, 4677-4684]. The results define a minimal mechanism: M.K + ATP in equilibrium with (M).K.ATP in equilibrium with (M).K.ADP.Pi in equilibrium with M.K.ADP + Pi in equilibrium with M.K + ADP, where M, K, and Pi represent microtubules, kinesin, and inorganic phosphate, respectively, with k+1 = 0.8-3 microM-1 s-1, k-1 = 100-300 s-1, k+2 = 70-120 s-1, k+4 = 10-20 s-1, and k+3 >> k-2 and k+3 >> k+4. Conditions were as follows: 25 degrees C, 20 mM HEPES, pH 7.2 with KOH, 5 mM magnesium acetate, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM potassium acetate, 1 mM DTT. The experiments presented do not determine the step in the cycle where kinesin dissociates from the microtubule or the step at which kinesin reassociates with the microtubule; therefore, the steps that may represent kinesin as the free enzyme are indicated by (M). A burst of ADP product formation was observed during the first turnover of the enzyme in the acid-quench experiments that define the ATP hydrolysis transient. The observation of the burst demonstrates that product release is rate limiting even in the presence of saturating microtubule concentrations. The pulse-chase experiments define the time course of ATP binding to the microtubule-K401 complex. At low ATP concentrations, ATP binding limits the rate of the burst. However, at high concentrations of ATP, ATP binding is faster than the rate of ATP hydrolysis with k+2 = 70-120 s-1. The amplitude of the burst of the ATP binding transient reached a maximum of 0.7 per site at saturating concentrations of ATP and microtubules. The amplitude of less than 1 is attributed to the fast k(off) for ATP (k-1 = 100-300 s-1) that leads to a partitioning of the M.K.ATP complex between ATP hydrolysis (k+2) and ATP release (k-1). These results indicate that ATP binds weakly to the M.K complex (Kd,ATP app approximately 100 microM). ADP release (k+4 = 10-20 s-1) is rate limiting during steady-state turnover, indicating that microtubules activate the kinesin ATPase by increasing k(off),ADP from 0.01 s-1 in the absence of microtubules to 10-20 s-1 at saturating microtubule concentrations.
- Liu GQ, Cai G, Del Casino C, Tiezzi A, Cresti M
- Kinesin-related polypeptide is associated with vesicles from Corylus avellana pollen.
- Cell Motil Cytoskeleton. 1994; 29: 155-66
- Display abstract
A 100-kDa polypeptide with microtubule-interacting properties was identified in a Golgi vesicle-enriched fraction from Corylus avellana pollen. The k71s23 antibody (directed to the kinesin heavy chain from bovine brain) [Tiezzi et al., 1992: Cell Motil. Cytoskeleton 21:132-137] localized the polypeptide on the external surface of membrane-bounded organelles. Some 100-kDa-containing vesicles copelleted with microtubules (polymerized from purified bovine brain tubulin) either in presence or absence of 5 mM AMPPNP, but they could be released by 10 mM ATP or 0.5 M KCl. The pollen microtubule-interacting protein, salt-extracted from membranes and partially purified by gel filtration, exhibited an ATPase activity (16.2 nmolPi/mg/min) which could be stimulated about 2-fold (32.5 nmolPi/mg/min) by addition of bovine brain microtubules. We suppose that the 100-kDa polypeptide is part of a molecular complex showing properties of the kinesin class.
- Vernon GG, Woolley DM
- Direct evidence for tension development between flagellar doublet microtubules.
- Exp Cell Res. 1994; 215: 390-4
- Display abstract
In this study of isolated ribbons of flagellar doublet microtubules, we demonstrate that a resistance to sliding exists in the interdoublet gap. By photolytically releasing ATP from caged ATP, it has been possible to follow closely the responses of individual specimens. Distortion of the helical superstructure of the doublets, most often by a reduction in helical pitch, is interpreted as revealing the development of tension between doublets. Tension does not develop in the presence of vanadate.
- Berliner E, Mahtani HK, Karki S, Chu LF, Cronan JE Jr, Gelles J
- Microtubule movement by a biotinated kinesin bound to streptavidin-coated surface.
- J Biol Chem. 1994; 269: 8610-5
- Display abstract
Kinesin, an ATP-dependent microtubule motor, can be studied in vitro in motility assays where the kinesin is nonspecifically adsorbed to a surface. However, adsorption can inactivate kinesin and may alter its reaction kinetics. We therefore prepared a biotinated kinesin derivative, K612-BIO, and characterized its activity in solution and when bound to streptavidin-coated surfaces. K612-BIO consists of the N-terminal 612 amino acids of the Drosophila kinesin alpha subunit linked to the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The C-terminal domain directs the efficient post-translational biotination of the protein. We expressed K612-BIO at high levels using the baculovirus expression vector system and purified it to near-homogeneity. The expressed protein is completely soluble, and > 90% is bound by streptavidin. K612-BIO steady-state ATPase kinetics (KM,ATP = 24 microM, K0.5, microtubule = 0.61 mg ml-1, Vmax = approximately 25 s-1 head-1, 25 degrees C) are similar to those reported for intact kinesin. ATPase kinetics are not affected by the addition of streptavidin. Enzyme bound to a surface coated with streptavidin drove microtubule gliding in the presence of 2 mM ATP at 750 +/- 130 nm s-1 (26 degrees C). Activity was abolished by pretreatment of the surface with biotin, indicating that the microtubule movements are due to specifically bound enzyme. Motility assays based on specific attachment of biotinated enzyme to streptavidin-coated surfaces will be useful for quantitative analysis of kinesin motility and may provide a way to detect activity in kinesin derivatives or kinesin-like proteins that have not yet been shown to move microtubules.
- Dabrowska R
- [Cytoplasmic motor proteins]
- Postepy Biochem. 1994; 40: 96-105
- McIlvain JM Jr, Burkhardt JK, Hamm-Alvarez S, Argon Y, Sheetz MP
- Regulation of kinesin activity by phosphorylation of kinesin-associated proteins.
- J Biol Chem. 1994; 269: 19176-82
- Display abstract
The mechanochemical motor proteins of the kinesin and cytoplasmic dynein families play important roles in microtubule-based intracellular motility. Although movement and distribution of organelles like secretory granules, vesicles, endoplasmic reticulum, and chromosomes depend on the activity of these motor proteins, little is known about the regulation of this movement. We report here that the hyperphosphorylation of components of the kinesin complex by treatment with okadaic acid increases kinesin motor activity at least 2-fold. The stimulation was observed using both a granule motility assay and a microtubule gliding assay, indicating that phosphorylation enhances the activity of the motor itself, rather than the affinity of the motor for membrane organelles. Under stimulatory conditions, three proteins that co-purify with kinesin (with mobilities of 150, 79, and 73 kDa) are consistently hyperphosphorylated. Dephosphorylation of these proteins reduces kinesin activity to basal levels. Therefore, we conclude that kinesin motor activity is directly modulated by the phosphorylation state of kinesin-associated proteins.
- Huang TG, Suhan J, Hackney DD
- Drosophila kinesin motor domain extending to amino acid position 392 is dimeric when expressed in Escherichia coli.
- J Biol Chem. 1994; 269: 16502-7
- Display abstract
A truncated domain of the alpha-subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP. This domain (designated DKH392) extends to amino acid 392 and contains the complete N-terminal region of kinesin which is highly conserved between species. The DKH392 construct includes an additional 52 amino acids beyond the minimal motor domain of 340 amino acid residues which has been previously characterized as DKH340 (Huang, T.-G., and Hackney, D. D. (1994) J. Biol. Chem. 269, 16493-16501). The s20,w values for DKH340 and DKH392 are 3.3 and 5.2 S and the D20,w values are 7.7 x 10(-7) and 4.9 x 10(-7) cm3 s-1, respectively. These results indicate that DKH340 is a monomer in solution, but DKH392 is a dimer. In the presence of adenosine 5-(beta,gamma-imido)triphosphate, DKH392 binds to microtubules with a stoichiometry of two head domains (one DKH392 dimer) per tubulin heterodimer in contrast to the tight binding of one DKH340 per tubulin heterodimer. Electron microscopy indicates that both DKH340 monomers and DKH392 dimers decorate microtubules with a periodicity of 8 nm.
- Harbury PB, Kim PS, Alber T
- Crystal structure of an isoleucine-zipper trimer.
- Nature. 1994; 371: 80-3
- Display abstract
Subunit oligomerization in many proteins is mediated by short coiled-coil motifs. These motifs share a characteristic seven-amino-acid repeat containing hydrophobic residues at the first (a) and fourth (d) positions. Despite this common pattern, different sequences form two-, three- and four-stranded helical ropes. We have investigated the basis for oligomer choice by characterizing variants of the GCN4 leucine-zipper dimerization domain that adopt trimeric or tetrameric structures in response to mutations at the a and d positions. We now report the high-resolution X-ray crystal structure of an isoleucine-containing mutant that folds into a parallel three-stranded, alpha-helical coiled coil. In contrast to the dimer and tetramer structures, the interior packing of the trimer can accommodate beta-branched residues in the most preferred rotamer at both hydrophobic positions. Compatibility of the shape of the core amino acids with the distinct packing spaces in the two-, three- and four-stranded conformations appears to determine the oligomerization state of the GCN4 leucine-zipper variants.
- Allan V
- Organelle movement. Dynactin: portrait of a dynein regulator.
- Curr Biol. 1994; 4: 1000-2
- Display abstract
Recent studies of dynactin, a protein complex implicated in regulating the cytoplasmic motor protein dynein, reveal that the complex contains a specialized actin filament and may also interact with microtubules.
- Malik F, Brillinger D, Vale RD
- High-resolution tracking of microtubule motility driven by a single kinesin motor.
- Proc Natl Acad Sci U S A. 1994; 91: 4584-8
- Display abstract
Kinesin is a microtubule-based motor protein that contains two identical force-generating subunits. The kinesin binding sites along the microtubule lie 8 nm apart (the dimension of the tubulin dimer), which implies that kinesin must translocate a minimum distance of 8 nm per hydrolysis cycle. Measurements of kinesin's microtubule-stimulated ATPase activity (approximately 20 ATP per sec) and velocity of transport (approximately 0.6 micron/sec), however, suggest that the net distance moved per ATP (approximately 30 nm) may be greater than one tubulin dimer under zero load conditions. To explore how kinesin translocates during its ATPase cycle, we constructed a microscope capable of tracking movement with 1-nm resolution at a bandwidth of 200 Hz and used this device to examine microtubule movement driven by a single kinesin motor. Regular stepwise movements were not observed in displacement traces of moving microtubules, although Brownian forces acting on elastic elements within the kinesin motor precluded detection of steps that were < 12 nm. Though individual steps of approximately 16 nm were occasionally observed, their infrequent occurrence suggests that kinesin rarely moves abruptly by distances of two or more tubulin subunits during its ATP hydrolysis cycle. Instead it is more likely that kinesin moves forward by the distance of only a single tubulin subunit under zero load conditions.
- Hoyt MA
- Cellular roles of kinesin and related proteins.
- Curr Opin Cell Biol. 1994; 6: 63-8
- Display abstract
Kinesin is but one member of a large superfamily of microtubule-based motor proteins. This diverse group of motors drives a number of essential subcellular movements, including transport of membranous organelles and mitotic spindle functions. Recent observations have revealed examples of functional cooperativity and antagonism between different kinesin-related motors.
- Coy DL, Howard J
- Organelle transport and sorting in axons.
- Curr Opin Neurobiol. 1994; 4: 662-7
- Display abstract
Recent advances of the past year in the field of organelle transport have documented the existence of numerous kinesin-related proteins and the presence of multiple conventional kinesins within neurons. Biochemical and genetic mutant analyses indicate that different kinesin superfamily members transport different organelles. In addition to microtubule-based systems, actin filaments and myosin motors are associated with organelle transport in axons. The great diversity of motor proteins suggests that they may play a role in sorting, in addition to transport.
- Hunt AJ, Gittes F, Howard J
- The force exerted by a single kinesin molecule against a viscous load.
- Biophys J. 1994; 67: 766-81
- Display abstract
Kinesin is a motor protein that uses the energy derived from the hydrolysis of ATP to power the transport of organelles along microtubules. To probe the mechanism of this chemical-to-mechanical energy transduction reaction, the movement of microtubules across glass surfaces coated with kinesin was perturbed by raising the viscosity of the buffer solution. When the viscosity of the solution used in the low density motility assay was increased approximately 100-fold through addition of polysaccharides and polypeptides, the longer microtubules, which experienced a larger drag force from the fluid, moved more slowly than the shorter ones. The speed of movement of a microtubule depended linearly on the drag force loading the motor. At the lowest kinesin density, where dilution experiments indicated that the movement was caused by a single kinesin molecule, extrapolation of the linear relationship yielded a maximum time-averaged drag force of 4.2 +/- 0.5 pN per motor (mean +/- experimental SE). The magnitude of the force argues against one type of "ratchet" model in which the motor is hypothesized to rectify the diffusion of the microtubule; at high viscosity, diffusion is too slow to account for the observed speeds. On the other hand, our data are consistent with models in which force is a consequence of strain developed in an elastic element within the motor; these models include a different "ratchet" model (of the type proposed by A. F. Huxley in 1957) as well as "power-stroke" models.
- Howard J
- Wrestling with kinesin.
- Nature. 1993; 364: 390-1
- Howard J
- Molecular motors. One giant step for kinesin.
- Nature. 1993; 365: 696-7
- Svoboda K, Schmidt CF, Schnapp BJ, Block SM
- Direct observation of kinesin stepping by optical trapping interferometry.
- Nature. 1993; 365: 721-7
- Display abstract
Do biological motors move with regular steps? To address this question, we constructed instrumentation with the spatial and temporal sensitivity to resolve movement on a molecular scale. We deposited silica beads carrying single molecules of the motor protein kinesin on microtubules using optical tweezers and analysed their motion under controlled loads by interferometry. We find that kinesin moves with 8-nm steps.
- Urrutia R, Murphy DB, Kachar B, McNiven MA
- Kinesin-mediated vesicular transport in a biochemically defined assay.
- Methods Cell Biol. 1993; 39: 253-66
- Display abstract
Here we have described simple and reproducible methods to observe kinesin-mediated vesicle and microtubule movements under defined conditions using video microscopy. We are optimistic that this assay will provide a useful tool to study kinesin function, regulation, and dynamic physical interactions with membranous organelles and microtubules.
- Howard J, Hunt AJ, Baek S
- Assay of microtubule movement driven by single kinesin molecules.
- Methods Cell Biol. 1993; 39: 137-47
- Shimizu T, Toyoshima YY, Vale RD
- Use of ATP analogs in motor assays.
- Methods Cell Biol. 1993; 39: 167-77
- Rodionov VI, Gyoeva FK, Tanaka E, Bershadsky AD, Vasiliev JM, Gelfand VI
- Microtubule-dependent control of cell shape and pseudopodial activity is inhibited by the antibody to kinesin motor domain.
- J Cell Biol. 1993; 123: 1811-20
- Display abstract
One of the major functions of cytoplasmic microtubules is their involvement in maintenance of asymmetric cell shape. Microtubules were considered to perform this function working as rigid structural elements. At the same time, microtubules play a critical role in intracellular organelle transport, and this fact raises the possibility that the involvement of microtubules in maintenance of cell shape may be mediated by directed transport of certain cellular components to a limited area of the cell surface (e.g., to the leading edge) rather than by their functioning as a mechanical support. To test this hypothesis we microinjected cultured human fibroblasts with the antibody (called HD antibody) raised against kinesin motor domain highly conserved among the different members of kinesin superfamily. As was shown before this antibody inhibits kinesin-dependent microtubule gliding in vitro and interferes with a number of microtubule-dependent transport processes in living cells. Preimmune IgG fraction was used for control experiments. Injections of fibroblasts with HD antibody but not with preimmune IgG significantly reduced their asymmetry, resulting in loss of long processes and elongated cell shape. In addition, antibody injection suppressed pseudopodial activity at the leading edge of fibroblasts moving into an experimentally made wound. Analysis of membrane organelle distribution showed that kinesin antibody induced clustering of mitochondria in perinuclear region and their withdrawal from peripheral parts of the cytoplasm. HD antibody does not affect either density or distribution of cytoplasmic microtubules. The results of our experiments show that many changes of phenotype induced in cells by microtubule-depolymerizing agents can be mimicked by the inhibition of motor proteins, and therefore microtubule functions in maintaining of the cell shape and polarity are mediated by motor proteins rather than by being provided by rigidity of tubulin polymer itself.
- Travis J
- Innovative techniques on display at Boston meeting.
- Science. 1993; 261: 1112-3
- Gilbert SP, Johnson KA
- Expression, purification, and characterization of the Drosophila kinesin motor domain produced in Escherichia coli.
- Biochemistry. 1993; 32: 4677-84
- Display abstract
The Drosophila kinesin heavy-chain gene was truncated to obtain the N-terminal 401 amino acid motor domain (designated K401) containing both the microtubule and ATP binding sites. The plasmid construct with the truncated kinesin gene was used to transform Escherichia coli. After induction, K401 was expressed as soluble kinesin protein at high levels and purified to homogeneity in milligram quantities. The purified protein was active and behaved as native kinesin with respect to its steady-state kinetic properties: K401 demonstrated a very low ATPase activity (kcat = 0.01 s-1) which was stimulated approximately 1000-fold by the addition of microtubules (kcat = 10 s-1; K0.5,MT = 0.9 microM tubulin; Km,ATP = 31 microM). Like native kinesin, K401 when purified contained ADP tightly bound at its active site, and the release of ADP from the active site occurred at a rate equal to the steady-state ATPase kcat. Active-site measurements using [alpha-32P]ATP demonstrated a stoichiometry of one ATPase site per K401 molecule. Like native kinesin, K401 can also hydrolyze MgGTP, and in the presence of microtubules, the rate of hydrolysis was increased dramatically from 0.03 to 16 s-1 (K0.5,MT = 2 microM tubulin; Km,GTP = 3.5 mM). These results establish that an active kinesin motor domain can be bacterially expressed and that this domain, the N-terminal 401 amino acids of the Drosophila kinesin heavy chain without light chains or additional eukaryotic factors, has full catalytic activity with microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)
- Gauger AK, Goldstein LS
- The Drosophila kinesin light chain. Primary structure and interaction with kinesin heavy chain.
- J Biol Chem. 1993; 268: 13657-66
- Display abstract
Kinesin light chain (KLC) complexes with the kinesin heavy chain (KHC) to form native kinesin. Proposed functions of KLC include coupling of cargo to KHC or modulation of KHC ATPase activity. In this paper we use the KHC tail, which binds specifically to KLC in blot overlays, as a probe to clone a cDNA encoding KLC from a Drosophila expression library. The identified clone encodes a protein with 70% amino acid identity to rat KLC. Drosophila KLC is predicted to form an alpha-helical coiled-coil between residues 34 and 129, followed by five imperfect tandem repeats of unknown function and a sixth shorter motif. These repeats are highly conserved across species. The Drosophila KLC gene is located at 69D on the third chromosome and is widely expressed, with 1.8-kb transcripts in most tissues, and slightly smaller transcripts in gonads. Finally, we present evidence that the heptad repeats of KLC are required for interaction with the KHC tail. Since the KHC tail used in our assay includes about 20 heptad repeats, this result suggests that KHC and KLC interact via coiled-coils. Such an interaction could provide stability to the KHC-KLC complex in vivo.
- Goldstein LS
- With apologies to scheherazade: tails of 1001 kinesin motors.
- Annu Rev Genet. 1993; 27: 319-51
- Nakata T, Sato-Yoshitake R, Okada Y, Noda Y, Hirokawa N
- Thermal drift is enough to drive a single microtubule along its axis even in the absence of motor proteins.
- Biophys J. 1993; 65: 2504-10
- Display abstract
One-dimensional diffusion of microtubules (MTs), a back-and-forth motion of MTs due to thermal diffusion, was reported in dynein motility assay. The interaction between MTs and dynein that allows such motion was implicated in its importance in the force generating cycle of dynein ATPase cycle. However, it was not known whether the phenomenon is special to motor proteins. Here we show two independent examples of one-dimensional diffusion of MTs in the absence of motor proteins. Dynamin, a MT-activated GTPase, causes a nucleotide dependent back-and-forth movement of single MT up to 1 micron along the longitudinal axes, although the MT never showed unidirectional consistent movement. Quantitative analysis of the motion and its nucleotide condition indicates that the motion is due to a thermal driven diffusion, restricted to one dimension, under the weak interaction between MT and dynamin. However, specific protein-protein interaction is not essential for the motion, because similar back-and-forth movement of MT was achieved on coverslips coated with only 0.8% methylcellulose. Both cases demonstrate that thermal diffusion could provide a considerable sliding of MTs only if MTs are restricted on the surface appropriately.
- Chandra R, Salmon ED, Erickson HP, Lockhart A, Endow SA
- Structural and functional domains of the Drosophila ncd microtubule motor protein.
- J Biol Chem. 1993; 268: 9005-13
- Display abstract
Nonclaret disjunctional (ncd) is a kinesin-related microtubule motor protein that is required for proper chromosome distribution in Drosophila. Despite its sequence similarity to kinesin heavy chain, ncd translocates with the opposite polarity as kinesin, toward microtubule minus ends. We have expressed different regions of the protein in bacteria and analyzed the proteins for function. Results indicate that ncd consists of three domains: a basic, proline-rich N-terminal "tail," a central alpha-helical coiled-coil stalk, and a C-terminal motor domain. The ncd N terminus proteins bundle microtubules in motility assays and show ATP-independent binding to microtubules in solution. Truncated proteins, lacking the tail but containing the predicted motor domain and differing lengths of the stalk, did not support microtubule gliding in in vitro assays but showed microtubule-stimulated MgATPase activity in solution. Addition of a nonspecific N terminus to two of the truncated proteins restored directional gliding and rotation of microtubules in motility assays, demonstrating that these properties map to the predicted mechanochemical domain of ncd. Physical properties of the C terminus proteins indicate that the stalk region is important for dimerization and that the ncd protein probably exists and functions as a dimer.
- Hogan CJ, Wein H, Wordeman L, Scholey JM, Sawin KE, Cande WZ
- Inhibition of anaphase spindle elongation in vitro by a peptide antibody that recognizes kinesin motor domain.
- Proc Natl Acad Sci U S A. 1993; 90: 6611-5
- Display abstract
Isolated central spindles or spindles in detergent-permeabilized cells from the diatom Cylindrotheca fusiformis can undergo ATP-dependent reactivation of spindle elongation in vitro. We have used a peptide antibody raised against a 10-amino acid portion common to the kinesin superfamily motor domain to look for kinesin-like motor activity during anaphase B of mitosis. The peptide antibody localizes to central spindles. Upon ATP reactivation of spindle elongation, antigens recognized by the antibody are associated exclusively with the central spindle midzone where antiparallel microtubules of each half-spindle overlap. The antibody recognizes several polypeptides by immunoblot using isolated spindle extracts. One of these polypeptides behaves like kinesin with respect to nucleotide-specific binding to and release from taxol-stabilized microtubules. Preincubation of the spindle model with the peptide antibody inhibits subsequent ATP reactivation of spindle elongation. Coincubation of the peptide antibody with peptide antigen rescues spindle function. These results support a role for kinesin-related protein(s) in spindle elongation (anaphase B) of mitosis and suggest that one or several polypeptides that we have identified in spindle extracts may fulfill this function.
- Sawin KE, Endow SA
- Meiosis, mitosis and microtubule motors.
- Bioessays. 1993; 15: 399-407
- Display abstract
A framework for understanding the complex movements of mitosis and meiosis has been provided by the recent discovery of microtubule motor proteins, required for the proper distribution of chromosomes or the structural integrity of the mitotic or meiotic spindle. Although overall features of mitosis and meiosis are often assumed to be similar in mechanism, it is now clear that they differ in several important aspects. These include spindle structure and assembly, and timing of chromosome segregation to opposite poles. Here we review progress in the functional characterization of several newly identified microtubule motor proteins, emphasizing their possible roles in spindle structure and function.
- Hunt AJ, Howard J
- Kinesin swivels to permit microtubule movement in any direction.
- Proc Natl Acad Sci U S A. 1993; 90: 11653-7
- Display abstract
Kinesin is a motor protein that uses the energy derived from ATP hydrolysis to transport organelles along microtubules. By analyzing the thermal fluctuation of microtubules tethered to glass surfaces by single molecules of kinesin, we have measured the torsional flexibility of the motor protein. The torsional stiffness of kinesin, (117 +/- 19) x 10(-24) N.m.rad-1 (mean +/- SEM), is so low that one kT of energy (approximately 4.1 x 10(-21) J at room temperature) is sufficient to twist a kinesin molecule through more than 360 degrees from its resting orientation. Consistent with this flexibility, motility assays show that one or more kinesin molecules can move a microtubule equally well in any direction. These results explain how a motor on the surface of an organelle can rapidly bind to and capture a microtubule irrespective of the organelle's orientation. Furthermore, the flexibility ensures that several motors can efficiently work together even though they are randomly oriented on the surface of an organelle rather than being in precise arrays like the motors of muscle and cilia.
- Liu CH, Higgins RJ, Buster D, Sanborn JR, Wilson BW
- The effect of organophosphates on a chicken brain or sea urchin egg kinesin-driven microtubule motility assay.
- Toxicol Lett. 1993; 69: 239-47
- Display abstract
The effect of neuropathic and non-neuropathic organophosphates (OPs) and acrylamide on an in vitro kinesin-driven microtubule (MT) motility assay was compared. The goal of the study was to determine whether this in vitro assay could confirm that a mechanism of action of neuropathic OPs was to impair kinesin activity and, therefore, possibly fast axonal anterograde transport (FAAT) in vivo. For our study, kinesin from chicken brain (CK) and sea urchin egg (SUK) was initially purified. Western immunoblotting confirmed the close antigenic homology between CK and SUK, using a mouse monoclonal sea urchin kinesin heavy chain-specific antibody (SUK 4). In the presence of microtubules (MTs) and MgATP, both CK- and SUK-driven MT movement was measured using a video-enhanced differential interference contrast microscope system with computer-assisted analysis. Using this assay system, we then tested separately the effect of two neuropathic OPs (diisopropylfluorophosphate (DFP) and phenyl saligenin phosphate (PSP)) and a non-neuropathic OP (paraoxon (PO)) each at a concentration of 10(-2) M at 27 degrees C. Additionally, we tested acrylamide (10(-2) M), since it is one of the best-characterized neurotoxins impairing FAAT in vivo. Our results demonstrated that none of these compounds significantly affected kinesin-driven MT motility in vitro compared to the standard controls. Further, this assay system was thus not able to discriminate between the neuropathic and non-neuropathic effect of these OPs.
- Stewart RJ, Thaler JP, Goldstein LS
- Direction of microtubule movement is an intrinsic property of the motor domains of kinesin heavy chain and Drosophila ncd protein.
- Proc Natl Acad Sci U S A. 1993; 90: 5209-13
- Display abstract
The kinesin heavy chain and the ncd (non-claret disjunctional) gene product of Drosophila are microtubule-associated motor proteins related by sequence similarity within an approximately 340-aa domain. Despite the sequence similarity, the kinesin heavy chain and ncd protein move in opposite directions on microtubules. To investigate the molecular basis for direction of movement, we created a series of truncated kinesin heavy chain and ncd proteins. We found that the conserved domain of both proteins has microtubule motor activity, although the efficiency with which ATP hydrolysis is coupled to microtubule movement declines dramatically with increasing truncation. Further, the direction of movement is intrinsic to the conserved motor domains, rather than being a consequence of domain organization or adjacent sequences.
- Ray S, Meyhofer E, Milligan RA, Howard J
- Kinesin follows the microtubule's protofilament axis.
- J Cell Biol. 1993; 121: 1083-93
- Display abstract
We tested the hypothesis that kinesin moves parallel to the microtubule's protofilament axis. We polymerized microtubules with protofilaments that ran either parallel to the microtubule's long axis or that ran along shallow helical paths around the cylindrical surface of the microtubule. When gliding across a kinesin-coated surface, the former microtubules did not rotate. The latter microtubules, those with supertwisted protofilaments, did rotate; the pitch and handedness of the rotation accorded with the supertwist measured by electron cryo-microscopy. The results show that kinesin follows a path parallel to the protofilaments with high fidelity. This implies that the distance between consecutive kinesin-binding sites along the microtubule must be an integral multiple of 4.1 nm, the tubulin monomer spacing along the protofilament, or a multiple of 8.2 nm, the dimer spacing.
- Andrews SB, Gallant PE, Leapman RD, Schnapp BJ, Reese TS
- Single kinesin molecules crossbridge microtubules in vitro.
- Proc Natl Acad Sci U S A. 1993; 90: 6503-7
- Display abstract
Kinesin is a cytoplasmic motor protein that moves along microtubules and can induce microtubule bundling and sliding in vitro. To determine how kinesin mediates microtubule interactions, we determined the shapes and mass distributions of squid brain kinesin, taxol-stabilized microtubules (squid and bovine), and adenosine 5'-[beta, gamma-imido]triphosphate-stabilized kinesin-microtubule complexes by high-resolution metal replication and by low-temperature, low-dose dark-field scanning transmission electron microscopy of unfixed, directly frozen preparations. Mass mapping by electron microscopy revealed kinesins loosely attached to the carbon support as asymmetrical dumbbell-shaped molecules, 40-52 nm long, with a mass of 379 +/- 15 kDa. The mass distribution and shape of these molecules suggest that these images represent kinesin in a shortened conformation. Kinesin-microtubule complexes were organized as bundles of linearly arrayed microtubules, stitched together at irregular intervals by cross-bridges typically < or = 25 nm long. The crossbridges had a mass of 360 +/- 15 kDa, consistent with one kinesin per crossbridge. These results suggest that kinesin has a second microtubule binding site in addition to the known site on the motor domain of the heavy chain; this second site may be located near the C terminus of the heavy chains or on the associated light chains. Thus, kinesin could play a role in either crosslinking or sliding microtubules.
- Leibler S, Huse DA
- Porters versus rowers: a unified stochastic model of motor proteins.
- J Cell Biol. 1993; 121: 1357-68
- Display abstract
We present a general phenomenological theory for chemical to mechanical energy transduction by motor enzymes which is based on the classical "tight-coupling" mechanism. The associated minimal stochastic model takes explicitly into account both ATP hydrolysis and thermal noise effects. It provides expressions for the hydrolysis rate and the sliding velocity, as functions of the ATP concentration and the number of motor enzymes. It explains in a unified way many results of recent in vitro motility assays. More importantly, the theory provides a natural classification scheme for the motors: it correlates the biochemical and mechanical differences between "porters" such as cellular kinesins or dyneins, and "rowers" such as muscular myosins or flagellar dyneins.
- Malekzadeh-Hemmat K, Gendry P, Launay JF
- Rat pancreas kinesin: identification and potential binding to microtubules.
- Cell Mol Biol (Noisy-le-grand). 1993; 39: 279-85
- Display abstract
We have demonstrated the presence of kinesin in the secretory pancreatic tissue using SDS-PAGE, immunoblot and immunoelectron microscopy techniques. Polyclonal antibodies were raised against the rat brain kinesin heavy chain and affinity-purified. Immunoblot studies showed that these antibodies were bound to a 116 kDa protein found in rat pancreas crude extracts and in partially purified kinesin fractions. Kinesin identification was also performed by a cosedimentation procedure based on its strong binding to microtubules in the presence of sodium fluoride. The microtubule-kinesin complex was observed by immunoelectron microscopy gold staining. The reversible association of kinesin with microtubules was generated by MgATP.
- Taylor EW
- Cell motility. Variations on the theme of movement.
- Nature. 1993; 361: 115-6
- Romberg L, Vale RD
- Chemomechanical cycle of kinesin differs from that of myosin.
- Nature. 1993; 361: 168-70
- Display abstract
Motor proteins move unidirectionally along cytoskeletal polymers by coupling translocation to cycles of ATP hydrolysis. The energy from ATP is required both to generate force and to dissociate the motor-filament complex in order to begin a new chemomechanical cycle. For myosin, force production is associated with phosphate release following ATP hydrolysis, whereas dissociation of actomyosin is tightly coupled to the binding of ATP. Dynein, a microtubule motor, uses a similar cycle, suggesting that all cytoskeletal motors might operate by a common mechanism. Here we investigate kinesin's chemomechanical cycle by assaying microtubule movement by single kinesin molecules when intermediate states in the hydrolysis cycle are prolonged with ATP analogues or inhibitors. In contrast to myosin and dynein, kinesin with bound ADP dissociates from microtubules during translocation, whereas kinesin with unhydrolysed nucleotide remains tightly associated with the polymer. These findings imply that kinesin converts ATP energy into mechanical work by a pathway distinct from that of myosin or dynein.
- Song YH, Mandelkow E
- Recombinant kinesin motor domain binds to beta-tubulin and decorates microtubules with a B surface lattice.
- Proc Natl Acad Sci U S A. 1993; 90: 1671-5
- Display abstract
We have expressed the recombinant squid kinesin head domain in Escherichia coli and studied its interaction with microtubules. The head is active as a microtubule-stimulated ATPase and binds to microtubules, but it does not support microtubule gliding by itself. The head binds to both microtubules and depolymerized tubulin. In each case the zero-length crosslinker 1-ethyl-3-[3-dimethylamino)propyl] carbodiimide induces a bond specifically to beta- but not alpha-tubulin. The head decorates brain microtubules with an 8-nm axial spacing. Thus the stoichiometry is one kinesin head per tubulin dimer. The lattice is that of flagellar B-tubules, implying that reassembled microtubules are not symmetric. Moreover, the A- and B-tubules of intact flagellar outer doublets are both decorated with a B lattice. This suggests that the B lattice is a general property of microtubules.
- Hirokawa N
- Axonal transport and the cytoskeleton.
- Curr Opin Neurobiol. 1993; 3: 724-31
- Display abstract
Great advances in the field of axonal transport have been made in the past year, including the identification of new molecular motors associated with microtubules and actin. In addition, studies on the mechanisms of bidirectional fast axonal transport have clarified new aspects of this process, such as the isolation of a kinesin-binding protein, kinectin, and the finding that phosphorylation regulates kinesin's dissociation from membranous organelles. New approaches to studying slow transport of cytoskeletal proteins have provided further evidence that the axonal cytoskeleton in mammalian systems is largely stationary, although a dynamic exchange occurs between polymers and a small pool of moving subunits.
- Skoufias DA, Scholey JM
- Cytoplasmic microtubule-based motor proteins.
- Curr Opin Cell Biol. 1993; 5: 95-104
- Display abstract
A multitude of microtubule-based motors drives diverse forms of intracellular transport and generates forces for maintaining the dynamic structural organization of cytoplasm. Recent work has illuminated the functions and mechanisms of action of some microtubule motors, and appears to have uncovered unforseen functional interactions between tubulin-based and actin-based systems.
- Rothwell SW, Deal CC, Pinto J, Wright DG
- Affinity purification and subcellular localization of kinesin in human neutrophils.
- J Leukoc Biol. 1993; 53: 372-80
- Display abstract
Studies of granule-microtubule interactions in human neutrophils have suggested that mechanochemical ATPases such as kinesin or dynein may play a role in granule mobilization during neutrophil activation by inflammatory signals. In this study we show that proteins extracted from the surface of neutrophil granules, found previously to contain microtubule-dependent ATPase activity, caused microtubules polymerized from phosphocellulose-purified rat brain tubulin to move across glass slides. Antibodies were generated against peptides based on two regions of the amino acid sequence of Drosophila kinesin: the ATPase active site (amino acids 86-99) in the head of the kinesin heavy chain and the tail of the heavy chain (residues 913-933). These antibodies were found to recognize kinesin in rat brain extracts as well as kinesin-like polypeptides in extracts of human neutrophils. Furthermore, when used in immunoaffinity chromatography, these antibodies permitted the isolation of a protein from neutrophil granule extracts that was recognized by Drosophila kinesin antibodies. Subcellular localization by immunofluorescence microscopy showed this protein to be associated principally with the cytoplasmic granules of neutrophils.
- Lopez LA, Sheetz MP
- Steric inhibition of cytoplasmic dynein and kinesin motility by MAP2.
- Cell Motil Cytoskeleton. 1993; 24: 1-16
- Display abstract
Using several in vitro motility assays, we found that motility driven by the microtubule (MT) motors, kinesin and cytoplasmic dynein, could be inhibited by MAP2 but not by tau protein or the MT-binding proteolytic fragment of MAP2. In MT gliding assays, even the presence of one MAP2 molecule per sixty-nine tubulin dimers caused an inhibition of about 75% of MT motility at low concentrations of both motors. The percent inhibition of motility decreased with increasing concentration of either motor, suggesting that the inhibition was the result of competition for access to the MT surface. The decrease in the number of moving MTs with MAP2 was correlated with an increase in the frequency of release of moving MTs from the motor-coated glass. In assays of in vitro vesicular organelle motility and formation of ER networks, the presence of MAP2 inhibited small vesicle movements and to a lesser extent ER network formation. To determine if competition for specific sites on the MT or coating of the MT surface inhibited motility, we used tau protein and the chymotryptic MT-binding fragments of MAP2 to coat MTs. No inhibition was observed and there was even an increase in the number of attached and moving MTs in the gliding assay with tau-coated MTs. Because MAP2, tau and the chymotryptic MT-binding fragments of MAP2 bind to the same domain on tubulin, masking of the MT surface sites does not appear responsible for the inhibition of motility by MAP2. Rather, we suggest that the sidearm of MAP2 interfered with the interaction of motors with MTs and caused a dramatic increase in the rate of MT release. In vivo, MAP2 could play a major role in the generation of cellular polarity even at substoichiometric levels by inhibiting transport on microtubules in specific domains of the cytoplasm.
- Hirokawa N
- Mechanism of axonal transport. Identification of new molecular motors and regulations of transports.
- Neurosci Res. 1993; 18: 1-9
- Display abstract
New molecular motors associated with microtubules and actin have been uncovered very recently. Furthermore, studies of the mechanisms of bidirectional fast axonal transports have clarified new aspects of these processes, such as identification of a kinesin binding protein (kinectin) and regulation of kinesin dissociation from membranous organelles by phosphorylation. These will lead to a more precise understanding of the mechanisms of axonal transports. Concerning the mechanism of the slow transport of cytoskeletal proteins, new approaches have provided further evidence that the axonal cytoskeleton in mammalian systems is largely stationary while dynamic exchanges occur between polymer and a small pool of moving subunits.
- Cole DG, Chinn SW, Wedaman KP, Hall K, Vuong T, Scholey JM
- Novel heterotrimeric kinesin-related protein purified from sea urchin eggs.
- Nature. 1993; 366: 268-70
- Display abstract
Kinesin heavy chain and kinesin-related polypeptides (KRPs) comprise a family of motor proteins with diverse intracellular transport functions. Using pan-kinesin peptide antibodies that react with these proteins, we have previously purified from sea urchin eggs a trimeric microtubule-binding and bundling protein, KRP (85/95) (ref. 8) comprising subunits of M(r) 115,000 (115K), 95K and 85K. We report here that kinesin-related genes encode the 85K and 95K subunits, and that the protein can be immunoprecipitated from cytosol as a trimeric complex using an 85K monoclonal antibody. We also find that purified KRP(85/95) directs movements towards the 'plus' ends of microtubules. To our knowledge, this protein is the first kinesin-related motor to be purified from its natural host cell in a native multimeric state.
- Hall K, Cole DG, Yeh Y, Scholey JM, Baskin RJ
- Force-velocity relationships in kinesin-driven motility.
- Nature. 1993; 364: 457-9
- Display abstract
Kinesin is a microtubule-based motor protein that uses energy released from Mg-ATP hydrolysis to generate force for the movement of intracellular membranes towards the fast-growing (plus) ends of microtubule tracks in cells. Kinesin-driven microtubule movement can be visualized and quantified using light microscope motility assays but our understanding of how kinesin generates force and motion is incomplete. Here we report the use of a centrifuge microscope to obtain force-velocity curves for kinesin-driven motility and to estimate that the maximal isometric force generated per kinesin is 0.12 +/- 0.03 pN per molecule.
- Walker RA, Sheetz MP
- Cytoplasmic microtubule-associated motors.
- Annu Rev Biochem. 1993; 62: 429-51
- Howard J
- Kinesin ATPase.
- Nature. 1993; 364: 396-396
- Harrison BC, Marchese-Ragona SP, Gilbert SP, Cheng N, Steven AC, Johnson KA
- Decoration of the microtubule surface by one kinesin head per tubulin heterodimer.
- Nature. 1993; 362: 73-5
- Display abstract
Kinesin, a microtubule-dependent ATPase, is believed to be involved in anterograde axonal transport. The kinesin head, which contains both microtubule and ATP binding sites, has the necessary components for the generation of force and motility. We have used saturation binding and electron microscopy to examine the interaction of the kinesin motor domain with the microtubule surface and found that binding saturated at one kinesin head per tubulin heterodimer. Both negative staining and cryo-electron microscopy revealed a regular pattern of kinesin bound to the microtubule surface, with an axial repeat of 8 nm. Optical diffraction analysis of decorated microtubules showed a strong layer-line at this spacing, confirming that one kinesin head binds per tubulin heterodimer. The addition of Mg-ATP to the microtubule-kinesin complex resulted in the complete dissociation of kinesin from the microtubule surface.
- Suda H, Taylor TW
- Intermolecular forces between the motor protein and the filament.
- J Theor Biol. 1993; 161: 39-50
- Display abstract
Intermolecular forces between motor proteins and filaments were evaluated on the basis of the experimental data of an in vitro motility assay by considering the molecular friction in the movement system. The molecular friction was caused by a different mechanism from that of the hydrodynamic drag. However, the molecular frictional forces apparently gave the same expression as the hydrodynamic frictional forces. The resulting equation was very effective in examining the physical properties of the weak interaction in the dynein-microtubules system from basic experiments carried out by Vale et al. (1989). From careful analysis of their experimental data, it was concluded that the hydrodynamic friction was not dominant, even in the weak binding state. The electrostatic interaction between dynein-heads and microtubules in the weak binding state was analyzed by applying the DLVO (Derjaguin-Landau-Verway-Overbeek) theory in colloid science through the ionic dependence of one-dimensional diffusion. The interacting distance between charges which took part in the weak adhesion was estimated to be 3 nm. In the present study, the molecular mechanism of the sliding velocity was also investigated for the myosin-actin filaments and the kinesin-microtubules systems by fitting the ATP-dependence and the ionic dependence in ATP-driven active sliding.
- Kuo SC, Sheetz MP
- Force of single kinesin molecules measured with optical tweezers.
- Science. 1993; 260: 232-4
- Display abstract
Isometric forces generated by single molecules of the mechanochemical enzyme kinesin were measured with a laser-induced, single-beam optical gradient trap, also known as optical tweezers. For the microspheres used in this study, the optical tweezers was spring-like for a radius of 100 nanometers and had a maximum force region at a radius of approximately 150 nanometers. With the use of biotinylated microtubules and special streptavidin-coated latex microspheres as handles, microtubule translocation by single squid kinesin molecules was reversibly stalled. The stalled microtubules escaped optical trapping forces of 1.9 +/- 0.4 piconewtons. The ability to measure force parameters of single macromolecules now allows direct testing of molecular models for contractility.
- Vale RD
- Measuring single protein motors at work.
- Science. 1993; 260: 169-70
- de Cuevas M, Tao T, Goldstein LS
- Evidence that the stalk of Drosophila kinesin heavy chain is an alpha-helical coiled coil.
- J Cell Biol. 1992; 116: 957-65
- Display abstract
Kinesin is a mechanochemical enzyme composed of three distinct domains: a globular head domain, a rodlike stalk domain, and a small globular tail domain. The stalk domain has sequence features characteristic of alpha-helical coiled coils. To gain insight into the structure of the kinesin stalk, we expressed it from a segment of the Drosophila melanogaster kinesin heavy chain gene and purified it from Escherichia coli. When observed by EM, this protein formed a rodlike structure 40-55 nm long that was occasionally bent at a hingelike region near the middle of the molecule. An additional EM study and a chemical cross-linking study showed that this protein forms a parallel dimer and that the two chains are in register. Finally, using circular dichroism spectroscopy, we showed that this protein is approximately 55-60% alpha-helical in physiological aqueous solution at 25 degrees C, and approximately 85-90% alpha-helical at 4 degrees C. From these results, we conclude that the stalk of kinesin heavy chain forms an alpha-helical coiled coil structure. The temperature dependence of the circular dichroism signal has two major transitions, at 25-30 degrees C and at 45-50 degrees C, which suggests that a portion of the alpha-helical structure in the stalk is less stable than the rest. By producing the amino-terminal (coil 1) and carboxy-terminal (coil 2) halves of the stalk separately in E. coli, we showed that the region that melts below 30 degrees C lies within coil 1, while the majority of coil 2 melts above 45 degrees C. We suggest that this difference in stability may play a role in the force-generating mechanism or regulation of kinesin.
- Cyr JL, Brady ST
- Molecular motors in axonal transport. Cellular and molecular biology of kinesin.
- Mol Neurobiol. 1992; 6: 137-55
- Display abstract
Neurons require a large amount of intracellular transport. Cytoplasmic polypeptides and membrane-bounded organelles move from the perikaryon, down the length of the axon, and to the synaptic terminals. This movement occurs at distinct rates and is termed axonal transport. Axonal transport is divided into the slow transport of cytoplasmic proteins including glycolytic enzymes and cytoskeletal structures and the fast transport of membrane-bounded organelles along linear arrays of microtubules. The polypeptide compositions of the rate classes of axonal transport have been well characterized, but the underlying molecular mechanisms of this movement are less clear. Progress has been particularly slow toward understanding force-generation in slow transport, but recent developments have provided insight into the molecular motors involved in fast axonal transport. Recent advances in the cellular and molecular biology of one fast axonal transport motor, kinesin, have provided a clearer understanding of organelle movement along microtubules. The availability of cellular and molecular probes for kinesin and other putative axonal transport motors have led to a reevaluation of our understanding of intracellular motility.
- Komatsu M et al.
- Isolation of kinesin from rat liver.
- Gastroenterol Jpn. 1992; 27: 793-793
- Yu H, Toyoshima I, Steuer ER, Sheetz MP
- Kinesin and cytoplasmic dynein binding to brain microsomes.
- J Biol Chem. 1992; 267: 20457-64
- Display abstract
Movement of cellular organelles in a directional manner along polar microtubules is driven by the motor proteins, kinesin and cytoplasmic dynein. The binding of these proteins to a microsomal fraction from embryonic chicken brain is investigated here. Both motors exhibit saturation binding to the vesicles, and proteolysis of vesicle membrane proteins abolishes binding. The maximal binding for kinesin is 12 +/- 1.7 and 43 +/- 2 pmol per mg of vesicle protein with or without 1 mM ATP, respectively. The maximal binding for cytoplasmic dynein is 55 +/- 3.8 and 73 +/- 3.7 pmol per mg of vesicle protein with or without ATP, respectively. These values correspond to 1-6 sites per vesicle of 100-nm diameter. The nonhydrolyzable ATP analog, adenyl-5'-yl imidodiphosphate (AMP-PNP), inhibited kinesin binding to vesicles but increased kinesin binding to microtubules. An antibody to the kinesin light chain also inhibited vesicle binding to kinesin. In the absence but not presence of ATP, competition between the two motors for binding was observed. We suggest that there are two distinguishable binding sites for kinesin and cytoplasmic dynein on these organelles in the presence of ATP and a shared site in the absence of ATP.
- Sadhu A, Taylor EW
- A kinetic study of the kinesin ATPase.
- J Biol Chem. 1992; 267: 11352-9
- Display abstract
The mechanism of kinesin ATPase has been investigated by transient state kinetic analysis. The results satisfy the scheme [formula: see text] where T, D, and P(i) refer to nucleotide tri- and diphosphate and inorganic phosphate, respectively. The nucleotide-binding steps were measured by the fluorescence enhancement of mant (2'-(3')-O-(N-methylanthraniloyl)-ATP and mant-ADP. The initial rapid equilibrium binding steps (1) and (6) are followed by isomerizations (k2 = 170 +/- 30 s-1 at 20 degrees C, k-5 greater than 100 s-1). The increase in fluorescence is 20-25% larger for K.T** than K.D*. The rate constant of the hydrolysis step k3 is 6-7 s-1. The fluorescence decreases after formation of K.T** at a rate of 7-10 s-1. This change could occur in step 3 or in step 4 if k4 much greater than k3. The value of k4 is larger than 0.1 s-1. The steady state rate is 0.003 s-1 which agrees with the rate of ADP dissociation (k5). Step 5 is rate limiting in the scheme in agreement with the conclusion of Hackney (Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314-6318) that ADP dissociation is the rate-limiting step.
- Endow SA, Titus MA
- Genetic approaches to molecular motors.
- Annu Rev Cell Biol. 1992; 8: 29-66
- Bloom GS
- Motor proteins for cytoplasmic microtubules.
- Curr Opin Cell Biol. 1992; 4: 66-73
- Display abstract
It has been thought that motile structures within the cell are driven toward the plus and minus ends of microtubules by the ATPases, kinesin and dynein, respectively. Recently obtained data indicate that this model is far too simplistic. Kinesin is now understood to be one representative of a family of proteins. Another member of the kinesin family has been found to generate force toward the microtubule minus end. Evidence for either a bidirectional dynein, or closely related retrograde and anterograde forms of dynein has also received potent new support. The discovery of a third potential microtubule motor, the GTPase, 'dynamin', complicates matters further.
- Aizawa H, Sekine Y, Takemura R, Zhang Z, Nangaku M, Hirokawa N
- Kinesin family in murine central nervous system.
- J Cell Biol. 1992; 119: 1287-96
- Display abstract
In neuronal axons, various kinds of membranous components are transported along microtubules bidirectionally. However, only two kinds of mechanochemical motor proteins, kinesin and brain dynein, had been identified as transporters of membranous organelles in mammalian neurons. Recently, a series of genes that encode proteins closely related to kinesin heavy chain were identified in several organisms including Schizosaccharomyces pombe, Aspergillus niddulans, Saccharomyces cerevisiae, Caenorhabditus elegans, and Drosophila. Most of these members of the kinesin family are implicated in mechanisms of mitosis or meiosis. To address the mechanism of intracellular organelle transport at a molecular level, we have cloned and characterized five different members (KIF1-5), that encode the microtubule-associated motor domain homologous to kinesin heavy chain, in murine brain tissue. Homology analysis of amino acid sequence indicated that KIF1 and KIF5 are murine counterparts of unc104 and kinesin heavy chain, respectively, while KIF2, KIF3, and KIF4 are as yet unidentified new species. Complete amino acid sequence of KIF3 revealed that KIF3 consists of NH2-terminal motor domain, central alpha-helical rod domain, and COOH-terminal globular domain. Complete amino acid sequence of KIF2 revealed that KIF2 consists of NH2-terminal globular domain, central motor domain, and COOH-terminal alpha-helical rod domain. This is the first identification of the kinesin-related protein which has its motor domain at the central part in its primary structure. Northern blot analysis revealed that KIF1, KIF3, and KIF5 are expressed almost exclusively in murine brain, whereas KIF2 and KIF4 are expressed in brain as well as in other tissues. All these members of the kinesin family are expressed in the same type of neurons, and thus each one of them may transport its specific organelle in the murine central nervous system.
- Navone F et al.
- Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells.
- J Cell Biol. 1992; 117: 1263-75
- Display abstract
To understand the interactions between the microtubule-based motor protein kinesin and intracellular components, we have expressed the kinesin heavy chain and its different domains in CV-1 monkey kidney epithelial cells and examined their distributions by immunofluorescence microscopy. For this study, we cloned and sequenced cDNAs encoding a kinesin heavy chain from a human placental library. The human kinesin heavy chain exhibits a high level of sequence identity to the previously cloned invertebrate kinesin heavy chains; homologies between the COOH-terminal domain of human and invertebrate kinesins and the nonmotor domain of the Aspergillus kinesin-like protein bimC were also found. The gene encoding the human kinesin heavy chain also contains a small upstream open reading frame in a G-C rich 5' untranslated region, features that are associated with translational regulation in certain mRNAs. After transient expression in CV-1 cells, the kinesin heavy chain showed both a diffuse distribution and a filamentous staining pattern that coaligned with microtubules but not vimentin intermediate filaments. Altering the number and distribution of microtubules with taxol or nocodazole produced corresponding changes in the localization of the expressed kinesin heavy chain. The expressed NH2-terminal motor and the COOH-terminal tail domains, but not the alpha-helical coiled coil rod domain, also colocalized with microtubules. The finding that both the kinesin motor and tail domains can interact with cytoplasmic microtubules raises the possibility that kinesin could crossbridge and induce sliding between microtubules under certain circumstances.
- Leopold PL, McDowall AW, Pfister KK, Bloom GS, Brady ST
- Association of kinesin with characterized membrane-bounded organelles.
- Cell Motil Cytoskeleton. 1992; 23: 19-33
- Display abstract
The family of molecular motors known as kinesin has been implicated in the translocation of membrane-bounded organelles along microtubules, but relatively little is known about the interaction of kinesin with organelles. In order to understand these interactions, we have examined the association of kinesin with a variety of organelles. Kinesin was detected in purified organelle fractions, including synaptic vesicles, mitochondria, and coated vesicles, using quantitative immunoblots and immunoelectron microscopy. In contrast, isolated Golgi membranes and nuclear fractions did not contain detectable levels of kinesin. These results demonstrate that the organelle binding capacity of kinesin is selective and specific. The ability to purify membrane-bounded organelles with associated kinesin indicates that at least a portion of the cellular kinesin has a relatively stable association with membrane-bounded organelles in the cell. In addition, immunoelectron microscopy of mitochondria revealed a patch-like pattern in the kinesin distribution, suggesting that the organization of the motor on the organelle membrane may play a role in regulating organelle motility.
- Hackney DD
- Kinesin and myosin ATPases: variations on a theme.
- Philos Trans R Soc Lond B Biol Sci. 1992; 336: 13-7
- Display abstract
The enzymes kinesin and myosin are examples of molecular motors which couple ATP hydrolysis to directed movement of biological structures. Myosin has been extensively studied and its structure and mechanism of coupling are known in detail. Much less is known about kinesin, but many of its major properties are similar to those of myosin. Both enzymes have two catalytic head groups at the end of a long alpha-helical rod. The head groups contain the sites for ATP hydrolysis and interaction with their respective partners for movement (microtubules or F-actin). In each case the binding and hydrolysis of ATP is rapid and the steady state ATPase rate is limited by a slow step in the region of product release. This slow release of product is accelerated by interaction with actin or microtubules coupled to changes in binding affinity. As there is no evidence for a close evolutionary link between kinesin and myosin, these and other similarities may represent convergence to set of common functional properties which are constrained by the requirements of protein structure and the use of ATP hydrolysis as a source of energy. It will be of particular interest to determine if these common properties are also shared by the large number of divergent proteins which have recently been discovered to possess a domain which is homologous to the head group of kinesin.
- Vale RD, Malik F, Brown D
- Directional instability of microtubule transport in the presence of kinesin and dynein, two opposite polarity motor proteins.
- J Cell Biol. 1992; 119: 1589-96
- Display abstract
Kinesin and dynein are motor proteins that move in opposite directions along microtubules. In this study, we examine the consequences of having kinesin and dynein (ciliary outer arm or cytoplasmic) bound to glass surfaces interacting with the same microtubule in vitro. Although one might expect a balance of opposing forces to produce little or no net movement, we find instead that microtubules move unidirectionally for several microns (corresponding to hundreds of ATPase cycles by a motor) but continually switch between kinesin-directed and dynein-directed transport. The velocities in the plus-end (0.2-0.3 microns/s) and minus-end (3.5-4 microns/s) directions were approximately half those produced by kinesin (0.5 microns/s) and ciliary dynein (6.7 microns/s) alone, indicating that the motors not contributing to movement can interact with and impose a drag upon the microtubule. By comparing two dyneins with different duty ratios (percentage of time spent in a strongly bound state during the ATPase cycle) and varying the nucleotide conditions, we show that the microtubule attachment times of the two opposing motors as well as their relative numbers determine which motor predominates in this assay. Together, these findings are consistent with a model in which kinesin-induced movement of a microtubule induces a negative strain in attached dyneins which causes them to dissociate before entering a force-generating state (and vice versa); reversals in the direction of transport may require the temporary dissociation of the transporting motor from the microtubule. The bidirectional movements described here are also remarkably similar to the back-and-forth movements of chromosomes during mitosis and membrane vesicles in fibroblasts. These results suggest that the underlying mechanical properties of motor proteins, at least in part, may be responsible for reversals in microtubule-based transport observed in cells.
- Tiezzi A, Moscatelli A, Cai G, Bartalesi A, Cresti M
- An immunoreactive homolog of mammalian kinesin in Nicotiana tabacum pollen tubes.
- Cell Motil Cytoskeleton. 1992; 21: 132-7
- Display abstract
A cytoskeletal apparatus is involved in the movement of vesicles, organelles, and gametes in the pollen tube. The function of microfilaments has been defined quite precisely, but the role of microtubules needs to be further clarified. On the basis of immunological and biochemical investigations, we have identified a polypeptide showing common properties with kinesin, a microtubule-based motor mainly described in nonplant tissues, in the pollen tube of Nicotiana tabacum. Like mammalian kinesin, the kinesin-immunoreactive homolog from Nicotiana tabacum pollen tubes binds to mammalian microtubules in an AMP-PNP dependent manner. The kinesin-like component is likely to be involved in the movement of vesicular material in the growing pollen tube.
- Vale RD
- Microtubule motors: many new models off the assembly line.
- Trends Biochem Sci. 1992; 17: 300-4
- Display abstract
A far greater variety of microtubule-based motors populate the interior of most eukaryotic cells than was ever imagined, and the inventory of these proteins is growing each year. The discovery of new motors, however, has raised many questions of how cells use their arsenal of force-generating machines. The ability to apply genetics, bacterial expression, biochemistry and in vitro motility assays to study motor proteins provides new opportunities for examining these problems at a molecular level.
- Green LA, Kaplan MP, Liem RK
- Kinesin heavy chain from bovine brain and Drosophila appear to be highly homologous molecules.
- J Neurosci Res. 1991; 28: 151-5
- Display abstract
A microtubule-enriched fraction was prepared from bovine white matter, and kinesin and other microtubule-associated proteins were extracted from taxol-stabilized microtubules by homogenization and ultracentrifugation in the presence of nucleotides (guanosine triphosphate and adenosine triphosphate). The kinesin-enriched fractions were subjected to preparative SDS-PAGE, and the band representing the kinesin heavy chain was excised, homogenized, and subjected to partial enzymatic digestion with Staphylococcus aureus V8 protease. Four peptides were selected for sequence analysis and compared to the previously published sequence for the Drosophila kinesin heavy chain (Yang JT, Laymon RA, Goldstein LSB, Cell 56:879-889, 1989). All four peptides matched closely with portions of the Drosophila sequence corresponding to the central, alpha-helical domain. Total amino acid composition analysis of bovine kinesin heavy chain also reveals a high degree of homology to the Drosophila sequence.
- Schliwa M, Shimizu T, Vale RD, Euteneuer U
- Nucleotide specificities of anterograde and retrograde organelle transport in Reticulomyxa are indistinguishable.
- J Cell Biol. 1991; 112: 1199-203
- Display abstract
Membrane-bound organelles move bidirectionally along microtubules in the freshwater ameba, Reticulomyxa. We have examined the nucleotide requirements for transport in a lysed cell model and compared them with kinesin and dynein-driven motility in other systems. Both anterograde and retrograde transport in Reticulomyxa show features characteristic of dynein but not of kinesin-powered movements: organelle transport is reactivated only by ATP and no other nucleoside triphosphates; the Km and Vmax of the ATP-driven movements are similar to values obtained for dynein rather than kinesin-driven movement; and of 15 ATP analogues tested for their ability to promote organelle transport, only 4 of them did. This narrow specificity resembles that of dynein-mediated in vitro transport and is dissimilar to the broad specificity of the kinesin motor (Shimizu, T., K. Furusawa, S. Ohashi, Y. Y. Toyoshima, M. Okuno, F. Malik, and R. D. Vale. 1991. J. Cell Biol. 112: 1189-1197). Remarkably, anterograde and retrograde organelle transport cannot be distinguished at all with respect to nucleotide specificity, kinetics of movement, and the ability to use the ATP analogues. Since the "kinetic fingerprints" of the motors driving transport in opposite directions are indistinguishable, the same type of motor(s) may be involved in the two directions of movement.
- Song YH, Heins S, Mandelkow E, Mandelkow EM
- Aluminum fluoride, microtubule stability, and kinesin rigor.
- J Cell Sci Suppl. 1991; 14: 147-50
- Display abstract
Aluminum fluoride may be used both to stabilize microtubules and to induce strong binding of kinesin, thus circumventing the need for taxol and AMP-PNP in kinesin preparations.
- Endow SA
- The emerging kinesin family of microtubule motor proteins.
- Trends Biochem Sci. 1991; 16: 221-5
- Display abstract
A family of proteins related to the microtubule motor, kinesin, is emerging. Members of this family, which includes both plus- and minus-end motors, are involved in nuclear functions such as nuclear fusion after karyogamy, spindle pole-body separation and chromosome segregation, as well as in transport in neuronal cells.
- Vallee R
- Chromosome kinetics. Movement on two fronts.
- Nature. 1991; 351: 187-8
- Buster D, Scholey JM
- Purification and assay of kinesin from sea urchin eggs and early embryos.
- J Cell Sci Suppl. 1991; 14: 109-15
- Display abstract
This paper describes the procedures used to purify the microtubule motor, kinesin, from mitotic cells, namely sea urchin eggs and cleavage stage embryos, and describes methods for assaying its motor activity.
- Weiss DG, Seitz-Tutter D, Langford GM
- Characteristics of the motor responsible for the gliding of native microtubules from squid axoplasm.
- J Cell Sci Suppl. 1991; 14: 157-61
- Display abstract
Nucleotide-dependent movement of native microtubules (nMTs) in squid axoplasm has biochemical and biophysical characteristics of kinesin-driven motility. However, the high vanadate and N-ethylmaleimide sensitivity and the velocity demonstrate that the properties of the native motile system differ considerably from those of purified kinesin preparations.
- Brady ST, Pfister KK
- Kinesin interactions with membrane bounded organelles in vivo and in vitro.
- J Cell Sci Suppl. 1991; 14: 103-8
- Display abstract
The ability of kinesin to interact with microtubules in a nucleotide-dependent manner and mediate microtubule-based motility has received the greatest amount of attention to date. Several lines of experimentation are now beginning to examine the interaction with membrane-bounded organelles. Immunochemical, biochemical and morphological approaches have shown that kinesin is associated with some, but not all, classes of membrane-bounded organelles found in cells. Similarly, evidence suggests that the distal portion of the rod and the tail portions of the kinesin heavy chain as well as the kinesin light chains may be important for the interaction with membrane surfaces. As a substantial amount of information about the molecular structure and biochemistry of kinesin has become available, the functional implications of interactions with membrane structures in vivo are being addressed.
- Marya PK, Fraylich PE, Flood CR, Rao R, Eagles PA
- Studies using a fluorescent analogue of kinesin.
- J Cell Sci Suppl. 1991; 14: 139-42
- Display abstract
The microtubule motor protein kinesin has been conjugated with 5-iodoacetamido fluorescein (5-IAF). The analogue, AF-kinesin, supports organelle motility and the movement of microtubules.
- Saxton WM, Hicks J, Goldstein LS, Raff EC
- Kinesin heavy chain is essential for viability and neuromuscular functions in Drosophila, but mutants show no defects in mitosis.
- Cell. 1991; 64: 1093-102
- Display abstract
The in vivo function of the microtubule motor protein kinesin was examined in Drosophila using genetics and immunolocalization. Kinesin heavy chain mutations (khc) cause abnormal behavior and lethality. Mutant larvae exhibit loss of mobility and tactile responsiveness in the most posterior segments, followed by general paralysis and death during larval or pupal development. Adults homozygous for a temperature-sensitive allele also exhibit a loss in mobility and sensory responses. The data indicate that kinesin function is essential and suggest that kinesin has an important role in the neuromuscular system, perhaps as a motor for axonal transport. The possibility of more general cellular functions remains open, but observation of embryogenesis and morphogenesis in khc mutants suggests that mitosis and the cell cycle can proceed in spite of impaired kinesin function. Immunolocalization suggests that kinesin may have some general cellular functions but that it is not a major component of mitotic spindles.
- Kuo SC, Gelles J, Steuer E, Sheetz MP
- A model for kinesin movement from nanometer-level movements of kinesin and cytoplasmic dynein and force measurements.
- J Cell Sci Suppl. 1991; 14: 135-8
- Display abstract
Our detailed measurements of the movements of kinesin- and dynein-coated latex beads have revealed several important features of the motors which underlie basic mechanical aspects of the mechanisms of motor movements. Kinesin-coated beads will move along the paths of individual microtubule protofilaments with high fidelity and will pause at 4 nm intervals along the microtubule axis under low ATP conditions. In contrast, cytoplasmic dynein-coated beads move laterally across many protofilaments as they travel along the microtubule, without any regular pauses, suggesting that the movements of kinesin-coated beads are not an artefact of the method. These kinesin bead movements suggest a model for kinesin movement in which the two heads walk along an individual protofilament in a hand-over-hand fashion. A free head would only be able to bind to the next forward tubulin subunit on the protofilament and its binding would pull off the trailing head to start the cycle again. This model is consistent with the observed cooperativity between the heads and with the movement by single dimeric molecules. Several testable predictions of the model are that kinesin should be able to bind to both alpha and beta tubulin and that the length of the neck region of the molecule should control the off-axis motility. In this article, we describe the technology for measuring nanometer-level movements and the force generated by the kinesin molecule.
- McDonald HB, Stewart RJ, Goldstein LS
- The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor.
- Cell. 1990; 63: 1159-65
- Display abstract
The Drosophila ncd gene is required for chromosome segregation during female meiosis. Previous analyses suggested that the ncd gene encoded a protein with sequence similarity to the kinesin motor domain, which suggested that, like kinesin, the ncd protein might be a plus end-directed microtubule motor. Here we describe the expression of ncd protein in E. coli and the initial characterization of the ncd protein's motor properties. The ncd protein is indeed a microtubule motor, but the polarity of movement is minus end directed. The ncd protein also has microtubule bundling activity. These findings limit possible models for the in vivo functions of the ncd protein and suggest that motor proteins with similar sequence can generate movement in opposite directions along a microtubule.
- Cohn SA
- The mechanochemistry of kinesin. A review.
- Mol Chem Neuropathol. 1990; 12: 83-94
- Display abstract
The mechanochemical protein kinesin is believed to play an important role in intracellular vesicle movements, including the anterograde motion of axoplasmic transport. This article reviews some of the pharmacological and biochemical information about kinesin, particularly with respect to the properties of nucleotide-dependent microtubule binding, microtubule-activated ATPase activity, and kinesin-driven microtubule translocation. The implications of this information on the mechanochemical mechanisms of kinesin are discussed and a brief comparison of kinesin with two other mechanochemical proteins, myosin and dynein, is also given.
- Malik F, Vale R
- Cell biology. A new direction for kinesin.
- Nature. 1990; 347: 713-4
- Block SM, Goldstein LS, Schnapp BJ
- Bead movement by single kinesin molecules studied with optical tweezers.
- Nature. 1990; 348: 348-52
- Display abstract
Kinesin, a mechanoenzyme that couples ATP hydrolysis to movement along microtubules, is thought to power vesicle transport and other forms of microtubule-based motility. Here, microscopic silica beads were precoated with carrier protein, exposed to low concentrations of kinesin, and individually manipulated with a single-beam gradient-force optical particle trap ('optical tweezers') directly onto microtubules. Optical tweezers greatly improved the efficiency of the bead assay, particularly at the lowest kinesin concentrations (corresponding to approximately 1 molecule per bead). Beads incubated with excess kinesin moved smoothly along a microtubule for many micrometres, but beads carrying from 0.17-3 kinesin molecules per bead, moved, on average, only about 1.4 microns and then spontaneously released from the microtubule. Application of the optical trap directly behind such moving beads often pulled them off the microtubule and back into the centre of the trap. This did not occur when a bead was bound by an AMP.PNP-induced rigor linkage, or when beads were propelled by several kinesin molecules. Our results are consistent with a model in which kinesin detaches briefly from the microtubule during a part of each mechanochemical cycle, rather than a model in which kinesin remains bound at all times.
- Hollenbeck PJ, Swanson JA
- Radial extension of macrophage tubular lysosomes supported by kinesin.
- Nature. 1990; 346: 864-6
- Display abstract
The centrifugal elongation of membranes to form extended tubular structures is a widespread form of intracellular organelle movement. Tubular lysosomes and the endoplasmic reticulum, for example, undergo such extension in association with microtubules, and this process has been mimicked in vitro by combining purified microtubules with isolated membranes and the mechanochemical ATPase kinesin. This, along with evidence that kinesin is associated with the endoplasmic reticulum, has led to the suggestion that kinesin provides the motive force for the formation and maintenance of elongated tubulovesicular structures in cells. We have addressed this hypothesis in murine macrophages, which have prominent tubular lysosomes whose form depends on the integrity of microtubules. Here we report that two antikinesin antibodies which disrupt in vitro motility will each cause centripetal collapse of the array of tubular lysosomes when scrape-loaded into macrophages. To our knowledge this provides the first in vivo evidence that kinesin is responsible for extension of tubulovesicular structures along microtubules.
- Schnapp BJ, Crise B, Sheetz MP, Reese TS, Khan S
- Delayed start-up of kinesin-driven microtubule gliding following inhibition by adenosine 5'-[beta,gamma-imido]triphosphate.
- Proc Natl Acad Sci U S A. 1990; 87: 10053-7
- Display abstract
Kinesin is a microtubule-activated ATPase that moves objects toward the plus end of microtubules and makes microtubules glide along a glass surface. Here we investigate a remarkable effect of the nonhydrolyzable analogue of ATP, adenosine 5'-[beta,gamma-imido]triphosphate (p[NH]ppA), on kinesin-driven microtubule gliding. Microtubule gliding that has been blocked by rapid replacement of ATP with p[NH]ppA requires 1-2 min of exposure to ATP before microtubule gliding resumes. This latency is not shortened by prolonged washing of p[NH]ppA-blocked microtubules in nucleotide-free buffer for up to 15 min, suggesting that ATP binding to a second nucleotide binding site on kinesin triggers the release of bound p[NH]ppA. To test this hypothesis, the release of [3H]p[NH]ppA from kinesin-microtubule complexes was followed in parallel biochemical assays. In nucleotide-free buffer, the bound p[NH]ppA was released over several hours from the complexes. However, addition of ATP caused the release of p[NH]ppA from the kinesin-microtubule complexes within 2 min, which was similar to the latent period for start-up of microtubule gliding after p[NH]ppA inhibition. The stoichiometry of p[NH]ppA bound per kinesin heavy chain at saturation was estimated to be approximately 1:2. These results suggest a model in which each molecule of kinesin has at least two nucleotide binding sites that alternately bind nucleotide.
- Spudich JA
- Optical trapping: Motor molecules in motion.
- Nature. 1990; 348: 284-5
- Marya PK, Fraylich PE, Eagles PA
- Characterization of an active, fluorescein-labelled kinesin.
- Eur J Biochem. 1990; 193: 39-45
- Display abstract
Kinesin was isolated from bovine intradural nerve roots and conjugated with 5-(iodoacetamido)fluorescein. The modified kinesin (AF-kinesin) supports the movement of organelles along microtubules at rates comparable with those obtained using unmodified kinesin. AF-kinesin was purified by high-performance liquid chromatography. SDS/PAGE analysis of the purified fraction showed the presence of a fluorescent band at the position of the 125-kDa kinesin heavy chain. This protein promoted microtubule gliding with MgATP and with MgGTP at rates comparable to those of unlabelled kinesin. AF-kinesin had a fluorescein/protein ratio of one. Video microscopy at low light levels was used to monitor the interactions between the analogue and microtubules. AF-kinesin binds to microtubules in the presence of adenosine 5'-[beta, gamma-imino]triphosphate or ADP. Brief incubation of the microtubule. AF-kinesin complex with 10 mM ATP or GTP completely removes the labelled molecule. AF-kinesin can be inactivated in its ability to cause microtubule gliding by irradiating it with light that bleaches the bound fluorophore. When the protein is damaged in this way it still binds to microtubules and does so in the presence of ATP.
- Ashkin A, Schutze K, Dziedzic JM, Euteneuer U, Schliwa M
- Force generation of organelle transport measured in vivo by an infrared laser trap.
- Nature. 1990; 348: 346-8
- Display abstract
Organelle transport along microtubules is believed to be mediated by organelle-associated force-generating molecules. Two classes of microtubule-based organelle motors have been identified: kinesin and cytoplasmic dynein. To correlate the mechanochemical basis of force generation with the in vivo behaviour of organelles, it is important to quantify the force needed to propel an organelle along microtubules and to determine the force generated by a single motor molecule. Measurements of force generation are possible under selected conditions in vitro, but are much more difficult using intact or reactivated cells. Here we combine a useful model system for the study of organelle transport, the giant amoeba Reticulomyxa, with a novel technique for the non-invasive manipulation of and force application to subcellular components, which is based on a gradient-force optical trap, also referred to as 'optical tweezers'. We demonstrate the feasibility of using controlled manipulation of actively translocating organelles to measure direct force. We have determined the force driving a single organelle along microtubules, allowing us to estimate the force generated by a single motor to be 2.6 x 10(-7) dynes.
- Yang JT, Saxton WM, Stewart RJ, Raff EC, Goldstein LS
- Evidence that the head of kinesin is sufficient for force generation and motility in vitro.
- Science. 1990; 249: 42-7
- Display abstract
Kinesin is a mechanochemical protein that converts the chemical energy in adenosine triphosphate into mechanical force for movement of cellular components along microtubules. The regions of the kinesin molecule responsible for generating movement were determined by studying the heavy chain of Drosophila kinesin, and its truncated forms, expressed in Escherichia coli. The results demonstrate that (i) kinesin heavy chain alone, without the light chains and other eukaryotic factors, is able to induce microtubule movement in vitro, and (ii) a fragment likely to contain only the kinesin head is also capable of inducing microtubule motility. Thus, the amino-terminal 450 amino acids of kinesin contain all the basic elements needed to convert chemical energy into mechanical force.
- Gelfand VI
- Cytoplasmic microtubular motors.
- Curr Opin Cell Biol. 1989; 1: 63-6
- Yang JT, Laymon RA, Goldstein LS
- A three-domain structure of kinesin heavy chain revealed by DNA sequence and microtubule binding analyses.
- Cell. 1989; 56: 879-89
- Display abstract
The structure and function of kinesin heavy chain from D. melanogaster have been studied using DNA sequence analysis and analysis of the properties of truncated kinesin heavy chain synthesized in vitro. Analysis of the sequence suggests the existence of a 50 kd globular amino-terminal domain that contains an ATP binding consensus sequence, followed by another 50-60 kd domain that has sequence characteristics consistent with the ability to fold into an alpha helical coiled coil. The properties of amino- and carboxy-terminally truncated kinesin heavy chains synthesized in vitro reveal that a 60 kd amino-terminal fragment has the nucleotide-dependent microtubule binding activities of the intact kinesin heavy chain, and hence is likely to be a "motor" domain. Finally, the sequence data indicate the presence of a small carboxy-terminal domain. Because it is located at the end of the molecule away from the putative "motor" domain, we propose that this domain is responsible for interactions with other proteins, vesicles, or organelles. These data suggest that kinesin has an organization very similar to that of myosin even though there are no obvious sequence similarities between the two molecules.
- Howard J, Hudspeth AJ, Vale RD
- Movement of microtubules by single kinesin molecules.
- Nature. 1989; 342: 154-8
- Display abstract
Kinesin is a motor protein that uses energy derived from ATP hydrolysis to move organelles along microtubules. Using a new technique for measuring the movement produced in vitro by individual kinesin molecules, it is shown that a single kinesin molecule can move a microtubule for several micrometers. New information about the mechanism of force generation by kinesin is presented.
- McCaffrey G, Vale RD
- Identification of a kinesin-like microtubule-based motor protein in Dictyostelium discoideum.
- EMBO J. 1989; 8: 3229-34
- Display abstract
Dictyostelium discoideum, a unicellular eukaryote amenable to both biochemical and genetic dissection, provides an attractive system for studying microtubule-based transport. In this work, we have identified microtubule-based motor activities in Dictyostelium cell extracts and have partially purified a protein that induces microtubule translocation along glass surfaces. This protein, which sediments at approximately 9S in sucrose density gradients and is composed of a 105 kd polypeptide, generates anterograde movement along microtubules that is insensitive to 5 mM NEM (N-ethyl-maleimide) but sensitive to 200 microM vanadate, and has similar nucleotide-dependent microtubule binding properties to those of kinesins purified from mammals, sea urchin and Drosophila. This kinesin-like molecule from Dictyostelium, however, is immunologically distinct from bovine and squid neuronal kinesins and supports microtubule movement on glass at four-fold greater velocities (2.0 versus 0.5 microns/sec). Furthermore, AMP-PNP (adenylyl imidodiphosphate), which promotes attachment of previously characterized kinesins to microtubules, decreases the affinity of the Dictyostelium kinesin homolog for microtubules. Thus, an AMP-PNP-induced rigor binding may not be a characteristic of kinesins from lower eukaryotes.
- Gelles J, Schnapp BJ, Sheetz MP
- Tracking kinesin-driven movements with nanometre-scale precision.
- Nature. 1988; 331: 450-3
- Display abstract
Several enzyme complexes drive cellular movements by coupling free energy-liberating chemical reactions to the production of mechanical work. A key goal in the study of these systems is to characterize at the molecular level mechanical events associated with individual reaction steps in the catalytic cycles of single enzyme molecules. Ideally, one would like to measure movements driven by single (or a few) enzyme molecules with sufficient temporal resolution and spatial precision that these events can be directly observed. Kinesin, a force-generating ATPase involved in microtubule-based intracellular organelle transport, will drive the unidirectional movement of microscopic plastic beads along microtubules in vitro. Under certain conditions, a few (less than or equal to 10) kinesin molecules may be sufficient to drive either bead movement or organelle transport. Here we describe a method for determining precise positional information from light-microscope images. The method is applied to measure kinesin-driven bead movements in vitro with a precision of 1-2 nm. Our measurements reveal basic mechanical features of kinesin-driven movements along the microtubule lattice, and place significant constraints on possible molecular mechanisms of movement.
- Schroer TA, Schnapp BJ, Reese TS, Sheetz MP
- The role of kinesin and other soluble factors in organelle movement along microtubules.
- J Cell Biol. 1988; 107: 1785-92
- Display abstract
Kinesin is a force-generating ATPase that drives the sliding movement of microtubules on glass coverslips and the movement of plastic beads along microtubules. Although kinesin is suspected to participate in microtubule-based organelle transport, the exact role it plays in this process is unclear. To address this question, we have developed a quantitative assay that allows us to determine the ability of soluble factors to promote organelle movement. Salt-washed organelles from squid axoplasm exhibited a nearly undetectable level of movement on purified microtubules. Their frequency of movement could be increased greater than 20-fold by the addition of a high speed axoplasmic supernatant. Immunoadsorption of kinesin from this supernatant decreased the frequency of organelle movement by more than 70%; organelle movements in both directions were markedly reduced. Surprisingly, antibody purified kinesin did not promote organelle movement either by itself or when it was added back to the kinesin-depleted supernatant. This result suggested that other soluble factors necessary for organelle movement were removed along with kinesin during immunoadsorption of the supernatant. A high level of organelle motor activity was recovered in a high salt eluate of the immunoadsorbent that contained only little kinesin. On the basis of these results we propose that organelle movement on microtubules involves other soluble axoplasmic factors in addition to kinesin.
- Huitorel P
- From cilia and flagella to intracellular motility and back again: a review of a few aspects of microtubule-based motility.
- Biol Cell. 1988; 63: 249-58
- Display abstract
Ciliary or flagellar movement is the model of microtubule-dependent motility, the best studied at the molecular level. It is based on the relative sliding of outer doublets of microtubules that are linked at their proximal end to the basal structure and interconnected by associated proteins, among which dynein ATPase is at the origin of the movement. It is regulated from inside and outside media by various diffusible factors such as Ca2+, cyclic adenosine monophosphate (cAMP), polypeptides and so on (see other conferences presented during this meeting). Other motility processes are based on microtubules: vesicle and organelle transport through the cytoplasm (axonal flow in neurons, pigment granule movements in fish chromatophores, movements of particles along heliozoan axopods, etc.) could be mediated by microtubule motors such as kinesin or MAP 1C. Kinesin and MAP 1C, like dynein, are proteins that bind to microtubules and show an ATPase activity associated with force production. They differ from each other by their structure, and biochemical and pharmacological properties. The movements of chromosomes during mitosis and meiosis have long been studied, but are still poorly understood at the molecular level; this topic will be discussed in the light of recent data. Other constituents of the cytoskeleton are certainly involved in cellular motility: actin microfilaments and their motor myosin, intermediate filaments, non-actin filaments, all organized around the Microtubule Organizing Center (MTOC). As more information becomes available, it seems increasingly obvious that these various networks are closely interconnected and that each component probably modulates, resists, or favors properties of its partners, contributing to cellular and intracellular motility.
- Penningroth SM, Rose PM, Peterson DD
- Evidence that the 116 kDa component of kinesin binds and hydrolyzes ATP.
- FEBS Lett. 1987; 222: 204-10
- Display abstract
Kinesin was prepared from bovine brain as described previously for studies of translocation. A major component of kinesin, (116 kDa) was shown to undergo specific photocrosslinking with [alpha-32P]ATP, indicating it was an ATP-binding polypeptide. A low ATPase activity associated with kinesin was stimulated up to 5-fold by microtubules to a specific activity of 14 nmol . min-1 . mg-1. N-Ethylmaleimide inhibited both [alpha-32P]ATP binding to the 116 kDa polypeptide and microtubule-stimulated ATPase activity, suggesting that the 116 kDa polypeptide was the catalytic subunit of kinesin. Though the ATPase activity associated with kinesin is low, it may be sufficient to support motility assuming it is coupled to the velocity of translocation.
- Porter ME et al.
- Characterization of the microtubule movement produced by sea urchin egg kinesin.
- J Biol Chem. 1987; 262: 2794-802
- Display abstract
We have used an in vitro assay to characterize some of the motile properties of sea urchin egg kinesin. Egg kinesin is purified via 5'-adenylyl imidodiphosphate-induced binding to taxol-assembled microtubules, extraction from the microtubules in ATP, and gel filtration chromatography (Scholey, J. M., Porter, M. E., Grissom, P. M., and McIntosh, J. R. (1985) Nature 318, 483-486). This partially purified kinesin is then adsorbed to a glass coverslip, mixed with microtubules and ATP, and viewed by video-enhanced differential interference contrast microscopy. The microtubule translocating activity of the purified egg kinesin is qualitatively similar to the analogous activity observed in crude extracts of sea urchin eggs and resembles the activity of neuronal kinesin with respect to both the maximal rate (greater than 0.5 micron/s) and the direction of movement. Axonemes glide on a kinesin-coated coverslip toward their minus ends, and kinesin-coated beads translocate toward the plus ends of centrosome microtubules. Sea urchin egg kinesin is inhibited by high concentrations of SH reagents ([N-ethylmaleimide] greater than 3-5 mM), vanadate greater than 50 microM, and [nonhydrolyzable nucleotides] greater than or equal to [MgATP]. The nucleotide requirement of sea urchin egg kinesin is fairly broad (ATP greater than GTP greater than ITP), and the rate of microtubule movement increases in a saturable fashion with the [ATP]. We conclude that the motile activity of egg kinesin is indistinguishable from that of neuronal kinesin. We propose that egg kinesin may be associated with microtubule-based motility in vivo.
- Sheetz MP, Vale R, Schnapp B, Schroer T, Reese T
- Vesicle movements and microtubule-based motors.
- J Cell Sci Suppl. 1986; 5: 181-8
- Display abstract
The movements of many cytoplasmic vesicles follow the paths of microtubules, some moving in one direction and others moving in the opposite direction on the same microtubule. Recently we have isolated one cytoplasmic motor, kinesin, and defined another, the axoplasmic retrograde factor, both of which are capable of powering anionic latex beads in both directions along polar microtubule arrays. Evidence summarized here supports but does not prove the hypothesis that kinesin and the retrograde motors are indeed responsible for powering vesicle movements.
- Hill TL
- Kinetic diagram and free energy diagram for kinesin in microtubule-related motility.
- Proc Natl Acad Sci U S A. 1986; 83: 3326-30
- Display abstract
The theoretical formalism that shows how biochemistry (ATPase activity) is related to mechanics in muscle contraction can be extended to the role of kinesin in microtubule-related motility. The main features added are the freedom of kinesin molecules to come and go from the motility complex and the small number of operative kinesin molecules in some systems. The starting points for this kind of approach are the kinetic diagram of biochemical states and the corresponding free energy diagram for these states. These topics are introduced and discussed here in relation to those systems that are presumed to use kinesin.
- Crowley PH, Benham CJ, Lenhart SM, Morgan JL
- Peripheral doublet microtubules and wave generation in eukaryotic flagella.
- J Theor Biol. 1981; 93: 769-84