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NORMAL GENOMIC

• Danino D, Hinshaw JE
• Dynamin family of mechanoenzymes.
• Curr Opin Cell Biol. 2001; 13: 454-60
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• The dynamin family of proteins is continually growing, and in recent years members have been localized to areas of mitochondrial fission, plant phragmoplasts and chloroplasts, and viral ribonucleoprotein complexes. All the dynamin-like proteins examined to-date appear to assemble into oligomers, such as rings or spirals; however, it remains to be determined if a global mechanism of action exists. Even the role of dynamin in vesicle formation remains controversial as to whether it behaves as a molecular switch or as a mechanochemical enzyme.

• Binns DD et al.
• The mechanism of GTP hydrolysis by dynamin II: a transient kinetic study.
• Biochemistry. 2000; 39: 7188-96
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• Dynamin II is a 98 kDa protein (870 amino acids) required for the late stages of clathrin-mediated endocytosis. The GTPase activity of dynamin is required for its function in the budding stages of receptor-mediated endocytosis and synaptic vesicle recycling. This activity is stimulated when dynamin self-associates on multivalent binding surfaces, such as microtubules and anionic liposomes. We first investigated the oligomeric state of dynamin II by analytical ultracentrifuge sedimentation equilibrium measurements at high ionic strength and found that it was best described by a monomer-tetramer equilibrium. We then studied the intrinsic dynamin GTPase mechanism by using a combination of fluorescence stopped-flow and HPLC methods using the fluorescent analogue of GTP, mantdGTP (2'-deoxy-3'-O-(N-methylanthraniloyl) guanosine-5'-triphosphate), under the same ionic strength conditions. The results are interpreted as showing that mantdGTP binds to dynamin in a two-step mechanism. The dissociation constant of mantdGTP binding to dynamin, calculated from the ratio of the off-rate to the on-rate (k(off)/k(on)), was 0.5 &mgr;M. Cleavage of mantdGTP then occurs to mantdGDP and P(i) followed by the rapid release of mantdGDP and P(i). No evidence of reversibility of hydrolysis was observed. The cleavage step itself is the rate-limiting step in the mechanism. This mechanism more closely resembles that of the Ras family of proteins involved in cell signaling than the myosin ATPase involved in cellular motility.

• Burger KN, Demel RA, Schmid SL, de Kruijff B
• Dynamin is membrane-active: lipid insertion is induced by phosphoinositides and phosphatidic acid.
• Biochemistry. 2000; 39: 12485-93
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• Dynamin is a large GTPase involved in the regulation of membrane constriction and fission during receptor-mediated endocytosis. Dynamin contains a pleckstrin-homology domain which is essential for endocytosis and which binds to anionic phospholipids. Here, we show for the first time that dynamin is a membrane-active molecule capable of penetrating into the acyl chain region of membrane lipids. Lipid penetration is strongly stimulated by phosphatidic acid (PA), phosphatidylinositol 4-phosphate, and phosphatidylinositol 4, 5-bisphosphate. Though binding is more efficient in the presence of the phosphoinositides, a much larger part of the dynamin molecule penetrates into PA-containing mixed-lipid systems. Thus, local lipid metabolism will dramatically influence dynamin-lipid interactions, and dynamin-lipid interactions are likely to play an important role in dynamin-dependent endocytosis. Our data suggest that dynamin is directly involved in membrane destabilization, a prerequisite to membrane fission.

• Daly C, Sugimori M, Moreira JE, Ziff EB, Llinas R
• Synaptophysin regulates clathrin-independent endocytosis of synaptic vesicles.
• Proc Natl Acad Sci U S A. 2000; 97: 6120-5
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• The GTPase dynamin I is required for synaptic vesicle (SV) endocytosis. Our observation that dynamin binds to the SV protein synaptophysin in a Ca(2+)-dependent fashion suggested the possibility that a dynamin/synaptophysin complex functions in SV recycling. In this paper we show that disruption of the dynamin/synaptophysin interaction by peptide injection into the squid giant synapse preterminal results in a decrease in transmitter release during high-frequency stimulation, indicating an inhibition of SV recycling. Electron microscopy of these synapses reveals a depletion of SVs, demonstrating a block of vesicle retrieval after fusion. In addition, we observed an increase in clathrin-coated vesicles, indicating that the peptide does not block clathrin-dependent endocytosis. We conclude that the dynamin/synaptophysin complex functions in a clathrin-independent mechanism of SV endocytosis that is required for efficient synaptic transmission.

• Gad H et al.
• Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin.
• Neuron. 2000; 27: 301-12
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• Coordination between sequential steps in synaptic vesicle endocytosis, including clathrin coat formation, fission, and uncoating, appears to involve proteinprotein interactions. Here, we show that compounds that disrupt interactions of the SH3 domain of endophilin with dynamin and synaptojanin impair synaptic vesicle endocytosis in a living synapse. Two distinct endocytic intermediates accumulated. Free clathrin-coated vesicles were induced by a peptide-blocking endophilin's SH3 domain and by antibodies to the proline-rich domain (PRD) of synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. We suggest that the SH3 domain of endophilin participates in both fission and uncoating and that it may be a key component of a molecular switch that couples the fission reaction to uncoating.

• McNiven MA, Kim L, Krueger EW, Orth JD, Cao H, Wong TW
• Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape.
• J Cell Biol. 2000; 151: 187-98
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• The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin-SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)DeltaPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.

• Qualmann B, Kelly RB
• Syndapin isoforms participate in receptor-mediated endocytosis and actin organization.
• J Cell Biol. 2000; 148: 1047-62
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• Syndapin I (SdpI) interacts with proteins involved in endocytosis and actin dynamics and was therefore proposed to be a molecular link between the machineries for synaptic vesicle recycling and cytoskeletal organization. We here report the identification and characterization of SdpII, a ubiquitously expressed isoform of the brain-specific SdpI. Certain splice variants of rat SdpII in other species were named FAP52 and PACSIN 2. SdpII binds dynamin I, synaptojanin, synapsin I, and the neural Wiskott-Aldrich syndrome protein (N-WASP), a stimulator of Arp2/3 induced actin filament nucleation. In neuroendocrine cells, SdpII colocalizes with dynamin, consistent with a role for syndapin in dynamin-mediated endocytic processes. The src homology 3 (SH3) domain of SdpI and -II inhibited receptor-mediated internalization of transferrin, demonstrating syndapin involvement in endocytosis in vivo. Overexpression of full-length syndapins, but not the NH(2)-terminal part or the SH3 domains alone, had a strong effect on cortical actin organization and induced filopodia. This syndapin overexpression phenotype appears to be mediated by the Arp2/3 complex at the cell periphery because it was completely suppressed by coexpression of a cytosolic COOH-terminal fragment of N-WASP. Consistent with a role in actin dynamics, syndapins localized to sites of high actin turnover, such as filopodia tips and lamellipodia. Our results strongly suggest that syndapins link endocytosis and actin dynamics.

• Dong J, Misselwitz R, Welfle H, Westermann P
• Expression and purification of dynamin II domains and initial studies on structure and function.
• Protein Expr Purif. 2000; 20: 314-23
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• Dynamin II, a large GTP-binding protein, is involved in endocytosis and in vesicle formation at the trans-Golgi network. To further elucidate functions of dynamin II, the pleckstrin homology domain (PHD), the proline-rich domain (PRD), and the C-terminal part of dynamin II (dynamin(500-870)) were expressed in Escherichia coli. The PHD, tagged C-terminally by a (His)(6) peptide, was expressed to 15% of cellular proteins and could be purified on nickel-chelating agarose. On the contrary, the PRD and dynamin(500-870) had to be tagged with a (His)(6) peptide at the N-terminus to bind to nickel-chelating agarose. Additional tagging with the S-peptide, which forms a stable complex with immobilized S-protein, allowed removal of strongly interacting E. coli proteins. Circular dichroic spectra indicate a structured recombinant PHD with a secondary structure content similar to that of the known PHD from dynamin I. The N-terminally tagged, recombinant PRD is unfolded but nevertheless binds specifically to the SH3 domain of amphiphysin II as well as to proteins extracted from rat brain. The described methods are suitable to isolate functionally active domains of dynamin II in sufficient amount and purity for further studies.

• Binns DD et al.
• Correlation between self-association modes and GTPase activation of dynamin.
• J Protein Chem. 1999; 18: 277-90
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• The GTPase activity of dynamin is obligatorily coupled, by a mechanism yet unknown, to the internalization of clathrin-coated endocytic vesicles. Dynamin oligomerizes in vitro and in vivo and both its mechanical and enzymatic activities appear to be mediated by this self-assembly. In this study we demonstrate that dynamin is characterized by a tetramer/monomer equilibrium with an equilibrium constant of 1.67 x 10(17) M(-3). Stopped-flow fluorescence experiments show that the association rate constant for 2'(3')-O-N-methylanthraniloyl (mant)GTP is 7.0 x 10(-5) M(-1) s(-1) and the dissociation rate constant is 2.1 s(-1), whereas the dissociation rate constant for mantdeoxyGDP is 93 s(-1). We also demonstrate the cooperativity of dynamin binding and GTPase activation on a microtubule lattice. Our results indicate that dynamin self-association is not a sufficient condition for the expression of maximal GTPase activity, which suggests that dynamin molecules must be in the proper conformation or orientation if they are to form an active oligomer.

• Okamoto M, Schoch S, Sudhof TC
• EHSH1/intersectin, a protein that contains EH and SH3 domains and binds to dynamin and SNAP-25. A protein connection between exocytosis and endocytosis?
• J Biol Chem. 1999; 274: 18446-54
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• In yeast two-hybrid screens for proteins that bind to SNAP-25 and may be involved in exocytosis, we isolated a protein called EHSH1 (for EH domain/SH3 domain-containing protein). Cloning of full-length cDNAs revealed that EHSH1 is composed of an N-terminal region with two EH domains, a central region that is enriched in lysine, leucine, glutamate, arginine, and glutamine (KLERQ domain), and a C-terminal region comprised of five SH3 domains. The third SH3 domain is alternatively spliced. Data bank searches demonstrated that EHSH1 is very similar to Xenopus and human intersectins and to human SH3P17. In addition, we identified expressed sequence tags that encode a second isoform of EHSH1, called EHSH2. EHSH1 is abundantly expressed in brain and at lower levels in all other tissues tested. In binding studies, we found that the central KLERQ domain of EHSH1 binds to recombinant or native brain SNAP-25 and SNAP-23. The C-terminal SH3 domains, by contrast, quantitatively interact with dynamin, a protein involved in endocytosis. Dynamin strongly binds to the alternatively spliced central SH3 domain (SH3C) and the two C-terminal SH3 domains (SH3D and SH3E) but not to the N-terminal SH3 domains (SH3A and SH3B). Immunoprecipitations confirmed that both dynamin and SNAP-25 are complexed to EHSH1 in brain. Our data suggest that EHSH1/intersectin may be a novel adaptor protein that couples endocytic membrane traffic to exocytosis. The ability of multiple SH3 domains in EHSH1 to bind to dynamin suggests that EHSH1 can cluster several dynamin molecules in a manner that is regulated by alternative splicing.

• McPherson PS
• Regulatory role of SH3 domain-mediated protein-protein interactions in synaptic vesicle endocytosis.
• Cell Signal. 1999; 11: 229-38
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• Src homology (SH) 3 domains are small modules found in a diverse array of proteins. The presence of an SH3 domain confers upon its resident protein the ability to interact with specific proline-rich sequences in protein binding partners. A major focus of research has highlighted a role for SH3 domain-mediated interactions in the regulation of signal transduction events. However, more recent data has suggested an important function for SH3 domains in vesicular trafficking. This review will focus on this newly emerging role with a particular emphasis on the molecular components involved in synaptic vesicle endocytosis and the regulatory role of SH3 domain-mediated protein-protein interactions in this process.

• Ringstad N et al.
• Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis.
• Neuron. 1999; 24: 143-54
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• Endophilin/SH3p4 is a protein highly enriched in nerve terminals that binds the GTPase dynamin and the polyphosphoinositide phosphatase synaptojanin, two proteins implicated in synaptic vesicle endocytosis. We show here that antibody-mediated disruption of endophilin function in a tonically stimulated synapse leads to a block in the invagination of clathrin-coated pits adjacent to the active zone and therefore to a block of synaptic vesicle recycling. We also show that in a cell-free system, endophilin is not associated with clathrin coats and is a functional partner of dynamin. Our findings suggest that endophilin is part of a biochemical machinery that acts in trans to the clathrin coat from early stages to vesicle fission.

• Lai MM et al.
• The calcineurin-dynamin 1 complex as a calcium sensor for synaptic vesicle endocytosis.
• J Biol Chem. 1999; 274: 25963-6
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• Exocytosis of synaptic vesicles is calcium-dependent, with synaptotagmin serving as the calcium sensor. Endocytosis of synaptic vesicles has also been postulated as a calcium-dependent process; however, an endocytic calcium sensor has not been found. We now report a physical association between the calcium-dependent phosphatase calcineurin and dynamin 1, a component of the synaptic endocytic machinery. The calcineurin-dynamin 1 interaction is calcium-dependent, with an EC(50) for calcium in the range of 0.1-0. 4 microM. Disruption of the calcineurin-dynamin 1 interaction inhibits clathrin-mediated endocytosis. Thus, the calcium-dependent formation of the calcineurin-dynamin 1 complex, delivered to the other endocytic coat proteins, provides a calcium-sensing mechanism that facilitates endocytosis.

• Li J, Schwarz TL
• Genetic evidence for an equilibrium between docked and undocked vesicles.
• Philos Trans R Soc Lond B Biol Sci. 1999; 354: 299-306
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• By using the shibire mutation to block endocytosis in a temperature-dependent fashion, we have manipulated the number of synaptic vesicles in a nerve terminal and have observed a remarkable proportionality of the number of quanta released to the size of the total vesicle pool. In the experiments described below we determine that approximately 0.3% of the vesicle pool is released per stimulus. The data suggest that the pool of readily releasable docked vesicles does not represent the saturation of a limiting number of release sites, but instead represents a subset of vesicles that is in equilibrium with the larger pool of vesicles. Before presenting this data and the significance of the finding for the regulation of neurotransmission, we will briefly review the use of Drosophila genetics as a tool for dissecting synaptic transmission.

• Kasai K, Shin HW, Shinotsuka C, Murakami K, Nakayama K
• Dynamin II is involved in endocytosis but not in the formation of transport vesicles from the trans-Golgi network.
• J Biochem (Tokyo). 1999; 125: 780-9
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• Dynamins are a family of approximately 100-kDa GTPases that are thought to play a pivotal role in the formation of endocytic coated vesicles. There are three dynamin genes in mammals: dynamin I is neuron-specific, dynamin II shows ubiquitous expression, and dynamin III is expressed in testis, brain, and lung. However, most studies on the functions of dynamins to date have been restricted to dynamin I. In the present study, we show that, like dynamin I, dynamin II is involved in receptor-mediated endocytosis. While this study was in progress, Jones et al. [Jones, S.M., Howell, K.E., Henley, J.R., Cao, H., and McNiven, M.A. (1998) Science 279, 573-577] reported that dynamin II is localized in the trans-Golgi network (TGN) and involved in the formation of constitutive transport vesicles and clathrin-coated vesicles from this compartment. However, immunofluorescence analyses and experiments using cells transfected with dominant-negative dynamin II failed to show any evidence for localization of dynamin II in the TGN or for its involvement in vesicle formation from this compartment. Our data thus indicate that dynamin II is involved in endocytosis but not in the formation of transport vesicles from the TGN.

• Qualmann B, Roos J, DiGregorio PJ, Kelly RB
• Syndapin I, a synaptic dynamin-binding protein that associates with the neural Wiskott-Aldrich syndrome protein.
• Mol Biol Cell. 1999; 10: 501-13
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• The GTPase dynamin has been clearly implicated in clathrin-mediated endocytosis of synaptic vesicle membranes at the presynaptic nerve terminal. Here we describe a novel 52-kDa protein in rat brain that binds the proline-rich C terminus of dynamin. Syndapin I (synaptic, dynamin-associated protein I) is highly enriched in brain where it exists in a high molecular weight complex. Syndapin I can be involved in multiple protein-protein interactions via a src homology 3 (SH3) domain at the C terminus and two predicted coiled-coil stretches. Coprecipitation studies and blot overlay analyses revealed that syndapin I binds the brain-specific proteins dynamin I, synaptojanin, and synapsin I via an SH3 domain-specific interaction. Coimmunoprecipitation of dynamin I with antibodies recognizing syndapin I and colocalization of syndapin I with dynamin I at vesicular structures in primary neurons indicate that syndapin I associates with dynamin I in vivo and may play a role in synaptic vesicle endocytosis. Furthermore, syndapin I associates with the neural Wiskott-Aldrich syndrome protein, an actin-depolymerizing protein that regulates cytoskeletal rearrangement. These characteristics of syndapin I suggest a molecular link between cytoskeletal dynamics and synaptic vesicle recycling in the nerve terminal.

• Staples RR, Ramaswami M
• Functional analysis of dynamin isoforms in Drosophila melanogaster.
• J Neurogenet. 1999; 13: 119-43
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• Dynamin and dynamin-like proteins are required for endocytosis, synaptic vesicle recycling and membrane trafficking. From the shibire locus in Drosophila melanogaster, six different isoforms of dynamin are generated by alternative splicing. However, the roles of the individual isoforms in cellular processes are unknown. To investigate functional differences among the dynamin isoforms, transgenic lines were generated that individually expressed each of 3 different isoforms under UASGAL4 control. The expression of the isoforms was controlled by neural promoter (elav)-driven GAL4, or by a shibire-promoter driven GAL4 transgene. Reporter gene expression indicated that the shi promoter is active during embryogenesis, and in larvae, pupae, and adults in a pattern consistent with normal dynamin expression. To assay for the ability of dynamin isoforms to function in vivo, the isoforms expressed via these GAL4 drivers were tested for the ability to rescue shibire phenotypes. When expressed at very high levels all individual isoforms tested rescued the temperature-sensitive paralytic phenotype of shi(ts2) flies; however, this rescue was partial, suggesting that no single tested isoform is sufficient for synaptic vesicle recycling in vivo. When tested for ability to rescue lethality induced by heat-pulsing larvae during development, shi- promoter driven expression of individual isoforms conferred significant resistance to heat treatment during larval development. However, all 3 isoforms were unable to rescue the lethality of shi12-12B mutants which are severely hypomorphic (or null) for shibire function. Taken together, these observations suggest that individual shibire isoforms have specific molecular activities in vivo.

• Barylko B et al.
• Synergistic activation of dynamin GTPase by Grb2 and phosphoinositides.
• J Biol Chem. 1998; 273: 3791-7
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• Hydrolysis of GTP by dynamin is essential for budding clathrin-coated vesicles from the plasma membrane. Two distinct domains of dynamin are implicated in the interactions with dynamin GTPase activators. Microtubules and Grb2 bind to the carboxyl-terminal proline/arginine-rich domain (PRD), whereas phosphoinositides bind to the pleckstrin homology (PH) domain. In this study we tested the effect of different phosphoinositides on dynamin GTPase activity and found that the best activator is phosphatidylinositol 4,5-bisphosphate followed by 1-O-(1, 2-di-O-palmitoyl-sn-glycerol-3-benzyloxyphosphoryl)-D-myo-inositol 3,4,5-triphosphate. Phosphatidylinositol 4-phosphate was a weak activator and phosphatidylinositol 3,4-bisphosphate did not activate GTPase at all. We then addressed the question of whether both domains of dynamin, PRD and PH, can be engaged simultaneously, and determined the effects of dual occupancy on dynamin GTPase activity. We found that Grb2 and phosphatidylinositol 4,5-bisphosphate together increased the dynamin GTPase activity up to 4-fold higher than that obtained by these activators tested separately, and also reduced the dynamin concentration required for half-maximal activities by 3-fold. These results indicate that both stimulators can bind to dynamin simultaneously resulting in superactivation of dynamin GTPase activity. We propose that SH3-containing proteins such as Grb2 bind to the dynamin PRD to target it to clathrin-coated pits and prime it for superactivation by phosphoinositides.

• Volchuk A, Narine S, Foster LJ, Grabs D, De Camilli P, Klip A
• Perturbation of dynamin II with an amphiphysin SH3 domain increases GLUT4 glucose transporters at the plasma membrane in 3T3-L1 adipocytes. Dynamin II participates in GLUT4 endocytosis.
• J Biol Chem. 1998; 273: 8169-76
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• The GLUT4 glucose transporter continuously recycles between the cell surface and an endosomal compartment in adipocytes. Insulin decreases the rate of GLUT4 endocytosis in addition to increasing its exocytosis. Endocytosis of the transporter is thought to occur at least in part via the clathrin-mediated endocytic system. The protein dynamin is involved in the final stages of clathrin-coated vesicle formation. Here we show that the dynamin II isoform is expressed in 3T3-L1 adipocytes and is present in isolated plasma membrane and low density microsomal fractions. Insulin reduced the levels of dynamin II associated with the plasma membrane by about half, raising the possibility that the hormone may reduce GLUT4 endocytosis by removing dynamin from the cell surface. A fusion protein containing the amphiphysin SH3 domain selectively bound dynamin II from 3T3-L1 adipocyte cell lysates. Microinjection of the fusion protein into these cells inhibited transferrin endocytosis and increased the levels of GLUT4 at the cell surface. Glutathione S-transferase alone, the SH3 domains of spectrin and Crk, and a mutated amphiphysin SH3 domain unable to bind dynamin II did not affect GLUT4 distribution. However, a peptide containing the dynamin II sequence that binds amphiphysin increased the surface presence of GLUT4. Moreover, in cells first treated with insulin to externalize GLUT4, the dynamin peptide, but not an unrelated control peptide, inhibited GLUT4 internalization upon insulin removal. These results suggest that interactions of dynamin II with amphiphysin may play an important role in GLUT4 endocytosis. We hypothesize that insulin may reduce GLUT4 endocytosis by regulating the function of dynamin II at the cell surface, as part of the mechanism to increase glucose uptake.

• Mundigl O, Ochoa GC, David C, Slepnev VI, Kabanov A, De Camilli P
• Amphiphysin I antisense oligonucleotides inhibit neurite outgrowth in cultured hippocampal neurons.
• J Neurosci. 1998; 18: 93-103
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• Amphiphysin I is an SH3 domain-containing neuronal protein, enriched in axon terminals, which was reported to act as a physiological binding partner for dynamin I in synaptic vesicle endocytosis. Rvs167 and Rvs161, the yeast homologs of amphiphysin I, have been implicated in endocytosis, actin function, and cell polarity. Now we have explored the possibility that amphiphysin I also may have a role in actin dynamics and cell polarity by testing the effect of amphiphysin I suppression on neurite outgrowth. Freshly plated hippocampal neurons were exposed to antisense oligonucleotides via a new delivery system based on a polycationic amphipathic polymer, PS980. Western blot analysis revealed that amphiphysin I levels steadily increased with neuronal differentiation, whereas in antisense-treated cultures amphiphysin I levels were reduced to approximately 10% of control levels at 48 hr. Concomitantly, a collapse of growth cones and a severe inhibition of neurite outgrowth and axon formation were observed. A similar effect was observed previously after dynamin I suppression in the same culture system (). We also have found that amphiphysin I and dynamin I colocalize in developing neurons at all developmental stages and that a pool of both proteins is colocalized with actin patches at the leading edge of growth cones. Our findings suggest a conserved role of the amphiphysin protein family in the dynamics of the cortical cell cytoskeleton and provide new evidence for a close functional link between amphiphysin I and dynamin I.

• Schmid SL, McNiven MA, De Camilli P
• Dynamin and its partners: a progress report.
• Curr Opin Cell Biol. 1998; 10: 504-12
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• Dynamin's role in clathrin-mediated endocytosis is now well established. Here we review new evidence from the past two years for the function of dynamin and related GTPases in other Intracellular trafficking events. We then summarize current information on the domain structure and function of this multidomain GTPase. Finally, we describe dynamin partners and their function in the context of clathrin-mediated endocytosis.

• Altschuler Y et al.
• Redundant and distinct functions for dynamin-1 and dynamin-2 isoforms.
• J Cell Biol. 1998; 143: 1871-81
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• A role for dynamin in clathrin-mediated endocytosis is now well established. However, mammals express three closely related, tissue-specific dynamin isoforms, each with multiple splice variants. Thus, an important question is whether these isoforms and splice variants function in vesicle formation from distinct intracellular organelles. There are conflicting data as to a role for dynamin-2 in vesicle budding from the TGN. To resolve this issue, we compared the effects of overexpression of dominant-negative mutants of dynamin-1 (the neuronal isoform) and dynamin-2 (the ubiquitously expressed isoform) on endocytic and biosynthetic membrane trafficking in HeLa cells and polarized MDCK cells. Both dyn1(K44A) and dyn2(K44A) were potent inhibitors of receptor-mediated endocytosis; however neither mutant directly affected other membrane trafficking events, including transport mediated by four distinct classes of vesicles budding from the TGN. Dyn2(K44A) more potently inhibited receptor-mediated endocytosis than dyn1(K44A) in HeLa cells and at the basolateral surface of MDCK cells. In contrast, dyn1(K44A) more potently inhibited endocytosis at the apical surface of MDCK cells. The two dynamin isoforms have redundant functions in endocytic vesicle formation, but can be targeted to and function differentially at subdomains of the plasma membrane.

• Wigge P et al.
• Amphiphysin heterodimers: potential role in clathrin-mediated endocytosis.
• Mol Biol Cell. 1997; 8: 2003-15
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• Amphiphysin (Amph) is a src homology 3 domain-containing protein that has been implicated in synaptic vesicle endocytosis as a result of its interaction with dynamin. In a screen for novel members of the amphiphysin family, we identified Amph2, an isoform 49% identical to the previously characterized Amph1 protein. The subcellular distribution of this isoform parallels Amph1, both being enriched in nerve terminals. Like Amph1, a role in endocytosis at the nerve terminal is supported by the rapid dephosphorylation of Amph2 on depolarization. Importantly, the two isoforms can be coimmunoprecipitated from the brain as an equimolar complex, suggesting that the two isoforms act in concert. As determined by cross-linking of brain extracts, the Amph1-Amph2 complex is a 220- to 250-kDa heterodimer. COS cells transfected with either Amph1 or Amph2 show greatly reduced transferrin uptake, but coexpression of the two proteins rescues this defect, supporting a role for the heterodimer in clathrin-mediated endocytosis. Although the src homology 3 domains of both isoforms interact with dynamin, the heterodimer can associate with multiple dynamin molecules in vitro and activates dynamin's GTPase activity. We propose that it is an amphiphysin heterodimer that drives the recruitment of dynamin to clathrin-coated pits in endocytosing nerve terminals.

• Ringstad N, Nemoto Y, De Camilli P
• The SH3p4/Sh3p8/SH3p13 protein family: binding partners for synaptojanin and dynamin via a Grb2-like Src homology 3 domain.
• Proc Natl Acad Sci U S A. 1997; 94: 8569-74
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• The GTPase dynamin I and the inositol 5-phosphatase synaptojanin are nerve terminal proteins implicated in synaptic vesicle recycling. Both proteins contain COOH-terminal proline-rich domains that can interact with a variety of Src homology 3 (SH3) domains. A major physiological binding partner for dynamin I and synaptojanin in the nervous system is amphiphysin I, an SH3 domain-containing protein also concentrated in nerve terminals. We have used the proline-rich tail of synaptojanin to screen a rat brain library by the two-hybrid method to identify additional interacting partners of synaptojanin. Three related proteins containing SH3 domains that are closely related to the SH3 domains of Grb2 were isolated: SH3p4, SH3p8, and SH3p13. Further biochemical studies demonstrated that the SH3p4/8/13 proteins bind to both synaptojanin and dynamin I. The SH3p4/8/13 transcripts are differentially expressed in tissues: SH3p4 mRNA was detected only in brain, SH3p13 mRNA was present in brain and testis, and the SH3p8 transcript was detected at similar levels in multiple tissues. Members of the SH3p4/8/13 protein family were found to be concentrated in nerve terminals, and pools of synaptojanin and dynamin I were coprecipitated from brain extracts with antibodies recognizing SH3p4/8/13. These findings underscore the important role of SH3-mediated interactions in synaptic vesicle recycling.

• Wigge P, Vallis Y, McMahon HT
• Inhibition of receptor-mediated endocytosis by the amphiphysin SH3 domain.
• Curr Biol. 1997; 7: 554-60
• Display abstract

• BACKGROUND: Receptor-mediated endocytosis appears to require the GTP-binding protein dynamin, but the process by which dynamin is recruited to clathrin-coated pits remains unclear. Dynamin contains several proline-rich clusters that bind to Src homology 3 (SH3) domains, which are short modules found in many signalling proteins and which mediate protein-protein interactions. Amphiphysin, a protein that is highly expressed in the brain, interacts with dynamin in vitro, as do Grb2 and many other SH3 domain-containing proteins. In this study, we examined the role of amphiphysin in receptor-mediated endocytosis in vivo. RESULTS: To address the importance of the amphiphysin SH3 domain in dynamin recruitment, we used a transferrin and epidermal growth factor (EGF) uptake assay in COS-7 fibroblasts. Amphiphysin is present in these cells at a low level and indeed in other peripheral tissues. Confocal immunofluorescence revealed that cells transfected with the amphiphysin SH3 domain showed a potent blockade in receptor-mediated endocytosis. To test whether the cellular target of amphiphysin is dynamin, COS-7 cells were contransfected with both dynamin and the amphiphysin SH3 domain; here, transferrin uptake was efficiently rescued. Importantly, the SH3 domains of Grb2, phospholipase C gamma and spectrin all failed to exert any effect on endocytosis. The mechanism of amphiphysin action in recruiting dynamin was additionally tested in vitro: amphiphysin could associate with both dynamin and alpha-adaptin simultaneously, further supporting a role for amphiphysin in endocytosis. CONCLUSIONS: Our results suggest that the SH3 domain of amphiphysin recruits dynamin to coated pits in vivo, probably via plasma membrane adaptor complexes. We propose that amphiphysin is not only required for synaptic-vesicle endocytosis, but might also be a key player in dynamin recruitment in all cells undergoing receptor-mediated endocytosis.

• Seugnet L, Simpson P, Haenlin M
• Requirement for dynamin during Notch signaling in Drosophila neurogenesis.
• Dev Biol. 1997; 192: 585-98
• Display abstract

• Singling out of a unique neural precursor from a group of equivalent cells, during Drosophila neurogenesis, involves Notch-mediated lateral signaling. During this process, activation of the Notch signaling pathway leads to repression of neural development. Disruption of this signaling pathway results in the development of an excess of neural cells. The loss of activity of dynamin, which is encoded by the gene shibire and is required for endocytosis, results in a similar phenotype. Here we have investigated the requirement of shibire function for Notch signaling during the segregation of sensory bristles on the notum of the fly. Overexpression of different constitutively active forms of Notch in shibire mutant flies indicates that shibire function is not necessary for transduction of the signal downstream of Notch, even when the receptor is integrated in the plasma membrane. However, when wild-type Notch is activated by its ligand Delta, dynamin is required in both signaling and receiving cells for normal singling out of precursors. This suggests an active role of the signaling cell for ligand-mediated receptor endocytosis in the case of transmembrane ligands. We discuss the possible implications of these results for normal functioning of Notch-mediated lateral signaling.

• Warnock DE, Baba T, Schmid SL
• Ubiquitously expressed dynamin-II has a higher intrinsic GTPase activity and a greater propensity for self-assembly than neuronal dynamin-I.
• Mol Biol Cell. 1997; 8: 2553-62
• Display abstract

• To begin to understand mechanistic differences in endocytosis in neurons and nonneuronal cells, we have compared the biochemical properties of the ubiquitously expressed dynamin-II isoform with those of neuron-specific dynamin-I. Like dynamin-I, dynamin-II is specifically localized to and highly concentrated in coated pits on the plasma membrane and can assemble in vitro into rings and helical arrays. As expected, the two closely related isoforms share a similar mechanism for GTP hydrolysis: both are stimulated in vitro by self-assembly and by interaction with microtubules or the SH3 domain-containing protein, grb2. Deletion of the C-terminal proline/arginine-rich domain from either isoform abrogates self-assembly and assembly-dependent increases in GTP hydrolysis. However, dynamin-II exhibits a approximately threefold higher rate of intrinsic GTP hydrolysis and higher affinity for GTP than dynamin-I. Strikingly, the stimulated GTPase activity of dynamin-II can be >40-fold higher than dynamin-I, due principally to its greater propensity for self-assembly and the increased resistance of assembled dynamin-II to GTP-triggered disassembly. These results are consistent with the hypothesis that self-assembly is a major regulator of dynamin GTPase activity and that the intrinsic rate of GTP hydrolysis reflects a dynamic, GTP-dependent equilibrium of assembly and disassembly.

• Cremona O, De Camilli P
• Synaptic vesicle endocytosis.
• Curr Opin Neurobiol. 1997; 7: 323-30
• Display abstract

• Exocytosis of synaptic vesicles is followed rapidly by reinternalization and recycling of their membranes. Recent studies have confirmed the key role of clathrin-mediated endocytosis in synaptic vesicle reformation and have identified new proteins that participate in this process. In addition, growing evidence suggests that lipids, primarily phosphoinositides, play an important role in synaptic vesicle recycling.

• Leprince C et al.
• A new member of the amphiphysin family connecting endocytosis and signal transduction pathways.
• J Biol Chem. 1997; 272: 15101-5
• Display abstract

• Src homology 3 (SH3) domains are conserved modules which participate in protein interaction by recognizing proline-rich motifs on target molecules. To identify new SH3-containing proteins, we performed a two-hybrid screen with a proline-rich region of human SOS-1. One of the specific SOS-1 interacting clones that were isolated from a mouse brain cDNA library defines a new protein that was named amphiphysin 2 because of its homology to the previously reported amphiphysin. Amphiphysin 2 is expressed in a number of mouse tissues through multiple RNA transcripts. Here, we report the amino acid sequence of a brain form of amphiphysin 2 (BRAMP2) encoded by a 2. 5-kilobase mRNA. BRAMP2 associates in vitro with elements of the endocytosis machinery such as alpha-adaptin and dynamin. On a biosensor surface, the BRAMP2/dynamin interaction appeared to be direct and partly dependent on a proline-rich sequence of dynamin. Association with dynamin was also observed in PC12 cells after cell stimulation with nerve growth factor, suggesting that amphiphysin 2 may be connected to receptor-dependent signaling pathways. This hypothesis is strengthened by the ability of BRAMP2 to interact with the p21(ras) exchange factor SOS, in vitro, as a possible point of interconnection between the endocytic and signaling pathways.

• Liu JP
• [Discovery of dynamin and the mechanism of vesicle recirculation in nerve terminal]
• Sheng Li Ke Xue Jin Zhan. 1997; 28: 55-7

• Ramjaun AR, Micheva KD, Bouchelet I, McPherson PS
• Identification and characterization of a nerve terminal-enriched amphiphysin isoform.
• J Biol Chem. 1997; 272: 16700-6
• Display abstract

• Amphiphysin is a nerve terminal-enriched protein thought to function in synaptic vesicle endocytosis, in part through Src homology 3 (SH3) domain-mediated interactions with dynamin and synaptojanin. Here, we report the characterization of a novel amphiphysin isoform (termed amphiphysin II) that was identified through a homology search of the data base of expressed sequence tags. Antibodies specific to amphiphysin II recognize a 90-kDa protein on Western blot that is brain-specific and highly enriched in nerve terminals. Like amphiphysin (now referred to as amphiphysin I), amphiphysin II binds to dynamin and synaptojanin through its SH3 domain. Further, both proteins bind directly to clathrin in an SH3 domain-independent manner. Taken together, these data suggest that amphiphysin II may participate with amphiphysin I in the regulation of synaptic vesicle endocytosis.

• Lin HC, Barylko B, Achiriloaie M, Albanesi JP
• Phosphatidylinositol (4,5)-bisphosphate-dependent activation of dynamins I and II lacking the proline/arginine-rich domains.
• J Biol Chem. 1997; 272: 25999-6004
• Display abstract

• Dynamins comprise a family of GTPases that participate in the early stages of endocytosis. The GTPase activity of neuronal specific dynamin I is stimulated by microtubules, negatively charged phospholipid vesicles, and Src homology 3-containing proteins, including Grb2. These activators were previously shown to bind to a proline/arginine-rich domain (PRD) in the carboxyl-terminal region of the enzyme. Dynamin II, which is ubiquitously expressed, had not been purified or characterized previously. In this study, the enzymatic properties of rat dynamin II and of D746, a dynamin II truncation mutant lacking the PRD, have been characterized. Dynamin II has a higher basal activity than dynamin I, but the two types of dynamin are stimulated similarly by microtubules, Grb2, and phospholipids. D746 is not activated by microtubules or Grb2, highlighting the significance of the PRD for these interactions, but it is activated by phospholipid vesicles containing phosphatidylserine or phosphatidylinositol-4,5- bisphosphate. Moreover, in contrast to previous reports, the PRD appears not to be required for phospholipid-stimulated self-assembly of dynamin, which is a key element in the regulation of its activity. Similar results were obtained with bovine brain dynamin I that had been subjected to limited proteolytic digestion to remove the PRD. Our data highlight the potential involvement of dynamin pleckstrin homology domains in the regulation of GTPase activity by phospholipids.

• Shupliakov O et al.
• Synaptic vesicle endocytosis impaired by disruption of dynamin-SH3 domain interactions.
• Science. 1997; 276: 259-63
• Display abstract

• The proline-rich COOH-terminal region of dynamin binds various Src homology 3 (SH3) domain-containing proteins, but the physiological role of these interactions is unknown. In living nerve terminals, the function of the interaction with SH3 domains was examined. Amphiphysin contains an SH3 domain and is a major dynamin binding partner at the synapse. Microinjection of amphiphysin's SH3 domain or of a dynamin peptide containing the SH3 binding site inhibited synaptic vesicle endocytosis at the stage of invaginated clathrin-coated pits, which resulted in an activity-dependent distortion of the synaptic architecture and a depression of transmitter release. These findings demonstrate that SH3-mediated interactions are required for dynamin function and support an essential role of clathrin-mediated endocytosis in synaptic vesicle recycling.

• Liu JP, Zhang QX, Baldwin G, Robinson PJ
• Calcium binds dynamin I and inhibits its GTPase activity.
• J Neurochem. 1996; 66: 2074-81
• Display abstract

• Synaptic vesicle recycling is a neuronal specialization of endocytosis that requires the GTPase activity of dynamin I and is triggered by membrane depolarization and Ca2+ entry. To establish the relationship between dynamin I GTPase activity and Ca2+, we used purified dynamin I and analyzed its interaction with Ca2+ in vitro. We report that Ca2+ bound to dynamin I and this was abolished by deletion of dynamin's C-terminal tail. Phosphorylation of dynamin I by protein kinase C promoted formation of a dynamin I tetramer and increased Ca2+ binding to the protein. Moreover, Ca2+ inhibited dynamin I GTPase activity after stimulation by phosphorylation or by phospholipids but not after stimulation with a GST-SH3 fusion protein containing the SH3 domain of phosphoinositide 3-kinase. These results suggest that in resting nerve terminals, phosphorylation of dynamin I by protein kinase C converts it to a tetramer that functions as a Ca(2+)-sensing protein. By binding to Ca2+, dynamin I GTPase activity is specifically decreased, possibly to regulate synaptic vesicle recycling.

• Kelly RB
• Endocytosis. Ringing necks with dynamin.
• Nature. 1995; 374: 116-7

• De Camilli P, Takei K, McPherson PS
• The function of dynamin in endocytosis.
• Curr Opin Neurobiol. 1995; 5: 559-65
• Display abstract

• Temperature-sensitive shibire mutants of Drosophila melanogaster become rapidly paralyzed upon a shift to the restrictive temperature, which is due to a block in synaptic vesicle endocytosis. The shibire gene encodes the GTPase dynamin. Recent studies have shown that dynamin forms rings at the neck of invaginated clathrin-coated pits, and have suggested that a conformational change in the ring, which correlates with GTP hydrolysis, plays an essential role in vesicle fission.

• Sontag JM, Fykse EM, Ushkaryov Y, Liu JP, Robinson PJ, Sudhof TC
• Differential expression and regulation of multiple dynamins.
• J Biol Chem. 1994; 269: 4547-54
• Display abstract

• Dynamin is a GTP-, microtubule-, and phospholipid-binding protein that is expressed primarily in brain. In Drosophila, the shibire gene encodes a homologue of dynamin; mutations in this gene result in a defect in endocytosis, suggesting a function for dynamin in endocytic membrane traffic. In the present study we show that there are at least two distinct dynamin genes in mammals whose products are referred to as dynamins I and II. The two dynamins are similar to each other (79% identity) and are both equally homologous to the Drosophila shibire gene product (66% identity). The highest degree of identity between dynamins is observed in their N-terminal halves, whereas their C termini exhibit little homology. Transcripts of both dynamin genes are subject to at least two alternative splicing events, the first of which is identically found in both dynamins, whereas the second site of alternative splicing is different between the two types of dynamins. The first alternatively spliced sequence of the dynamins consists of an interior region that is present in two distinct but homologous forms in both dynamins, suggesting alternative use of exons in both genes at identical positions. The second site of alternative splicing results in the generation of different C termini in dynamin I and in the inclusion or exclusion of an interior four-amino acid sequence in dynamin II. The two dynamins exhibit remarkable differences in their tissue distribution and regulation. Dynamin I is almost exclusively expressed in the central nervous system. Conversely, dynamin II is expressed ubiquitously in all tissues tested. Previous studies revealed that the GTPase activity of dynamin I is regulated by phosphorylation by protein kinase C in nerve terminals. Expression of dynamins I and II by transfection in COS cells demonstrates that only dynamin I but not dynamin II is a substrate for protein kinase C. Our data suggest a specialization in the endocytic functions and the regulation of dynamins between neural and non-neural tissues in mammals.

• Robinson PJ, Liu JP, Powell KA, Fykse EM, Sudhof TC
• Phosphorylation of dynamin I and synaptic-vesicle recycling.
• Trends Neurosci. 1994; 17: 348-53
• Display abstract

• In nerve terminals, neurotransmitters are packaged in synaptic vesicles, and released by exocytosis. Empty synaptic vesicles are rapidly recycled for reuse by endocytosis. Much progress has been made in identifying the proteins involved in synaptic-vesicle trafficking, but the mechanism and regulation of endocytosis have largely remained an enigma. One approach to defining regulatory proteins that might be involved is to study stimulus-dependent phosphorylation events in nerve terminals. This has led to the identification of dephosphin, which is quantitatively dephosphorylated by nerve-terminal depolarization. Sequencing reveals that dephosphin is identical with dynamin I, a GTP-binding protein that functions in endocytosis. Phosphorylation and dephosphorylation of nerve-terminal dynamin I/dephosphin regulates its intrinsic GTPase activity in parallel with the regulation of synaptic-vesicle recycling. Therefore, phosphorylation and dephosphorylation of dynamin I might provide a Ca(2+)-dependent switch for endocytosis in the synaptic-vesicle pathway.

• Tuma PL, Collins CA
• Activation of dynamin GTPase is a result of positive cooperativity.
• J Biol Chem. 1994; 269: 30842-7
• Display abstract

• Dynamin is a GTP-binding protein thought to be involved in the early stages of endocytosis. Presently, it is not known how dynamin GTP binding and hydrolysis are related to its role in this process. We previously characterized the ability of acidic phospholipid vesicles and microtubules to strongly stimulate the GTPase activity of purified brain dynamin. In a further analysis of dynamin enzymatic properties, we have found that the increase of dynamin GTP hydrolysis in the presence of activating agent depends on enzyme concentration. At low enzyme concentration, little or no activation is observed. Plots of dynamin GTPase activity with increasing enzyme concentration in the presence of either activating agent are strongly sigmoidal, indicating that positive cooperativity is responsible for the increased activity observed. A Hill coefficient of 2.3 was determined, implying that at least two dynamin molecules associate for maximal GTPase activity. No cooperative effects in GTP binding were observed. Linear transformation of reaction velocity versus enzyme concentration data indicate an apparent Km for dynamin-dynamin interactions of 37 nM, which is significantly lower than the physiological concentration of dynamin in brain. These results suggest that cooperative interactions between dynamin molecules are responsible for the apparent activation of GTPase observed and are likely involved in dynamin function in vivo.

• Herskovits JS, Burgess CC, Obar RA, Vallee RB
• Effects of mutant rat dynamin on endocytosis.
• J Cell Biol. 1993; 122: 565-78
• Display abstract

• Dynamin is a 100-kD microtubule-activated GTPase. Recent evidence has revealed a high degree of sequence homology with the product of the Drosophila gene shibire, mutations in which block the recycling of synaptic vesicles and, more generally, the formation of coated and non-coated vesicles at the plasma membrane. We have now transfected cultured mammalian COS-7 cells with both wild-type and mutant dynamin cDNAs. Point mutations in the GTP-binding consensus sequence elements of dynamin equivalent to dominant negative mutations in ras, and an NH2-terminal deletion of the entire GTP-binding domain of dynamin, block transferrin uptake and alter the distribution of clathrin heavy chain and alpha-, but not gamma-, adaptin. COOH-terminal deletions reverse these effects, identifying this portion of dynamin as a site of interaction with other components of the endocytic pathway. Over-expression of neither wild-type nor mutant forms of dynamin affected the distribution of microtubules. These results demonstrate a specific role for dynamin and for GTP in the initial stages of receptor-mediated endocytosis.

• Guan K, Farh L, Marshall TK, Deschenes RJ
• Normal mitochondrial structure and genome maintenance in yeast requires the dynamin-like product of the MGM1 gene.
• Curr Genet. 1993; 24: 141-8
• Display abstract

• The isolation and characterization of MGM1, an yeast gene with homology to members of the dynamin gene family, is described. The MGM1 gene is located on the right arm of chromosome XV between STE4 and PTP2. Sequence analysis revealed a single open reading frame of 902 residues capable of encoding a protein with an approximate molecular mass of 101 kDa. Loss of MGM1 resulted in slow growth on rich medium, failure to grow on non-fermentable carbon sources, and loss of mitochondrial DNA. The mitochondria also appeared abnormal when visualized with an antibody to a mitochondrial-matrix marker. MGM1 encodes a dynamin-like protein involved in the propagation of functional mitochondria in yeast.

• Tuma PL, Stachniak MC, Collins CA
• Activation of dynamin GTPase by acidic phospholipids and endogenous rat brain vesicles.
• J Biol Chem. 1993; 268: 17240-6
• Display abstract

• Dynamin is a GTPase thought to play a role in endocytosis based on genetic analysis of its homolog in Drosophila melanogaster shibire. Previous studies have stressed an in vitro association with microtubules, though additional evidence suggests that dynamin associates with membranous organelles. In an analysis of the enzymatic and membrane binding properties of dynamin, we have found that the acidic phospholipids, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, are able to stimulate GTP hydrolysis in a manner similar to activation previously shown with microtubules. A neutral phospholipid, phosphatidylcholine, had no effect on dynamin GTPase. Activation of dynamin was biphasic, with a decrease in activity back to basal levels with increased concentrations of either microtubules or liposomes. A comparison between GTPase stimulation induced by microtubules and that by phospholipids suggests that ionic interactions between the basic C-terminal domain of dynamin and the negatively charged microtubule or phospholipid head group are important. In support of this, GTPase stimulation by these agents in combination was not additive. A salt-extracted membrane fraction from brain tissue also activated dynamin GTPase, though to a lower extent than pure phospholipids. These results suggest that membrane components could be responsible for some aspects of the regulation of dynamin function in vivo.

• Robinson PJ et al.
• Dynamin GTPase regulated by protein kinase C phosphorylation in nerve terminals.
• Nature. 1993; 365: 163-6
• Display abstract

• Dynamin is a microtubule-binding protein with a microtubule-activated GTPase activity. The gene encoding dynamin is mutated in shibire, a Drosophila mutant defective in endocytosis in nerve terminals and other cells. These observations place dynamin into two distinct functional contexts, suggesting roles in microtubule-based motility or in endocytosis. We report here that dynamin is identical to the neuronal phosphoprotein dephosphin (P96), originally identified by its stimulus-dependent dephosphorylation in nerve terminals. Dynamin is a protein doublet of M(r) 94 and 96K arising by alternative splicing of its primary transcript. In the nerve terminal, both forms of dynamin are phosphorylated by protein kinase C (PKC) and are quantitatively dephosphorylated on excitation. In vitro, dynamin is also phosphorylated by casein kinase II which inhibits PKC phosphorylation. Phosphorylation by PKC but not by casein kinase II enhances the GTPase activity of dynamin 12-fold. The dynamins are therefore a group of nerve terminal phosphoproteins whose GTPase is regulated by phosphorylation in parallel with synaptic vesicle recycling. The regulation of dynamin GTPase could serve as the trigger for the rapid endocytosis of synaptic vesicles after exocytosis.

• Vallee RB, Shpetner HS
• Cell biology. Dynamin in synaptic dynamics.
• Nature. 1993; 365: 107-8

• Vallee RB, Herskovits JS, Aghajanian JG, Burgess CC, Shpetner HS
• Dynamin, a GTPase involved in the initial stages of endocytosis.
• Ciba Found Symp. 1993; 176: 185-93
• Display abstract

• Dynamin is a high molecular mass (100 kDa) GTPase which binds to and co-purifies with microtubules. Molecular cloning of rat brain dynamin has revealed the three well-established consensus sequence elements for GTP binding within the N-terminal third of the protein, as well as sequence similarity within this region to the interferon-inducible antiviral Mx proteins, the product of the yeast membrane sorting gene VPS1, and the product of the yeast mitochondrial replication gene MGM1. More extensive sequence similarity between rat dynamin and the product of the Drosophila gene shibire, which is involved in endocytosis, has also been found. In in vitro assays microtubules strongly stimulate the dynamin GTPase. This effect can be reversed by removal of the dynamin C-terminus using papain, which abolishes microtubule binding. Overexpression of mutant forms of dynamin in vivo using Cos-7 cells inhibits transferrin uptake and alters the distribution of clathrin and of alpha-adaptin, but not gamma-adaptin. Deletion of the C-terminus of mutant forms of dynamin abolishes these effects. Together these results suggest a critical role for dynamin in the early stages of endocytosis. It is uncertain whether microtubules interact with dynamin in vivo or whether the in vitro effects of microtubules mimic the effects of other regulatory elements in vivo.

• Vater CA, Raymond CK, Ekena K, Howald-Stevenson I, Stevens TH
• The VPS1 protein, a homolog of dynamin required for vacuolar protein sorting in Saccharomyces cerevisiae, is a GTPase with two functionally separable domains.
• J Cell Biol. 1992; 119: 773-86
• Display abstract

• The product of the VPS1 gene, Vps1p, is required for the sorting of soluble vacuolar proteins in the yeast Saccharomyces cerevisiae. We demonstrate here that Vps1p, which contains a consensus tripartite motif for guanine nucleotide binding, is capable of binding and hydrolyzing GTP. Vps1p is a member of a subfamily of large GTP-binding proteins whose members include the vertebrate Mx proteins, the yeast MGM1 protein, the Drosophila melanogaster shibire protein, and dynamin, a bovine brain protein that bundles microtubules in vitro. Disruption of microtubules did not affect the fidelity or kinetics of vacuolar protein sorting, indicating that Vps1p function is not dependent on microtubules. Based on mutational analyses, we propose a two-domain model for Vps1p function. When VPS1 was treated with hydroxylamine, half of all mutations isolated were found to be dominant negative with respect to vacuolar protein sorting. All of the dominant-negative mutations analyzed further mapped to the amino-terminal half of Vps1p and gave rise to full-length protein products. In contrast, recessive mutations gave rise to truncated or unstable protein products. Two large deletion mutations in VPS1 were created to further investigate Vps1p function. A mutant form of Vps1p lacking the carboxy-terminal half of the protein retained the capacity to bind GTP and did not interfere with sorting in a wild-type background. A mutant form of Vps1p lacking the entire GTP-binding domain interfered with vacuolar protein sorting in wild-type cells. We suggest that the amino-terminal domain of Vps1p provides a GTP-binding and hydrolyzing activity required for vacuolar protein sorting, and the carboxy-terminal domain mediates Vps1p association with an as yet unidentified component of the sorting apparatus.

• Maeda K, Nakata T, Noda Y, Sato-Yoshitake R, Hirokawa N
• Interaction of dynamin with microtubules: its structure and GTPase activity investigated by using highly purified dynamin.
• Mol Biol Cell. 1992; 3: 1181-94
• Display abstract

• We purified a large amount of dynamin with high enzymatical activity from rat brain tissue by a new procedure. Dynamin 0.48 mg was obtained from 20 g of rat brain. The purity of dynamin was almost 98%. Dynamin plays a role of GTPase rather than ATPase. In the absence of microtubules, Michaelis constant (Km) and maximum velocity (Vmax) for dynamin GTPase were 370 microM and 0.25 min-1, respectively, and in their presence, both were significantly accelerated up to 25 microM and 5.5 min-1. On the other hand, the ATPase activity was very low in the absence of microtubules, and even in their presence, Km and Vmax for dynamin ATPase were 0.2 mM and 0.91 min-1. Despite slow GTPase turnover rate in the absence of microtubules, binding of GTP and its nonhydrolizing analogues was very fast, indicating that GTP binding step is not rate limiting. Dynamin did not cause a one-directional consistent microtubule sliding movement just like kinesin or dynein in the presence of 2 mM ATP or 2 mM GTP. We observed the molecular structure of dynamin with low-angle rotary shadowing technique and revealed that the dynamin molecule is globular in shape. Gel filtration assay revealed that these globules were the oligomers of 100-kDa dynamin polypeptide. Dynamin bound to microtubules with a 1:1 approximately 1.2 molar ratio in the absence of GTP. Quick-freeze deep-etch electron microscopy of the dynamin-microtubule complex showed that dynamin decorates the surface of microtubules helically, like a screw bolt, very orderly and tightly with 11.4 +/- 0.9 (SD)nm period. Contrary to the previous report, microtubules make bundles by the attachment of the dynamin helixes around each adjacent microtubule, and no cross-bridge formation was observed.

• Maguire ME et al.
• Mg2+ transporting P-type ATPases of Salmonella typhimurium. Wrong way, wrong place enzymes.
• Ann N Y Acad Sci. 1992; 671: 244-55

• Vallee RB
• Dynamin: motor protein or regulatory GTPase.
• J Muscle Res Cell Motil. 1992; 13: 493-6

• Nakata T, Iwamoto A, Noda Y, Takemura R, Yoshikura H, Hirokawa N
• Predominant and developmentally regulated expression of dynamin in neurons.
• Neuron. 1991; 7: 461-9
• Display abstract

• We have cloned a cDNA for dynamin, a 100 kd microtubule-associated motor protein whose 5' region contains a GTP-binding motif homologous to that of the Mx proteins, from a rat brain library and analyzed its expression. Dynamin mRNA is 3.6 kb and is preferentially expressed in the brain after postnatal day 7, parallel to the developmental increase of the protein. In situ hybridization revealed high levels of dynamin transcripts in neural cells in the cerebellar cortex, hippocampus (particularly in the CA3 area), and cerebral cortex. The transcripts appeared in cerebellar granular cells only after they had ceased dividing and had migrated to the inner granular layer. We show that dynamin is expressed predominantly in neural cells after elongation of their processes, suggesting a role especially in mature neurons.

• Obar RA, Shpetner HS, Vallee RB
• Dynamin: a microtubule-associated GTP-binding protein.
• J Cell Sci Suppl. 1991; 14: 143-5
• Display abstract

• We recently identified dynamin as a third nucleotide-sensitive microtubule-associated protein in brain tissue, in addition to kinesin and cytoplasmic dynein. Molecular cloning analysis has revealed that dynamin contains the three consensus elements characteristic of GTP-binding proteins, and biochemical results support a role for GTP in dynamin function. Dynamin is also homologous to the Mx proteins, involved in interferon-induced viral resistance, and the product of the yeast VPS1 gene, involved in vacuolar protein sorting. These results identify a novel class of GTP-utilizing proteins, with apparently diverse functions.

• Chen MS et al.
• Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis.
• Nature. 1991; 351: 583-6
• Display abstract

• Dynamin was discovered in bovine brain tissue as a nucleotide-sensitive microtubule-binding protein of relative molecular mass 100,000. It was found to cross-link microtubules into highly ordered bundles, and appeared to have a role in intermicrotubule sliding in vitro. Cloning and sequencing of rat brain dynamin complementary DNA identified an N-terminal region of about 300 amino acids which contained the three consensus elements characteristic of GTP-binding proteins. Extensive homology was found between this domain and the mammalian Mx proteins which are involved in interferon-induced viral resistance, and with the product of the VPS1 locus in Saccharomyces cerevisiae, which has been implicated both in membrane protein sorting, and in meiotic spindle pole separation. Dynamin-containing microtubule bundles were not observed in an immunofluorescence study of cultured mammalian cells, but a role for a GTP-requiring protein in intermicrotubule sliding during mitosis in plants has been reported. We report here that Drosophila melanogaster contains multiple tissue-specific and developmentally-regulated forms of dynamin, which are products of the shibire locus previously implicated in endocytic protein sorting.

• Shpetner HS, Vallee RB
• Purification and characterization of dynamin.
• Methods Enzymol. 1991; 196: 192-201

• Hollenbeck PJ
• Cell motility. Dynamin joins the family.
• Nature. 1990; 347: 229-229

• Scaife R, Margolis RL
• Biochemical and immunochemical analysis of rat brain dynamin interaction with microtubules and organelles in vivo and in vitro.
• J Cell Biol. 1990; 111: 3023-33
• Display abstract

• We have purified a 100-kD rat brain protein that has microtubule cross-linking activity in vitro, and have determined that it is dynamin, a putative microtubule-associated motility protein. We find that dynamin appears to be specific to neuronal tissue where it is present in both soluble and particulate tissue fractions. In the cytosol it is abundant, representing as much as 1.5% of the total extractable protein. Dynamin appears to be in particulate material due to association with a distinct subcellular membrane fraction. Surprisingly, by immunofluorescence analysis of PC12 cells we find that dynamin is distributed uniformly throughout the cytoplasm with no apparent microtubule association in either interphase, mitotic, or taxol-treated cells. Upon nerve growth factor (NGF) induction of PC12 cell differentiation into neurons, dynamin levels increase approximately twofold. In the cell body, the distribution of dynamin again remains clearly distinct from that of tubulin, and in axons, where microtubules are numerous and ordered into bundles, dynamin staining is sparse and punctate. On the other hand, in the most distal domain of growth cones, where there are relatively few microtubules, dynamin is particularly abundant. The dynamin staining of neurites is abolished by extraction of the cells with detergent under conditions that preserve microtubules, suggesting that dynamin in neurites is associated with membranes. We conclude that dynamin is a neuronal protein that is specifically associated with as yet unidentified vesicles. It is possible, but unproven, that it may link vesicles to microtubules for transport in differentiated axons.

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