SMART MODE:

NORMAL GENOMIC

• Galkin VE, Orlova A, Lukoyanova N, Wriggers W, Egelman EH
• Actin depolymerizing factor stabilizes an existing state of F-actin and can change the tilt of F-actin subunits.
• J Cell Biol. 2001; 153: 75-86
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• Proteins in the actin depolymerizing factor (ADF)/cofilin family are essential for rapid F-actin turnover, and most depolymerize actin in a pH-dependent manner. Complexes of human and plant ADF with F-actin at different pH were examined using electron microscopy and a novel method of image analysis for helical filaments. Although ADF changes the mean twist of actin, we show that it does this by stabilizing a preexisting F-actin angular conformation. In addition, ADF induces a large ( approximately 12 degrees ) tilt of actin subunits at high pH where filaments are readily disrupted. A second ADF molecule binds to a site on the opposite side of F-actin from that of the previously described ADF binding site, and this second site is only largely occupied at high pH. All of these states display a high degree of cooperativity that appears to be an integral part of F-actin.

• Weaver AM et al.
• Cortactin promotes and stabilizes Arp2/3-induced actin filament network formation.
• Curr Biol. 2001; 11: 370-4
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• Cortactin is a c-src substrate associated with sites of dynamic actin assembly at the leading edge of migrating cells. We previously showed that cortactin binds to Arp2/3 complex, the essential molecular machine for nucleating actin filament assembly. In this study, we demonstrate that cortactin activates Arp2/3 complex based on direct visualization of filament networks and pyrene actin assays. Strikingly, cortactin potently inhibited the debranching of filament networks. When cortactin was added in combination with the active VCA fragment of N-WASp, they synergistically enhanced Arp2/3-induced actin filament branching. The N-terminal acidic and F-actin binding domains of cortactin were both necessary to activate Arp2/3 complex. These results support a model in which cortactin modulates actin filament dendritic nucleation by two mechanisms, (1) direct activation of Arp2/3 complex and (2) stabilization of newly generated filament branch points. By these mechanisms, cortactin may promote the formation and stabilization of the actin network that drives protrusion at the leading edge of migrating cells.

• Ono S
• The Caenorhabditis elegans unc-78 gene encodes a homologue of actin-interacting protein 1 required for organized assembly of muscle actin filaments.
• J Cell Biol. 2001; 152: 1313-9
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• Assembly and maintenance of myofibrils require dynamic regulation of the actin cytoskeleton. In Caenorhabditis elegans, UNC-60B, a muscle-specific actin depolymerizing factor (ADF)/cofilin isoform, is required for proper actin filament assembly in body wall muscle (Ono, S., D.L. Baillie, and G.M. Benian. 1999. J. Cell Biol. 145:491--502). Here, I show that UNC-78 is a homologue of actin-interacting protein 1 (AIP1) and functions as a novel regulator of actin organization in myofibrils. In unc-78 mutants, the striated organization of actin filaments is disrupted, and large actin aggregates are formed in the body wall muscle cells, resulting in defects in their motility. Point mutations in unc-78 alleles change conserved residues within different WD repeats of the UNC-78 protein and cause less severe phenotypes than a deletion allele, suggesting that these mutations partially impair the function of UNC-78. UNC-60B is normally localized in the diffuse cytoplasm and to the myofibrils in wild type but mislocalized to the actin aggregates in unc-78 mutants. Similar Unc-78 phenotypes are observed in both embryonic and adult muscles. Thus, AIP1 is an important regulator of actin filament organization and localization of ADF/cofilin during development of myofibrils.

• Chen J, Godt D, Gunsalus K, Kiss I, Goldberg M, Laski FA
• Cofilin/ADF is required for cell motility during Drosophila ovary development and oogenesis.
• Nat Cell Biol. 2001; 3: 204-9
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• The driving force behind cell motility is the actin cytoskeleton. Filopodia and lamellipodia are formed by the polymerization and extension of actin filaments towards the cell membrane. This polymerization at the barbed end of the filament is balanced by depolymerization at the pointed end, recycling the actin in a 'treadmilling' process. One protein involved in this process is cofilin/actin-depolymerizing factor (ADF), which can depolymerize actin filaments, allowing treadmilling to occur at an accelerated rate. Cofilin/ADF is an actin-binding protein that is required for actin-filament disassembly, cytokinesis and the organization of muscle actin filaments. There is also evidence that cofilin/ADF enhances cell motility, although a direct requirement in vivo has not yet been shown. Here we show that Drosophila cofilin/ADF, which is encoded by the twinstar (tsr) gene, promotes cell movements during ovary development and oogenesis. During larval development, cofilin/ADF is required for the cell rearrangement needed for formation of terminal filaments, stacks of somatic cells that are important for the initiation of ovarioles. It is also required for the migration of border cells during oogenesis. These results show that cofilin/ADF is an important regulator of actin-based cell motility during Drosophila development.

• Smertenko AP et al.
• Interaction of pollen-specific actin-depolymerizing factor with actin.
• Plant J. 2001; 25: 203-12
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• We have examined the interaction of recombinant lily pollen ADF, LlADF1, with actin and found that whilst it bound both G- and F-actin, it had a much smaller effect on the polymerization and depolymerization rate constants than the maize vegetative ADF, ZmADF3. An antiserum specific to pollen ADF, antipADF, was raised and used to localize pollen ADF in daffodil--a plant in which massive reorganizations of the actin cytoskeleton have been seen to occur as pollen enters and exits dormancy. We show, for the first time, an ADF decorating F-actin in cells that did not result from artificial increase in ADF concentration. In dehydrated pollen this ADF : actin array is replaced by actin : ADF rodlets and aggregates of actin, which presumably act as a storage form of actin during dormancy. In germinated pollen ADF has no specific localization, except when an adhesion is made at the tip where actin and ADF now co-localize. These activities of pollen ADF are discussed with reference to the activities of ZmADF3 and other members of the ADF/cofilin group of proteins.

• Taunton J
• Actin filament nucleation by endosomes, lysosomes and secretory vesicles.
• Curr Opin Cell Biol. 2001; 13: 85-91
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• Intracellular pathogens such as Listeria monocytogenes and vaccinia virus propel themselves through the cytoplasm of mammalian cells by nucleating actin filaments. Recently, actin assembly has also been shown to power the movement of intracellular vesicles, and this may be a mechanism underlying endomembrane movement in a variety of physiological contexts. Surprisingly, class I myosins have been found to play important roles in both actin nucleation and endomembrane trafficking.

• Provost P, Doucet J, Hammarberg T, Gerisch G, Samuelsson B, Radmark O
• 5-Lipoxygenase interacts with coactosin-like protein.
• J Biol Chem. 2001; 276: 16520-7
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• We have recently identified coactosin-like protein (CLP) in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait. In this report, we demonstrate a direct interaction between 5LO and CLP. 5LO associated with CLP, which was expressed as a glutathione S-transferase fusion protein, in a dose-dependent manner. Coimmunoprecipitation experiments using epitope-tagged 5LO and CLP proteins transiently expressed in human embryonic kidney 293 cells revealed the presence of CLP in 5LO immunoprecipitates. In reciprocal experiments, 5LO was detected in CLP immunoprecipitates. Non-denaturing polyacrylamide gel electrophoresis and cross-linking experiments showed that 5LO binds CLP in a 1:1 molar stoichiometry in a Ca(2+)-independent manner. Site-directed mutagenesis suggested an important role for lysine 131 of CLP in mediating 5LO binding. In view of the ability of CLP to bind 5LO and filamentous actin (F-actin), we determined whether CLP could physically link 5LO to actin filaments. However, no F-actin-CLP.5LO ternary complex was observed. In contrast, 5LO appeared to compete with F-actin for the binding of CLP. Moreover, 5LO was found to interfere with actin polymerization. Our results indicate that the 5LO-CLP and CLP-F-actin interactions are mutually exclusive and suggest a modulatory role for 5LO in actin dynamics.

• Dong CH, Xia GX, Hong Y, Ramachandran S, Kost B, Chua NH
• ADF proteins are involved in the control of flowering and regulate F-actin organization, cell expansion, and organ growth in Arabidopsis.
• Plant Cell. 2001; 13: 1333-46
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• Based mostly on the results of in vitro experiments, ADF (actin-depolymerizing factor) proteins are thought to be key modulators of the dynamic organization of the actin cytoskeleton. The few studies concerned with the in vivo function of ADF proteins that have been reported to date were performed almost exclusively using single-cell systems and have failed to produce consistent results. To investigate ADF functions in vivo and during the development of multicellular organs, we generated transgenic Arabidopsis plants that express a cDNA encoding an ADF protein (AtADF1) in the sense or the antisense orientation under the control of a strong constitutively active promoter. Selected lines with significantly altered levels of AtADF protein expression were characterized phenotypically. Overexpression of AtADF1 resulted in the disappearance of thick actin cables in different cell types, caused irregular cellular and tissue morphogenesis, and reduced the growth of cells and organs. In contrast, reduced AtADF expression promoted the formation of actin cables, resulted in a delay in flowering, and stimulated cell expansion as well as organ growth. These results are consistent with the molecular functions of ADF as predicted by in vitro studies, support the global roles of ADF proteins during the development of a multicellular organism, and demonstrate that these proteins are key regulators of F-actin organization, flowering, and cell and organ expansion in Arabidopsis.

• Goode BL, Rodal AA, Barnes G, Drubin DG
• Activation of the Arp2/3 complex by the actin filament binding protein Abp1p.
• J Cell Biol. 2001; 153: 627-34
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• The actin-related protein (Arp) 2/3 complex plays a central role in assembly of actin networks. Because distinct actin-based structures mediate diverse processes, many proteins are likely to make spatially and temporally regulated interactions with the Arp2/3 complex. We have isolated a new activator, Abp1p, which associates tightly with the yeast Arp2/3 complex. Abp1p contains two acidic sequences (DDW) similar to those found in SCAR/WASp proteins. We demonstrate that mutation of these sequences abolishes Arp2/3 complex activation in vitro. Genetic studies indicate that this activity is important for Abp1p functions in vivo. In contrast to SCAR/WASp proteins, Abp1p binds specifically to actin filaments, not monomers. Actin filament binding is mediated by the ADF/cofilin homology (ADF-H) domain of Abp1p and is required for Arp2/3 complex activation in vitro. We demonstrate that Abp1p recruits Arp2/3 complex to the sides of filaments, suggesting a novel mechanism of activation. Studies in yeast and mammalian cells indicate that Abp1p is involved functionally in endocytosis. Based on these results, we speculate that Abp1p may link Arp2/3-mediated actin assembly to a specific step in endocytosis.

• Mun JH et al.
• Two closely related cDNAs encoding actin-depolymerizing factors of petunia are mainly expressed in vegetative tissues.
• Gene. 2000; 257: 167-76
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• Actin-depolymerizing factor (ADF) is one of the small actin-binding proteins that regulate actin dynamics in cells. We have isolated two cDNA clones, PhADF1 and PhADF2, encoding ADF from cDNA libraries constructed from petal protoplast cultures and flowers of Petunia hybrida. PhADF1 and PhADF2 encode polypeptides of 139 and 143 amino acids with a calculated molecular mass of 16.04 and 16.51kDa, respectively. Co-sedimentation assay showed that the recombinant PhADF1 protein produced in Escherichia coli binds to F-actin at pH7. 0 and preferentially depolymerizes it at pH8.0. Gene tree analysis indicates that the plant ADF family can be grouped into four classes, and PhADFs are included in class I. Southern blot analyses revealed that one or two copies of PhADF genes are present in petunia genome, and several other related isoforms also exist. Northern blot analyses indicated that PhADF1 and PhADF2 are closely related and abundantly expressed in every plant organ except pollen. In addition, they are highly accumulated in mature vegetative tissue (petal, leaf, and stem). Our results indicate that the transcription of petunia ADF genes is differentially regulated by developmental signals.

• Lueck A, Yin HL, Kwiatkowski DJ, Allen PG
• Calcium regulation of gelsolin and adseverin: a natural test of the helix latch hypothesis.
• Biochemistry. 2000; 39: 5274-9
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• The gelsolin family of actin filament binding proteins have highly homologous structures. Gelsolin and adseverin, also known as scinderin, are the most similar members of this family, with adseverin lacking a C-terminal helix found in gelsolin. This helix has been postulated to serve as a calcium-sensitive latch, keeping gelsolin inactive. To test this hypothesis, we have analyzed the kinetics of severing by gelsolin, adseverin, and a gelsolin truncate which lacks the C-terminal latch. We find that the relationship between severing rate and calcium ion concentration differs between gelsolin and adseverin, and suggest that calcium controls one rate-limiting step in the activation of adseverin and two in the activation of gelsolin. In contrast, both proteins are activated equally by protons, and have identical severing kinetics at pHs below 6.3. The temperature sensitivity of severing by adseverin and gelsolin is remarkably different, with gelsolin increasing its severing rate 8-fold per 10 degrees C increase in temperature and adseverin increasing its rate only 2-fold per 10 degrees C increase in temperature. Analysis of the gelsolin construct lacking the C-terminal helix demonstrates that this helix is responsible for the regulatory differences between gelsolin and adseverin. These results support the C-terminal latch hypothesis for the calcium ion activation of gelsolin.

• Wear MA, Schafer DA, Cooper JA
• Actin dynamics: assembly and disassembly of actin networks.
• Curr Biol. 2000; 10: 8915-8915
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• Assembly of branched actin filament networks at the leading edge of migrating cells requires stimulation of the Arp2/3 complex by WASp proteins, in concert with the WASp activators Cdc42, PIP(2) and profilin. Network disassembly and debranching appears to be linked to actin-bound ATP hydrolysis as filaments age.

• Blanchoin L, Pollard TD, Mullins RD
• Interactions of ADF/cofilin, Arp2/3 complex, capping protein and profilin in remodeling of branched actin filament networks.
• Curr Biol. 2000; 10: 1273-82
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• BACKGROUND: Cellular movements are powered by the assembly and disassembly of actin filaments. Actin dynamics are controlled by Arp2/3 complex, the Wiskott-Aldrich syndrome protein (WASp) and the related Scar protein, capping protein, profilin, and the actin-depolymerizing factor (ADF, also known as cofilin). Recently, using an assay that both reveals the kinetics of overall reactions and allows visualization of actin filaments, we showed how these proteins co-operate in the assembly of branched actin filament networks. Here, we investigated how they work together to disassemble the networks. RESULTS: Actin filament branches formed by polymerization of ATP-actin in the presence of activated Arp2/3 complex were found to be metastable, dissociating from the mother filament with a half time of 500 seconds. The ADF/cofilin protein actophorin reduced the half time for both dissociation of gamma-phosphate from ADP-Pi-actin filaments and debranching to 30 seconds. Branches were stabilized by phalloidin, which inhibits phosphate dissociation from ADP-Pi-filaments, and by BeF3, which forms a stable complex with ADP and actin. Arp2/3 complex capped pointed ends of ATP-actin filaments with higher affinity (Kd approximately 40 nM) than those of ADP-actin filaments (Kd approximately 1 microM), explaining why phosphate dissociation from ADP-Pi-filaments liberates branches. Capping protein prevented annealing of short filaments after debranching and, with profilin, allowed filaments to depolymerize at the pointed ends. CONCLUSIONS: The low affinity of Arp2/3 complex for the pointed ends of ADP-actin makes actin filament branches transient. By accelerating phosphate dissociation, ADF/cofilin promotes debranching. Barbed-end capping proteins and profilin allow dissociated branches to depolymerize from their free pointed ends.

• Liu ZJ, Vysotski ES, Chen CJ, Rose JP, Lee J, Wang BC
• Structure of the Ca2+-regulated photoprotein obelin at 1.7 A resolution determined directly from its sulfur substructure.
• Protein Sci. 2000; 9: 2085-93
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• The crystal structure of the photoprotein obelin (22.2 kDa) from Obelia longissima has been determined and refined to 1.7 A resolution. Contrary to the prediction of a peroxide, the noncovalently bound substrate, coelenterazine, has only a single oxygen atom bound at the C2-position. The protein-coelenterazine 2-oxy complex observed in the crystals is photo-active because, in the presence of calcium ion, bioluminescence emission within the crystal is observed. This structure represents only the second de novo protein structure determined using the anomalous scattering signal of the sulfur substructure in the crystal. The method used here is theoretically different from that used for crambin in 1981 (4.72 kDa) and represents a significant advancement in protein crystal structure determination.

• Qian Y, Baisden JM, Zot HG, Van Winkle WB, Flynn DC
• The carboxy terminus of AFAP-110 modulates direct interactions with actin filaments and regulates its ability to alter actin filament integrity and induce lamellipodia formation.
• Exp Cell Res. 2000; 255: 102-13
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• The actin filament-associated protein AFAP-110 is an SH2/SH3 binding partner for Src. AFAP-110 contains several protein-binding motifs in its amino terminus and has been hypothesized to function as an adaptor molecule that could link signaling proteins to actin filaments. Recent studies using deletional mutagenesis demonstrated that AFAP-110 can alter actin filament integrity in SV40 transformed Cos-1 cells. Thus, AFAP-110 may be positioned to modulate the effects of Src upon actin filaments. In this report, we sought to determine whether (a) AFAP-110 could interact with actin filaments directly and (b) deletion mutants could affect actin filament integrity and cell shape in untransformed fibroblast cells. The data demonstrate that the carboxy terminus of AFAP-110 is both necessary and sufficient for actin filament association, in vivo and in vitro. Analysis of the carboxy terminus revealed a mean 40% similarity with other known actin-binding motifs, indicating a mechanism for binding to actin filaments. AFAP-110 can also induce lamellipodia formation. Contiguous with the alpha-helical, actin-binding motif is an alpha-helical, leucine zipper motif. Deletion of the leucine zipper motif (AFAP(Deltalzip)) followed by cellular expression enabled AFAP(Deltalzip) to alter actin filament integrity and cell shape in untransformed cells as evidenced by the induction of lamellipodia formation. We hypothesize that AFAP-110 may be an important signaling protein that can directly modulate changes in actin filament integrity and induce lamellipodia formation.

• Karpova TS, Reck-Peterson SL, Elkind NB, Mooseker MS, Novick PJ, Cooper JA
• Role of actin and Myo2p in polarized secretion and growth of Saccharomyces cerevisiae.
• Mol Biol Cell. 2000; 11: 1727-37
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• We examined the role of the actin cytoskeleton in secretion in Saccharomyces cerevisiae with the use of several quantitative assays, including time-lapse video microscopy of cell surface growth in individual living cells. In latrunculin, which depolymerizes filamentous actin, cell surface growth was completely depolarized but still occurred, albeit at a reduced level. Thus, filamentous actin is necessary for polarized secretion but not for secretion per se. Consistent with this conclusion, latrunculin caused vesicles to accumulate at random positions throughout the cell. Cortical actin patches cluster at locations that correlate with sites of polarized secretion. However, we found that actin patch polarization is not necessary for polarized secretion because a mutant, bee1Delta(las17Delta), which completely lacks actin patch polarization, displayed polarized growth. In contrast, a mutant lacking actin cables, tpm1-2 tpm2Delta, had a severe defect in polarized growth. The yeast class V myosin Myo2p is hypothesized to mediate polarized secretion. A mutation in the motor domain of Myo2p, myo2-66, caused growth to be depolarized but with only a partial decrease in the level of overall growth. This effect is similar to that of latrunculin, suggesting that Myo2p interacts with filamentous actin. However, inhibition of Myo2p function by expression of its tail domain completely abolished growth.

• Trifaro JM, Rose SD, Marcu MG
• Scinderin, a Ca2+-dependent actin filament severing protein that controls cortical actin network dynamics during secretion.
• Neurochem Res. 2000; 25: 133-44
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• Secretory vesicles are localized in specific compartments within neurosecretory cells. These are different pools in which vesicles are in various states of releasability. The transit of vesicles between compartments is controlled and regulated by Ca2+, scinderin and the cortical F-actin network. Cortical F-actin disassembly is produced by the filament severing activity of scinderin. This Ca2+-dependent activity of scinderin together with its Ca2+-independent actin nucleating activity, control cortical F-actin dynamics during the secretory cycle. A good understanding of the interaction of actin with scinderin and of the role of this protein in secretion has been provided by the analysis of the molecular structure of scinderin together with the use of recombinant proteins corresponding to its different domains.

• Sameshima M, Kishi Y, Osumi M, Mahadeo D, Cotter DA
• Novel actin cytoskeleton: actin tubules.
• Cell Struct Funct. 2000; 25: 291-5
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• In spores of Dictyostelium discoideum three actin filaments are bundled to form a novel tubular structure and the tubules are then organized into rods. These tubular structures we will term actin tubules. Actin tubules are reconstructed from the supernatant of spore homogenates, while the usual actin filaments were bundled after incubation of supernatants from growing cells. Alpha-actinin, ABP-120 and EF-1alpha are not essential for rod formation. Cofilin is a component of the cytoplasmic rods but few cofilin molecules are included in the nuclear rods. The viability of spores lacking actin rods is very low, and the spore shape is round instead of capsular. The rods can be fragmented by pressure, indicating that the rods may be effective in absorbing physical pressure. The complex organization of actin filaments, actin tubules and rods may be required for spores to achieve complete dormancy and maintain viability.

• Bonet C, Ternent D, Maciver SK, Mozo-Villarias A
• Rapid formation and high diffusibility of actin-cofilin cofilaments at low pH.
• Eur J Biochem. 2000; 267: 3378-84
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• Cofilin is a small actin-binding protein that is known to bind both F-actin and G-actin, severing the former. The interaction of cofilin with actin is pH-sensitive, F-actin being preferentially bound at low pH and G-actin at higher pH, within the physiological range. Diffusion coefficients of F-actin with cofilin were measured by the fluorescence recovery after photobleaching (FRAP) technique. This has the potential for simultaneous and direct measurement of average polymer length via the average diffusion coefficient of the polymers (DLM) as well as the fraction of polymerized actin, fLM, present in solution. In the range of cofilin-actin ratios up to 1 : 1 and at both pH 6.5 and pH 8.0, the diffusion coefficients of the polymers increased with the amount of cofilin present in the complex, in a co-operative manner to a plateau. We interpret this as indicating co-operative binding/severing and that filaments less than a certain length cannot be severed further. Under the conditions used here, filaments were found to be more motile at pH 6.5 than at pH 8.0. At pH 8.0, some actin is expected to be sequestered as ADP-actin-cofilin complexes, with the remaining actin being present as long slowly diffusing filaments. At pH 6.5, however, cofilin binds to F-actin to form short rapidly diffusing cofilaments. These filaments form very rapidly from cofilin-actin monomeric complexes, possibly indicating that this complex is able to polymerize without dissociation. These findings may be relevant to the nuclear import of actin-cofilin complexes.

• Pantaloni D, Boujemaa R, Didry D, Gounon P, Carlier MF
• The Arp2/3 complex branches filament barbed ends: functional antagonism with capping proteins.
• Nat Cell Biol. 2000; 2: 385-91
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• The Arp2/3 complex is an essential regulator of actin polymerization in response to signalling and generates a dendritic array of filaments in lamellipodia. Here we show that the activated Arp2/3 complex interacts with the barbed ends of filaments to initiate barbed-end branching. Barbed-end branching by Arp2/3 quantitatively accounts for polymerization kinetics and for the length correlation of the branches of filaments observed by electron microscopy. Filament branching is visualized at the surface of Listeria in a reconstituted motility assay. The functional antagonism between the Arp2/3 complex and capping proteins is essential in the maintenance of the steady state of actin assembly and actin-based motility.

• Hajkova L, Nyman T, Lindberg U, Karlsson R
• Effects of cross-linked profilin:beta/gamma-actin on the dynamics of the microfilament system in cultured cells.
• Exp Cell Res. 2000; 256: 112-21
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• There is evidence that the profilin:actin complex is the immediate precursor in the formation of actin filaments in cells. This paper describes the cell morphology and microfilament distribution after microinjection of covalently cross-linked profilin:beta/gamma-actin (PxA) in two different cell lines. Injected cells were either kept unstimulated or stimulated with platelet-derived growth factor (PDGF) before fixation and visualization of filamentous actin. After injection of low doses of PxA, the cells displayed an actin organization characterized by a clearance of diffuse fluorescence from a region immediately interior of ruffling edges and the appearance of small dots of fluorescence in the same region. At higher concentrations, PxA effectively inhibited outgrowth of lamellae and microspikes, and there was a drastic reduction of actin staining in the zone behind the advancing edge. This effect is reminiscent of the effect of cytochalasin B on fibroblasts and the growth cone of neuronal cells. As in these cases, there remained a rim of actin-dependent fluorescence on the very edge of the membrane lamella, particularly in the PxA-treated fibroblasts. The interference of PxA with the formation of surface structures was pronounced after PDGF stimulation. Here, PxA effectively eliminated the enhancement of the ruffling activity in the cell edges and on the dorsal surface of the cells. In contrast to PxA, injection of non-cross-linked profilin:beta/gamma-actin had no apparent effect on cell morphology and microfilament distribution except for an increased concentration of filamentous actin in one of the cell lines.

• Pope BJ, Gonsior SM, Yeoh S, McGough A, Weeds AG
• Uncoupling actin filament fragmentation by cofilin from increased subunit turnover.
• J Mol Biol. 2000; 298: 649-61
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• The actin depolymerizing factor (ADF)/cofilin family of proteins interact with actin monomers and filaments in a pH-sensitive manner. When ADF/cofilin binds F-actin it induces a change in the helical twist and fragmentation; it also accelerates the dissociation of subunits from the pointed ends of filaments, thereby increasing treadmilling or depolymerization. Using site-directed mutagenesis we characterized the two actin-binding sites on human cofilin. One target site was chosen because we previously showed that the villin head piece competes with ADF for binding to F-actin. Limited sequence homology between ADF/cofilin and the part of the villin headpiece essential for actin binding suggested an actin-binding site on cofilin involving a structural loop at the opposite end of the molecule to the alpha-helix already implicated in actin binding. Binding through the alpha-helix is primarily to monomeric actin, whereas the loop region is specifically involved in filament association. We have characterized the actin binding properties of each site independently of the other. Mutation of a single lysine residue in the loop region abolishes binding to filaments, but not to monomers. Using the mutation analogous to the phosphorylated form of cofilin (S3D), we show that filament binding is inhibited at physiological ionic strength but not under low salt conditions. At low ionic strength, this mutant induces both the twist change and fragmentation characteristic of wild-type cofilin, but does not activate subunit dissociation. The results suggest a two-site binding to filaments, initiated by association through the loop site, followed by interaction with the adjacent subunit through the "helix" site at the opposite end of the molecule. Together, these interactions induce twist and fragmentation of filaments, but the twist change itself is not responsible for the enhanced rate of actin subunit release from filaments.

• Cooper JA, Schafer DA
• Control of actin assembly and disassembly at filament ends.
• Curr Opin Cell Biol. 2000; 12: 97-103
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• The most important discovery in the field is that the Arp2/3 complex nucleates assembly of actin filaments with free barbed ends. Arp2/3 also binds the sides of actin filaments to create a branched network. Arp2/3's nucleation activity is stimulated by WASP family proteins, some of which mediate signaling from small G-proteins. Listeria movement caused by actin polymerization can be reconstituted in vitro using purified proteins: Arp2/3 complex, capping protein, actin depolymerizing factor/cofilin, and actin. actin depolymerizing factor/cofilin increases the rate at which actin subunits leave pointed ends, and capping protein caps barbed ends.

• Southwick FS
• Gelsolin and ADF/cofilin enhance the actin dynamics of motile cells.
• Proc Natl Acad Sci U S A. 2000; 97: 6936-8

• Bowman GD, Nodelman IM, Hong Y, Chua NH, Lindberg U, Schutt CE
• A comparative structural analysis of the ADF/cofilin family.
• Proteins. 2000; 41: 374-84
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• Actin-depolymerizing factor (ADF) and cofilin define a family of actin-binding proteins essential for the rapid turnover of filamentous actin in vivo. Here we present the 2.0 A crystal structure of Arabidopsis thaliana ADF1 (AtADF1), the first plant crystal structure from the ADF/cofilin (AC) family. Superposition of the four AC isoform structures permits an accurate sequence alignment that differs from previously reported data for the location of vertebrate-specific inserts and reveals a contiguous, vertebrate-specific surface opposite the putative actin-binding surface. Extending the structure-based sequence alignment to include 30 additional isoforms indicates three major groups: vertebrates, plants, and "other eukaryotes." Within these groups, several structurally conserved residues that are not conserved throughout the entire AC family have been identified. Residues that are highly conserved among all isoforms tend to cluster around the tryptophan at position 90 and a structurally conserved kink in alpha-helix 3. Analysis of surface character shows the presence of a hydrophobic patch and a highly conserved acidic cluster, both of which include several residues previously implicated in actin binding.

• Ho YS, Burden LM, Hurley JH
• Structure of the GAF domain, a ubiquitous signaling motif and a new class of cyclic GMP receptor.
• EMBO J. 2000; 19: 5288-99
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• GAF domains are ubiquitous motifs present in cyclic GMP (cGMP)-regulated cyclic nucleotide phosphodiesterases, certain adenylyl cyclases, the bacterial transcription factor FhlA, and hundreds of other signaling and sensory proteins from all three kingdoms of life. The crystal structure of the Saccharomyces cerevisiae YKG9 protein was determined at 1.9 A resolution. The structure revealed a fold that resembles the PAS domain, another ubiquitous signaling and sensory transducer. YKG9 does not bind cGMP, but the isolated first GAF domain of phosphodiesterase 5 binds with K:(d) = 650 nM. The cGMP binding site of the phosphodiesterase GAF domain was identified by homology modeling and site-directed mutagenesis, and consists of conserved Arg, Asn, Lys and Asp residues. The structural and binding studies taken together show that the cGMP binding GAF domains form a new class of cyclic nucleotide receptors distinct from the regulatory domains of cyclic nucleotide-regulated protein kinases and ion channels.

• Blanchoin L, Robinson RC, Choe S, Pollard TD
• Phosphorylation of Acanthamoeba actophorin (ADF/cofilin) blocks interaction with actin without a change in atomic structure.
• J Mol Biol. 2000; 295: 203-11
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• LIM-kinase activated by GST-Pak1 phosphorylates Acanthamoeba actophorin stoichiometrically and specifically on serine 1. The atomic structure of phosphorylated actophorin determined by X-ray crystallography is essentially identical with the structure of unphosphorylated actophorin. We compared biochemical properties of phosphorylated actophorin, unphosphorylated actophorin and mutants of actophorin with serine 1 replaced by aspartic acid or alanine. Phosphorylation strongly inhibits interaction of actophorin with Mg-ADP- or Mg-ATP-actin monomers and Mg-ADP-actin filaments, so Ser1 phosphorylation directly blocks interaction of actin-depolymerizing factor (ADF)/cofilin proteins with actin. About 30 % of actophorin is phosphorylated in live amoebas grown in suspension culture. Phosphorylation of ADF/cofilin proteins by LIM-kinase or other enzymes will tend to stabilize actin filaments by inhibiting the ability of these proteins to sever and depolymerize older actin filaments that have hydrolyzed their bound ATP and dissociated the phosphate.

• Meberg PJ
• Signal-regulated ADF/cofilin activity and growth cone motility.
• Mol Neurobiol. 2000; 21: 97-107
• Display abstract

• It is becoming increasingly evident that proteins of the actin depolymerizing factor (ADF)/cofilin family are essential regulators of actin turnover required for many actin-based cellular processes, including motility. ADF can increase actin turnover by either increasing the rate of actin filament treadmilling or by severing actin filaments. In neurons ADF is highly expressed in neuronal growth cones and its activity is regulated by many signals that affect growth cone motility. In addition, increased activity of ADF causes an increase in neurite extension. ADF activity is inhibited upon phosphorylation by LIM kinases (LIMK), kinases activated by members of the Rho family of small GTPases. ADF become dephosphorylated downstream of signal pathways that activate PI-3 kinase or increase levels of intracellular calcium. The growth-regulating effects of ADF together with its ability to be regulated by a wide variety of guidance cues, suggest that ADF may regulate growth cone advance and navigation.

• Meberg PJ, Bamburg JR
• Increase in neurite outgrowth mediated by overexpression of actin depolymerizing factor.
• J Neurosci. 2000; 20: 2459-69
• Display abstract

• Growth cone motility is regulated by changes in actin dynamics. Actin depolymerizing factor (ADF) is an important regulator of actin dynamics, and extracellular signal-induced changes in ADF activity may influence growth cone motility and neurite extension. To determine this directly, we overexpressed ADF in primary neurons and analyzed neurite lengths. Recombinant adenoviruses were constructed that express wild-type Xenopus ADF/cofilin [XAC(wt)], as well as two mutant forms of XAC, the active but nonphosphorylatable XAC(A3) and the less active, pseudophosphorylated XAC(E3). XAC expression was detectable on Western blots 24 hr after infection and peaked at 3 d in cultured rat cortical neurons. Peak expression was approximately 75% that of endogenous ADF. XAC(wt) expression caused a slight increase in growth cone area and filopodia but decreased filopodia numbers on neurite shafts. At maximal XAC levels, neurite lengths increased >50% compared with controls infected with a green fluorescent protein-expressing adenovirus. Increased neurite extension was directly related to the expression of active XAC. Expression of the XAC(E3) mutant did not increase neurite extension, whereas expression of the XAC(A3) mutant increased neurite extension but to a lesser extent than XAC(wt), which was partially phosphorylated. XAC expression had minimal, if any, impact on F-actin levels and did not result in compensatory changes in the expression of endogenous ADF or actin. However, F-actin turnover appeared to increase based on F-actin loss after treatment with drugs that block actin polymerization. These results provide direct evidence that increased ADF activity promotes process extension and neurite outgrowth.

• Chen H, Bernstein BW, Bamburg JR
• Regulating actin-filament dynamics in vivo.
• Trends Biochem Sci. 2000; 25: 19-23
• Display abstract

• The assembly and disassembly (i.e. turnover) of actin filaments in response to extracellular signals underlie a wide variety of basic cellular processes such as cell division, endocytosis and motility. The bulk turnover of subunits is 100-200 times faster in cells than with pure actin, suggesting a complex regulation in vivo. Significant progress has been made recently in identifying and clarifying the roles of several cellular proteins that coordinately regulate actin-filament turnover.

• Gutsche-Perelroizen I, Lepault J, Ott A, Carlier MF
• Filament assembly from profilin-actin.
• J Biol Chem. 1999; 274: 6234-43
• Display abstract

• Profilin plays a major role in the assembly of actin filament at the barbed ends. The thermodynamic and kinetic parameters for barbed end assembly from profilin-actin have been measured turbidimetrically. Filament growth from profilin-actin requires MgATP to be bound to actin. No assembly is observed from profilin-CaATP-actin. The rate constant for association of profilin-actin to barbed ends is 30% lower than that of actin, and the critical concentration for F-actin assembly from profilin-actin units is 0.3 microM under physiological ionic conditions. Barbed ends grow from profilin-actin with an ADP-Pi cap. Profilin does not cap the barbed ends and is not detectably incorporated into filaments. The EDC-cross-linked profilin-actin complex (PAcov) both copolymerizes with F-actin and undergoes spontaneous self-assembly, following a nucleation-growth process characterized by a critical concentration of 0.2 microM under physiological conditions. The PAcov polymer is a helical filament that displays the same diffraction pattern as F-actin, with layer lines at 6 and 36 nm. The PAcov filaments bound phalloidin with the same kinetics as F-actin, bound myosin subfragment-1, and supported actin-activated ATPase of myosin subfragment-1, but they did not translocate in vitro along myosin-coated glass surfaces. These results are discussed in light of the current models of actin structure.

• McGough A, Chiu W
• ADF/cofilin weakens lateral contacts in the actin filament.
• J Mol Biol. 1999; 291: 513-9
• Display abstract

• Observed in vivo motility rates can only be accounted for if the rate of actin filament treadmilling in cells is considerably greater than has been quantified for purified actin in vitro. ADF/cofilin is uniquely suited to promote actin dynamics in cells, owing to its remarkable ability to change actin filament structure. In earlier work we showed that human cofilin chanRges filament twist by about 5 degrees per subunit and suggested that this contributes to increased filament turnover. Our initial structural modeling provided some insights into how the longitudinal actin-actin contacts might be disrupted following cofilin-induced twisting. Here we present direct evidence that cofilin also disrupts lateral actin-actin contacts in the filament and suggest a model showing how this could contribute to cofilin's novel effects on actin filament dynamics and assembly.

• Svitkina TM, Borisy GG
• Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia.
• J Cell Biol. 1999; 145: 1009-26
• Display abstract

• The leading edge (approximately 1 microgram) of lamellipodia in Xenopus laevis keratocytes and fibroblasts was shown to have an extensively branched organization of actin filaments, which we term the dendritic brush. Pointed ends of individual filaments were located at Y-junctions, where the Arp2/3 complex was also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments suggested that the Arp2/3 complex also provided protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin was excluded from the distal 0.4 micrometer++ of the lamellipodial network of keratocytes and in fibroblasts it was located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Our evidence supports a dendritic nucleation model (Mullins, R.D., J.A. Heuser, and T.D. Pollard. 1998. Proc. Natl. Acad. Sci. USA. 95:6181-6186) for lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex.

• Loisel TP, Boujemaa R, Pantaloni D, Carlier MF
• Reconstitution of actin-based motility of Listeria and Shigella using pure proteins.
• Nature. 1999; 401: 613-6
• Display abstract

• Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, alpha-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.

• Cheng D, Marner J, Rubenstein PA
• Interaction in vivo and in vitro between the yeast fimbrin, SAC6P, and a polymerization-defective yeast actin (V266G and L267G).
• J Biol Chem. 1999; 274: 35873-80
• Display abstract

• A mutant yeast actin (GG) has decreased hydrophobicity in a subdomain 3/4 hydrophobic plug believed to be involved in a hydrophobic cross-strand "plug-pocket" interaction necessary for actin filament stability. This actin will not polymerize in vitro but is compatible with cell viability. We have assessed the ability of Sac6p, the yeast homologue of the actin filament stabilizing and bundling protein fimbrin, to restore polymerization in vitro and to facilitate GG-actin function in vivo. Sac6p rescues GG-actin polymerization at 25 degrees C but not at 4 degrees C. The actin polymerizes into bundles at room temperature with a fimbrin:actin molar ratio of 1:4. At this ratio, every actin monomer contacts a Sac6p actin binding domain. Following cold-induced depolymerization, actin/Sac6p mixtures repolymerize beginning at 15 degrees C instead of the 25 degrees C required for de novo assembly, because of the presence of residual actin-Sac6p nuclei. Generation of haploid Deltasac6/GG-actin cells from either diploid or haploid cells was unsuccessful. The facile isolation of cells with either mutation alone indicates a synthetic lethal relationship between this actin allele and the SAC6 gene. Sac6p may allow GG-actin function in vivo by stabilizing the actin in bundles thereby helping maintain sufficient levels of an otherwise destabilized actin monomer within the cell.

• Ressad F, Didry D, Egile C, Pantaloni D, Carlier MF
• Control of actin filament length and turnover by actin depolymerizing factor (ADF/cofilin) in the presence of capping proteins and ARP2/3 complex.
• J Biol Chem. 1999; 274: 20970-6
• Display abstract

• The effect of Arabidopsis thaliana ADF1 and human ADF on the number of filaments in F-actin solutions has been examined using a seeded polymerization assay. ADF did not sever filaments in a catalytic fashion, but decreased the steady-state length distribution of actin filaments in correlation with its effect on actin dynamics. The increase in filament number was modest as compared with the large increase in filament turnover. ADF did not decrease the length of filaments shorter than 1 micrometer. ADF promoted the rapid turnover of gelsolin-capped filaments in a manner dependent on the number of pointed ends. To explain these results, we propose that, as a consequence of the cooperative binding of ADF to F-actin, two populations of energetically different filaments coexist in solution pending a flux of subunits from one to the other. The ADF-decorated filaments depolymerize rapidly from their pointed ends, while undecorated filaments polymerize. ADF also promotes rapid turnover of gelsolin-capped filaments in the presence of the pointed end capper Arp2/3 complex. It is shown that the Arp2/3 complex steadily generates new barbed ends in solutions of gelsolin-capped filaments, which represents an important aspect of its function in actin-based motility.

• Ono S
• Purification and biochemical characterization of actin from Caenorhabditis elegans: its difference from rabbit muscle actin in the interaction with nematode ADF/cofilin.
• Cell Motil Cytoskeleton. 1999; 43: 128-36
• Display abstract

• Biochemical analysis of cytoskeletal proteins of the nematode Caenorhabditis elegans can be combined with a vast resource of genetic information in order to understand the regulation and function of the cytoskeleton in vivo. Here, I report an improved and efficient method to purify actin from wild-type C. elegans and characterization of its biochemical properties. The purified actin was highly pure and free of several known actin-binding proteins. G-actin was polymerized into F-actin in a similar kinetic process to rabbit muscle actin. G-actin interacted with bovine DNase I and inhibited its activity. However, UNC-60B, an isoform of ADF/cofilin in C. elegans, showed a marked depolymerizing activity on C. elegans actin but not on rabbit muscle actin. The results indicate that C. elegans actin shares common biochemical properties with rabbit muscle actin, while actin-binding proteins can interact with C. elegans actin in a distinct manner from rabbit muscle actin.

• Weber A
• Actin binding proteins that change extent and rate of actin monomer-polymer distribution by different mechanisms.
• Mol Cell Biochem. 1999; 190: 67-74
• Display abstract

• Actin binding proteins control actin assembly and disassembly by altering the critical concentration and by changing the kinetics of polymerization. All of these control mechanisms in some way or the other make use of the energy of hydrolysis of actin-bound ATP. Capping of barbed filament ends increases the critical concentration as long as ATP hydrolysis maintains a difference in the actin monomer binding constants of the two ends. A further increase in the critical concentration on adding a second cap, tropomodulin, to the other, pointed filament end also requires ATP hydrolysis as described by the model presented here. Changes in the critical concentration are amplified into much larger changes of the monomer pool by actin sequestering proteins, provided their actin binding equilibrium constants fall within a relatively narrow range around the values for the two critical concentrations of actin. Cofilin greatly speeds up treadmilling, which requires ATP hydroysis, by increasing the rate constant of depolymerization. Profilin increases the rate of elongation at the barbed filament end, coupled to a lowering of the critical concentration, only if ATP hydrolysis makes profilin binding to the barbed end independent of its binding constant for actin monomers.

• Sept D, Elcock AH, McCammon JA
• Computer simulations of actin polymerization can explain the barbed-pointed end asymmetry.
• J Mol Biol. 1999; 294: 1181-9
• Display abstract

• Computer simulations of actin polymerization were performed to investigate the role of electrostatic interactions in determining polymerization rates. Atomically detailed models of actin monomers and filaments were used in conjunction with a Brownian dynamics method. The simulations were able to reproduce the measured barbed end association rates over a range of ionic strengths and predicted a slower growing pointed end, in agreement with experiment. Similar simulations neglecting electrostatic interactions indicate that configurational and entropic factors may actually favor polymerization at the pointed end, but electrostatic interactions remove this trend. This result would indicate that polymerization at the pointed end is not only limited by diffusion, but faces electrostatic forces that oppose binding. The binding of the actin depolymerizing factor (ADF) and G-actin complex to the end of a filament was also simulated. In this case, electrostatic steering effects lead to an increase in the simulated association rate. Together, the results indicate that simulations provide a realistic description of both polymerization and the binding of more complex structures to actin filaments.

• Bamburg JR
• Proteins of the ADF/cofilin family: essential regulators of actin dynamics.
• Annu Rev Cell Dev Biol. 1999; 15: 185-230
• Display abstract

• Ubiquitous among eukaryotes, the ADF/cofilins are essential proteins responsible for the high turnover rates of actin filaments in vivo. In vertebrates, ADF and cofilin are products of different genes. Both bind to F-actin cooperatively and induce a twist in the actin filament that results in the loss of the phalloidin-binding site. This conformational change may be responsible for the enhancement of the off rate of subunits at the minus end of ADF/cofilin-decorated filaments and for the weak filament-severing activity. Binding of ADF/cofilin is competitive with tropomyosin. Other regulatory mechanisms in animal cells include binding of phosphoinositides, phosphorylation by LIM kinases on a single serine, and changes in pH. Although vertebrate ADF/cofilins contain a nuclear localization sequence, they are usually concentrated in regions containing dynamic actin pools, such as the leading edge of migrating cells and neuronal growth cones. ADF/cofilins are essential for cytokinesis, phagocytosis, fluid phase endocytosis, and other cellular processes dependent upon actin dynamics.

• Carlier MF, Ressad F, Pantaloni D
• Control of actin dynamics in cell motility. Role of ADF/cofilin.
• J Biol Chem. 1999; 274: 33827-30

• Huang L, Wong TY, Lin RC, Furthmayr H
• Replacement of threonine 558, a critical site of phosphorylation of moesin in vivo, with aspartate activates F-actin binding of moesin. Regulation by conformational change.
• J Biol Chem. 1999; 274: 12803-10
• Display abstract

• Point and deletion mutants of moesin were examined for F-actin binding by blot overlay and co-sedimentation, and for intra- and intermolecular interactions with N- and C-terminal domains with yeast two-hybrid and in vitro binding assays. Wild-type moesin molecules interact poorly with F-actin and each other, and bind neither C- nor N-terminal fragments. Interaction with F-actin is strongly enhanced by replacement of Thr558 with aspartate (T558D), by deletion of 11 N-terminal residues (DelN11), by deletion of the entire N-terminal membrane-binding domain of both wild type and T558D mutant molecules, and by exposure to phosphatidylinositol 4, 5-diphosphate. Activation of F-actin binding is accompanied by changes in inter- and intramolecular domain interactions. The T558D mutation renders moesin capable of binding wild type but not mutated (T558D) C-terminal or wild type N-terminal fragments. The interaction between the latter two is prevented. DelN11 truncation enables binding of wild type N and C domain fragments. These changes suggest that the T558D mutation, mimicking phosphorylation of Thr558, promotes F-actin binding by disruption of interdomain interactions between N and C domains and exposure of the high affinity F-actin binding site in the C-terminal domain. Oscillation between activated and resting state could thus provide the structural basis for transient interactions between moesin and the actin cytoskeleton in protruding and retracting microextensions.

• Huang M, Yang C, Schafer DA, Cooper JA, Higgs HN, Zigmond SH
• Cdc42-induced actin filaments are protected from capping protein.
• Curr Biol. 1999; 9: 979-82
• Display abstract

• Each actin filament has a pointed and a barbed end, however, filament elongation occurs primarily at the barbed end. Capping proteins, by binding to the barbed end, can terminate this elongation. The rate of capping depends on the concentration of capping protein [1], and thus, if capping terminates elongation, the length of filaments should vary inversely with the concentration of capping protein. In cell extracts, such as those derived from neutrophils, new actin filaments can be nucleated by addition of GTPgammaS-activated Cdc42 (a small GTPase of the Rho family). To determine whether elongation of these filaments is terminated by capping, we manipulated the concentration of capping protein, the major calcium-independent capping protein in neutrophils, and observed the effects on filament lengths. Depletion of 70% of the capping protein from extracts increased the mean length of filaments elongated from spectrin-actin seeds (very short actin filaments with free barbed ends) but did not increase the mean length of filaments induced by Cdc42. Furthermore, doubling the concentration of capping protein in cell extracts by adding pure capping protein did not decrease the mean length of filaments induced by Cdc42. These results suggest that the barbed ends of Cdc42-induced filaments are protected from capping by capping protein.

• Sinha S, Rappu P, Lange SC, Mantsala P, Zalkin H, Smith JL
• Crystal structure of Bacillus subtilis YabJ, a purine regulatory protein and member of the highly conserved YjgF family.
• Proc Natl Acad Sci U S A. 1999; 96: 13074-9
• Display abstract

• The yabJ gene in Bacillus subtilis is required for adenine-mediated repression of purine biosynthetic genes in vivo and codes for an acid-soluble, 14-kDa protein. The molecular mechanism of YabJ is unknown. YabJ is a member of a large, widely distributed family of proteins of unknown biochemical function. The 1.7-A crystal structure of YabJ reveals a trimeric organization with extensive buried hydrophobic surface and an internal water-filled cavity. The most important finding in the structure is a deep, narrow cleft between subunits lined with nine side chains that are invariant among the 25 most similar homologs. This conserved site is proposed to be a binding or catalytic site for a ligand or substrate that is common to YabJ and other members of the YER057c/YjgF/UK114 family of proteins.

• Iida K, Yahara I
• Cooperation of two actin-binding proteins, cofilin and Aip1, in Saccharomyces cerevisiae.
• Genes Cells. 1999; 4: 21-32
• Display abstract

• BACKGROUND: Cofilin is a low-molecular weight actin-modulating protein, and is structurally and functionally conserved among eukaryotes. Cofilin is encoded by COF1 in Saccharomyces cerevisiae, and is essential for cell viability. Cofilin binds to and severs actin filaments in vitro, and also enhances their depolymerization. A partner protein that cooperates with cofilin in vivo has not been identified. RESULTS: When COF1 was over-expressed in yeast cells under the GAL1 promoter in a medium containing galactose as a sole carbon source, the cells did not survive. These results indicate that cells can grow only when the expression of cofilin is appropriately regulated. Several temperature sensitive (ts-) mutants were independently created by the random mutagenesis of COF1 with hydroxylamine. Mutated amino acids in ts-mutants were mapped in the sequences that were presumed to be involved in actin binding. A gene on a multicopy plasmid which suppresses the ts-phenotype of cof1-101, a typical ts-cofilin mutant, was isolated. The suppressor gene, SCF1, was found to be identical to AIP1, a gene encoding an actin-interacting protein. Although SCF1/AIP1 is not essential for cell viability, a combination of cof1-101 and Deltascf1/aip1 is synthetic lethal. Immunofluorescence staining of a wild-type strain using anti-Aip1 antibodies revealed that Aip1 was distributed in cortical actin patches where cofilin was also co-localized. Thick and long fibres stained with anti-cofilin antibody were detected in Deltascf1/aip1 cells, but not in SCF1/AIP1 cells. CONCLUSIONS: These results suggest the cooperative modulation of the actin cytoskeleton by cofilin and Aip1.

• Rodal AA, Tetreault JW, Lappalainen P, Drubin DG, Amberg DC
• Aip1p interacts with cofilin to disassemble actin filaments.
• J Cell Biol. 1999; 145: 1251-64
• Display abstract

• Actin interacting protein 1 (Aip1) is a conserved component of the actin cytoskeleton first identified in a two-hybrid screen against yeast actin. Here, we report that Aip1p also interacts with the ubiquitous actin depolymerizing factor cofilin. A two-hybrid-based approach using cofilin and actin mutants identified residues necessary for the interaction of actin, cofilin, and Aip1p in an apparent ternary complex. Deletion of the AIP1 gene is lethal in combination with cofilin mutants or act1-159, an actin mutation that slows the rate of actin filament disassembly in vivo. Aip1p localizes to cortical actin patches in yeast cells, and this localization is disrupted by specific actin and cofilin mutations. Further, Aip1p is required to restrict cofilin localization to cortical patches. Finally, biochemical analyses show that Aip1p causes net depolymerization of actin filaments only in the presence of cofilin and that cofilin enhances binding of Aip1p to actin filaments. We conclude that Aip1p is a cofilin-associated protein that enhances the filament disassembly activity of cofilin and restricts cofilin localization to cortical actin patches.

• Goode BL et al.
• Coronin promotes the rapid assembly and cross-linking of actin filaments and may link the actin and microtubule cytoskeletons in yeast.
• J Cell Biol. 1999; 144: 83-98
• Display abstract

• Coronin is a highly conserved actin-associated protein that until now has had unknown biochemical activities. Using microtubule affinity chromatography, we coisolated actin and a homologue of coronin, Crn1p, from Saccharomyces cerevisiae cell extracts. Crn1p is an abundant component of the cortical actin cytoskeleton and binds to F-actin with high affinity (Kd 6 x 10(-9) M). Crn1p promotes the rapid barbed-end assembly of actin filaments and cross-links filaments into bundles and more complex networks, but does not stabilize them. Genetic analyses with a crn1Delta deletion mutation also are consistent with Crn1p regulating filament assembly rather than stability. Filament cross-linking depends on the coiled coil domain of Crn1p, suggesting a requirement for Crn1p dimerization. Assembly-promoting activity is independent of cross-linking and could be due to nucleation and/or accelerated polymerization. Crn1p also binds to microtubules in vitro, and microtubule binding is enhanced by the presence of actin filaments. Microtubule binding is mediated by a region of Crn1p that contains sequences (not found in other coronins) homologous to the microtubule binding region of MAP1B. These activities, considered with microtubule defects observed in crn1Delta cells and in cells overexpressing Crn1p, suggest that Crn1p may provide a functional link between the actin and microtubule cytoskeletons in yeast.

• Rich P, Fisher N
• Generic features of quinone-binding sites.
• Biochem Soc Trans. 1999; 27: 561-5

• Aizawa H, Katadae M, Maruya M, Sameshima M, Murakami-Murofushi K, Yahara I
• Hyperosmotic stress-induced reorganization of actin bundles in Dictyostelium cells over-expressing cofilin.
• Genes Cells. 1999; 4: 311-24
• Display abstract

• BACKGROUND: Cofilin is a low-molecular weight actin-modulating protein, which binds to, severs, and depolymerizes actin filaments in vitro. Aip1, an actin-interacting protein, was recently identified as a product of a gene on a multicopy plasmid which suppresses the temperature-sensitive phenotype of a cofilin mutant in Saccharomyces cerevisiae. Actin cytoskeleton plays an essential role in resistance to hyperosmotic stress in Dictyostelium discoideum. The roles of cofilin and Aip1 in this resistance are not known. RESULTS: In response to hyperosmotic stress, D. discoideum cells round up. This stress-induced morphological change involves the redistribution of cofilin, together with actin filaments, into cortical contractile portions of the cells, followed by their contraction. Over-expression of cofilin increases and thickens cortical actin bundles in cells. The bundles become tight and are reorganized into a ring-shaped structure in response to hyperosmotic stress. The ring structure of actin bundles had two characteristic bands across them; bright and dark bands, heavily stained and not stained with phalloidin. In the bundles, straight filaments with a diameter of 5.3-nm were aligned parallel by cross-bridge structures. In cells lacking the myosin-II heavy chain, the bundles, which were induced by an over-expression of cofilin, shortened and became straight following hyperosmotic stress, forming a polygonal structure. D. discoideum Aip1/Wrp2 enhanced the severing of actin filaments by cofilin in vitro and colocalized with cofilin in cells, including those that were over-expressing cofilin before and after exposure to hyperosmotic stress. CONCLUSIONS: Cofilin plays a pivotal role in concert with Aip1/Wrp2 in the reorganization of actin architectures into bundles that contract in a myosin-II-independent manner, in response to hyperosmotic stress.

• Moriyama K, Yahara I
• Two activities of cofilin, severing and accelerating directional depolymerization of actin filaments, are affected differentially by mutations around the actin-binding helix.
• EMBO J. 1999; 18: 6752-61
• Display abstract

• The biochemical activities of cofilin are controversial. We demonstrated that porcine cofilin severs actin filaments and accelerates monomer release at the pointed ends. At pH 7.1, 0.8 &mgr;M cofilin cut filaments (2.2 &mgr;M actin) about every 290 subunits and increased the depolymerization rate 6.4-fold. A kink in the major alpha-helix of cofilin is thought to constitute a contact site for actin. Side chain hydroxyl groups of Ser119, Ser120 and Tyr82 in cofilin form hydrogen bonds with main chain carbonyl moieties from the helix, causing the kink. We eliminated side chain hydroxyls by Ser-->Ala and/or Tyr-->Phe mutagenesis. Severing and depolymerization-enhancing activities were reduced dramatically in an Ala120 mutant, whereas the latter was decreased in a Phe82 mutant with a relatively small effect on severing, suggesting different structural bases for the two activities of cofilin. The Ala120-equivalent mutation in yeast cofilin affected cell growth, whereas that of the Phe82-equivalent had no effect in yeast. These results indicate the physiological significance of the severing activity of cofilin that is brought about by the kink in the helix.

• Bamburg JR, McGough A, Ono S
• Putting a new twist on actin: ADF/cofilins modulate actin dynamics.
• Trends Cell Biol. 1999; 9: 364-70
• Display abstract

• The actin-depolymerizing factor (ADF)/cofilins are a family of essential actin regulatory proteins, ubiquitous among eukaryotes, that enhance the turnover of actin by regulating the rate constants of polymerization and depolymerization at filament ends, changing the twist of the filament and severing actin filaments. Genetic and cell-biological studies have shown that an ADF/cofilin is required to drive the high turnover of the actin cytoskeleton observed in vivo. The activity of ADF/cofilin is regulated by a variety of mechanisms, including specific phosphorylation and dephosphorylation. This review addresses aspects of ADF/cofilin structure, dynamics, regulation and function.

• Eicken C, Krebs B, Sacchettini JC
• Catechol oxidase - structure and activity.
• Curr Opin Struct Biol. 1999; 9: 677-83
• Display abstract

• Recently determined structures of copper-containing plant catechol oxidase in three different catalytic states have provided new insights into the mechanism of this enzyme and its relationship to other copper type-3 proteins. Moreover, the active site of catechol oxidase has been found to be structurally conserved with the oxygen-binding site of a molluscan hemocyanin.

• Maciver SK, Pope BJ, Whytock S, Weeds AG
• The effect of two actin depolymerizing factors (ADF/cofilins) on actin filament turnover: pH sensitivity of F-actin binding by human ADF, but not of Acanthamoeba actophorin.
• Eur J Biochem. 1998; 256: 388-97
• Display abstract

• Actin depolymerizing factor (ADF) from vertebrates and actophorin from Acanthamoeba castellanii are members of a protein family that bind monomeric and polymeric actin and have been shown by microscopy to sever filaments. Here, we compare the properties of recombinant human ADF and actophorin using rabbit muscle actin. ADF binds tenfold more strongly than actophorin to monomeric actin (G-actin)-ATP, and both bind co-operatively to F-actin. ADF decorates filaments below pH 7.3 and induces substantial depolymerization at higher pH values [Hawkins, M., Pope, B., Maciver, S. K. & Weeds, A. G. (1993) Human actin depolymerizing factor mediates a pH-sensitive destruction of actin filaments, Biochemistry 32, 9985-9993], but, at all pH values tested, actophorin binds to filaments in a similar manner to ADF at pH 6.5. Both proteins increase the depolymerization rate at the pointed ends of gelsolin-capped filaments, but the effect of ADF is more marked at pH 8.0. Both proteins accelerate the nucleating activity when mixed with filamentous actin (F-actin), but not with gelsolin-capped filaments, and they rapidly decrease the lengths of filaments as evidenced by electron microscopy. Both of these effects are best explained by a weak severing activity. Our results are discussed in relation to earlier models and to the structural changes observed when ADF binds F-actin [McGough, A., Pope, B., Chiu, W. & Weeds, A. (1997) Cofilin changes the twist of F-actin: implications for actin filament dynamics and cellular function, J. Cell Biol. 138, 771-781]. We also discuss the relevance of these observations to their possible roles in facilitating actin turnover in cells, thereby regulating filament dynamics in cell motility.

• McGough A, Chiu W, Way M
• Determination of the gelsolin binding site on F-actin: implications for severing and capping.
• Biophys J. 1998; 74: 764-72
• Display abstract

• Gelsolin is a six-domain protein that regulates actin assembly by severing, capping, and nucleating filaments. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on F-actin. To obtain fully decorated filaments under severing conditions, we have studied a derivative (G2-6) that has a reduced severing efficiency compared to gelsolin. A three-dimensional reconstruction of G2-6:F-actin was obtained by electron cryomicroscopy and helical reconstruction. The structure shows that gelsolin bridges two longitudinally associated monomers when it binds the filament. The F-actin binding region of G2-6 is centered axially at subdomain 3 and radially between subdomains 1 and 3 of the upper actin monomer. Our results suggest that for severing to occur, both gelsolin and actin undergo large conformational changes.

• Asakura T et al.
• Isolation and characterization of a novel actin filament-binding protein from Saccharomyces cerevisiae.
• Oncogene. 1998; 16: 121-30
• Display abstract

• We purified a novel actin filament (F-actin)-binding protein from the soluble fraction of Saccharomyces cerevisiae by successive column chromatographies by use of the 125I-labeled F-actin blot overlay method. The purified protein showed a minimum Mr of about 140 kDa on SDS-polyacrylamide gel electrophoresis and we named it ABP140. A search with the partial amino acid sequences of ABP140 against the Saccharomyces Genome Database revealed that the open reading frame of the ABP140 gene (ABP140) corresponded to YOR239W fused with YOR240W by the +1 translational frame shift. The encoded protein consisted of 628 amino acids with a calculated Mr of 71,484. The recombinant protein interacted with F-actin and showed the activity to crosslink F-actin into a bundle. Indirect immunofluorescence study demonstrated that ABP140 was colocalized with both cortical actin patches and cytoplasmic actin cables in intact cells. However, elimination of ABP140 by gene disruption did not show a deleterious effect on cell growth or affect the organization of F-actin. These results indicate that ABP140 is not required for cell growth but may be involved in the reorganization of F-actin in the budding yeast.

• Smertenko AP, Jiang CJ, Simmons NJ, Weeds AG, Davies DR, Hussey PJ
• Ser6 in the maize actin-depolymerizing factor, ZmADF3, is phosphorylated by a calcium-stimulated protein kinase and is essential for the control of functional activity.
• Plant J. 1998; 14: 187-93
• Display abstract

• Maize actin-depolymerizing factor, ZmADF, binds both G- and F-actin and enhances in vitro actin dynamics. Evidence from studies on vertebrate ADF/cofilin supports the view that this class of protein responds to intracellular and extracellular signals and causes actin reorganization. As a test to determine whether such signal-responsive pathways existed in plants, this study addressed the ability of maize ADF to be phosphorylated and the likely effects of such phosphorylation on its capacity to modulate actin dynamics. It is shown that maize ADF3 (ZmADF3) can be phosphorylated by a calcium-stimulated protein kinase present in a 40-70% ammonium sulphate fraction of a plant cell extract. Phosphorylation is shown to be on Ser6, which is only one of nine amino acids that are fully conserved among the ADF/cofilin proteins across distantly related species. In addition, an analogue of phosphorylated ZmADF3 created by mutating Ser6 to Asp6 (zmadf3-4) does not bind G- or F-actin and has little effect on the enhancement of actin dynamics. These results are discussed in context of the previously observed actin reorganization in root hair cells.

• Belmont LD, Drubin DG
• The yeast V159N actin mutant reveals roles for actin dynamics in vivo.
• J Cell Biol. 1998; 142: 1289-99
• Display abstract

• Actin with a Val 159 to Asn mutation (V159N) forms actin filaments that depolymerize slowly because of a failure to undergo a conformational change after inorganic phosphate release. Here we demonstrate that expression of this actin results in reduced actin dynamics in vivo, and we make use of this property to study the roles of rapid actin filament turnover. Yeast strains expressing the V159N mutant (act1-159) as their only source of actin have larger cortical actin patches and more actin cables than wild-type yeast. Rapid actin dynamics are not essential for cortical actin patch motility or establishment of cell polarity. However, fluid phase endocytosis is defective in act1-159 strains. act1-159 is synthetically lethal with cofilin and profilin mutants, supporting the conclusion that mutations in all of these genes impair the polymerization/ depolymerization cycle. In contrast, act1-159 partially suppresses the temperature sensitivity of a tropomyosin mutant, and the loss of cytoplasmic cables seen in fimbrin, Mdm20p, and tropomyosin null mutants, suggesting filament stabilizing functions for these actin-binding proteins. Analysis of the cables in these double-mutant cells supports a role for fimbrin in organizing cytoplasmic cables and for Mdm20p and tropomyosin in excluding cofilin from the cables.

• Mullins RD, Kelleher JF, Xu J, Pollard TD
• Arp2/3 complex from Acanthamoeba binds profilin and cross-links actin filaments.
• Mol Biol Cell. 1998; 9: 841-52
• Display abstract

• The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a Kd of 7 microM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motile Acanthamoeba, and its localization is distinct from that of alpha-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells.

• Taglicht D, Michaelis S
• Saccharomyces cerevisiae ABC proteins and their relevance to human health and disease.
• Methods Enzymol. 1998; 292: 130-62

• Silva AM, Lee AY, Erickson JW, Goldberg DE
• Structural analysis of plasmepsin II. A comparison with human aspartic proteases.
• Adv Exp Med Biol. 1998; 436: 363-73

• Goode BL, Drubin DG, Lappalainen P
• Regulation of the cortical actin cytoskeleton in budding yeast by twinfilin, a ubiquitous actin monomer-sequestering protein.
• J Cell Biol. 1998; 142: 723-33
• Display abstract

• Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)-twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.

• Ressad F et al.
• Kinetic analysis of the interaction of actin-depolymerizing factor (ADF)/cofilin with G- and F-actins. Comparison of plant and human ADFs and effect of phosphorylation.
• J Biol Chem. 1998; 273: 20894-902
• Display abstract

• The thermodynamics and kinetics of actin interaction with Arabidopsis thaliana actin-depolymerizing factor (ADF)1, human ADF, and S6D mutant ADF1 protein mimicking phosphorylated (inactive) ADF are examined comparatively. ADFs interact with ADP.G-actin in rapid equilibrium (k+ = 155 microM-1.s-1 and k- = 16 s-1 at 4 degreesC under physiological ionic conditions). The kinetics of interaction of plant and human ADFs with F-actin are slower and exhibit kinetic cooperativity, consistent with a scheme in which the initial binding of ADF to two adjacent subunits of the filament nucleates a structural change that propagates along the filament, allowing faster binding of ADF in a "zipper" mode. ADF binds in a non-cooperative faster process to gelsolin-capped filaments or to subtilisin-cleaved F-actin, which are structurally different from standard filaments (Orlova, A., Prochniewicz, E., and Egelman, E. H. (1995) J. Mol. Biol. 245, 598-607). In contrast, the binding of phalloidin to F-actin cooperatively inhibits its interaction with ADF. The ADF-facilitated nucleation of ADP.actin self-assembly indicates that ADF stabilizes lateral interactions in the filament. Plant and human ADFs cause only partial depolymerization of F-actin at pH 8, consistent with identical functions in enhancing F-actin dynamics. Phosphorylation does not affect ADF activity per se, but decreases its affinity for actin by 20-fold.

• Laine RO, Phaneuf KL, Cunningham CC, Kwiatkowski D, Azuma T, Southwick FS
• Gelsolin, a protein that caps the barbed ends and severs actin filaments, enhances the actin-based motility of Listeria monocytogenes in host cells.
• Infect Immun. 1998; 66: 3775-82
• Display abstract

• The actin-based motility of Listeria monocytogenes requires the addition of actin monomers to the barbed or plus ends of actin filaments. Immunofluorescence micrographs have demonstrated that gelsolin, a protein that both caps barbed ends and severs actin filaments, is concentrated directly behind motile bacteria at the junction between the actin filament rocket tail and the bacterium. In contrast, CapG, a protein that strictly caps actin filaments, fails to localize near intracellular Listeria. To explore the effect of increasing concentrations of gelsolin on bacterial motility, NIH 3T3 fibroblasts stably transfected with gelsolin cDNA were infected with Listeria. The C5 cell line containing 2.25 times control levels of gelsolin supported significantly higher velocities of bacterial movement than did control fibroblasts (mean +/- standard error of the mean, 0.09 +/- 0.003 micro(m)/s [n = 176] versus 0.05 +/- 0.003 micro(m)/s [n = 65]). The rate of disassembly of the Listeria-induced actin filament rocket tail was found to be independent of gelsolin content. Therefore, if increases in gelsolin content result in increases in Listeria-induced rocket tail assembly rates, a positive correlation between gelsolin content and tail length would be expected. BODIPY-phalloidin staining of four different stably transfected NIH 3T3 fibroblast cell lines confirmed this expectation (r = 0.92). Rocket tails were significantly longer in cells with a high gelsolin content. Microinjection of gelsolin 1/2 (consisting of the amino-terminal half of native gelsolin) also increased bacterial velocity by more than 2.2 times. Microinjection of CapG had no effect on bacterial movement. Cultured skin fibroblasts derived from gelsolin-null mice were capable of supporting intracellular Listeria motility at velocities comparable to those supported by wild-type skin fibroblasts. These experiments demonstrated that the surface of Listeria contains a polymerization zone that can block the barbed-end-capping activity of both gelsolin and CapG. The ability of Listeria to uncap actin filaments combined with the severing activity of gelsolin can accelerate actin-based motility. However, gelsolin is not absolutely required for the actin-based intracellular movement of Listeria because its function can be replaced by other actin regulatory proteins in gelsolin-null cells, demonstrating the functional redundancy of the actin system.

• Bergdoll M, Eltis LD, Cameron AD, Dumas P, Bolin JT
• All in the family: structural and evolutionary relationships among three modular proteins with diverse functions and variable assembly.
• Protein Sci. 1998; 7: 1661-70
• Display abstract

• The crystal structures of three proteins of diverse function and low sequence similarity were analyzed to evaluate structural and evolutionary relationships. The proteins include a bacterial bleomycin resistance protein, a bacterial extradiol dioxygenase, and human glyoxalase I. Structural comparisons, as well as phylogenetic analyses, strongly indicate that the modern family of proteins represented by these structures arose through a rich evolutionary history that includes multiple gene duplication and fusion events. These events appear to be historically shared in some cases, but parallel and historically independent in others. A significant early event is proposed to be the establishment of metal-binding in an oligomeric ancestor prior to the first gene fusion. Variations in the spatial arrangements of homologous modules are observed that are consistent with the structural principles of three-dimensional domain swapping, but in the unusual context of the formation of larger monomers from smaller dimers or tetramers. The comparisons support a general mechanism for metalloprotein evolution that exploits the symmetry of a homooligomeric protein to originate a metal binding site and relies upon the relaxation of symmetry, as enabled by gene duplication, to establish and refine specific functions.

• Ono S, Benian GM
• Two Caenorhabditis elegans actin depolymerizing factor/cofilin proteins, encoded by the unc-60 gene, differentially regulate actin filament dynamics.
• J Biol Chem. 1998; 273: 3778-83
• Display abstract

• The Caenorhabditis elegans unc-60 gene encodes two actin depolymerizing factor/cofilin proteins which are implicated in the regulation of actin filament assembly in body wall muscle. We examined the interaction of recombinant UNC-60A and B proteins with actin and found that they differentially regulate actin filament dynamics. Co-pelleting assays with F-actin showed that UNC-60A depolymerized but did not remain bound to F-actin, whereas UNC-60B bound to but did not depolymerize F-actin. In the pH range of 6.8-8.0, the apparent activities of UNC-60A and B did not change although UNC-60A showed greater actin-depolymerizing activity at higher pH. These activities were further confirmed by a light scattering assay and electron microscopy. The effects of these proteins on actin polymerization were quite different. UNC-60A inhibited polymerization in a concentration-dependent manner. On the other hand, UNC-60B strongly inhibited the nucleation process but accelerated the following elongation step. However, an excess amount of UNC-60B increased the amount of unpolymerized actin. These results indicate that UNC-60A depolymerizes actin filaments and inhibits actin polymerization, whereas UNC-60B strongly binds to F-actin without depolymerizing it and, through binding to G-actin, changes the rate of actin polymerization depending on the UNC-60B:actin ratio. These data suggest that the two UNC-60 isoforms play differential roles in regulating actin filament dynamics in vivo.

• Meberg PJ, Ono S, Minamide LS, Takahashi M, Bamburg JR
• Actin depolymerizing factor and cofilin phosphorylation dynamics: response to signals that regulate neurite extension.
• Cell Motil Cytoskeleton. 1998; 39: 172-90
• Display abstract

• The actin assembly-regulating activity of actin depolymerizing factor (ADF)/ cofilin is inhibited by phosphorylation. Studies were undertaken to characterize the signaling pathways and phosphatases involved in activating phosphorylated ADF (pADF), emphasizing signals related to neuronal process extension. Western blots using antibodies to ADF and cofilin, as well as an ADF/cofilin phosphoepitope-specific antibody characterized in this paper, were used to measure changes in the phosphorylation state and phosphate turnover of ADF/cofilin in response to inhibitors and agents known to influence growth cone motility. Increases in both [Ca2+]i and cAMP levels induced rapid pADF dephosphorylation in HT4 and cortical neurons. Calcium-dependent dephosphorylation depended on the activation of protein phosphatase 2B (PP2B), while cAMP-dependent dephosphorylation was likely through activation of PP1. Growth factors such as NGF and insulin also induced rapid pADF/pcofilin dephosphorylation, with NGF-stimulated dephosphorylation in PC12 cells correlated with the translocation of ADF/cofilin to ruffling membranes. Of special interest was the finding that the rate of phosphate turnover on both pADF and pcofilin could be enhanced by growth factors without changing net pADF levels, demonstrating that growth factors can activate bifurcating pathways that promote both phosphorylation and dephosphorylation of ADF/cofilin. All experimental results indicated that dynamics of phosphorylation on ADF and cofilin are coordinately regulated. Signals that decreased pADF levels are associated with increased process extension, while agents that increased pADF levels, such as lysophosphatidic acid, inhibit process extension. These data indicate that dephosphorylation/activation of pADF is a significant response to the activation of signal pathways that regulate actin dynamics and alter cell morphology and neuronal outgrowth.

• Lazo ND, Downing DT
• Amyloid fibrils may be assembled from beta-helical protofibrils.
• Biochemistry. 1998; 37: 1731-5

• Du J, Frieden C
• Kinetic studies on the effect of yeast cofilin on yeast actin polymerization.
• Biochemistry. 1998; 37: 13276-84
• Display abstract

• The effect of yeast cofilin on the kinetics of polymerization of yeast actin has been examined at 20 degrees C at both pH 8.0 and 6.6. In the absence of cofilin, the kinetic data may be described by a simple nucleation-elongation mechanism. Kinetic data in the presence of cofilin suggests a complex dependence on the cofilin concentration. At low cofilin-to-actin ratios, cofilin increases the rate of polymerization in a way best fit by assuming filament fragmentation. The apparent fragmentation rate constants increase with increasing cofilin concentration leveling off above a cofilin-to-actin ratio of 1:8 and are independent of pH. At higher cofilin-to-actin ratios, a nonpolymerizable cofilin-G-actin complex forms resulting in a decreased rate of polymerization. The data from fluorescence photobleaching recovery experiments at low cofilin-to-actin ratios are consistent with the presence of severed filaments at both pH 8 and 6.6. However, at pH 8 and a cofilin-to-actin ratio of 1:16, about 40-50% of the total actin is present as G-actin after polymerization while at pH 6.6 little or no G-actin is present at the same cofilin-to-actin ratio. The results suggest some cooperativity with respect to cofilin binding to filamentous actin which may be pH dependent.

• Wriggers W, Tang JX, Azuma T, Marks PW, Janmey PA
• Cofilin and gelsolin segment-1: molecular dynamics simulation and biochemical analysis predict a similar actin binding mode.
• J Mol Biol. 1998; 282: 921-32
• Display abstract

• An understanding of the actin-depolymerizing function attributed to members of the ADF/cofilin/destrin superfamily requires a structural model of these proteins in complex with actin. As a step toward defining actin-cofilin interactions, the complex of yeast cofilin with monomeric actin was predicted, starting with the actin-gelsolin segment-1 binding mode recently suggested for the actin-destrin complex. After refinement by molecular dynamics simulation, the structure of cofilin converged in a new binding mode that required only minimal changes induced in the actin-cofilin interface. The predicted complex exhibits strong interactions between the N termini of actin and cofilin, mediated by a salt bridge of cofilin Arg3 with actin Asp1. The forming of this salt bridge could be prevented by the phosphorylation of cofilin Ser4, which is believed to inhibit cofilin depolymerization activity. Recent mutagenesis studies, crosslinking experiments and peptide binding studies are consistent with the predicted model of the actin-cofilin complex. The structural homology between cofilin and gelsolin segment-1 binding to actin was confirmed experimentally by two types of competitive binding assays.

• Didry D, Carlier MF, Pantaloni D
• Synergy between actin depolymerizing factor/cofilin and profilin in increasing actin filament turnover.
• J Biol Chem. 1998; 273: 25602-11
• Display abstract

• The mechanism of control of the steady state of actin assembly by actin depolymerizing factor (ADF)/cofilin and profilin has been investigated. Using Tbeta4 as an indicator of the concentration of ATP-G-actin, we show that ADF increases the concentration of ATP-G-actin at steady state. The measured higher concentration of ATP-G-actin is quantitatively consistent with the increase in treadmilling, caused by the large increase in the rate of depolymerization from the pointed ends induced by ADF (Carlier, M.-F. , Laurent, V., Santolini, J., Didry, D., Melki, R., Xia, G.-X., Hong, Y., Chua, N.-H., and Pantaloni, D. (1997) J. Cell Biol. 136, 1307-1322). Experiments demonstrate that profilin synergizes with ADF to further enhance the turnover of actin filaments up to a value 125-fold higher than in pure F-actin solutions. Profilin and ADF act at the two ends of filaments in a complementary fashion to increase the processivity of treadmilling. Using the capping protein CapZ, we show that ADF increases the number of filaments at steady state by 1. 3-fold, which cannot account for the 25-fold increase in turnover rate. Computer modeling of the combined actions of ADF and profilin on the dynamics of actin filaments using experimentally determined rate constants generates a distribution of the different actin species at steady state, which is in quantitative agreement with the data.

• Ayscough KR
• In vivo functions of actin-binding proteins.
• Curr Opin Cell Biol. 1998; 10: 102-11
• Display abstract

• Actin and actin-binding proteins have been identified in eukaryotic organisms across the evolutionary spectrum. Although many actin-binding proteins have been purified and studied in vitro, our understanding of the in vivo functions of these proteins has, until recently, lagged behind. In the past year, in vivo studies, especially those using genetic approaches, have led to significant advances in our understanding of how actin-binding proteins function in a cellular environment.

• Blanchoin L, Pollard TD
• Interaction of actin monomers with Acanthamoeba actophorin (ADF/cofilin) and profilin.
• J Biol Chem. 1998; 273: 25106-11
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• Acanthamoeba actophorin is a member of ADF/cofilin family that binds both actin monomers and filaments. We used fluorescence anisotropy to study the interaction of actin monomers with recombinant actophorin labeled with rhodamine on a cysteine substituted for Serine-88. Labeled actophorin retains its affinity for actin and ability to reduce the low shear viscosity of actin filaments. At physiological ionic strength, actophorin binds Mg-ADP-actin monomers (Kd = 0.1 microM) 40 times stronger than Mg-ATP-actin monomers. When bound to actin monomers, actophorin has no effect on elongation at either end of actin filaments by Mg-ATP-actin and slightly increases the rate of elongation at both ends by Mg-ADP-actin. Thus actophorin does not sequester actin monomers. Sedimentation equilibrium ultracentrifugation shows that actophorin and profilin compete for binding actin monomers. Actophorin and profilin have opposite effects on the rate of exchange of nucleotide bound to actin monomers. Despite the high affinity of actophorin for ADP-actin, physiological concentrations of profilin overcome the inhibition of ADP exchange by actophorin. Profilin rapidly recycles ADP-actin back to the profilin-ATP-actin pool ready for elongation of actin filaments.

• Xu J, Wirtz D, Pollard TD
• Dynamic cross-linking by alpha-actinin determines the mechanical properties of actin filament networks.
• J Biol Chem. 1998; 273: 9570-6
• Display abstract

• We used smooth muscle alpha-actinin to evaluate the contribution of cross-linker dynamics to the mechanical properties of actin filament networks. Recombinant actin-binding domain (residues 2-269) binds actin filaments with a Kd of 1 microM at 25 degrees C, 20 times stronger than actin-binding domain produced by thermolysin digestion of native alpha-actinin (residues 25-257). Between 8 and 25 degrees C the rate constants for recombinant actin-binding domain to bind to (0.8-2.7 microM-1 s-1) and dissociate from (0.2-2.4 s-1) actin filaments depend on temperature. At 8 degrees C actin filaments cross-linked with alpha-actinin are stiff and nearly solid, whereas at 25 degrees C the mechanical properties approach those of actin filaments alone. In these experiments, high actin concentrations kept most of the alpha-actinin bound to actin and temperature varied a single parameter, cross-linker dynamics, because the mechanical properties of pure actin filaments (a viscoelastic gel) or biotinylated actin filaments cross-linked irreversibly by avidin (a stiff viscoelastic solid) depend little on temperature. These results show that the rate of exchange of dynamic cross-links between actin filaments is an important determinant of the mechanical properties of the networks.

• Wahlberg MH
• The distribution of F-actin during the development of Diphyllobothrium dendriticum (Cestoda).
• Cell Tissue Res. 1998; 291: 561-70
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• The distribution of actin filaments in all developmental stages of the tapeworm Diphyllobothrium dendriticum was studied. It is the first investigation of the placement of microfilaments during the development of a flatworm, and the results show that actin filaments, in all developmental stages, can be found in the subtegument and the flame cells. Muscle fibers possibly corresponding to the longitudinal, transversal, and dorsoventral muscles of the adult and plerocercoid were already detected in the procercoid. Concerning the adult worm, a new set of longitudinal fibers in the peripheral parts of the adult proglottid was found. The ducts of the protonephridial system and the vitellarias were seen to be surrounded by longitudinally oriented actin filaments, while the uterine ducts and the vagina were encircled by microfilaments. Prominent layers of circular muscle fibers surrounded the cirrus and the seminal vesicle, and radial fibers were also detected. Areas faintly stained with TRITC-phalloidin were found in the developing germ cells, the cells of the genital anlage, vitelline cells, the tegument, and the main nerve cords. None of these structures were autofluorescent, which is also true concerning the intensively labeled oncospheral hooks.

• Puius YA, Mahoney NM, Almo SC
• The modular structure of actin-regulatory proteins.
• Curr Opin Cell Biol. 1998; 10: 23-34
• Display abstract

• Filamentous actin structures possess unique biophysical and biochemical properties and are required for cell locomotion, cell division, compartmentalization and morphological processes. The site-specific assembly and disassembly of these structures are directed by actin-regulatory proteins. This article reviews how structural studies are now defining the atomic details of small modular domains present in actin-regulatory proteins responsible for crosslinking, severing and capping of actin filaments, as well as for localization of actin filament assembly. These studies have identified three modular strategies for the design of proteins that regulate the actin cytoskeleton.

• Arai A, Nakazawa T
• Arrangement of actin filaments and cytoplasmic granules in the sea urchin egg after TPA treatment.
• Cell Motil Cytoskeleton. 1998; 39: 21-30
• Display abstract

• Elongation of microvilli and formation of actin filaments after treatment with a phorbol ester, TPA, were investigated in unfertilized eggs of Hemicentrotus pulcherrimus. Microvilli on the egg surface were examined by scanning electron microscopy. Actin filaments in the cortical layer of the eggs were observed by fluorescence microscopy using rhodamine-labeled phalloidin. The actin molecules were polymerized and bundled to form long filaments inside the cortical layer of eggs after TPA treatment. Arrangement of the actin filaments was followed by spiral elongation of microvilli. Transmission electron microscopic studies showed that the cortical granules under the cell membrane of sea urchin eggs were transferred after TPA treatment from the surface to the interior of the cell [Ciapa et al., 1988: Dev. Biol. 128:142-149]. This movement of the cortical granules was inhibited by cytochalasin B, but not by nocodazole. Furthermore, the distribution of clear granules was changed following TPA treatment. From these results we conclude that intracellular actin filaments may cause the transport of cortical granules and clear granules into the central area of the egg by the activation of protein kinase C. The possible involvement of actin in the inward displacement of granules might be the result of the rearrangement of actin filaments in the cortical layer.

• Arima K, Imanaka M, Okuzono S, Kazuta Y, Kotani S
• Evidence for structural differences between the two highly homologous actin-regulatory proteins, destrin and cofilin.
• Biosci Biotechnol Biochem. 1998; 62: 215-20
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• The amino acid sequences of destrin and cofilin are very similar (84% homology) throughout the entire range of proteins, but they have different functions. In this study, we constructed a new cofilin expression plasmid, which had high expression frequency, and the structures of destrin and cofilin were analyzed by limited proteolysis and circular dichroism (CD). When destrin was digested by trypsin, two fragments of 17.0 kDa and 9.2 kDa were obtained, whereas only one 8.4 kDa fragment was obtained from cofilin. In spite of the overall sequence homology, an N-terminal amino acid sequence analyses of the fragments revealed the cleavage sites on destrin and cofilin to be different. These results suggest that destrin and cofilin differ in their overall tertiary folds. Cofilin showed activity similar to destrin at high pH values, although no pH-dependent structural change in cofilin was confirmed by using limited proteolysis and CD.

• Goldmann WH, Tempel M, Sprenger I, Isenberg G, Ezzell RM
• Viscoelasticity of actin-gelsolin networks in the presence of filamin.
• Eur J Biochem. 1997; 246: 373-9
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• Cross-linking of actin filaments by filamin by means of frequency-dependent rheology yields an increase in the filament's elasticity and stiffness. Higher cross-linker (filamin) ratios are required for mean actin-filament lengths of 5-6 microm than for random-length distribution of actin filaments. The loss modulus (i.e. the viscous portion) in the region of the internal-chain dynamics [G"(omega) approximately omega(alpha)] is influenced by the cross-linking of filaments, and with an increasing molar ratio of filamin/actin a reduction of alpha is observed. Rheological measurements reveal that actin networks are already formed at the polymerizing stage at a molar ratio of filamin/actin of less than 1:100, and electron micrographs show phase separation of actin/filament networks of low density and of actin/filament bundles.

• Kuang B, Rubenstein PA
• The effects of severely decreased hydrophobicity in a subdomain 3/4 loop on the dynamics and stability of yeast G-actin.
• J Biol Chem. 1997; 272: 4412-8
• Display abstract

• The hydrophobicity of the subdomain 3/4 hydrophobic loop (262-274) has been implicated to be essential for actin's function. We previously showed (Kuang, B., and Rubenstein, P. A. (1997) J. Biol. Chem. 272, 1237-1247) that a mutant yeast actin (V266G/L267G) with markedly decreased hydrophobicity in this loop conferred severe cold sensitivity to its polymerization. Here we further tested the mutational effect on the conformation and function of G-actin. This GG mutation caused no significant changes in overall secondary structure or in the microenvironment around actin's tryptophan residues, nor did it alter the dissociation constant of G-actin for ATP. However, it lowers the intrinsic ATPase activity and the melting temperature for Mg-GG actin from 51 to 33 degrees C and transforms the conformation of subdomain 2 and the central cleft of G-actin into an F-monomer-like structure. The results suggest that the hydrophobic plug may not only play a role in actin filament stabilization but also may be important for controlling the stability of G-actin and for promoting the conformational change of the monomer needed for addition to a growing actin filament.

• Rosenblatt J, Agnew BJ, Abe H, Bamburg JR, Mitchison TJ
• Xenopus actin depolymerizing factor/cofilin (XAC) is responsible for the turnover of actin filaments in Listeria monocytogenes tails.
• J Cell Biol. 1997; 136: 1323-32
• Display abstract

• In contrast to the slow rate of depolymerization of pure actin in vitro, populations of actin filaments in vivo turn over rapidly. Therefore, the rate of actin depolymerization must be accelerated by one or more factors in the cell. Since the actin dynamics in Listeria monocytogenes tails bear many similarities to those in the lamellipodia of moving cells, we have used Listeria as a model system to isolate factors required for regulating the rapid actin filament turnover involved in cell migration. Using a cell-free Xenopus egg extract system to reproduce the Listeria movement seen in a cell, we depleted candidate depolymerizing proteins and analyzed the effect that their removal had on the morphology of Listeria tails. Immunodepletion of Xenopus actin depolymerizing factor (ADF)/cofilin (XAC) from Xenopus egg extracts resulted in Listeria tails that were approximately five times longer than the tails from undepleted extracts. Depletion of XAC did not affect the tail assembly rate, suggesting that the increased tail length was caused by an inhibition of actin filament depolymerization. Immunodepletion of Xenopus gelsolin had no effect on either tail length or assembly rate. Addition of recombinant wild-type XAC or chick ADF protein to XAC-depleted extracts restored the tail length to that of control extracts, while addition of mutant ADF S3E that mimics the phosphorylated, inactive form of ADF did not reduce the tail length. Addition of excess wild-type XAC to Xenopus egg extracts reduced the length of Listeria tails to a limited extent. These observations show that XAC but not gelsolin is essential for depolymerizing actin filaments that rapidly turn over in Xenopus extracts. We also show that while the depolymerizing activities of XAC and Xenopus extract are effective at depolymerizing normal filaments containing ADP, they are unable to completely depolymerize actin filaments containing AMPPNP, a slowly hydrolyzible ATP analog. This observation suggests that the substrate for XAC is the ADP-bound subunit of actin and that the lifetime of a filament is controlled by its nucleotide content.

• DiNubile MJ, Huang S
• Capping of the barbed ends of actin filaments by a high-affinity profilin-actin complex.
• Cell Motil Cytoskeleton. 1997; 37: 211-25
• Display abstract

• Profilin, a ubiquitous 12 to 15-kDa protein, serves many functions, including sequestering monomeric actin, accelerating nucleotide exchange on actin monomers, decreasing the critical concentration of the barbed end of actin filaments, and promoting actin polymerization when barbed ends are free. Most previous studies have focused on profilin itself rather than its complex with actin. A high-affinity profilin-actin complex (here called profilactin) can be isolated from a poly-(L)-proline (PLP) column by sequential elution with 3 M and 7 M urea. Profilactin inhibited the elongation rate of pyrenyl-G-actin from filament seeds in a concentration- and time-dependent manner. Much greater inhibition of elongation was observed with spectrin-F-actin than gelsolin-F-actin seeds, suggesting that the major effect of profilactin was due to capping the barbed ends of actin filaments. Its dissociation constant for binding to filament ends was 0.3 microM; the on- and off-rate constants were estimated to be 1.7 x 10(3) M-1 s-1 and 4.5 x 10(-4) s-1, respectively. Purified profilin (obtained by repetitive applications to a PLP column and assessed by silver-stained polyacylamide gels) did not slow the elongation rate of pyrenyl-G-actin from filament seeds. Capping protein could not be detected by Western blotting in the profilactin preparation, but low concentrations of gelsolin did contaminate our preparation. However, prolonged incubation with either calcium or EGTA did not affect capping activity, implying that contaminating gelsolin-actin complexes were not primarily responsible for the observed capping activity. Reapplication of the profilactin preparation to PLP-coupled Sepharose removed both profilin and actin and concurrently eliminated its capping activity. Profilactin that was reapplied to uncoupled Sepharose retained its capping activity. Phosphatidylinositol-4,5-bisphosphate (PIP2) was the most potent phosphoinositol in reducing the capping activity of profilactin. Dissociation of the tight profilactin complex may serve as a unique mechanism by which profilin helps regulate actin filament growth.

• Carlier MF et al.
• Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility.
• J Cell Biol. 1997; 136: 1307-22
• Display abstract

• Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical-chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADP-bound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi-bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.

• Monaco HL
• Crystal structure of chicken riboflavin-binding protein.
• EMBO J. 1997; 16: 1475-83
• Display abstract

• The crystal structure of chicken egg white riboflavin-binding protein, determined to a resolution of 2.5 A, is the prototype of a family that includes other riboflavin- and folate-binding proteins. An unusual characteristic of these molecules is their high degree of cross-linking by disulfide bridges and, in the case of the avian proteins, the presence of stretches of highly phosphorylated polypeptide chain. The structure of chicken egg white riboflavin-binding protein is characterized by a ligand-binding domain and a phosphorylated motif. The ligand-binding domain has a fold that appears to be strongly conditioned by the presence of the disulfide bridges. The phosphorylated motif, essential for vitamin uptake, is made up of two helices found before and after the flexible phosphorylated region. The riboflavin molecule binds to the protein with the isoalloxazine ring stacked in between the rings of Tyr75 and Trp156. This geometry and the proximity of other tryptophans explain the fluorescent quenching observed when riboflavin binds to the protein.

• Maruta H
• [F-actin cappers]
• Gan To Kagaku Ryoho. 1997; 24: 1442-7
• Display abstract

• Members of a large protein family that cap the barbed (fast-growing) end of actin filament (F-actin) are called F-actin "Cappers". The first F-actin capper called Cap 28/31 is a heterodimer of 28 kDa and 31 kDa proteins, and was isolated from a soil amoeba called Acanthamoeba (Isenberg et al., 1980). F-actin cappers are present in any eucaryotes from yeast to human, and block actin polymerization by capping the fast-growing end of F-actin. In non-stimulated cells, most of the fast-growing ends of actin filaments are capped by an 1:1 complex of actin monomer (G-actin) and profilin, a PIP2-binding protein. When cells are stimulated by one of the mitogenic cytokines such as EGF and PDGF, Ras is activated, and consequently Rac is activated. Rac in turn activates PI-4 kinase which produces PIP2. PIP2 then binds profilin, and dissociates the profilin/G-actin complex, leading to uncapping of the fast-growing end of actin filament, and induces a rapid actin polymerization. Eventually, this results in the induction of membrane ruffling. We found that (1) the Ras/Rac-induced uncapping is required for oncogenicity of Ras, and (2) either capping at the fast-growing end by F-actin cappers such as tensin and cytochalasins, or sequestering PIP2 by PIP2-binders such as cofilin mutants (blocking the uncapping) is sufficient to suppress the malignant transformation caused by oncogenic Ras mutants such as v-Ha-Ras.

• Hernandez MC, Andres-Barquin PJ, Martinez S, Bulfone A, Rubenstein JL, Israel MA
• ENC-1: a novel mammalian kelch-related gene specifically expressed in the nervous system encodes an actin-binding protein.
• J Neurosci. 1997; 17: 3038-51
• Display abstract

• We have identified and characterized a novel murine gene, Ectoderm-Neural Cortex-1 (ENC-1), that is an early and highly specific marker of neural induction in vertebrates. ENC-1, which encodes a kelch family related protein, is expressed during early gastrulation in the prospective neuroectodermal region of the epiblast and later in development throughout the nervous system (NS). ENC-1 expression is highly dynamic and, after neurulation, preferentially defines prospective cortical areas. The only apparent expression of ENC-1 outside the NS is restricted to the rostral-most somitomere of the presomitic mesoderm, at the times corresponding to the epithelialization that precedes somite formation. Cellular expression of epitope-tagged ENC-1 shows extensive co-localization of ENC-1 with the actin cytoskeleton, and immunoprecipitation studies demonstrate a physical association between ENC-1 and actin. ENC-1 functions as an actin-binding protein that may be important in the organization of the actin cytoskeleton during neural fate specification and development of the NS.

• Welch MD, DePace AH, Verma S, Iwamatsu A, Mitchison TJ
• The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly.
• J Cell Biol. 1997; 138: 375-84
• Display abstract

• The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (p omplex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat-containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion.

• Jiang CJ, Weeds AG, Hussey PJ
• The maize actin-depolymerizing factor, ZmADF3, redistributes to the growing tip of elongating root hairs and can be induced to translocate into the nucleus with actin.
• Plant J. 1997; 12: 1035-43
• Display abstract

• The maize actin depolymerizing factor, ZmADF3, binds G- and F-actin, and increases in vitro actin dynamics. Polyclonal antibodies have been raised against ZmADF3 and these detect a single band of approximately 17 kDa in all maize tissues examined, with the exception of pollen. In the development of root hairs, the distribution of ZmADF3 is related to actin reorganization. In the early stages of hair development, ZmADF3 is distributed throughout the cytoplasm. As the hair emerges and the microfilament bundles redirect to the outgrowth there is a simultaneous redistribution of ZmADF3 which now concentrates at the tip of the emerging hair and remains in this position as elongation proceeds. These observations show that ZmADF3 localizes to a region where actin is being remodelled during tip growth. After cytochalasin D treatment which disrupts actin filaments, short rods of ZmADF3 and actin appear in the nucleus suggesting that ZmADF3 may function by guiding actin to sites of actin polymerization.

• Egelman EH
• New angles on actin dynamics.
• Structure. 1997; 5: 1135-7
• Display abstract

• Actin is now realised to play a dynamic role in muscle contraction and many cellular motility events that occur when the motor domain of myosin uses the energy of ATP hydrolysis to move along the actin filament. Optical and electron microscopic studies have led to seemingly contradictory pictures of actin filament dynamics.

• Milzani A, Dalledonne I, Vailati G, Colombo R
• Paraquat induces actin assembly in depolymerizing conditions.
• FASEB J. 1997; 11: 261-70
• Display abstract

• The molecular mechanism (or mechanisms) at the basis of paraquat (PQ) (a widely used herbicide) toxicity is far from being fully understood. Until now, two main points of view have emerged: 1) PQ-related cell injuries could be mediated by toxic oxygen free radicals coming from the metabolism of the herbicide by the microsomal enzyme system, and/or 2) PQ, by inducing mitochondrial swelling and breakage, could cause troubles in cell energy charge, then driving the cell to death. Recently, some of cytoskeletal structures (microtubules and microfilaments) have been proposed as further PQ cell targets. The microfilament system in particular seems to be markedly affected by the herbicide, but so far no direct evidence associates PQ to actin damage. In this study, experimental data are presented concerning the direct effect of PQ on actin dynamics in solution. We demonstrate that actin selectively binds PQ; moreover, PQ induces the formation of actin sopramolecular structures in depolymerizing medium (G-buffer). Furthermore, by the interactions with F-actin cross-linking proteins (alpha-actinin and filamin), FITC-phalloidin, and myosin subfragment 1 (S1), it is demonstrated that PQ-induced actin aggregates are undoubtedly built up by F-actin. Electron micrographs showed that PQ-induced actin polymers are very short and tend to aggregate one to another. This mutual cohesion leads to the steric blockage of polymer growing ends as suggested by nucleated actin polymerization assays. Sonication, by releasing F-actin fragments from short polymer aggregates, allows actin polymer ends to regain their growing ability.

• Barlow JN et al.
• Studies on non-haem ferrous-dependent oxygenases and oxidases.
• Biochem Soc Trans. 1997; 25: 86-90

• Ayscough KR, Stryker J, Pokala N, Sanders M, Crews P, Drubin DG
• High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A.
• J Cell Biol. 1997; 137: 399-416
• Display abstract

• We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2-5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site. LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization. Results show that at least two molecular pathways, one actin-dependent and the other actin-independent, underlie polarity development. The actin-dependent pathway localizes secretory vesicles and a putative vesicle docking complex to sites of cell surface growth, providing an explanation for the dependence of polarized cell surface growth on actin function. Unexpectedly, several proteins that function with actin during cell polarity development, including an unconventional myosin (Myo2p), calmodulin, and an actin-interacting protein (Bud6/Aip3p), achieved polarized localization by an actin-independent pathway, revealing interdependence among cell polarity pathways. Finally, transient actin depolymerization caused many cells to abandon one bud site or mating projection and to initiate growth at a second site. Thus, actin filaments are also required for maintenance of an axis of cell polarity.

• Furukawa R, Fechheimer M
• The structure, function, and assembly of actin filament bundles.
• Int Rev Cytol. 1997; 175: 29-90
• Display abstract

• The cellular organization, function, and molecular composition of selected biological systems with prominent actin filament bundles are reviewed. An overall picture of the great variety of functions served by actin bundles emerges from this overview. A unifying theme is that the actin cross-linking proteins are conserved throughout the eukaryotic kingdom and yet assembled in a variety of combinations to produce actin bundles of differing functions. Mechanisms of actin bundle formation in vitro are considered illustrating the variety of physical and chemical driving forces in this exceedingly complex process. Our limited knowledge regarding the formation of actin filament bundles in vivo is contrasted with the elegant biophysical studies performed in vitro but nonetheless reveals that interactions with membranes, nucleation sites, and other organizational components must contribute to formation of actin bundles in vivo.

• Minamide LS, Painter WB, Schevzov G, Gunning P, Bamburg JR
• Differential regulation of actin depolymerizing factor and cofilin in response to alterations in the actin monomer pool.
• J Biol Chem. 1997; 272: 8303-9
• Display abstract

• Myoblasts, transfected with a human gene encoding a beta-actin point mutation, down-regulate expression of actin depolymerizing factor (ADF) and its mRNA. Regulation is posttranscriptional. Expression of cofilin, a structurally similar protein, and profilin, CapG, and tropomodulin is not altered with increasing mutant beta-actin expression. Myoblasts expressing either human gamma-actin or the mutant beta-actin down-regulate the endogenous mouse actin genes to keep a constant level of actin mRNA, whereas the gamma-actin transfectants do not down-regulate ADF. Thus, ADF expression is regulated differently from actin expression. The mutant beta-actin binds to ADF with about the same affinity as normal actin; however, it does not assemble into normal actin filaments. The decrease in ADF expression correlates with an increase in the unassembled actin pool. When the actin monomer pool in untransfected myoblasts is increased 70% by treatment with latrunculin A, synthesis of ADF and actin are down-regulated compared with cofilin and 19 other proteins selected at random. Increasing the actin monomer pool also results in nearly complete phosphorylation of both ADF and cofilin. Thus, ADF and cofilin are coordinately regulated by posttranslational modification, but their expression is differentially regulated. Furthermore, expression of ADF is responsive to the utilization of actin by the cell.

• Su S, Gao YG, Zhang H, Terwilliger TC, Wang AH
• Analyses of the stability and function of three surface mutants (R82C, K69H, and L32R) of the gene V protein from Ff phage by X-ray crystallography.
• Protein Sci. 1997; 6: 771-80
• Display abstract

• The high-resolution crystal structure of the gene V protein (GVP) from the Ff filamentous phages (M13, fl, fd) has been solved recently for the wild-type and two surface mutant (Y41F and Y41H) proteins, leading to a plausible model for the polymeric GVP-ssDNA complex (Guan Y, Zhang H, Wang AHJ, 1995, Protein Sci 4:187-197). The model of the complex shows extensive contacts between neighboring dimer GVPs involving electrostatic interactions between the K69 from one and the D79 and R82 from the next dimer. In addition, hydrophobic interactions between the amino acids L32 and L44 from one and G23 from the next dimer also contribute to the dimer-dimer interactions. Mutations at the L32, K69, and R82 amino acid sites generally destabilize the protein and many of these affect the function of the phage. We have studied the structural effects of three mutant proteins involving those sites, i.e., L32R, K69H, and R82C, by X-ray crystallographic analysis at 2.0 A resolution. In L32R GVP, the structural perturbation is localized, whereas in K69H and R82C GVPs, some long-range effects are also detected in addition to the local perturbation. We have interpreted the protein stability and the functional properties associated with those mutations in terms of the observed structural perturbations.

• Sha B, Luo M
• Structure of a bifunctional membrane-RNA binding protein, influenza virus matrix protein M1.
• Nat Struct Biol. 1997; 4: 239-44
• Display abstract

• Matrix protein (M1) of influenza virus is a bifunctional protein that mediates the encapsidation of RNA-nucleoprotein cores into the membrane envelope. It is therefore required that M1 binds both membrane and RNA simultaneously. The X-ray crystal structure of the N-terminal portion of type A influenza virus M1-amino acid residues 2-158-has been determined at 2.08 A resolution at pH 4.0. The protein forms a dimer. A highly positively charged region on the dimer surface is suitably positioned to bind RNA while the hydrophobic surface opposite the RNA binding region may be involved in interactions with the membrane. The membrane-binding hydrophobic surface could be buried or exposed after a conformational change.

• Fedorov AA, Ball T, Mahoney NM, Valenta R, Almo SC
• The molecular basis for allergen cross-reactivity: crystal structure and IgE-epitope mapping of birch pollen profilin.
• Structure. 1997; 5: 33-45
• Display abstract

• BACKGROUND: The profilins are a group of ubiquitous actin monomer binding proteins that are responsible for regulating the normal distribution of filamentous actin networks in eukaryotic cells. Profilins also bind polyphosphoinositides, which can disrupt the profilin-action complex, and proline-rich ligands which localize profilin to sites requiring extensive actin filament accumulation. Profilins represent cross-reactive allergens for almost 20 % of all pollen allergic patients. RESULTS: We report the X-ray crystal structure of birch pollen profilin (BPP) at 2.4 resolution. The major IgE-reactive epitopes have been mapped and were found to cluster on the N- and C-terminal alpha helices and a segment of the protein containing two strands of the beta sheet. The overall fold of this protein is similar to that of the mammalian and amoeba profilins, however, there is a significant change in the orientation of the N-terminal alpha helix in BPP. This change in orientation alters the topography of a hydrophobic patch on the surface of the molecule, which is thought to be involved in the binding of proline-rich ligands. CONCLUSIONS: Profilin has been identified as an important cross-reactive allergen for patients suffering from multivalent type I allergy. The prevalent epitopic areas are located in regions with conserved sequence and secondary structure and overlap the binding sites for natural profilin ligands, indicating that the native ligand-free profilin acts as the original cross-sensitizing agent. Structural homology indicates that the basic features of the G actin-profilin interaction are conserved in all eukaryotic organisms, but suggests that mechanistic differences in the binding of proline-rich ligands may exist. The structure of BPP provides a molecular basis for understanding allergen cross-reactivity.

• Brenner C et al.
• Crystal structures of HINT demonstrate that histidine triad proteins are GalT-related nucleotide-binding proteins.
• Nat Struct Biol. 1997; 4: 231-8
• Display abstract

• Histidine triad nucleotide-binding protein (HINT), a dimeric purine nucleotide-binding protein from rabbit heart, is a member of the HIT (histidine triad) superfamily which includes HINT homologues and FHIT (HIT protein encoded at the chromosome 3 fragile site) homologues. Crystal structures of HINT-nucleotide complexes demonstrate that the most conserved residues in the superfamily mediate nucleotide binding and that the HIT motif forms part of the phosphate binding loop. Galactose-1-phosphate uridylyltransferase, whose deficiency causes galactosemia, contains tandem HINT domains with the same fold and mode of nucleotide binding as HINT despite having no overall sequence similarity. Features of FHIT, a diadenosine polyphosphate hydrolase and candidate tumour suppressor, are predicted from HINT-nucleotide structures.

• Lechler T, Li R
• In vitro reconstitution of cortical actin assembly sites in budding yeast.
• J Cell Biol. 1997; 138: 95-103
• Display abstract

• We have developed a biochemical approach for identifying the components of cortical actin assembly sites in polarized yeast cells, based on a permeabilized cell assay that we established for actin assembly in vitro. Previous analysis indicated that an activity associated with the cell cortex promotes actin polymerization in the bud. After inactivation by a chemical treatment, this activity can be reconstituted back to the permeabilized cells from a cytoplasmic extract. Fractionation of the extract revealed that the reconstitution depends on two sequentially acting protein factors. Bee1, a cortical actin cytoskeletal protein with sequence homology to Wiskott-Aldrich syndrome protein, is required for the first step of the reconstitution. This finding, together with the severe defects in actin organization associated with the bee1 null mutation, indicates that Bee1 protein plays a direct role in controlling actin polymerization at the cell cortex. The factor that acts in the second step of the reconstitution has been identified by conventional chromatography. It is composed of a novel protein, Pca1. Sequence analysis suggests that Pca1 has the potential to interact with SH3 domain-containing proteins and phospholipids.

• Fujita H et al.
• Characterization of gelsolin truncates that inhibit actin depolymerization by severing activity of gelsolin and cofilin.
• Eur J Biochem. 1997; 248: 834-9
• Display abstract

• Gelsolin is a calcium-activated actin-binding protein with six subdomains. The N-terminal (G1) domain is essential for actin-filament-severing activity while other domains within G2-3 position the protein on the filament side allowing G1 to sever. In order to generate reagents capable of competitively inhibiting endogenous gelsolin and, potentially, other actin filament regulatory protein, we expressed several truncates of gelsolin in Escherichia coli, and analyzed how they affected the in vitro activity of two different actin-binding proteins, gelsolin and cofilin. A Ca2+-sensitive truncate containing G2-6 inhibited the F-actin-depolymerizing activities of both gelsolin and cofilin, while a G2-3 truncate was less effective. Using two independent assays, our results support the idea that gelsolin truncates inhibit actin filament severing and do not markedly affect actin subunit dissociation kinetics. Cosedimentation assays in the presence of calcium demonstrate that the G2-6 truncate binds to F-actin more strongly than the G2-3 truncate consistent with a protection mechanism by conformational change of F-actin and/or competitive binding to actin filaments which depends upon the presence of actin filament binding domains.

• Obinata T et al.
• Low molecular-weight G-actin binding proteins involved in the regulation of actin assembly during myofibrillogenesis.
• Cell Struct Funct. 1997; 22: 181-9
• Display abstract

• We previously demonstrated that small G-actin binding proteins, cofilin, ADF and profilin, are involved in the actin dynamics during myofibrillogenesis (OBINATA, T. (1993). Int. Rev. Cytol., 143: 153-189.). To better understand how they are responsible for the regulation of actin assembly, the amounts of the actin-binding proteins were quantified by means of quantitative immunoblotting and compared with that of G-actin pool. The sum of the amounts of cofilin, ADF and profilin was insufficient at early developmental stages but sufficient at later stages to account for the pool of G-actin in muscle cells. We detected expression of thymosin beta 4 at a considerable level in young embryonic but not in adult skeletal muscles. We, therefore, conclude that the G-actin pool in young embryonic skeletal muscle is mainly due to cofilin, ADF, profilin and thymosin beta 4. Switching from a non-muscle-type (NM-) cofilin to a muscle-type (M-) cofilin was observed during muscle development of mammals. In order to clarify cofilin-dependent regulation of actin assembly in muscle cells, cofilin tagged with fluorescence dyes was introduced into C2 myoblasts by a micro injection method. The exogeneous cofilin, but not ADF, caused quick disassembly of actin filaments and accumulated in furrow region of dividing cells. The analogs of the unphosphorylated form (A3-cofilin) and the phosphorylated form (D3-cofilin) were prepared by converting Ser3, a regulatory phosphorylation site, to Ala or Asp. When A3-cofilin and D3-cofilin were injected into living cells, the former was concentrated at the membrane ruffles and cleavage furrow, while the latter showed only diffuse distribution in the cytoplasm. These results suggest that the subcellular distribution of cofilin as well as its interaction with actin in vivo is regulated by its phosphorylation and dephosphorylation.

• DiNubile MJ, Huang S
• High concentrations of phosphatidylinositol-4,5-bisphosphate may promote actin filament growth by three potential mechanisms: inhibiting capping by neutrophil lysates, severing actin filaments and removing capping protein-beta2 from barbed ends.
• Biochim Biophys Acta. 1997; 1358: 261-78
• Display abstract

• Cell locomotion requires rapid growth of cortical actin filaments whose barbed ends are capped in the resting cell. Phosphatidylinositol-4,5-bisphosphate (PIP2) may play a critical role as an intracellular messenger in cytoskeletal rearrangement after stimulation. We have examined the effects of PIP2 micelles on the Ca2+-independent actin filament capping activity in high speed supernatants of neutrophil lysates which we had previously demonstrated to be almost entirely due to capping protein-beta2, a homologue of cap Z. High concentrations of PIP2 totally prevented the capping of exogenous spectrin-F-actin seeds by dilute supernatants of neutrophil extracts. Capping could also be inhibited, albeit less effectively, by PIP and PI, but not by other phospholipids. When incubated with filaments in the absence of supernatant, PIP2 increased the number of growing ends. PIP2 also uncapped previously capped actin filaments, as demonstrated by incubating supernatant-capped and uncapped seeds with and without PIP2 and then comparing the initial elongation rates after addition of pyrenyl-G-actin. Incubation of capped seeds with high concentrations of PIP2 increased the number of free barbed ends to a level comparable to that of the uncapped seeds exposed to PIP2. PIP2 caused uncapping to occur too quickly to be explained simply by the off-rate of capping protein-beta2, implying that PIP2 interacted directly with capping protein on the filament ends. In fact, PIP2 transiently uncapped capped seeds in the presence of excess free capping protein. From our data, we estimate that millimolar concentrations of PIP2 (almost 100-fold higher than the amount predicted from the effective concentration in purified systems) would be required to inhibit all the capping protein-beta2 in the cytosol. This discrepancy probably results, in large part, from sequestration of PIP2 by other PIP2-binding proteins in the cytoplasm. If PIP2 mediates differential cytoskeletal growth after chemoattractant stimulation in vivo, very high concentrations may be required subjacent to the plasma membrane for regional severing and uncapping of actin filaments to occur quickly near the perturbed membrane.

• McGough A, Pope B, Chiu W, Weeds A
• Cofilin changes the twist of F-actin: implications for actin filament dynamics and cellular function.
• J Cell Biol. 1997; 138: 771-81
• Display abstract

• Cofilin is an actin depolymerizing protein found widely distributed in animals and plants. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on actin filaments. Cofilin binds filamentous (F)-actin cooperatively by bridging two longitudinally associated actin subunits. The binding site is centered axially at subdomain 2 of the lower actin subunit and radially at the cleft between subdomains 1 and 3 of the upper actin subunit. Our work has revealed a totally unexpected (and unique) property of cofilin, namely, its ability to change filament twist. As a consequence of this change in twist, filaments decorated with cofilin have much shorter 'actin crossovers' ( approximately 75% of those normally observed in F-actin structures). Although their binding sites are distinct, cofilin and phalloidin do not bind simultaneously to F-actin. This is the first demonstration of a protein that excludes another actin-binding molecule by changing filament twist. Alteration of F-actin structure by cofilin/ADF appears to be a novel mechanism through which the actin cytoskeleton may be regulated or remodeled.

• Neely MD, Macaluso E
• Motile areas of leech neurites are rich in microfilaments and two actin-binding proteins: gelsolin and profilin.
• Proc R Soc Lond B Biol Sci. 1997; 264: 1701-6
• Display abstract

• Cell motility is produced by changes in the dynamics and organization of actin filaments. The aim of the experiments described here was to test whether growing neurites contain two actin-binding proteins, gelsolin and profilin, that regulate polymerization of actin and affect non-neuronal cell motility. The distribution of gelsolin, profilin and the microfilaments was compared by immunocytochemistry of leech neurons growing in culture. We observed that microfilaments are enriched in the peripheral motile areas of the neurites. Both gelsolin and profilin are also concentrated in these regions. Gelsolin is abundant in filopodia and is associated with single identifiable microfilament bundles in lamellipodia. Profilin is not prominent in filopodia and shows a diffuse staining pattern in lamellipodia. The colocalization of gelsolin and profilin in motile, microfilament-rich areas supports the hypothesis that they synergistically regulate the actin dynamics that underlie neurite growth.

• Allen ML, Dobrowolski JM, Muller H, Sibley LD, Mansour TE
• Cloning and characterization of actin depolymerizing factor from Toxoplasma gondii.
• Mol Biochem Parasitol. 1997; 88: 43-52
• Display abstract

• We determined the predicted amino acid sequence of actin depolymerizing factor (ADF) from Toxoplasma gondii by sequencing the full-length cDNA. T. gondii ADF consists of 118 amino acids (calculated molecular weight 13,400) and shares a high degree of sequence similarity to other low molecular weight actin monomer sequestering proteins, especially Acanthamoeba actophorin, plant ADFs and yeast and vertebrate cofilin. ADF from T. gondii is smaller and does not contain a nuclear localization sequence like the related vertebrate proteins. Southern blot analysis indicates that T. gondii ADF is a single-copy gene. Homogeneous recombinant T. gondii ADF purified from E. coli is active in binding actin monomers and depolymerizing F-actin. Localization of ADF by immunofluorescence and immunoelectron microscopy indicates ADF is scattered throughout the cytoplasm and prominently localized beneath the plasma membrane in T. gondii.

• Symersky J, Monod M, Foundling SI
• High-resolution structure of the extracellular aspartic proteinase from Candida tropicalis yeast.
• Biochemistry. 1997; 36: 12700-10
• Display abstract

• The crystal structure of the secreted aspartic proteinase from Candida tropicalis yeast (SAPT) has been determined to 1.8 A resolution. The classic aspartic proteinase bilobal structure and domain topology is conserved in SAPT, with the substrate binding cleft situated between the two domains. Structural comparisons made with pepsin indicate that insertions and deletions in the primary sequence modify the SAPT structure to create a more spacious substrate binding cleft with altered specificity. An unexpected tetrapeptide has been found to occupy binding sites S1'-S3', and this suggests the order of release of peptide products in the catalytic mechanism of these enzymes. Structural features are considered with regard to previous substrate specificity data.

• Yamano A, Heo NH, Teeter MM
• Crystal structure of Ser-22/Ile-25 form crambin confirms solvent, side chain substate correlations.
• J Biol Chem. 1997; 272: 9597-600
• Display abstract

• It is not agreed that correlated positions of disordered protein side chains (substate correlations) can be deduced from diffraction data. The pure Ser-22/Ile-25 (SI form) crambin crystal structure confirms correlations deduced for the natural, mixed sequence form of crambin crystals. Physical separation of the mixed form into pure SI form and Pro-22/Leu-25 (PL form) crambin and the PL form crystal structure determination (Yamano, A., and Teeter, M. M. (1994) J. Biol. Chem. 269, 13956-13965) support the proposed (Teeter, M. M., Roe, S. M., and Heo, N. H. (1993) J. Mol. Biol. 230, 292-311) correlation model. Electron density of mixed form crambin crystals shows four possible pairs of side chain conformations for heterogeneous residue 22 and nearby Tyr-29 (2(2) = 4, two conformations for each of two side chains). One combination can be eliminated because of short van der Waals' contacts. However, only two alternates have been postulated to exist in mixed form crambin: Pro-22/Tyr-29A and Ser-22/Tyr-29B. In crystals of the PL form, Pro-22 and Tyr-29A are found to be in direct van der Waals' contact (Yamano, A., and Teeter, M. M. (1994) J. Biol. Chem. 269, 13956-13965). Comparison of the SI form structure with the mixed form electron density confirms that the fourth combination of side chains does not occur and that side chain correlations are mediated by water networks.

• Van Troys M, Dewitte D, Verschelde JL, Goethals M, Vandekerckhove J, Ampe C
• Analogous F-actin binding by cofilin and gelsolin segment 2 substantiates their structural relationship.
• J Biol Chem. 1997; 272: 32750-8
• Display abstract

• Cofilin is representative for a family of low molecular weight actin filament binding and depolymerizing proteins. Recently the three-dimensional structure of yeast cofilin and of the cofilin homologs destrin and actophorin were resolved, and a striking similarity to segments of gelsolin and related proteins was observed (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagaka, F. (1996) Cell 85, 1047-1055; Fedorov, A. A., Lappalainen, P., Fedorov, E. V., Drubin, D. G., and Almo, S. C. (1997) Nat. Struct. Biol. 4, 366-369; Leonard, S. A., Gittis, A. G., Petrella, E. C., Pollard, T. D., and Lattman, E. E. (1997) Nat. Struct. Biol. 4, 369-373). Using peptide mimetics, we show that the actin binding site stretches over the entire cofilin alpha-helix 112-128. In addition, we demonstrate that cofilin and its actin binding peptide compete with gelsolin segments 2-3 for binding to actin filaments. Based on these competition data, we propose that cofilin and segment 2 of gelsolin use a common structural topology to bind to actin and probably share a similar target site on the filament. This adds a functional dimension to their reported structural homology, and this F-actin binding mode provides a basis to further enlighten the effect of members of the cofilin family on actin filament dynamics.

• Carlier MF, Pantaloni D
• Control of actin dynamics in cell motility.
• J Mol Biol. 1997; 269: 459-67
• Display abstract

• Actin polymerization plays a major role in cell movement. The controls of actin sequestration/desequestration and of filament turnover are two important features of cell motility. Actin binding proteins use properties derived from the steady-state monomer-polymer cycle of actin in the presence of ATP, to control the F-actin/G-actin ratio and the turnover rate of actin filaments. Capping proteins and profilin regulate the size of the pools of F-actin and unassembled actin by affecting the steady-state concentration of ATP-G-actin. At steady state, the treadmilling cycle of actin filaments is fed by their disassembly from the pointed ends. It is regulated in two different ways by capping proteins and ADF, as follows. Capping proteins, in decreasing the number of growing barbed ends, increase their individual rate of growth and create a "funneled" treadmilling process. ADF/cofilin, in increasing the rate of pointed-end disassembly, increases the rate of filament turnover, hence the rate of barbed-end growth. In conclusion, capping proteins and ADF cooperate to increase the rate of actin assembly up to values that support the rates of actin-based motility processes.

• Tilney LG, Tilney MS, Guild GM
• Formation of actin filament bundles in the ring canals of developing Drosophila follicles.
• J Cell Biol. 1996; 133: 61-74
• Display abstract

• Growing the intracellular bridges that connect nurse cells with each o ther and to the developing oocyte is vital for egg development. These ring canals increase from 0.5 microns in diameter at stage 2 to 10 microns in diameter at stage 11. Thin sections cut horizontally as you would cut a bagel, show that there is a layer of circumferentially oriented actin filaments attached to the plasma membrane at the periphery of each canal. By decoration with subfragment 1 of myosin we find actin filaments of mixed polarities in the ring such as found in the "contractile ring" formed during cytokinesis. In vertical sections through the canal the actin filaments appear as dense dots. At stage 2 there are 82 actin filaments in the ring, by stage 6 there are 717 and by stage 10 there are 726. Taking into account the diameter, this indicates that there is 170 microns of actin filaments/canal at stage 2 (pi x 0.5 microns x 82), 14,000 microns at stage 9 and approximately 23,000 microns at stage 11 or one inch of actin filament! The density of actin filaments remains unchanged throughout development. What is particularly striking is that by stages 4-5, the ring of actin filaments has achieved its maximum thickness, even though the diameter has not yet increased significantly. Thereafter, the diameter increases. Throughout development, stages 2-11, the canal length also increases. Although the density (number of actin filaments/micron2) through a canal remains constant from stage 5 on, the actin filaments appear as a net of interconnected bundles. Further information on this net of bundles comes from studying mutant animals that lack kelch, a protein located in the ring canal that has homology to the actin binding protein, scruin. In this mutant, the actin filaments form normally but individual bundles that comprise the fibers of the net are not bound tightly together. Some bundles enter into the ring canal lumen but do not completely occlude the lumen. all these observations lay the groundwork for our understanding of how a noncontractile ring increases in thickness, diameter, and length during development.

• Kuhlman PA, Hughes CA, Bennett V, Fowler VM
• A new function for adducin. Calcium/calmodulin-regulated capping of the barbed ends of actin filaments.
• J Biol Chem. 1996; 271: 7986-91
• Display abstract

• Adducin is a membrane skeleton protein originally described in human erythrocytes that promotes the binding of spectrin to actin and also binds directly to actin and bundles actin filaments. Adducin is associated with regions of cell-cell contact in nonerythroid cells, where it is believed to play a role in regulating the assembly of the spectrin-actin membrane skeleton. In this study we demonstrate a novel function for adducin; it completely blocks elongation and depolymerization at the barbed (fast growing) ends of actin filaments, thus functioning as a barbed end capping protein (Kcap approximately 100 nM). This barbed end capping activity requires the intact adducin molecule and is not provided by the NH2-terminal globular head domains alone nor by the COOH-terminal extended tail domains, which were previously shown to contain the spectrin-actin binding, calmodulin binding, and phosphorylation sites. A novel difference between adducin and other previously described capping proteins is that it is down-regulated by calmodulin in the presence of calcium. The association of stoichiometric amounts of adducin with the short erythrocyte actin filaments in the membrane skeleton indicates that adducin could be the functional barbed end capper in erythrocytes and play a role in restricting actin filament length. Our experiments also suggest novel possibilities for calcium regulation of actin filament assembly by adducin in erythrocytes and at cell-cell contact sites in nonerythroid cells.

• Mills RG, Minamide LS, Yuan A, Bamburg JR, Bray JJ
• Slow axonal transport of soluble actin with actin depolymerizing factor, cofilin, and profilin suggests actin moves in an unassembled form.
• J Neurochem. 1996; 67: 1225-34
• Display abstract

• We examined the axonal transport of actin and its monomer binding proteins, actin depolymerizing factor, cofilin, and profilin, in the chicken sciatic nerve following injection of [35S]methionine into the lumbar spinal cord. At intervals up to 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Actin and its binding proteins were then isolated by affinity chromatography on DNase I-Sepharose and by one- and two-dimensional polyacrylamide gel electrophoresis. Fluorographic analysis showed that the specific activity of soluble actin was two to three times that of its particulate form and that soluble actin, cofilin, actin depolymerizing factor, and profilin were transported at similar rates in slow component b of axonal flow. Our data strongly support the view that the mobile form of actin in slow transport is soluble and that a substantial amount of this actin may travel as a complex with actin depolymerizing factor, cofilin, and profilin. Along labeled nerves the specific activity of the unphosphorylated form of actin depolymerizing factor, which binds actin, was not significantly different from that of its "inactive" phosphorylated form. This constancy in specific activity suggests that continuous inactivation and reactivation of actin depolymerizing factor occur during transport, which could contribute to the exchange of soluble actin with the filamentous actin pool.

• Perelroizen I, Didry D, Christensen H, Chua NH, Carlier MF
• Role of nucleotide exchange and hydrolysis in the function of profilin in action assembly.
• J Biol Chem. 1996; 271: 12302-9
• Display abstract

• Profilin, an essential G-actin-binding protein, has two opposite regulatory functions in actin filament assembly. It facilitates assembly at the barbed ends by lowering the critical concentration (Pantaloni, D., and Carlier, M.-F. (1993) Cell 75, 1007-1014); in contrast it contributes to the pool of unassembled actin when barbed ends are capped. We proposed that the first of these functions required an input of energy. How profilin uses the ATP hydrolysis that accompanies actin polymerization and whether the acceleration of nucleotide exchange on G-actin by profilin participates in its function in filament assembly are the issues addressed here. We show that 1) profilin increases the treadmilling rate of actin filaments in the presence of Mg2+ ions; 2) when filaments are assembled from CaATP-actin, which polymerizes in a quasireversible fashion, profilin does not promote assembly at the barbed ends and has only a G-actin-sequestering function; 3) plant profilins do not accelerate nucleotide exchange on G-actin, yet they promote assembly at the barbed end. The enhancement of nucleotide exchange by profilin is therefore not involved in its promotion of actin assembly, and the productive growth of filaments from profilin-actin complex requires the coupling of ATP hydrolysis to profilin-actin assembly, a condition fulfilled by Mg-actin, and not by Ca-actin.

• Chen X, Whitmire D, Bowen JP
• Xylanase homology modeling using the inverse protein folding approach.
• Protein Sci. 1996; 5: 705-8
• Display abstract

• Xylanase has been used in wood pulp bleaching in an effort to reduce chlorine release into the environment and pollution associated with paper production. The three-dimensional structure of xylanase is important to enable better understanding of the enzyme mechanism and to help design a more thermostable xylanase mutant. At the time this work was begun, there was no sequence homologous protein available for traditional sequence-based homology modeling. In order to circumvent this problem, the inverse protein folding approach was undertaken to find a suitable template structure. Model structures of Bacillus circulans xylanase were built based on the data-base search results of related proteins. The model structures were refined and compared to the recently solved xylanase X-ray crystal structure. The overall structural similarity between the theoretical model and experimental structure demonstrate the usefulness of this approach. Disagreement in folding topology, however, warrants further research into the inverse protein folding approach.

• Balasubramanian MK, Feoktistova A, McCollum D, Gould KL
• Fission yeast Sop2p: a novel and evolutionarily conserved protein that interacts with Arp3p and modulates profilin function.
• EMBO J. 1996; 15: 6426-37
• Display abstract

• Profilins bind to monomeric actin and also interact with ligands such as phosphoinositide 4,5-bisphosphate, the proline-rich protein VASP and a complex of four to six polypeptides identified in Acanthamoeba that includes two actin-related proteins. Here, we report the identification and characterization of an essential gene from Schizosaccharomyces pombe, sop2+, a mutation in which rescues the temperature-sensitive lethality of a profilin mutation, cdc3-124. The sop2-1 mutant is defective for cell elongation and septation, suggesting that it is involved in multiple cortical actin-requiring processes. Consistent with a role in actin cytoskeletal function, negative interactions have been identified between sop2-1 and act1-48, a mutant allele of actin. Sop2p is a novel 377 amino acid polypeptide with similarity to proteins of the beta-transducin repeat family. Sop2p-related proteins have been identified by sequencing projects in diverse species, and we have isolated a human cDNA highly related to sop2+, SOP2 Hs, which functionally complements the sop2-1 mutation. Sop2p proteins from all species contain peptide sequences identical or highly similar to two peptide sequences from an Acanthamoeba beta-transducin repeat protein present in the profilin binding complex. Biochemical analyses demonstrate that Sop2p is present in a complex which also contains the actin-related protein, Arp3p. Immunofluorescence studies reveal the presence of Sop2p in (i) punctate structures distributed throughout the cell, (ii) cables that extend the length of the cell, and (iii) a medial band in a small percentage of septating cells. Collectively these data demonstrate the interaction of Sop2p with Arp3p, profilin and actin.

• Ayscough KR, Drubin DG
• ACTIN: general principles from studies in yeast.
• Annu Rev Cell Dev Biol. 1996; 12: 129-60
• Display abstract

• Three of the most important questions concerning actin function are: (a) How does actin structure relate to actin function? (b) How does each of the numerous proteins that interact with actin contribute to actin cytoskeleton function in vivo? (c) How are the activities of these proteins regulated? Powerful molecular genetics combined with well-established biochemical techniques make the yeast Saccharomyces cerevisiae an ideal organism for studies aimed at answering these questions. The protein sequences and biochemical properties of actin and its interacting proteins and the pathways that regulate these interactions all appear to be conserved, indicating that principles elucidated from studies in yeast will apply to all eukaryotes. In this review, we highlight advances in our general understanding of actin properties, interactions with other proteins, and regulation of the actin cytoskeleton, derived from studies in the budding yeast S. cerevisiae.

• Kaiser DA, Pollard TD
• Characterization of actin and poly-L-proline binding sites of Acanthamoeba profilin with monoclonal antibodies and by mutagenesis.
• J Mol Biol. 1996; 256: 89-107
• Display abstract

• We characterized several deletion and substitution mutations of Acanthamoeba profilin and nine monoclonal antibodies to Acanthamoeba profilin. The results provide two independent lines of evidence about the binding sites for actin and poly-L-proline on the profilin molecule. This new evidence is consistent with the main conclusions about these binding sites from previous structural and mutagenic studies. Mutagenesis also revealed that the native structure of profilin is very sensitive to substitutions and deletions at the C terminus. For example, profilin with a deletion of the eight C-terminal residues has many of the physical properties of a molten globule, yet remarkably still binds to actin. This instability may account for the lack of function of similar mutants in yeast.

• Yahara I et al.
• A role of cofilin/destrin in reorganization of actin cytoskeleton in response to stresses and cell stimuli.
• Cell Struct Funct. 1996; 21: 421-4
• Display abstract

• 1. Cofilin is an essential actin-regulating protein widely distributed in all eucaryotes. The structure and function of cofilin are conserved during evolution. 2. Cofilin depolymerizes F-actin in vitro at alkaline pH and severs F-actin in vitro at pH lower than 7.3. Overexpression of cofilin in viable cells induced bundles of actin filaments suggesting that the severing activity rather than the actin-depolymerizing or monomeric actin-sequestering activity is physiologically significant in vivo. 3. The actin bundle formation induced by overexpression of cofilin is accompanied with an increase in cell motility of Dictyostelium cells. 4. In higher vertebrates, the actin-binding activity of cofilin is negatively regulated by phosphorylation on its Ser-3 residue. The actin-binding activity is essential for yeast cells to grow. 5. Stresses and various cell stimuli activate cofilin by inducing dephosphorylation of cofilin in resting vertebrate cells. 6. Cofilin has an nuclear localization signal sequence and translocates into the nucleus together with actin in response to various stresses. Functional roles of cofilin/actin in the nucleus remain to be elucidated. 7. Tertiary structure of destrin (cofilin) resembles that of gelsolin segment 1 and well explains its functions such as Ca(2+)-independent actin binding activity.

• Teubner A, Wegner A
• The rate of annealing of actin tropomyosin filaments depends strongly on the length of the filaments.
• Biochim Biophys Acta. 1996; 1297: 214-8
• Display abstract

• Actin tropomyosin filaments were sheared to produce short filaments. Following incubation for 0 to 10000 s annealing of the filaments was assayed by determination of the rate of polymerization of monomeric actin onto the filament ends. The rate of decrease of the concentration of filament ends was found to be proportional to its fourth power. In contrast, the rate of end-to-end association of actin filaments in the absence of tropomyosin was proportional to the square of the concentration of filament ends. The strong dependence on the filament length of the rate of annealing of actin tropomyosin filaments was interpreted by the model of Hill (Biophys. J., 44, 285-288 (1983)) who pointed out that the rate constant of end-to-end association of long rod-like filaments is expected to depend on the length of the filaments for sterical conditions.

• Sizonenko GI, Karpova TS, Gattermeir DJ, Cooper JA
• Mutational analysis of capping protein function in Saccharomyces cerevisiae.
• Mol Biol Cell. 1996; 7: 1-15
• Display abstract

• To investigate physiologic functions and structural correlates for actin capping protein (CP), we analyzed site-directed mutations in CAP1 and CAP2, which encode the alpha and beta subunits of CP in Saccharomyces cerevisiae. Mutations in four different regions caused a loss of CP function in vivo despite the presence of mutant protein in the cells. Mutations in three regions caused a complete loss of all aspects of function, including the actin distribution, viability with sac6, and localization of CP to actin cortical patches. Mutation of the fourth region led to partial loss of only one function-formation of actin cables. Some mutations retained function and exhibited the complete wild-type phenotype, and some mutations led to a complete loss of protein and therefore loss of function. The simplest hypothesis that can explain these results is that a single biochemical property is necessary for all in vivo functions. This biochemical property is most likely binding to actin filaments, because the nonfunctional mutant CPs no longer co-localize with actin filaments in vivo and because direct binding of CP to actin filaments has been well established by studies with purified proteins in vitro. More complex hypotheses, involving the existence of additional biochemical properties important for function, cannot be excluded by this analysis.

• Maun NA, Speicher DW, DiNubile MJ, Southwick FS
• Purification and properties of a Ca(2+)-independent barbed-end actin filament capping protein, CapZ, from human polymorphonuclear leukocytes.
• Biochemistry. 1996; 35: 3518-24
• Display abstract

• In human polymorphonuclear leukocytes (PMN), changes in the actin architecture are critical for the shape changes required for chemotaxis and phagocytosis. Barbed-end capping proteins are likely to regulate actin assembly in PMN. The previously identified barbed-end blocking proteins in PMN, gelsolin and CapG, require Ca(2+) to initiate capping of actin filaments. Because chemoattractants can stimulate PMN actin assembly by a calcium-independent signal transduction pathway, we sought to purify a calcium-independent barbed-end capping activity from PMN cytoplasmic extracts. A Ca(2+) -insensitive actin polymerization inhibitory activity was partially purified from human PMN [Southwick & Stossel (1981) J. Biol. Chem 256, 3030]. Using five column chromatography steps, we purified the protein to homogeneity as assessed by silver staining. Purification was associated with an increase in specific activity of greater than 40 X. Western blot analysis identified the protein as the nonmuscle isoform of the heterodimeric capping protein capZ. Human PMN capZ has an apparent disassociation constant of 3 nM for capping in the presence or absence of micromolar Ca(2+), as assessed by both pyrenylactin elongation and depolymerization assays. Similar to the activity reported for the actin polymerization inhibitor, activity of PMN capZ was inhibited by increasing the KC1 concentration from 0.1 M to 0.6 M. The capping function was also inhibited by phosphatidylinositol 4,5-bisphosphate (PIP(2)) micelles, with half-maximal inhibition occurring at 5.5 micrograms mL(-1). PMN capZ did not nucleate actin assembly, sequester actin monomers, or sever actin filaments. Quantitative Western blot analysis revealed that capZ levels corresponded to 0.7-1.0% of the total human PMN cytoplasmic protein. Given its abundance and high affinity for barbed filament ends, capZ is likely to play an important role in the calcium-independent regulation of actin filament assembly associated with PMN chemotaxis.

• Dufort PA, Lumsden CJ
• How profilin/barbed-end synergy controls actin polymerization: a kinetic model of the ATP hydrolysis circuit.
• Cell Motil Cytoskeleton. 1996; 35: 309-30
• Display abstract

• The role of ATP hydrolysis in the regulation of the actin cytoskeleton continues to be a subject of controversy. Since actin polymerization can occur in the absence of ATP, the energy of hydrolysis is not needed for filament assembly. Recent work has instead suggested a regulatory role for ATP in cytoskeletal remodeling. In particular, both profilin and free filament barbed ends have been shown to play major roles in the processing of ATP by actin. We have developed a new integrated kinetic model to examine how the maintenance of the pool of unpolymerized actin and the flux of actin subunits through filaments are controlled by profilin and free filament barbed ends through their interaction with ATP. An analysis of the model's steady states predicts how two novel regulatory pathways may regulate the cytoskeleton in vivo. Coordinated changes in the availability of both profilin and free barbed ends mediate the following regulatory effects: (1) both the nucleotide composition and the absolute amount of free G-actin can be changed separately or together to substantially alter the total amount of F-actin; and (2) uncapping the barbed ends of only a modest fraction of filaments causes all filaments to begin slowly depolymerizing from their pointed ends, resulting in the total depolymerization of the remaining capped filaments. We report that the phenomenon of treadmilling, wherein the barbed end growth of each filament is exactly balanced by pointed end loss at steady state, is only possible in the limiting case when all barbed ends are uncapped. The capping of any fraction of barbed ends increases the critical concentration of ATP-G-actin, causing the remaining free barbed ends to grow faster than their pointed ends can shrink. On the basis of these findings we propose a major revision to the treadmilling model for actin-based motility, in which the rapidly growing filaments with free barbed ends are continuously severed toward their rear followed by capping of the newly exposed barbed ends. This revised model, herein referred to as "treadsevering," allows sustained and rapid barbed end growth to occur indefinitely at a steady state provided a continuous input of ATP.

• Lopez I et al.
• Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins.
• Proc Natl Acad Sci U S A. 1996; 93: 7415-20
• Display abstract

• In pollen development, a dramatic reorganization of the actin cytoskeleton takes place during the passage of the pollen grain into dormancy and on activation of pollen tube growth. A role for actin-binding proteins is implicated and we report here the identification of a small gene family in maize that encodes actin depolymerizing factor (ADF)-like proteins. The ADF group of proteins are believed to control actin polymerization and depolymerization in response to both intracellular and extracellular signals. Two of the maize genes ZmABP1 and ZmABP2 are expressed specifically in pollen and germinating pollen suggesting that the protein products may be involved in pollen actin reorganization. A third gene, ZmABP3, encodes a protein only 56% and 58% identical to ZmABP1 and ZmABP2, respectively, and its expression is suppressed in pollen and germinated pollen. The fundamental biochemical characteristics of the ZmABP proteins has been elucidated using bacterially expressed ZmABP3 protein. This has the ability to bind monomeric actin (G-actin) and filamentous actin (F-actin). Moreover, it decreases the viscosity of polymerized actin solutions consistent with an ability to depolymerize filaments. These biochemical characteristics, taken together with the sequence comparisons, support the inclusion of the ZmABP proteins in the ADF group.

• Yeh J, Haarer BK
• Profilin is required for the normal timing of actin polymerization in response to thermal stress.
• FEBS Lett. 1996; 398: 303-7
• Display abstract

• We have used a fluorometric assay to determine the relative amounts of polymerized actin (F-actin) in wild-type and profilin mutant yeast cells. Our results indicate that profilin plays a role in maintaining normal F-actin levels in response to shifts to high temperature. Cells lacking profilin display a greater drop in F-actin levels upon such temperature shifts, and are slower to recover to initial F-actin levels than are wild-type cells. Interestingly, shifts to cold temperatures result in rapid increases of F-actin levels in wild-type and profilin null cells. We have further determined that shifting to high-osmolarity growth conditions causes a relatively slow decrease in F-actin levels in wild-type cells, and a small but rapid increase in the F-actin levels in profilin null cells. Profilin null cells contain normal concentrations of F-actin while growing exponentially at room temperature, indicating that profilin is not essential for maintaining F-actin concentrations during steady-state growth. Our data suggest that actin is inherently unstable in vivo at high temperatures, and that profilin helps to maintain actin in its filamentous state at these temperatures, perhaps by stimulating actin polymerization in a proper temporal and spatial fashion.

• Haynie DT, Ponting CP
• The N-terminal domains of tensin and auxilin are phosphatase homologues.
• Protein Sci. 1996; 5: 2643-6
• Display abstract

• Tensin, an actin filament capping protein, and auxilin, a component of receptor-mediated endocytosis, are known to have 350 residue regions of significant sequence similarity near their N-termini (Schroder et al., 1995, Eur J Biochem 228:297-304). Here we demonstrate that these regions are homologous, not only to each other, but also to the catalytic domain of a putative protein tyrosine phosphatase (PTP) from Saccharomyces cerevisiae and to other PTPs. We propose that the PTP-like portion of the homology region of tensin and auxilin represents a distinct domain. A detailed sequence comparison indicates that the PTP-like domain in tensin is unlikely to exhibit phosphatase activity, whereas in auxilin it may possess a different phosphatase specificity from tyrosine phosphatases. It is probable that the PTP-like domains in tensin and auxilin mediate binding interactions with phosphorylated polypeptides; they may therefore represent members of a distinct class of phosphopeptide recognition domain.

• Schafer DA, Jennings PB, Cooper JA
• Dynamics of capping protein and actin assembly in vitro: uncapping barbed ends by polyphosphoinositides.
• J Cell Biol. 1996; 135: 169-79
• Display abstract

• Bursts of actin polymerization in vivo involve the transient appearance of free barbed ends. To determine how rapidly barbed ends might appear and how long they might remain free in vivo, we studied the kinetics of capping protein, the major barbed end capper, binding to barbed ends in vitro. First, the off-rate constant for capping protein leaving a barbed end is slow, predicting a half-life for a capped barbed end of approximately 30 min. This half-life implies that cells cannot wait for capping protein to spontaneously dissociate from capped barbed ends in order to create free barbed ends. However, we find that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-mono-phosphate (PIP) cause rapid and efficient dissociation of capping protein from capped filaments. PIP2 is a strong candidate for a second messenger regulating actin polymerization; therefore, the ability of PIP2 to remove capping protein from barbed ends is a potential mechanism for stimulating actin polymerization in vivo. Second, the on-rate constant for capping protein binding to free barbed ends predicts that actin filaments could grow to the length of filaments observed in vivo during one lifetime. Third, capping protein beta-subunit isoforms did not differ in their actin binding properties, even in tests with different actin isoforms. A major hypothesis for why capping protein beta-subunit isoforms exist is thereby excluded. Fourth, the proposed capping protein regulators, Hsc70 and S100, had no effect on capping protein binding to actin in vitro.

• Sun HQ, Kwiatkowska K, Yin HL
• beta-Thymosins are not simple actin monomer buffering proteins. Insights from overexpression studies.
• J Biol Chem. 1996; 271: 9223-30
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• beta-Thymosins are the currently favored candidates for maintaining the large actin monomer (G-actin) pool in living cells. To determine if beta-thymosin behaves like a simple G-actin buffering agent in the complex environment of a cell, we overexpressed thymosin beta10 (Tbeta 10) in NIH3T3 cells and determined the effect on the monomer/polymer equilibrium. Tbeta 10 is the predominant beta-thymosin isoform in the NIH3T3 cell line, and it is present in approximately equal molar ratio to profilin and cofilin/actin depolymerizing factor, two other well characterized actin monomer binding proteins. Clonal cell lines that overexpressed three times more Tbeta 10 had 23-33% more polymerized actin than control cells, and the filaments appeared thicker after staining with fluorescent phalloidin. There was no change in total actin, profilin, and cofilin/actin depolymerizing factor content. The overexpressing cells were more motile; they spread faster and had higher chemotactic and wound healing activity. Assuming that there is no compensatory inactivation of the other classes of monomer binding proteins, our paradoxical observation can be accounted for quantitatively by a parallel in vitro study (Carlier, M.-F., Didry, D., Erk, I., Lepault, J., Van Troys, L., Vanderkekove, J., Perelroizen, I., Yin, H. L., Doi, Y., and Pantaloni, D., (1996) J. Biol. Chem. 271, 9231-9239). beta-Thymosin at levels comparable with that found in the overexpressing cells binds actin filaments and decreases the critical concentration (C(c)) for actin polymerization. This reduces the monomer buffering ability of beta-thymosin, so that above a certain threshold an incremental increase in thymosin does not lead to a corresponding increase in G-actin. Furthermore, the decrease in C(c) reduces the buffering capacity of the other actin monomer binding proteins. As a consequence, an increase in beta-thymosin does not necessarily result in a proportionate increase in actin monomer content in a complex environment containing other actin monomer binding proteins. The outcome depends on the level of beta-thymosin expression relative to the composition of the other actin monomer binding protein. Our results suggest that beta-thymosins are not simple actin buffering proteins and that their biphasic action may have physiological significance.

• Mylvaganam SE, Bonaventura C, Bonaventura J, Getzoff ED
• Structural basis for the root effect in haemoglobin.
• Nat Struct Biol. 1996; 3: 275-83
• Display abstract

• The remarkable ability of root effect haemoglobins to pump oxygen against high O2 gradients results from extreme, acid-induced reductions in O2 affinity and cooperativity. The long-sought mechanism for the root effect, revealed by the 2 angstrom crystal structure of the ligand-bound haemoglobin from Leiostomus xanthurus at pH 7.5, unexpectedly involves modulation of the R-state. Key residues strategically assemble positive-charge clusters across the allosteric beta1 beta2-interface in the R-state. At low pH, protonation of the beta N terminus and His 147(HC3)beta within these clusters is postulated to destabilize the R-state and promote the acid-triggered, allosteric R-->T switch with concomitant O2 release. Surprisingly, a set of residues specific to root effect haemoglobins recruit additional residues, conserved among most haemoglobins, to produce the root effect.

• Eddy RJ, Han J, Sauterer RA, Condeelis JS
• A major agonist-regulated capping activity in Dictyostelium is due to the capping protein, cap32/34.
• Biochim Biophys Acta. 1996; 1314: 247-59
• Display abstract

• Stimulation of starved Dictyostelium amoebae with the chemoattractant cAMP produces a rapid increase in actin nucleation activity at 5 seconds which is cotemporal with an increase in actin assembly and a decrease in Ca(2+)-insensitive capping activity [1]. Further characterization of this capping activity, called aginactin, led to the isolation of an Hsc70 [2]. Here, we demonstrate that purified aginactin contains both Hsc70 and the heterodimeric barbed-end capping protein, cap32/34. Immunoprecipitation of cap32/34 from purified aginactin removes all capping activity while immunoprecipitation of Hsc70 does not, indicating that the capping activity of aginactin is an intrinsic property of cap32/34. Gel filtration and immunoprecipitation assays fail to demonstrate the existence of a stable, high affinity complex between Hsc70 and cap32/34 in either lysate supernatants or aginactin pools but indicate the presence of a transient, ATP-sensitive interaction in cell lysates. Reconstitution experiments with purified Hsc70 and cap32/34 demonstrate that Hsc70 neither stimulates nor inhibits the capping activity of native cap32/34. Furthermore, we measured a Kd of approx. 0.8 nM for the binding of cap32/34 to barbed ends of actin filaments in the absence or presence of Hsc70, in agreement with Kd values measured for purified capping protein from other sources. We conclude, therefore, that cap32/34 is responsible for the capping activity called aginactin and that Hsc70 is not a regulatory cofactor for cap32/34 in Dictyostelium but may function as a chaperone during assembly of the cap32/34 heterodimer.

• Nagaoka R, Abe H, Obinata T
• Site-directed mutagenesis of the phosphorylation site of cofilin: its role in cofilin-actin interaction and cytoplasmic localization.
• Cell Motil Cytoskeleton. 1996; 35: 200-9
• Display abstract

• It has been demonstrated that the activity of ADF and cofilin, which constitute a functionally related protein family, is markedly altered by phosphorylation, and that the phosphorylation site is Ser 3 in their amino acid sequences [Agnew et al., 1995: J. Biol. Chem. 270:17582-17587; Moriyama et al., 1996: Genes Cells 1:73-86]. In order to clarify the function of the phosphorylated and unphosphorylated forms of cofilin in living cells especially in the process of cytokinesis, we generated analogs of the unphosphorylated form (A3-cofilin) and phosphorylated form (D3-cofilin) by converting the phosphorylation site (Ser 3) of cofilin to Ala and Asp, respectively. The mutated proteins were produced in an Escherichia coli expression system, and conjugated with fluorescent dyes. In in vitro functional assay, labeled A3-cofilin retained the authentic ability to bind to and sever F-actin, while labeled D3-cofilin failed to interact with actin. They were then injected into living cells to examine their cellular distribution. They exhibited distinct localization patterns in the cytoplasm; A3-cofilin was highly concentrated at the membrane ruffles and cleavage furrow, where endogenous cofilin is also known to be enriched. In contrast, D3-cofilin showed only diffuse distribution both in the cytoplasm and nucleus. These results suggest that the subcellular distribution of cofilin as well as its interacting with actin in vivo is regulated by its phosphorylation and dephosphorylation.

• Aizawa H, Sutoh K, Yahara I
• Overexpression of cofilin stimulates bundling of actin filaments, membrane ruffling, and cell movement in Dictyostelium.
• J Cell Biol. 1996; 132: 335-44
• Display abstract

• Cofilin is a low molecular weight actin-modulating protein whose structure and function are conserved among eucaryotes. Cofilin exhibits in vitro both a monomeric actin-sequestering activity and a filamentous actin-severing activity. To investigate in vivo functions of cofilin, cofilin was overexpressed in Dictyostelium discoideum cells. An increase in the content of D. discoideum cofilin (d-cofilin) by sevenfold induced a co-overproduction of actin by threefold. In cells over-expressing d-cofilin, the amount of filamentous actin but not that of monomeric actin was increased. Overexpressed d-cofilin co-sedimented with actin filaments, suggesting that the sequestering activity of d-cofilin is weak in vivo. The overexpression of d-cofilin increased actin bundles just beneath ruffling membranes where d-cofilin was co-localized. The overexpression of d-cofilin also stimulated cell movement as well as membrane ruffling. We have demonstrated in vitro that d-cofilin transformed latticework of actin filaments cross-linked by alpha-actinin into bundles probably by severing the filaments. D. discoideum cofilin may sever actin filaments in vivo and induce bundling of the filaments in the presence of cross-linking proteins so as to generate contractile systems involved in membrane ruffling and cell movement.

• Jung E, Fucini P, Stewart M, Noegel AA, Schleicher M
• Linking microfilaments to intracellular membranes: the actin-binding and vesicle-associated protein comitin exhibits a mannose-specific lectin activity.
• EMBO J. 1996; 15: 1238-46
• Display abstract

• Comitin is a 24 kDa actin-binding protein from Dictyostelium discoideum that is located primarily on Golgi and vesicle membranes. We have probed the molecular basis of comitin's interaction with both actin and membranes using a series of truncation mutants obtained by expressing the appropriate cDNA in Escherichia coli. Comitin dimerizes in solution; its principle actin-binding activity is located between residues 90 and 135. The N-terminal 135 'core' residues of comitin contain a 3-fold sequence repeat that is homologous to several monocotyledon lectins and which retains key residues that determine these lectins' three-dimensional structure and mannose binding. These repeats of comitin appear to mediate its interaction with mannose residues in glycoproteins or glycolipids on the cytoplasmic surface of membrane vesicles from D.discoideum, and comitin can be released from membranes with mannose. Our data indicate that comitin binds to vesicle membranes via mannose residues and, by way of its interaction with actin, links these membranes to the cytoskeleton.

• Kim E, Miller CJ, Reisler E
• Polymerization and in vitro motility properties of yeast actin: a comparison with rabbit skeletal alpha-actin.
• Biochemistry. 1996; 35: 16566-72
• Display abstract

• Actin purified from the yeast (Saccharomyces cerevisae) was polymerized faster than rabbit skeletal alpha-actin by MgCl2. The two actins polymerized at similar rates in the presence of CaCl2. Yeast actin, up to 25 microM, was not polymerized by KCl (100-300 mM); the monovalent salt also inhibited the MgCl2-induced polymerization of actin. The local structure of the subdomain-2 region in yeast actin filaments was probed by subtilisin and trypsin digestions. Loop 38-52 appeared more flexible and accessible to subtilisin in yeast than in rabbit actin. In contrast, tryptic digestions at Lys-61 and -68 occurred at the same rate for yeast and alpha-actin filaments. Modification of yeast actin by a sulfhydryl reagent CPM [7-(diethylamino)-3-(4'-maleimidophenyl)-4-methylcoumain] was specific to the Cys-374 residue; no labeling of a yeast actin mutant containing an alanine substitution for cysteine 374 was observed. The rates of Cys-374 labeling by CPM were similar for yeast and muscle actin, suggesting a similar environment for the C terminus in both polymers. In the in vitro motility assays, yeast actin required higher concentrations of heavy meromyosin (HMM) for its sliding than did the rabbit actin. At saturating concentrations of HMM, the sliding velocities of both actins were the same (3.0 microns/s). Relative forces generated by HMM with yeast and muscle actin were assessed by monitoring their in vitro motility in the presence of NEM-HMM load. The sliding of yeast actin was stopped at a level of external load (molar ratio NEM-HMM/HMM = 0.25) lower than that of muscle actin (NEM-HMM/HMM = 0.43), suggesting lower force production with yeast actin. These results are discussed in terms of the myosin cross-bridge cycle and actomyosin interactions.

• Ono S, Abe H, Obinata T
• Stimulus-dependent disorganization of actin filaments induced by overexpression of cofilin in C2 myoblasts.
• Cell Struct Funct. 1996; 21: 491-9
• Display abstract

• Actin depolymerizing factor (ADF)/cofilin is a widely distributed family of actin-binding proteins which regulate actin polymerization in a pH-dependent manner. In cultured cells, cofilin, as well as ADF, translocates from the cytoplasm into the nucleus together with actin and forms rod-like structures in response to heat shock or dimethylsulfoxide (DMSO) treatment. In order to study in vivo interaction of cofilin with actin, we examined the effects of cofilin overexpression on actin cytoskeleton in C2 myoblasts. Interestingly, no remarkable effect was observed on phalloidin-stained patterns in cells overexpressing cofilin as compared with normal cells. However, upon treatment with DMSO, cytoplasmic actin filaments were disrupted and intranuclear rod structures containing cofilin and actin were apparently larger and thicker in cells overexpressing cofilin than in normal cells. Heat shock also stimulated disruption of microfilaments and formation of both intranuclear and prominent cytoplasmic cofilin-actin rods in cofilin-transfected cells, suggesting that DMSO-treatment or heat shock triggers cofilin-actin interaction. We further found that a myosin ATPase inhibitor (BDM) induced a reduction in cytoplasmic staining with phalloidin in cofilin-transfected cells. The results suggest that myosin activity might be involved in the regulation of cofilin-actin interactions in vivo.

• Abe H, Obinata T, Minamide LS, Bamburg JR
• Xenopus laevis actin-depolymerizing factor/cofilin: a phosphorylation-regulated protein essential for development.
• J Cell Biol. 1996; 132: 871-85
• Display abstract

• Two cDNAs, isolated from a Xenopus laevis embryonic library, encode proteins of 168 amino acids, both of which are 77% identical to chick cofilin and 66% identical to chick actin-depolymerizing factor (ADF), two structurally and functionally related proteins. These Xenopus ADF/cofilins (XADs) differ from each other in 12 residues spread throughout the sequence but do not differ in charge. Purified GST-fusion proteins have pH-dependent actin-depolymerizing and F-actin-binding activities similar to chick ADF and cofilin. Similarities in the developmental and tissue specific expression, embryonic localization, and in the cDNA sequence of the noncoding regions, suggest that the two XACs arise from allelic variants of the pseudotetraploid X. laevis. Immunofluorescence localization of XAC in oocyte sections with an XAC-specific monoclonal antibody shows it to be diffuse in the cortical cytoplasm. After fertilization, increased immunostaining is observed in two regions: along the membrane, particularly that of the vegetal hemisphere, and at the interface between the cortical and animal hemisphere cytoplasm. The cleavage furrow and the mid-body structure are stained at the end of first cleavage. Neuroectoderm derived tissues, notochord, somites, and epidermis stain heavily either continuously or transiently from stages 18-34. A phosphorylated form of XAC (pXAC) was identified by 2D Western blotting, and it is the only species found in oocytes. Dephosphorylation of >60% of the pXAC occurs within 30 min after fertilization. Injection of one blastomere at the 2 cell stage, either with constitutively active XAC or with an XAC inhibitory antibody, blocked cleavage of only the injected blastomere in a concentration-dependent manner without inhibiting nuclear division. The cleavage furrow of eggs injected with constitutively active XAC completely regressed. Blastomeres injected with neutralized antibody developed normally. These results suggest that XAC is necessary for cytokinesis and that its activity must be properly regulated for cleavage to occur.

• Moriyama K, Iida K, Yahara I
• Phosphorylation of Ser-3 of cofilin regulates its essential function on actin.
• Genes Cells. 1996; 1: 73-86
• Display abstract

• BACKGROUND: Cofilin is a low-molecular weight actin-modulating protein, and is structurally and functionally conserved in eucaryotes from yeast to mammals. The functions of cofilin appear to be regulated by phosphorylation and dephosphorylation. RESULTS: A proteolytic study of phosphorylated porcine cofilin and expression of a mutated cofilin in cultured cells revealed that Ser-3 is the unique phosphorylation site. Phosphorylated cofilin was found not to bind to either F- or G-actin while unphosphorylated cofilin binds to both. S3D-cofilin, in which Ser-3 was replaced with Asp, did not bind in vitro to actin while S3A-cofilin did. The transient over-expression of wild-type or S3A-cofilin in cultured cells caused disruption of preexisting actin structures and induced cytoplasmic actin bundles. Heat shock-induced nuclear or NaCl buffer-induced cytoplasmic actin/cofilin rods contained the expressed cofilin. In contrast, the over-expression of S3D-cofilin did not alter the actin structures. Induced actin rods did not contain S3D-cofilin. S3D-porcine cofilin did not complement the lethality associated with delta cof1 mutations in Saccharomyces cerevisiae while wild-type and S3A-cofilin did. Furthermore, we found that S2A/S4D- and S2D/S4D-yeast cofilin mutants were not viable. CONCLUSION: We conclude that the function of cofilin is negatively regulated in vivo by phosphorylation of Ser-3 and that cells require the function of unphosphorylated cofilin for viability.

• Chik JK, Lindberg U, Schutt CE
• The structure of an open state of beta-actin at 2.65 A resolution.
• J Mol Biol. 1996; 263: 607-23
• Display abstract

• The structure of an "open state" of crystalline profilin:beta-actin has been solved to 2.65 A by X-ray crystallography. The open-state crystals, in 1.8 M potassium phosphate, have an expanded unit cell dimension in the c direction of 185.7 A compared with 171.9 A in the previously solved ammonium sulphate-stabilized "tight-state" structure. The unit cell change between the open and the tight states is accompanied by large subdomain movements in actin. Furthermore, the nucleotide in the open state is significantly more exposed to solvent, and local conformational changes in the hydrophobic pocket surrounding cysteine 374 occur during the transition to the tight state. Significant changes were observed at the N terminus and in the DNase-I binding loop. Neither the structure of profilin nor its contact with beta-actin are affected by the changes in the unit cell. Applying osmotic pressure to profilin:beta-actin crystals brings about a collapse of the unit cell comparable with that seen in the open to tight-state transition, enabling an estimate of the work required to cause this transformation of beta-actin in the crystals. The slight difference in energy between the open and collapsed states explains the extreme sensitivity of profilin:beta-actin crystals to changes in chemical and thermal environment.

• Furukawa R, Fechheimer M
• Role of the Dictyostelium 30 kDa protein in actin bundle formation.
• Biochemistry. 1996; 35: 7224-32
• Display abstract

• We have studied the formation of bundles in mixtures of actin with the Dictyostelium 30 kDa actin-bundling protein as a function of 30 kDa protein concentration, actin concentration, and filament length. The presence of the 30 kDa protein promotes formation of filament bundles at actin concentrations and filament lengths that are not spontaneously aligned into liquid crystalline domains in the absence of the 30 kDa protein. Bundle formation in the presence of the 30 kDa protein was observed over a broad range of actin filament lengths and concentrations. Bundling was filament length dependent, and short filaments were more efficiently bundled. Bundles formed at actin concentrations as low as 2 microM. The volume fraction of the bundled portion and concentrations of actin and the 30 kDa protein in the bundled portion were measured using a sedimentation assay. Bundles have concentrations of actin and 30 kDa protein that are 10-20 and 5-20 times, respectively, greater than that of the bulk solution. Computer modeling reveals that bundling of actin by a bundling protein increases both the mean length and the polydispersity of the length distribution, factors which lower the actin concentration required for spontaneous alignment within the bundle. We propose that entropy-driven spontaneous ordering may contribute to bundle formation in two ways. Bundling of actin creates longer aggregates with a more polydisperse length distribution in which actin aligns spontaneously within the bundle at very low concentrations. In addition, bundling creates locally high concentrations of actin within these aggregates that will spontaneously align, providing an additional driving force for bundle ordering.

• Nagaoka R, Minami N, Hayakawa K, Abe H, Obinata T
• Quantitative analysis of low molecular weight G-actin-binding proteins, cofilin, ADF and profilin, expressed in developing and degenerating chicken skeletal muscles.
• J Muscle Res Cell Motil. 1996; 17: 463-73
• Display abstract

• A large amount of G-actin is pooled in the cytoplasm of young embryonic skeletal muscle and, although its concentration is reduced as muscle develops, the total amount of actin in muscle cells increases remarkably. Three G-actin-binding proteins, cofilin, ADF and profilin, are known to be involved in creating the G-actin pool in the embryonic muscle. To better understand how they are responsible for the regulation of assembly and disassembly of actin in developing and degenerating muscles, we measured the amounts of the three G-actin-binding proteins by means of quantitative immunoblotting and compared them with that of G-actin. The sum of the amounts of the three actin-binding proteins was insufficient at early developmental stages but sufficient at later stages to account for the pool of G-actin in young muscle cells. It decreased in parallel with the decrease in the G-actin pool as muscle developed. Expression of thymosin beta 4, which is known to be extremely important for G-actin-sequestering in a variety of non-muscle cells, was detected at a considerable level in young embryonic but not in adult skeletal muscles according to Northern and Western blotting. In degenerating denervated and dystrophic muscles, cofilin and profilin, but not ADF, were significantly increased in amount. From these results, we conclude that the G-actin pool in young embryonic skeletal muscle is mainly due to cofilin, ADF, profilin and thymosin beta 4, but thymosin beta 4 as well as ADF becomes less important as muscle develops. Cofilin and profilin may also be involved in the redistribution of actin during myofibrillogenesis and in the process of actin disassembly in degenerating muscles.

• Marston SB, Fraser ID, Bing W, Roper G
• A simple method for automatic tracking of actin filaments in the motility assay.
• J Muscle Res Cell Motil. 1996; 17: 497-506
• Display abstract

• A great deal of quantitative information about the actomyosin interaction can be obtained from the basic Kron and Spudich in vitro motility assay provided that care is taken to obtain consistency between experiments and that the data is examined comprehensively and not selectively. From observations of filament movement under a wide variety of conditions we have formulated the hypothesis that a large number of filaments moving over a short time period is indistinguishable from fewer filaments moving over a longer sequence of frames. This has been used to devise a simple automation of filament detection procedures. A sequence of images is digitized through a frame-grabber. If successive pairs of frames are compared the program will search for and detect the new position of every filament and show its vector on screen. Velocity is calculated and shown as a frequency histogram. The program regularly detects over 100 filaments moving in each pair of frames; usually a sequence of up to 15 pairs of frames are studied yielding 500-1000 vectors in total. The algorithm cannot deal with filaments that meet, cross or divide, however, when filaments are moving less than 2 microns between frames this is only a small proportion of the whole. The program outputs fraction of filaments motile, mean velocity with standard deviation and density of filaments (filaments microns-2). A cumulative frequency histogram gives an immediate visual indication of the performance of the population of filaments. Direct comparisons show that the data produced by automatic tracking is indistinguishable from manual tracking apart from the small apparent velocity of non-mobile filaments. The detection process takes about 5 min and requires little skill or judgement. This can lead to great increases in the rate of data analysis in motility work.

• Moon A, Drubin DG
• The ADF/cofilin proteins: stimulus-responsive modulators of actin dynamics.
• Mol Biol Cell. 1995; 6: 1423-31

• Southwick FS
• Gain-of-function mutations conferring actin-severing activity to human macrophage cap G.
• J Biol Chem. 1995; 270: 45-8
• Display abstract

• Nonmuscle cell motility requires marked changes in the consistency and shape of the peripheral cytoplasm. These changes are regulated by a gel-sol transformation of the actin filament network, and actin filament-severing proteins are responsible for network solation. Macrophage Cap G, unlike all other proteins in the gelsolin family, caps but does not sever actin filaments. Two amino acid stretches in Cap G diverge markedly from the severing proteins: 84LNTLLGE and 124AFHKTS. Discrete mutations in Cap G have been generated to determine if these amino acid sequences are critical for actin filament severing. Conversion of 84LNTLLGE to the gelsolin actin-binding helix sequence (84LDDYLGG) renders Cap G capable of severing actin filaments (half-maximal severing, 1-2 microM). Adding a second set of mutations, converting 124AFHKTS to 124GFKHV, enhances severing by 10-fold (half-maximal severing, 0.1-0.2 microM). These experiments support a critical role for these two regions in actin filament severing and showcase the power of gain-of-function mutations in clarifying structure-function relationships.

• Dyer CE, Shuttleworth CA, Kielty CM
• Conformation and function of fibrillin 8-cysteine motifs.
• Biochem Soc Trans. 1995; 23: 506-506

• Nagaoka R, Abe H, Kusano K, Obinata T
• Concentration of cofilin, a small actin-binding protein, at the cleavage furrow during cytokinesis.
• Cell Motil Cytoskeleton. 1995; 30: 1-7
• Display abstract

• Cofilin is a small actin-binding protein which regulates actin polymerization in a pH-dependent manner. Immunofluorescence microscopy with a monoclonal antibody for cofilin revealed that this protein is temporarily concentrated at the contractile ring during cytokinesis. Cofilin appeared to accumulate rapidly at the contractile ring during late stages of furrowing, and was finally enriched at the midbody. The concentration of cofilin at the contractile ring was observed in several kinds of cultured cells. Furthermore, cofilin introduced into living cells by a microinjection method was also concentrated at the contractile ring. These results suggest that cofilin is involved in actin reorganization during cytokinesis.

• Gunsalus KC, Bonaccorsi S, Williams E, Verni F, Gatti M, Goldberg ML
• Mutations in twinstar, a Drosophila gene encoding a cofilin/ADF homologue, result in defects in centrosome migration and cytokinesis.
• J Cell Biol. 1995; 131: 1243-59
• Display abstract

• We describe the phenotypic and molecular characterization of twinstar (tsr), an essential gene in Drosophila melanogaster. Two P-element induced alleles of tsr (tsr1 and tsr2) result in late larval or pupal lethality. Cytological examination of actively dividing tissues in these mutants reveals defects in cytokinesis in both mitotic (larval neuroblast) and meiotic (larval testis) cells. In addition, mutant spermatocytes show defects in aster migration and separation during prophase/prometaphase of both meiotic divisions. We have cloned the gene affected by these mutations and shown that it codes for a 17-kD protein in the cofilin/ADF family of small actin severing proteins. A cDNA for this gene has previously been described by Edwards et al. (1994). Northern analysis shows that the tsr gene is expressed throughout development, and that the tsr1 and tsr2 alleles are hypomorphs that accumulate decreased levels of tsr mRNA. These findings prompted us to examine actin behavior during male meiosis to visualize the effects of decreased twinstar protein activity on actin dynamics in vivo. Strikingly, both mutants exhibit abnormal accumulations of F-actin. Large actin aggregates are seen in association with centrosomes in mature primary spermatocytes. Later, during ana/telophase of both meiotic divisions, aberrantly large and misshaped structures appear at the site of contractile ring formation and fail to disassemble at the end of telophase, in contrast with wild-type. We discuss these results in terms of possible roles of the actin-based cytoskeleton in centrosome movement and in cytokinesis.

• Metcalf P, Fusek M
• Cathepsin D crystal structures and lysosomal sorting.
• Adv Exp Med Biol. 1995; 362: 193-200

• Sun HQ, Kwiatkowska K, Yin HL
• Actin monomer binding proteins.
• Curr Opin Cell Biol. 1995; 7: 102-10
• Display abstract

• Small actin monomer binding proteins are essential components of the actin polymerization machinery. Originally thought of as passive buffers that prevent polymerization of actin monomers, recent discoveries elucidate how some actin monomer binding proteins can promote as well as inhibit polymerization, and how they cooperate to regulate actin assembly.

• DiNubile MJ, Cassimeris L, Joyce M, Zigmond SH
• Actin filament barbed-end capping activity in neutrophil lysates: the role of capping protein-beta 2.
• Mol Biol Cell. 1995; 6: 1659-71
• Display abstract

• A barbed-end capping activity was found in high speed supernates of neutrophils lysed in submicromolar calcium. In dilute supernate (> or = 100-fold dilution of cytoplasm), this activity accounted for most of the inhibition of barbed-end elongation of pyrenyl-G-actin from spectrin-F-actin seeds. Pointed-end elongation from gelsolin-capped F-actin seeds was not inhibited at comparable concentrations of supernate, thus excluding actin monomer sequestration as a cause of the observed inhibition. Most of the capping activity was due to capping protein-beta 2 (a homologue of cap Z). Thus, while immunoadsorption of > or = 95% of the gelsolin in the supernate did not decrease capping activity, immunoadsorption of capping protein-beta 2 reduced capping activity proportionally to the amount of capping protein-beta 2 adsorbed. Depletion of > 90% of capping protein-beta 2 from the supernate removed 90% of its capping activity. The functional properties of the capping activity were defined. The dissociation constant for binding to barbed ends (determined by steady state and kinetic analyses) was approximately 1-2 nM; the on-rate of capping was between 7 x 10(5) and 5 x 10(6) M-1 s-1; and the off-rate was approximately 2 x 10(-3) s-1. The concentration of capper free in the intact cell (determined by adsorption of supernate with spectrin-actin seeds) was estimated to be approximately 1-2 microM. Thus, there appeared to be enough high affinity capper to cap all the barbed ends in vivo. Nevertheless, immediately after lysis with detergent, neutrophils contained sites that nucleate barbed-end elongation of pyrenyl-G-actin. These barbed ends subsequently become capped with a time course and concentration dependence similar to that of spectrin-F-actin seeds in high speed supernates. These observations suggest that, despite the excess of high affinity capper, some ends either are not capped in vivo or are transiently uncapped upon lysis and dilution.

• Schafer DA, Cooper JA
• Control of actin assembly at filament ends.
• Annu Rev Cell Dev Biol. 1995; 11: 497-518
• Display abstract

• Actin filament polymerization involves exchange of subunits of filament ends, which can be controlled in vitro and in vivo by other proteins that bind actin filaments and inhibit subunit addition or loss at the ends. Proteins that bind to the barbed end, including capping protein, the gelsolin super-family, tensin, and profilin are discussed, as are proteins that bind to the pointed end, including tropomodulin and spectrin/band 4.1. Some proteins that bind along the sides of filaments and influence assembly at ends are also discussed. Functional roles in vivo are emphasized.

• Aizawa H, Sutoh K, Tsubuki S, Kawashima S, Ishii A, Yahara I
• Identification, characterization, and intracellular distribution of cofilin in Dictyostelium discoideum.
• J Biol Chem. 1995; 270: 10923-32
• Display abstract

• We identified and purified an actin monomer-binding protein of apparent molecular weight of 15,000 from Dictyostelium discoideum. The 15-kDa protein depolymerized actin filaments in a pH-dependent manner. The protein also had an activity to decrease apparent viscosity of actin solutions in a dose-dependent manner. This activity was inhibited by phosphatidyl inositides. Molecular cloning of genes encoding this protein revealed that the protein is 42% identical in its primary sequence to yeast cofilin. We concluded that the 15-kDa protein is cofilin of this organism. D. discoideum cells contain two cofilin genes (DCOF1 and DCOF2) whose nucleotide sequences were entirely identical in their exsons while the promoter and intron regions were different. Promoter assay experiments revealed that DCOF1 is expressed both in vegetative and differentiating cells and that DCOF2 is not expressed under any conditions examined. Gene disruption experiments suggested that DCOF1 might be essential for the proliferation of D. discoideum cells whereas the disruption of DCOF2 was proven not to alter any phenotypes. Indirect immunofluorescence microscopic observations showed that cofilin is distributed diffusely throughout cytoplasm in vegetative cells. In flattened cells under starvation stress, cofilin localized at dramatically reorganizing actin-cytoskeletons in ruffling membranes of the leading edge, but not at rigid actin meshwork in focal adhesion plaques. These results suggest that cofilin may be involved in dynamic reorganization of membranous actin cytoskeletons.

• Agnew BJ, Minamide LS, Bamburg JR
• Reactivation of phosphorylated actin depolymerizing factor and identification of the regulatory site.
• J Biol Chem. 1995; 270: 17582-7
• Display abstract

• Actin depolymerizing factor (ADF) occurs naturally in two forms, one of which contains a phosphorylated Ser and does not bind G-actin or depolymerize F-actin. Removal of this phosphate in vitro by alkaline phosphatase restores full F-actin depolymerizing activity. To identify the phosphorylation site, [32P]pADF was purified and digested with endoproteinase Lys-C. The digest contained only one 32P-labeled peptide. Further digestion with endoproteinase Asp-N and mass spectrometric analysis showed that this peptide came from the N terminus of ADF. Alkaline phosphatase treatment of one Asp-N peptide (mass 753) converted it to a peptide of mass 673, demonstrating that this peptide contains the phosphate group. Tandem mass spectrometric sequence analysis of this peptide identified the phosphorylated Ser as the encoded Ser3 (Ser2 in the processed protein). HeLa cells, transfected with either chick wild-type ADF cDNA or a cDNA mutated to code for Ala in place of Ser24 or Thr25, express and phosphorylate the exogenous ADF. Cells also expressed high levels of mutant ADF when Ser3 was deleted or converted to either Ala or Glu. However, none of these mutants was phosphorylated, confirming that Ser3 in the encoded ADF is the single in vivo regulatory site.

• Lipke PN, Chen MH, de Nobel H, Kurjan J, Kahn PC
• Homology modeling of an immunoglobulin-like domain in the Saccharomyces cerevisiae adhesion protein alpha-agglutinin.
• Protein Sci. 1995; 4: 2168-78
• Display abstract

• The Saccharomyces cerevisiae adhesion protein alpha-agglutinin is expressed by cells of alpha mating type. On the basis of sequence similarities, alpha-agglutinin has been proposed to contain variable-type immunoglobulin-like (IgV) domains. The low level of sequence similarity to IgV domains of known structure made homology modeling using standard sequence-based alignment algorithms impossible. We have therefore developed a secondary structure-based method that allowed homology modeling of alpha-aggulutinin domain III, the domain most similar to IgV domains. The model was assessed and where necessary refined to accommodate information obtained by biochemical and molecular genetic approaches, including the positions of a disulfide bond, glycosylation sites, and proteolytic sites. The model successfully predicted surface exposure of glycosylation and proteolytic sites, as well as identifying residues essential for binding activity. One side of the domain was predicted to be covered by carbohydrate residues. Surface accessibility and volume packing analyses showed that the regions of the model that have greatest sequence dissimilarity from the IgV consensus sequence are poorly structured in the biophysical sense. Nonetheless, the utility of the model suggests that these alignment and testing techniques should be of general use for building and testing of models of proteins that share limited sequence similarity with known structures.

• Pasmans SG, Aalbers M, van Ree R, Knol EF, Jansen HM, Aalberse RC
• IgE binding to a human profilin-containing fraction caused by contamination with yeast-derived material.
• J Allergy Clin Immunol. 1995; 96: 696-7

• Kapp OH, Moens L, Vanfleteren J, Trotman CN, Suzuki T, Vinogradov SN
• Alignment of 700 globin sequences: extent of amino acid substitution and its correlation with variation in volume.
• Protein Sci. 1995; 4: 2179-90
• Display abstract

• Seven-hundred globin sequences, including 146 nonvertebrate sequences, were aligned on the basis of conservation of secondary structure and the avoidance of gap penalties. Of the 182 positions needed to accommodate all the globin sequences, only 84 are common to all, including the absolutely conserved PheCD1 and HisF8. The mean number of amino acid substitutions per position ranges from 8 to 13 for all globins and 5 to 9 for internal positions. Although the total sequence volumes have a variation approximately 2-3%, the variation in volume per position ranges from approximately 13% for the internal to approximately 21% for the surface positions. Plausible correlations exist between amino acid substitution and the variation in volume per position for the 84 common and the internal but not the surface positions. The amino acid substitution matrix derived from the 84 common positions was used to evaluate sequence similarity within the globins and between the globins and phycocyanins C and colicins A, via calculation of pairwise similarity scores. The scores for globin-globin comparisons over the 84 common positions overlap the globin-phycocyanin and globin-colicin scores, with the former being intermediate. For the subset of internal positions, overlap is minimal between the three groups of scores. These results imply a continuum of amino acid sequences able to assume the common three-on-three alpha-helical structure and suggest that the determinants of the latter include sites other than those inaccessible to solvent.

• Dhanaraj RR et al.
• Protein engineering of surface loops: preliminary X-ray analysis of the CHY155-165RHI mutant.
• Adv Exp Med Biol. 1995; 362: 95-9

• Hug C et al.
• Capping protein levels influence actin assembly and cell motility in dictyostelium.
• Cell. 1995; 81: 591-600
• Display abstract

• Actin assembly is important for cell motility, but the mechanism of assembly and how it relates to motility in vivo is largely unknown. In vitro, actin assembly can be controlled by proteins, such as capping protein, that bind filament ends. To investigate the function of actin assembly in vivo, we altered the levels of capping protein in Dictyostelium cells and found changes in resting and chemoattractant-induced actin assembly that were consistent with the in vitro properties of capping protein in capping but not nucleation. Significantly, overexpressers moved faster and underexpressers moved slower than control cells. Mutants also exhibited changes in cytoskeleton architecture. These results provide insights into in vivo actin assembly and the role of the actin cytoskeleton in motility.

• Edwards KA, Montague RA, Shepard S, Edgar BA, Erikson RL, Kiehart DP
• Identification of Drosophila cytoskeletal proteins by induction of abnormal cell shape in fission yeast.
• Proc Natl Acad Sci U S A. 1994; 91: 4589-93
• Display abstract

• To clone metazoan genes encoding regulators of cell shape, we have developed a functional assay for proteins that affect the morphology of a simple organism, the fission yeast Schizosaccharomyces pombe. A Drosophila melanogaster cDNA library was constructed in an inducible expression vector and transformed into S. pombe. When expression of the Drosophila sequences was induced, aberrant cell shapes were found in 0.2% of the transformed colonies. Four severe phenotypes representing defects in cytokinesis and/or cell shape maintenance were examined further. Each displayed drastic and specific reorganizations of the actin cytoskeleton. Three of the cDNAs responsible for these defects appear to encode cytoskeletal components: the actin binding proteins profilin and cofilin/actin depolymerizing factor and a membrane-cytoskeleton linker of the ezrin/merlin family. These results demonstrate that a yeast phenotypic screen efficiently identifies conserved genes from more complex organisms and sheds light on their potential in vivo functions.

• Cedergren-Zeppezauer ES et al.
• Crystallization and structure determination of bovine profilin at 2.0 A resolution.
• J Mol Biol. 1994; 240: 459-75
• Display abstract

• Profilin regulates the behavior of the eukaryotic microfilament system through its interaction with non-filamentous actin. It also binds several ligands, including poly(L-proline) and the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Bovine profilin crystals (space group C2; a = 69.15 A, b = 34.59 A, c = 52.49 A; alpha = gamma = 90 degrees, beta = 92.56 degrees) were grown from a mixture of poly(ethylene glycol) 400 and ammonium sulfate. X-ray diffraction data were collected on an imaging plate scanner at the DORIS storage ring (DESY, Hamburg), and were phased by molecular replacement, using a search model derived from the 2.55 A structure of profilin complexed to beta-actin. The refined model of bovine profilin has a crystallographic R-factor of 16.5% in the resolution range 6.0 to 2.0 A and includes 128 water molecules, several of which form hydrogen bonds to stabilize unconventional turns. The structure of free bovine profilin is similar to that of bovine profilin complexed to beta-actin, and C alpha atoms from the two structures superimpose with an r.m.s. deviation of 1.25 A. This value is reduced to 0.51 A by omitting Ala1 and the N-terminal acetyl group, which lie at a profilin-actin interface in crystals of the complex. These residues display a strained conformation in crystalline profilin-actin but may allow the formation of a hydrogen bond between the N-acetyl carbonyl group of profilin and the phenol hydroxyl group of Tyr188 in actin. Several other actin-binding residues of profilin show different side-chain rotomer conformations in the two structures. The polypeptide fold of bovine profilin is generally similar to those observed by NMR for profilin from other sources, although the N terminus of Acanthamoeba profilin isoform I lies in a distorted helix and the C-terminal helix is less tilted with respect to the strands in the central beta-pleated sheet than is observed in bovine profilin. The majority of the aromatic residues in profilin are exposed to solvent and lie in either of two hydrophobic patches, neither of which takes part in an interface with actin. One of these patches is required for binding poly(L-proline) and contains an aromatic cluster comprising the highly conserved residues Trp3, Tyr6, Trp31 and Tyr139. In forming this cluster, Trp31 adopts a sterically strained rotamer conformation.(ABSTRACT TRUNCATED AT 400 WORDS)

• Fedorov AA, Magnus KA, Graupe MH, Lattman EE, Pollard TD, Almo SC
• X-ray structures of isoforms of the actin-binding protein profilin that differ in their affinity for phosphatidylinositol phosphates.
• Proc Natl Acad Sci U S A. 1994; 91: 8636-40
• Display abstract

• We determined the structures of Acanthamoeba profilin I and profilin II by x-ray crystallography at resolutions of 2.0 and 2.8 A, respectively. The polypeptide folds and the actin-binding surfaces of the amoeba profilins are very similar to those of bovine and human profilins. The electrostatic potential surfaces of the two Acanthamoeba isoforms differ. Two areas of high positive potential on the surface of profilin II are candidate binding sites for phosphatidylinositol phosphates. The proximity of these sites to the actin binding site provides an explanation for the competition between actin and lipids for binding profilin.

• Ono S, Minami N, Abe H, Obinata T
• Characterization of a novel cofilin isoform that is predominantly expressed in mammalian skeletal muscle.
• J Biol Chem. 1994; 269: 15280-6
• Display abstract

• Cofilin is an actin-modulating protein of 20 kDa, which is widely distributed throughout muscle and non-muscle cells. By means of immunoblotting combined with two-dimensional gel electrophoresis, we found that two cofilin variants, muscle type (M-type) and non-muscle type (NM-type), exist in mammals, while a single isoform exists in chickens. During in vitro myogenesis of mouse C2 cells, expression of the M-type cofilin was upregulated. To better understand the nature of the M-type cofilin, we cloned cDNAs encoding M-type cofilin from the cDNA library of C2 myotubes and determined the entire sequence. The deduced peptide sequence contained a nuclear localization signal and a putative actin-binding sequence as reported in NM-type cofilin. The sequence showed 81% identity in the amino acid residues with the mouse NM-type cofilin sequence and, interestingly, higher homology (96% identity) with that of chicken cofilin. The mRNA encoding M-type cofilin, though it contains two variants that differ in the size of their 3'-non-coding sequences, was detected predominantly in heart, skeletal muscle, C2 myotubes, and testis by Northern blotting, while the mRNA for NM-type cofilin was seen in a variety of non-muscle tissues. The presence of the muscle type isoform of cofilin strongly suggests that cofilin is deeply involved in the regulation of actin function not only in non-muscle cells but also in muscle cells.

• Finkel T, Theriot JA, Dise KR, Tomaselli GF, Goldschmidt-Clermont PJ
• Dynamic actin structures stabilized by profilin.
• Proc Natl Acad Sci U S A. 1994; 91: 1510-4
• Display abstract

• We describe the production and analysis of clonal cell lines in which we have overexpressed human profilin, a small ubiquitous actin monomer binding protein, to assess the role of profilin on actin function in vivo. The concentration of filamentous actin is increased in cells with higher profilin levels, and actin filament half-life measured in these cells is directly proportional to the steady-state profilin concentration. The distribution of actin filaments is altered by profilin overexpression. While parallel actin bundles crossing the cells are virtually absent in cells overexpressing profilin, the submembranous actin network of these cells is denser than in control cells. These results suggest that in vivo profilin regulates the stability, and thereby distribution, of specific dynamic actin structures.

• Maciver SK, Weeds AG
• Actophorin preferentially binds monomeric ADP-actin over ATP-bound actin: consequences for cell locomotion.
• FEBS Lett. 1994; 347: 251-6
• Display abstract

• Actophorin from Acanthamoeba castellanii severs actin filaments and sequesters actin monomers. Here we report that actophorin binds ADP-bound monomers with higher affinity than ATP-bound monomers. Actophorin is therefore much less efficient at severing actin filaments in the presence of ADP compared to ATP, particularly taking account of the higher critical concentration in ADP. Monomer binding is also reduced in the presence of 25 mM inorganic phosphate (which is assumed to form ADP.Pi-actin). These findings are discussed in the light of observations on the nucleotide specificity of other monomer binding proteins and related to the role of actin in lamellar protrusion and cell locomotion.

• Ito J, Masuda M, Tanaka R
• Sialosylcholesterol effects on reconstitution of microfilament and glia filament.
• J Neurochem. 1994; 62: 235-9
• Display abstract

• The effects of alpha-sialosylcholesterol (alpha-SC) on formation of either microfilament or glia filament of rat astrocytes were investigated using a reconstitution system. Polymerization of the depolymerized microfilament preparation that had been extracted from a crude cytoskeletal fraction of rat astrocytes, in the presence of 100 mM KCl and 10 mM MgCl2, was suppressed in a dose-dependent manner by alpha-SC. alpha-SC inhibited polymerization of G-actin in a similar manner. The intensity of alpha-SC inhibition of G-actin polymerization was as great as that of microfilament polymerization, suggesting that the inhibition of microfilament polymerization by alpha-SC was due to the direct action of alpha-SC on actin, the main component of microfilament. alpha-SC depolymerized partly the polymerized microfilament preparation, which resembled F-actin (microfilament-like filaments). alpha-SC suppressed, in a dose-dependent manner, polymerization of a glia filament preparation that had been extracted from astrocyte cytoskeletons in the presence of phalloidin. An increase in the amount of added alpha-SC (up to 15 microM) decreased the amount of the larger glia filament-like filaments, which were 10 nm thick and centrifuged down at 16,000 g for 30 min, and increased that of smaller ones precipitated only after centrifugation at 100,000 g for 1 h. The lower the concentration of the depolymerized glia filament extract, the greater was the inhibition by alpha-SC of the polymerization. alpha-SC repressed polymerization of vimentin, the dominant component of glia filament. Vimentin polymerization was more strongly inhibited by alpha-SC than polymerization of glia filament was.(ABSTRACT TRUNCATED AT 250 WORDS)

• Mulholland J, Preuss D, Moon A, Wong A, Drubin D, Botstein D
• Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane.
• J Cell Biol. 1994; 125: 381-91
• Display abstract

• We characterized the yeast actin cytoskeleton at the ultrastructural level using immunoelectron microscopy. Anti-actin antibodies primarily labeled dense, patchlike cortical structures and cytoplasmic cables. This localization recapitulates results obtained with immunofluorescence light microscopy, but at much higher resolution. Immuno-EM double-labeling experiments were conducted with antibodies to actin together with antibodies to the actin binding proteins Abp1p and cofilin. As expected from immunofluorescence experiments, Abp1p, cofilin, and actin colocalized in immuno-EM to the dense patchlike structures but not to the cables. In this way, we can unambiguously identify the patches as the cortical actin cytoskeleton. The cortical actin patches were observed to be associated with the cell surface via an invagination of plasma membrane. This novel cortical cytoskeleton-plasma membrane interface appears to consist of a fingerlike invagination of plasma membrane around which actin filaments and actin binding proteins are organized. We propose a possible role for this unique cortical structure in wall growth and osmotic regulation.

• Vinh DB, Drubin DG
• A yeast TCP-1-like protein is required for actin function in vivo.
• Proc Natl Acad Sci U S A. 1994; 91: 9116-20
• Display abstract

• We previously identified the ANC2 gene in a screen for mutations that enhance the defects caused by yeast actin mutations. Here we report that ANC2 is an essential gene that encodes a member of the TCP-1 family. TCP-1-related proteins are subunits of cytosolic heteromeric protein complexes referred to as chaperonins. These complexes can bind to newly synthesized actin and tubulin in vitro and can convert these proteins into an assembly-competent state. We show that anc2-1 mutants contain abnormal and disorganized actin structures, are defective in cellular morphogenesis, and are hypersensitive to the microtubule inhibitor benomyl. Furthermore, overexpression of wild-type Anc2p ameliorates defects in actin organization and cell growth caused by actin overproduction. Mutations in BIN2 and BIN3, two other genes that encode TCP-1-like proteins, also enhance the phenotypes of actin mutants. Taken together, these findings demonstrate that TCP-1-like proteins are required for actin and tubulin function in vivo.

• Staiger CJ, Yuan M, Valenta R, Shaw PJ, Warn RM, Lloyd CW
• Microinjected profilin affects cytoplasmic streaming in plant cells by rapidly depolymerizing actin microfilaments.
• Curr Biol. 1994; 4: 215-9
• Display abstract

• BACKGROUND: Cytoplasmic streaming is a conspicuous feature of plant cell behaviour, in which organelles and vesicles shuttle along cytoplasmic strands that contain actin filaments. The mechanisms that regulate streaming and the formation of actin filament networks are largely unknown, but in all likelihood involve actin-binding proteins. The monomeric actin-binding protein, profilin, is a key regulator of actin-filament dynamics in animal cells and it has recently been identified in plants as a pollen allergen. We set out to determine whether plant profilin can act as a monomeric actin-binding protein and influence actin dynamics in plant cells in vivo. RESULTS: Recombinant birch-pollen profilin was purified by polyproline affinity chromatography and microinjected into Tradescantia blossfeldiana stamen hair cells. After profilin injection, a rapid and irreversible change in cellular organization and streaming was observed: within 1-3 minutes the transvacuolar cytoplasmic strands became thinner and snapped, and cytoplasmic streaming ceased. Fluorescein-labelled-phalloidin staining confirmed that this was due to depolymerization of actin filaments. To confirm that the effects observed were due to sequestration of monomeric actin, another monomeric actin-binding protein, DNase I, was injected and found to produce comparable results. CONCLUSIONS: Profilin can act as a potent regulator of actin organization in living plant cells. Its rapid effect on the integrity of cytoplasmic strands and cytoplasmic streaming supports a model in which organelle movements depend upon microfilaments that exist in dynamic equilibrium with the pool of monomeric actin.

• Angelastro JM, Purich DL
• Phosphorylation states of actin filament adenine nucleotides in detergent-extracted neuronal cytoskeletal fractions.
• Biochem Biophys Res Commun. 1994; 201: 1490-4
• Display abstract

• High performance liquid chromatography of nucleotides from Triton X-100 cytoskeletal extracts has permitted analysis of the ATP and ADP content of actin filaments isolated intact from PC12 pheochromocytoma cells. We observed that the adenine nucleotide content matched the actin content of these cytoskeletal extracts, a finding consistent with the unit stoichiometry of nucleotide binding. Efficient assembly-linked ATP hydrolysis occurs in vivo, and based on a boundary hydrolysis model for nucleotide-promoted assembly, the observed ADP/ATP ratio indicates that the average microfilament in nonmuscle cells has 2-4 ATP-actin molecules at its growing end. Studies with NB41A3 neuroblastoma cells indicate that the ATP content of assembled actin filaments is about 3-4 times lower.

• Moon AL, Janmey PA, Louie KA, Drubin DG
• Cofilin is an essential component of the yeast cortical cytoskeleton.
• J Cell Biol. 1993; 120: 421-35
• Display abstract

• We have biochemically identified the Saccharomyces cerevisiae homologue of the mammalian actin binding protein cofilin. Cofilin and related proteins isolated from diverse organisms are low molecular weight proteins (15-20 kD) that possess several activities in vitro. All bind to monomeric actin and sever filaments, and some can stably associate with filaments. In this study, we demonstrate using viscosity, sedimentation, and actin assembly rate assays that yeast cofilin (16 kD) possesses all of these properties. Cloning and sequencing of the S. cerevisiae cofilin gene (COF1) revealed that yeast cofilin is 41% identical in amino acid sequence to mammalian cofilin and, surprisingly, has homology to a protein outside the family of cofilin-like proteins. The NH2-terminal 16kD of Abp1p, a 65-kD yeast protein identified by its ability to bind to actin filaments, is 23% identical to yeast cofilin. Immunofluorescence experiments showed that, like Abp1p, cofilin is associated with the membrane actin cytoskeleton. A complete disruption of the COF1 gene was created in diploid cells. Sporulation and tetrad analysis revealed that yeast cofilin has an essential function in vivo. Although Abp1p shares sequence similarity with cofilin and has the same distribution as cofilin in the cell, multiple copies of the ABP1 gene cannot compensate for the loss of cofilin. Thus, cofilin and Abp1p are structurally related but functionally distinct components of the yeast membrane cytoskeleton.

• Hayden SM, Miller PS, Brauweiler A, Bamburg JR
• Analysis of the interactions of actin depolymerizing factor with G- and F-actin.
• Biochemistry. 1993; 32: 9994-10004
• Display abstract

• Chick actin depolymerizing factor (ADF) is an actin binding protein previously shown to rapidly depolymerize actin filaments in vitro, yielding a 1:1 complex of ADF and actin monomer. Here we show that ADF protects actin monomer from denaturation by EDTA by inhibiting the exchange of actin-bound nucleotide. Under low ionic strength conditions, the approximate dissociation constant (KD) for the ADF-actin complex determined from exchange of nucleotide (1,N6-etheno-ATP) is about 150 and is calcium-independent. Addition of ADF to monomeric actin inhibits actin assembly as well as the ATP hydrolysis that normally accompanies assembly. Complex formation is demonstrated between ADF and actin containing either ATP, ADP, or AMPPNP as the bound nucleotide. A KD of 0.1-0.2 microM was calculated for both the ADF-ATP-actin and ADF-AMPPNP-actin complexes, whereas the KD for the ADF-ADP-actin complex is about 1.3 microM. ADF can either depolymerize or cosediment with F-actin in a stoichiometric fashion, but these reciprocal activities are pH-dependent. At pHs between 6.5 and 7.1, ADF cosediments with F-actin and demonstrates only weak depolymerizing activity. ADF binding is cooperative and saturates at a 1:1 ADF:actin molar ratio. At pHs between 7.1 and 7.7, ADF shows increasing depolymerizing activity and less F-actin binding. At pH 8.0, ADF depolymerizes F-actin in a stoichiometric manner. Both the F-actin binding and the depolymerizing activities of ADF are inhibited by phalloidin.(ABSTRACT TRUNCATED AT 250 WORDS)

• Hawkins M, Pope B, Maciver SK, Weeds AG
• Human actin depolymerizing factor mediates a pH-sensitive destruction of actin filaments.
• Biochemistry. 1993; 32: 9985-93
• Display abstract

• ADF (actin depolymerizing factor) is an M(r) 19,000 actin-binding protein present in many vertebrate tissues and particularly abundant in neuronal cells. We have cloned human ADF and here show it to be identical in sequence to porcine destrin. Human ADF expressed in Escherichia coli behaves like native ADF from porcine brain. It binds to G-actin at pH 8 with a 1:1 stoichiometry and Kd approximately 0.2 microM, thereby sequestering monomers and preventing polymerization. It does not cosediment with F-actin at this pH, but severs actin filaments in a calcium-insensitive manner. The severing activity is only about 0.1% efficient. By contrast, at pH values below 7, ADF binds to actin filaments in a highly cooperative manner and at a 1:1 ratio to filament subunits. When the pH is raised to 8.0, the decorated filaments are rapidly severed and depolymerized.

• Quirk S et al.
• Primary structure of and studies on Acanthamoeba actophorin.
• Biochemistry. 1993; 32: 8525-33
• Display abstract

• We determined the amino acid sequence of the actin monomer binding/actin filament severing protein actophorin from Acanthamoeba castellanii by automated Edman degradation of peptide fragments and by sequencing of full-length cDNA. Actophorin consists of 138 amino acids (calculated molecular weight of 15,543) and shares a high degree of sequence similarity to other low molecular weight actin monomer sequestering proteins, especially vertebrate cofilin, vertebrate actin depolymerizing factor/destrin, and echinoderm depactin. Actophorin is smaller and does not contain a nuclear localization sequence like the related vertebrate proteins. Southern blot analysis indicates that actophorin is a single-copy gene; however, Northern blots show two distinct mRNA species of 1 and 0.9 kb in size. Homogeneous recombinant actophorin purified from Escherichia coli is indistinguishable from the native protein in its physical properties and in biochemical assays of its interaction with actin, but is less reactive with three monoclonal antibodies raised against the native protein. The NH2 terminus of native actophrin is blocked, while the initiating methionine residue is removed from recombinant actophorin. This difference has no measurable effect on activity. By fluorescent antibody staining of Acanthamoeba, actophorin colocalizes with actin filaments in the cortical cytoplasm, especially at the leading edge of the cell. Additionally, actophorin binds phosphatidylinositol 4',5'-bisphosphate. The recombinant actophorin forms X-ray diffraction quality crystals of superior quality in poly(ethylene glycol)/2-propanol and, like the native crystal form, belongs to space group P2(1)2(1)2(1).

• Iida K, Moriyama K, Matsumoto S, Kawasaki H, Nishida E, Yahara I
• Isolation of a yeast essential gene, COF1, that encodes a homologue of mammalian cofilin, a low-M(r) actin-binding and depolymerizing protein.
• Gene. 1993; 124: 115-20
• Display abstract

• We have cloned a Saccharomyces cerevisiae gene (COF1) encoding a low-M(r) actin-binding protein of 143 amino acid (aa) residues (yeast cofilin; Cof); its aa sequence is 35% identical to porcine Cof. The yeast recombinant Cof produced in Escherichia coli exhibited in vitro activities on actin filaments similar to those of mammalian and avian Cof. Gene disruption and tetrad analysis showed that gene COF1 is essential for yeast cell growth. Expression of the cDNA of porcine Cof or destrin (Des), the latter a Cof-related protein, complemented the cof1 null allele in yeast cells.

• Zigmond SH
• Recent quantitative studies of actin filament turnover during cell locomotion.
• Cell Motil Cytoskeleton. 1993; 25: 309-16
• Display abstract

• Cell locomotion depends on polymerization and depolymerization of filamentous actin. Net polymerization at the cell front occurs fast enough to fill the extending lamellipod, and since total F-actin is essentially constant over time, depolymerization must equal polymerization. Indeed, the fastest moving cell types have the highest rates of depolymerization. Accounting for the high rate of depolymerization raises several problems. One is that net depolymerization requires the concentration of G-actin to be low (below the critical concentration), but rapid polymerization (occurring < 1 micron away) requires the concentration of G-actin to be high (well above the critical concentration). This may be accomplished by spatial compartmentalization of factors that favor polymerization or depolymerization, and/or by proteins that bind G-actin and prevent spontaneous polymerization while allowing barbed-end elongation. A second problem is that depolymerization proceeds faster than would seem possible from studies of F-actin in vitro (as calculated from number and lengths of filaments present and in vitro rate constants). Rapid depolymerization may be accomplished by filament cutters or by cytoplasmic components (as yet undiscovered) that increase the rate of depolymerization.

• Morgan TE, Lockerbie RO, Minamide LS, Browning MD, Bamburg JR
• Isolation and characterization of a regulated form of actin depolymerizing factor.
• J Cell Biol. 1993; 122: 623-33
• Display abstract

• Actin depolymerizing factor (ADF) is an 18.5-kD protein with pH-dependent reciprocal F-actin binding and severing/depolymerizing activities. We previously showed developing muscle down-regulates ADF (J. R. Bamburg and D. Bray. 1987. J. Cell Biol. 105: 2817-2825). To further study this process, we examined ADF expression in chick myocytes cultured in vitro. Surprisingly, ADF immunoreactivity increases during the first 7-10 d in culture. This increase is due to the presence of a new ADF species with higher relative molecular weight which reacts identically to brain ADF with antisera raised against either brain ADF or recombinant ADF. We have purified both ADF isoforms from myocytes and have shown by peptide mapping and partial sequence analysis that the new isoform is structurally related to ADF. Immunoprecipitation of both isoforms from extracts of cells prelabeled with [32P]orthophosphate showed that the new isoform is radiolabeled, predominantly on a serine residue, and hence is called pADF. pADF can be converted into a form which comigrates with ADF on 1-D and 2-D gels by treatment with alkaline phosphatase. pADF has been quantified in a number of cells and tissues where it is present from approximately 18% to 150% of the amount of unphosphorylated ADF. pADF, unlike ADF, does not bind to G-actin, or affect the rate or extent of actin assembly. Four ubiquitous protein kinases failed to phosphorylate ADF in vitro suggesting that ADF phosphorylation in vivo is catalyzed by a more specific kinase. We conclude that the ability to regulate ADF activity is important to muscle development since myocytes have both pre- and posttranslational mechanisms for regulating ADF activity. The latter mechanism is apparently a general one for cell regulation of ADF activity.

• Jockusch BM, Wiegand C, Temm-Grove CJ, Nikolai G
• Dynamic aspects of microfilament-membrane attachments.
• Symp Soc Exp Biol. 1993; 47: 253-66
• Display abstract

• Microfilament-membrane attachment sites are complex structures that are essential for tissue differentiation in animals. In this article, we focus on the assembly and dynamics of such contact sites as seen in two cell types differentiating in cultures of the embryonic chicken heart, cardiocytes and fibroblasts. Concentrating on the cytoplasmic domain, we refer to previous biochemical, light, and electron microscopic studies on the structure and dynamics of these regions and supplement them with our own recent data. Although many details are still to be elucidated, we would like to propose the following model. Actin, alpha-actinin and vinculin are the major structural components of all microfilament-membrane contacts. Various subtypes of junctions are characterised by additional structural components or by specific isoforms. Temporal regulation of contact sites is linked to assembly and disassembly of microfilaments and might be controlled by special regulatory proteins. Finally, the cytoplasmic domains of junctional complexes may serve as structural matrices for the positioning of proteins involved in signal transduction pathways.

• Shapland C, Hsuan JJ, Totty NF, Lawson D
• Purification and properties of transgelin: a transformation and shape change sensitive actin-gelling protein.
• J Cell Biol. 1993; 121: 1065-73
• Display abstract

• We have purified the transformation and shape change sensitive isoform of an actin associated polypeptide doublet previously described by us (Shapland, C., P. Lowings, and D. Lawson. 1988. J. Cell Biol. 107:153-161) and have shown that it is evolutionarily conserved as far back as yeast. The purified protein: (a) binds directly to actin filaments at a ratio of 1:6 actin monomers, with a binding constant (Ka) of approximately 7.5 x 10(5) M-1; and (b) causes actin filament gelation within 2 min. Although these activities are controlled by ionic strength (and may be mediated by positively charged amino acid residues) the molecule remains as a monomer irrespective of ionic conditions. EM reveals that the addition of this protein to actin filaments converts them from a loose, random distribution into a tangled, cross-linked meshwork within 1 min, and discrete tightly aggregated foci after 10 min. By use of an "add-back" cell permeabilization system we can rebind this molecule specifically to actin filaments in cells from which it has previously been removed. Since the protein is transformation sensitive and gels actin, we have named it transgelin.

• Aspenstrom P, Schutt CE, Lindberg U, Karlsson R
• Mutations in beta-actin: influence on polymer formation and on interactions with myosin and profilin.
• FEBS Lett. 1993; 329: 163-70
• Display abstract

• Two beta-actin mutants, one with proline 38 replaced with alanine (P38A) and the other with cysteine-374 replaced with serine (C374S), as well as the wild-type beta-actin, were expressed in the yeast, S. cerevisiae, purified to homogeneity, and analyzed in vitro for polymerizability and interaction with DNase I, myosin, and profilin. Both mutations interfered with the polymerization of the actin, and with its interaction with myosin. The C374S mutation had the most pronounced effect; it reduced the polymerizability of the actin, abolished its binding to profilin, and filaments containing this mutation moved at reduced rates in the in vitro 'motility assay'. The ATPase activity measured in solutions containing myosin subfragment 1 was similar for both the mutant and wild-type actins.

• Valenta R et al.
• Identification of profilin as an actin-binding protein in higher plants.
• J Biol Chem. 1993; 268: 22777-81
• Display abstract

• Profilin is a low molecular weight protein involved in the organization of the mammalian and protozoan cytoskeleton as well as in signal transduction. In this study, profilin is identified as an actin-binding protein in higher plants which is present in monocot and dicot angiosperms. Birch pollen profilin and actin can be copurified as a complex, and purified recombinant birch profilin can be used as an affinity matrix to obtain birch pollen actin. The binding of 125I-labeled recombinant birch pollen profilin to plant and animal actins can be blocked by profilin-specific antibodies that react with different epitopes of birch profilin. One of the blocking antibodies was raised against the 25 COOH-terminal amino acids indicating the importance of this region in the profilactin complex formation.

• Pantaloni D, Carlier MF
• How profilin promotes actin filament assembly in the presence of thymosin beta 4.
• Cell. 1993; 75: 1007-14
• Display abstract

• The role of profilin in the regulation of actin assembly has been reexamined. The affinity of profilin for ATP-actin appears 10-fold higher than previously thought. In the presence of ATP, the participation of the profilin-actin complex to filament elongation at the barbed end is linked to a decrease in the steady-state concentration of globular actin. This surprising effect is made possible by the involvement of the irreversible ATP hydrolysis accompanying actin polymerization. As a consequence, in the presence of thymosin beta 4 (T beta 4), low amounts of profilin promote extensive actin assembly off of the pool of actin-T beta 4 complex. When barbed ends are capped, profilin simply sequesters globular actin. A model is proposed for the function of profilin in actin-based motility.

• Hayakawa K et al.
• Increased expression of cofilin in dystrophic chicken and mouse skeletal muscles.
• J Biochem (Tokyo). 1993; 114: 582-7
• Display abstract

• A monoclonal antibody (McAb) specific for actin depolymerizing factor (ADF) was prepared. With this and previously prepared anti-cofilin McAb (MAB-22) and other antibodies, the expression of cofilin and ADF in the muscles of dystrophic (NH-413) chicken and dystrophic (C57BL/6J dy/dy) mice was compared with that in normal control animals by immunoblotting and immunocytochemical methods. Since cofilin expression is down-regulated during normal postnatal development of skeletal muscles [Abe et al. (1989) J. Biochem. 106, 696-702], cofilin was detected in the breast (pectoralis) muscle of normal adult chicken and the leg (femoris and tibialis anterior) muscles of normal mice only at a low level. ADF was not detectable in adult skeletal muscles. However, a significant increase of cofilin amount, but not of ADF amount, was observed in these muscles of the dystrophic animals, when the symptom of muscular dystrophy became evident. In order to localize cofilin in individual muscle fibers, serial cryosections of the dystrophic chicken muscle were examined with anti-cofilin antibody (MAB-22). The antibody stained cells of different size in the dystrophic muscle, indicating that cofilin expression was induced in the regenerating muscle cells as well as in the pre-existing myofibers. We suggest that cofilin is involved in disassembly or reorganization of actin in the dystrophic muscle.

• Abe H, Nagaoka R, Obinata T
• Cytoplasmic localization and nuclear transport of cofilin in cultured myotubes.
• Exp Cell Res. 1993; 206: 1-10
• Display abstract

• We used immunofluorescence methods to examine the cellular distribution of cofilin in chicken myotubes in primary culture. Cofilin showed mainly diffuse distribution in the cytoplasm except for rather strong staining around the nuclei and faint striated patterns along myofibrils, but did not stain inside the nuclei. Neither stress fiber-like structures nor myofibrils were clearly stained. In the presence of 10% dimethyl sulfoxide (DMSO), intranuclear actin-cofilin rods, which were composed of alpha-actin isoform and cofilin, were formed in all the nuclei of individual myotubes. In the cofilin sequence, a putative nuclear localization signal (NLS) was observed. We examined the NLS activity of this portion by using a synthetic peptide corresponding to the putative NLS. When the NLS peptide conjugated with bovine serum albumin was microinjected into the cytoplasm of myotubes, it was rapidly accumulated into the nuclei. The same result was obtained with in vitro a nuclear protein import assay system with digitonin-permeabilized myotubes. Therefore, we suggest that this portion is responsible for the nuclear transport of cofilin. In myotubes, the majority of cofilin was present in an unphosphorylated form and this form remained unchanged after the DMSO treatment. Thus, we suggest that the phosphorylation of cofilin itself is not directly involved in its nuclear transport at least in myotubes.

• Adams AE, Cooper JA, Drubin DG
• Unexpected combinations of null mutations in genes encoding the actin cytoskeleton are lethal in yeast.
• Mol Biol Cell. 1993; 4: 459-68
• Display abstract

• To understand the role of the actin cytoskeleton in cell physiology, and how actin-binding proteins regulate the actin cytoskeleton in vivo, we and others previously identified actin-binding proteins in Saccharomyces cerevisiae and studied the effect of null mutations in the genes for these proteins. A null mutation of the actin gene (ACT1) is lethal, but null mutations in the tropomyosin (TPM1), fimbrin (SAC6), Abp1p (ABP1), and capping protein (CAP1 and CAP2) genes have relatively mild or no effects. We have now constructed double and triple mutants lacking 2 or 3 of these actin-binding proteins, and studied the effect of the combined mutations on cell growth, morphology, and organization of the actin cytoskeleton. Double mutants lacking fimbrin and either Abp1p or capping protein show negative synthetic effects on growth, in the most extreme case resulting in lethality. All other combinations of double mutations and the triple mutant lacking tropomyosin, Abp1p, and capping protein, are viable and their phenotypes are similar to or only slightly more severe than those of the single mutants. Therefore, the synthetic phenotypes are highly specific. We confirmed this specificity by overexpression of capping protein and Abp1p in strains lacking fimbrin. Thus, while overexpression of these proteins has deleterious effects on actin organization in wild-type strains, no synthetic phenotype was observed in the absence of fimbrin. We draw two important conclusions from these results. First, since mutations in pairs of actin-binding protein genes cause inviability, the actin cytoskeleton of yeast does not contain a high degree of redundancy. Second, the lack of structural and functional homology among these genetically redundant proteins (fimbrin and capping protein or Abp1p) indicates that they regulate the actin cytoskeleton by different mechanisms. Determination of the molecular basis for this surprising conclusion will provide unique insights into the essential mechanisms that regulate the actin cytoskeleton.

• Sherratt JA, Lewis J
• Stress-induced alignment of actin filaments and the mechanics of cytogel.
• Bull Math Biol. 1993; 55: 637-54
• Display abstract

• Experimental evidence suggests that anisotropic stress induces alignment of intracellular actin filaments. We develop a model for this phenomenon, which includes a parameter reflecting the sensitivity of the microfilament network to changes in the stress field. When applied to a uniform cell sheet at rest, the model predicts that for sufficiently large values of the sensitivity parameter, all the actin filaments will spontaneously align in a single direction. Stress alignment can also be caused by a change in external conditions, and as an example of this we apply our model to the initial response of embryonic epidermis to wounding. Our solutions in this case are able to reflect the actin cable that has been found at the wound edge in recent experiments; the cable consists of microfilaments aligned with stress at the wound boundary of the epithelium. These applications suggest that stress-induced alignment of actin filaments could play a key role in some biological systems. This is the first attempt to include the alignment phenomenon in a mechanical model of cytogel.

• Bjorkegren C, Rozycki M, Schutt CE, Lindberg U, Karlsson R
• Mutagenesis of human profilin locates its poly(L-proline)-binding site to a hydrophobic patch of aromatic amino acids.
• FEBS Lett. 1993; 333: 123-6
• Display abstract

• The actin-binding protein, profilin, contains a src-homology (SH) 3-like fold (Schutt, C.E. et al., submitted), and its tight interaction with poly(L-proline) is reminiscent of the binding activity exhibited by SH3-domains. Here we demonstrate that replacements of aromatic amino acids in a hydrophobic patch on the surface of the profilin molecule abolish its poly(L-proline)-binding capacity. However, the location of this hydrophobic patch is found in another region of the molecule than that displaying structural similarities with SH3 domains.

• Brady RL, Lange G, Barclay AN
• Structural studies of CD4: crystal structure of domains 3 and 4 and their implication for the overall receptor structure.
• Biochem Soc Trans. 1993; 21: 958-63

• Ono S, Abe H, Nagaoka R, Obinata T
• Colocalization of ADF and cofilin in intranuclear actin rods of cultured muscle cells.
• J Muscle Res Cell Motil. 1993; 14: 195-204
• Display abstract

• Immunofluorescence microscopy revealed that two actin-binding proteins of low molecular weight with different functional activity, ADF and cofilin, are transported into nuclei of cultured myogenic cells to form rod structures there together with actin, when the cells were incubated in medium containing dimethylsulfoxide. In most cases, ADF and cofilin colocalized in the same nuclear actin rods, but ADF appeared to predominate in mononucleated cells, while cofilin was present in multinucleated myotubes. In some mononucleated cells, the nuclear actin rods were composed of ADF and actin but devoid of cofilin. An ADF homologue in mammals, destrin, was also translocated into nuclear actin rods under similar conditions. As a nuclear transport signal sequence exists in cofilin and ADF but not in actin, ADF and/or cofilin may be responsible for the nuclear import of actin in myogenic cells under certain conditions.

• Moriyama K, Yonezawa N, Sakai H, Yahara I, Nishida E
• Mutational analysis of an actin-binding site of cofilin and characterization of chimeric proteins between cofilin and destrin.
• J Biol Chem. 1992; 267: 7240-4
• Display abstract

• Cofilin and destrin are two related low molecular weight mammalian actin-binding proteins. Cofilin is an F-actin side-binding and pH-dependent actin-depolymerizing protein, and destrin is a pH-independent actin-depolymerizing protein. We have introduced a few point mutations within an actin-binding sequence of cofilin. Biochemical analyses of these mutant proteins have clearly shown that Lys112 and Lys114 of cofilin are crucially but differently involved in its interaction with actin and phosphatidylinositol 4,5-bisphosphate. This is the first example among actin-binding proteins whose point mutations inactivate their interaction with actin in vitro. We have also made and characterized a series of chimeric proteins between cofilin and destrin to identify the regions responsible for the pH dependence and the F-actin side binding activity of cofilin. Our results suggest that a central region consisting of 42 amino acid residues and a carboxyl-terminal quarter of cofilin are both involved in regulation of the pH-dependent actin depolymerizing activity and the activity to bind along F-actin.

• Amatruda JF, Gattermeir DJ, Karpova TS, Cooper JA
• Effects of null mutations and overexpression of capping protein on morphogenesis, actin distribution and polarized secretion in yeast.
• J Cell Biol. 1992; 119: 1151-62
• Display abstract

• CAP1, the gene encoding the alpha subunit of Saccharomyces cerevisiae capping protein, was cloned using a probe prepared by PCR with primers based on the amino acid sequence of purified alpha subunit peptides. The sequence is similar to that of capping protein alpha subunits of other species but not to that of the S. cerevisiae capping protein beta subunit or any other protein. Null mutants of capping protein, prepared by deletion of the coding region of CAP1 and CAP2 separately or together, are viable and have a similar phenotype. Deletion of the gene for one subunit leads to a loss of protein for the other subunit. The null mutant has a severe deficit of actin cables and an increased number of actin spots in the mother. Cells are round and relatively large. These features are heterogeneous within a population of cells and vary with genetic background. Overexpression of CAP1 and CAP2 also causes loss of actin cables and cell enlargement, as well as the additional traits of aberrant morphogenesis and cell wall thickening. Capping protein null strains and overexpression strains exhibited normal polarized secretion during bud growth as demonstrated by labeling with fluoresceinated Con A. Projection formation and chitin deposition in response to mating pheromone, mating efficiency, and bud site selection were also normal in capping protein null strains. In addition, bulk secretion of invertase was unimpaired. These data indicate that actin cables are not required for polarized secretion in S. cerevisiae.

• Amatruda JF, Cooper JA
• Purification, characterization, and immunofluorescence localization of Saccharomyces cerevisiae capping protein.
• J Cell Biol. 1992; 117: 1067-76
• Display abstract

• Capping protein binds the barbed ends of actin filaments and nucleates actin filament assembly in vitro. We purified capping protein from Saccharomyces cervisiae. One of the two subunits is the product of the CAP2 gene, which we previously identified as the gene encoding the beta subunit of capping protein based on its sequence similarity to capping protein beta subunits in chicken and Dictyostelium (Amatruda, J. F., J. F. Cannon, K. Tatchell, C. Hug, and J. A. Cooper. 1990. Nature (Lond.) 344:352-354). Yeast capping protein has activity in critical concentration and low-shear viscometry assays consistent with barbed-end capping activity. Like chicken capping protein, yeast capping protein is inhibited by PIP2. By immunofluorescence microscopy yeast capping protein colocalizes with cortical actin spots at the site of bud emergence and at the tips of growing buds and shmoos. In contrast, capping protein does not colocalize with actin cables or with actin rings at the site of cytokinesis.

• Pring M, Weber A, Bubb MR
• Profilin-actin complexes directly elongate actin filaments at the barbed end.
• Biochemistry. 1992; 31: 1827-36
• Display abstract

• We demonstrate that the profilin-G-actin complex can elongate actin filaments directly at the barbed end but cannot bind to the pointed end. During elongation, the profilin-actin complex binds to the barbed filament end, whereupon profilin is released, leaving the actin molecule behind. This was first proposed by Tilney [Tilney, L. G., et al. (1983) J. Cell Biol. 97, 112-124] and demonstrated by Pollard and Cooper [(1984) Biochemistry 23, 6631-6641] by electron microscopy. We show that a model without any outside energy supply, in contrast to the mechanism proposed by Pollard and Cooper, can be fitted to our and their [Kaiser et al. (1986) J. Cell Biol. 102, 221-226] findings. Input of outside energy is necessary only if profilin-mediated elongation continues after free G-actin has been lowered to or below the critical concentration observed at the barbed end in the absence of profilin.

• Chowdhury S, Smith KW, Gustin MC
• Osmotic stress and the yeast cytoskeleton: phenotype-specific suppression of an actin mutation.
• J Cell Biol. 1992; 118: 561-71
• Display abstract

• In the yeast Saccharomyces cerevisiae, actin filaments function to direct cell growth to the emerging bud. Yeast has a single essential actin gene, ACT1. Diploid cells containing a single copy of ACT1 are osmosensitive (Osms), i.e., they fail to grow in high osmolarity media (D. Shortle, unpublished observations cited by Novick, P., and D. Botstein. 1985. Cell. 40:415-426). This phenotype suggests that an underlying physiological process involving actin is osmosensitive. Here, we demonstrate that this physiological process is a rapid and reversible change in actin filament organization in cells exposed to osmotic stress. Filamentous actin was stained using rhodamine phalloidin. Increasing external osmolarity caused a rapid loss of actin filament cables, followed by a slower redistribution of cortical actin filament patches. In the recovery phase, cables and patches were restored to their original levels and locations. Strains containing an act1-1 mutation are both Osms and temperature-sensitive (Ts) (Novick and Botstein, 1985). To identify genes whose products functionally interact with actin in cellular responses to osmotic stress, we have isolated extragenic suppressors which revert only the Osms but not the Ts phenotype of an act1-1 mutant. These suppressors identify three genes, RAH1-RAH3. Morphological and genetic properties of a dominant suppressor mutation suggest that the product of the wild-type allele, RAH3+, is an actin-binding protein that interacts with actin to allow reassembly of the cytoskeleton following osmotic stress.

• Iida K, Matsumoto S, Yahara I
• The KKRKK sequence is involved in heat shock-induced nuclear translocation of the 18-kDa actin-binding protein, cofilin.
• Cell Struct Funct. 1992; 17: 39-46
• Display abstract

• The exposure of cultured mammalian cells to elevated temperatures induces the translocation of actin and cofilin into the nuclei and the formation of intranuclear bundles of actin filaments decorated by cofilin (actin/cofilin rods). Cofilin has a stretch of five basic amino acids, KKRKK, which was assumed to be the sequence involved in the heat shock-dependent accumulation of cofilin in nuclei. To examine this possibility, the site-directed mutagenesis technique was employed to alter the KKRKK sequence of cofilin to KTLKK and the mutated cofilin was expressed under the human beta-actin promoter in transfectants of mouse C3H-2K cell line. All the recombinants derived from porcine cofilin cDNA were constructed so as to possess an extra-nonapeptide at their N-termini when expressed; their intracellular distribution could, therefore, be discriminated from that of endogenous cofilin using the indirect immunofluorescence method with polyclonal antibodies directed against the extra-peptide. The results clearly showed that the mutated cofilin possessing KTLKK instead of KKRKK did not translocate into the nuclei in response to heat shock whereas a recombinant cofilin with the unaltered sequence of KKRKK responded to heat shock and formed intranuclear rods together with actin. Although in vitro actin binding experiments showed that KTLKK-cofilin has a weaker affinity to actin filaments than KKRKK-cofilin, KTLKK-cofilin was found to form cytoplasmic actin/cofilin rods when transformants were incubated in NaCl buffer. Furthermore, we have noted that endogenous cofilin present in cells expressing KTLKK-cofilin behaved normally, translocated into nuclei and formed intranuclear actin/cofilin rods upon heat shock. These results suggest that the KKRKK sequence of cofilin functions as a nuclear location signal upon heat shock.

• Theriot JA, Mitchison TJ
• Actin microfilament dynamics in locomoting cells.
• Nature. 1991; 352: 126-31
• Display abstract

• The dynamic behaviour of actin filaments has been directly observed in living, motile cells using fluorescence photoactivation. In goldfish epithelial keratocytes, the actin microfilaments in the lamellipodium remain approximately fixed relative to the substrate as the cell moves over them, regardless of cell speed. The rate of turnover of actin subunits in the lamellipodium is remarkably rapid. Cell movement is directly and tightly coupled to the formation of new actin filaments at the leading edge.

• Sheterline P, Handel SH, Hendry KA
• Reorganization and turnover of actin filament architectures in cells.
• Biochem Soc Trans. 1991; 19: 1120-4

• Maciver SK, Zot HG, Pollard TD
• Characterization of actin filament severing by actophorin from Acanthamoeba castellanii.
• J Cell Biol. 1991; 115: 1611-20
• Display abstract

• Actophorin is an abundant 15-kD actinbinding protein from Acanthamoeba that is thought to form a nonpolymerizable complex with actin monomers and also to reduce the viscosity of polymerized actin by severing filaments (Cooper et al., 1986. J. Biol. Chem. 261:477-485). Homologous proteins have been identified in sea urchin, chicken, and mammalian tissues. Chemical crosslinking produces a 1:1 covalent complex of actin and actophorin. Actophorin and profilin compete for crosslinking to actin monomers. The influence of actophorin on the steady-state actin polymer concentration gave a Kd of 0.2 microM for the complex of actophorin with actin monomers. Several new lines of evidence, including assays for actin filament ends by elongation rate and depolymerization rate, show that actophorin severs actin filaments both at steady state and during spontaneous polymerization. This is confirmed by direct observation in the light microscope and by showing that the effects of actophorin on the low shear viscosity of polymerized actin cannot be explained by monomer sequestration. The severing activity of actophorin is strongly inhibited by stoichiometric concentrations of phalloidin or millimolar concentrations of inorganic phosphate.

• Sheterline P, Handel SE, Molloy C, Hendry KA
• The nature and regulation of actin filament turnover in cells.
• Acta Histochem Suppl. 1991; 41: 303-9
• Display abstract

• Actin filaments in mammalian cells form a number of different architectures in conjunction with a number of different actin-binding proteins. In motile cells these complex architectural arrangements of actin filaments and associated proteins continuously adjust their 3-dimensional organisation to modify the shape and behaviour of cells in response to external information. Microinjection experiments with fluorescently-labelled actin monomers suggest that there is a continual exchange of monomers between the actin filaments and a soluble pool such that individual monomers exist for only a few minutes within polymers. These data suggest that remodelling of the actin filament architectures occurs by the continuous assembly of new filaments which is balanced by the disassembly of obsolete structures. The mechanisms driving and regulating the assembly and disassembly cycle are not yet clearly understood. The properties of the actin assembly ATPase in vitro suggest that the intrinsic exchange of monomers between polymers and the monomer pool is driven by the stoichiometric ATP hydrolysis which is uncoupled from monomer addition and leads to both treadmilling and to the potential for mechanisms analogous to the dynamic instability models proposed for microtubules. Because of the relatively rapid rate of ATP hydrolysis by monomers in the filament (k = 0.05-0.02/s), it is assumed that most of the F-actin in cells is in its ADP form. ADP-F-actin binds inorganic phosphate with a Kd close to that of cytoplasmic concentrations to form an ADP.Pi-F-actin form which has different kinetic, structural and behavioural properties to ADP-F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)

• Yonezawa N, Nishida E, Iida K, Kumagai H, Yahara I, Sakai H
• Inhibition of actin polymerization by a synthetic dodecapeptide patterned on the sequence around the actin-binding site of cofilin.
• J Biol Chem. 1991; 266: 10485-9
• Display abstract

• Cofilin is an F-actin side-binding and -depolymerizing protein with an apparent molecular mass of 21 kDa. By means of the end label fingerprinting method, the amino acid residue on cofilin sequence cross-linked to actin by zero length cross-linker, 1-ethyl-3-(3-dimethylamino propyl)carbodiimide, was identified as Lys112 and/or Lys114. A synthetic dodecapeptide patterned on the sequence around the actin-cross-linking site of cofilin (Trp104-Met115) inhibited the binding of cofilin to actin. Moreover, the dodecapeptide was found to be a potent inhibitor of actin polymerization. Thus, we conclude that the dodecapeptide sequence constitutes the region essential for the actin-binding and -depolymerizing activity of cofilin. A sequence similar to the dodecapeptide is found in other actin-depolymerizing proteins, destrin, actin-depolymerizing factor, and depactin. Therefore, the dodecapeptide sequence may be a consensus sequence essential for actin-binding and -depolymerizing activity in actin-depolymerizing proteins.

• Takagi T, Mabuchi I, Hosoya H, Furuhashi K, Hatano S
• Primary structure of profilins from two species of Echinoidea and Physarum polycephalum.
• Eur J Biochem. 1990; 192: 777-81
• Display abstract

• Profilin is a small G-actin-binding protein, the amino acid sequence of which was previously reported for calf, human, Acanthamoeba and yeast. Here the amino acid sequences of three profilins obtained from eggs of two species of Echinoidea, Clypeaster japonicus (order, Clypeasteroida) and Anthocidaris crassispina (order, Echinoida), and plasmodium of Physarum polycephalum were determined. Two echinoid profilins were composed of 139 amino acid residues, N-termini were acylated and the molecular mass was calculated to be 14.6 kDa, slightly larger than that of 13 kDa estimated by SDS/PAGE [Mabuchi, I. & Hosoya, H. (1982) Biomed. Res. 3, 465-476]. On the other hand, Physarum profilin was composed of 124 amino acid residues, the N-terminus was acylated, and the calculated molecular mass was 13132 Da. The sequences of C. japonicus and A. crassispina profilins were homologous (84% identical). However, the similarity of these profilins with those form other organisms was low. The sequence of Physarum profilin was homologous with Acanthamoeba profilin isoforms (51% identical) and with yeast profilin (42% identical), but not with other profilins. The relatively conservative sequence of profilins from yeast, Physarum, Acanthamoeba, echinoid eggs and mammalian cells was found in the N-terminal region, which was suggested to be a common actin-binding region. The C-terminal region was also conserved, although to a lesser extent than the N-terminal region.

• Hartmann H, Schleicher M, Noegel AA
• Heterodimeric capping proteins constitute a highly conserved group of actin-binding proteins.
• Dev Genet. 1990; 11: 369-76
• Display abstract

• The two subunits of the heterodimeric protein cap32/34, an actin-binding protein, are encoded by separate single-copy genes. We have established the genomic structure of both genes. A sequence comparison of cap32/34 with capZ from chicken skeletal muscle and two partially known sequences from Saccharomyces cerevisiae and Xenopus laevis show that heterodimeric capping proteins belong to a highly conserved group of actin-binding proteins. This conclusion is supported by the cross-reaction of polyclonal antibodies against cap32 and cap34 with proteins from lower and higher eukaryotes. In addition, a system is presented that allows the expression of truncated cap34 polypeptides under the control of the cap34 promoter.

• Drubin DG
• Actin and actin-binding proteins in yeast.
• Cell Motil Cytoskeleton. 1990; 15: 7-11

• Grazi E, Trombetta G, Guidoboni M
• Microfilament gel rigidity cooperates negatively with the binding of actin gelling proteins.
• Biochem Int. 1990; 21: 633-40
• Display abstract

• At 37 degrees C, in the presence of 0.1 M KC1 and 2 mM MgCl2, the binding of alpha-actinin to F-actin increases with the concentration of alpha-actinin but not with the concentration of F-actin. This implies that binding is determined by additional factors, beside the alpha-actinin - F-actin association constant. We propose that one of these factors is the rigidity of the gel, which cooperates negatively to the binding by increasing the work needed to bring two actin filaments at the reaction distance with alpha-actinin.

• Moriyama K et al.
• Destrin, a mammalian actin-depolymerizing protein, is closely related to cofilin. Cloning and expression of porcine brain destrin cDNA.
• J Biol Chem. 1990; 265: 5768-73
• Display abstract

• Destrin is a mammalian 19-kDa protein that rapidly depolymerizes F-actin in a stoichiometric manner. In this study, we isolated cDNA clones coding for destrin from a porcine brain cDNA library. The deduced amino acid sequence of destrin is 165 residues long and is very similar (71% identical) to that of cofilin, a widely distributed, pH-sensitive actin-modulating protein. Destrin contains a sequence nearly identical with the putative nuclear transport signal sequence of cofilin and a hexapeptide sequence identical with the amino-terminal sequence (residues 2-7) of tropomyosin, which is shown to be involved in cofilin binding to actin. Destrin, like cofilin, also has in its carboxyl-terminal portion a region homologous to the sequence shared by gelsolin, fragmin, and Acanthamoeba profilin. We have expressed destrin as well as cofilin in Escherichia coli, purified them, and examined their function in vitro. The two proteins were found to differ in their interaction with actin, like destrin and cofilin isolated from porcine brain. This suggests that the difference in the function of the two proteins results from the subtle difference in their amino acid sequence rather than possible differences in post-translational modifications. Northern blot analyses indicated that both destrin mRNA and cofilin mRNA are widely distributed in various tissues, but both mRNAs differ in their relative abundance among tissues.

• Yu FX, Johnston PA, Sudhof TC, Yin HL
• gCap39, a calcium ion- and polyphosphoinositide-regulated actin capping protein.
• Science. 1990; 250: 1413-5
• Display abstract

• The polymerization of actin filaments is involved in growth, movement, and cell division. It has been shown that actin polymerization is controlled by gelsolin, whose interactions with actin are activated by calcium ion (Ca2+) and inhibited by membrane polyphosphoinositides (PPI). A smaller Ca2(+)- and PPI-regulated protein, gCap39, which has 49% sequence identity with gelsolin, has been identified by cDNA cloning and protein purification. Like gelsolin, gCap39 binds to the fast-growing (+) end of actin filaments. However, gCap39 does not sever actin filaments and can respond to Ca2+ and PPI transients independently, under conditions in which gelsolin is ineffective. The coexistence of gCap39 with gelsolin should allow precise regulation of actin assembly at the leading edge of the cell.

• Amatruda JF, Cannon JF, Tatchell K, Hug C, Cooper JA
• Disruption of the actin cytoskeleton in yeast capping protein mutants.
• Nature. 1990; 344: 352-4
• Display abstract

• Capping protein controls the addition of actin subunits to the barbed end of actin filaments and nucleates actin polymerization in vitro. Capping protein has been identified in all eukaryotic cells examined so far; it is a heterodimer with subunits of relative molecular masses 32,000-36,000 (alpha-subunit) and 28,000-32,000 (beta-subunit). In skeletal muscle, capping protein (CapZ) probably binds the barbed ends of actin filaments at the Z line. The in vivo role of this protein in non-muscle cells is not known. We report here the characterization of CAP2, the single gene encoding the beta-subunit of capping protein in Saccharomyces cerevisiae. Yeast cells in which the CAP2 gene was disrupted by an insertion or a deletion had an abnormal actin distribution, including the loss of actin cables. The mutant cells were round and large, with a heterogeneous size distribution, and, although viable, grew more slowly than congenic wild-type cells. Chitin, a cell wall component restricted to the mother-bud junction in wild-type budding yeast, was found on the entire mother cell surface in the mutants. The phenotype of CAP2 disruption resembled that of temperature-sensitive mutations in the yeast actin gene ACT1, indicating that capping protein regulates actin-filament distribution in vivo.

• Adams ME, Minamide LS, Duester G, Bamburg JR
• Nucleotide sequence and expression of a cDNA encoding chick brain actin depolymerizing factor.
• Biochemistry. 1990; 29: 7414-20
• Display abstract

• Chick brain actin depolymerizing factor (ADF) is a 19-kDa protein that severs actin filaments and binds actin monomers. We have obtained a cDNA encoding ADF by screening a chick embryo lambda gt11 cDNA library with both a rabbit anti-ADF antiserum and two oligonucleotide probes. Several non-full-length clones of 636 bases and one full-length clone of 1886 bases were isolated and sequenced. The full-length cDNA encodes a protein of 165 amino acids with a calculated molecular weight of 18,520. The deduced amino acid sequence shows 73% identity with the porcine brain actin binding protein cofilin. The coding region of the ADF cDNA has been placed in an expression vector, and the resulting protein shows immunoreactivity with an anti-ADF antiserum but not with an anti-cofilin antibody. The expressed ADF has been purified and has an actin depolymerizing activity identical with that of brain ADF. Like cofilin, ADF contains a sequence similar to the nuclear transport signal sequence of the SV40 large T antigen and a calcium/calmodulin-dependent protein kinase II phosphorylation consensus sequence. Northern blots of both embryonic chick brain and muscle RNA revealed two ADF mRNAs of length 2.1 and 0.9 kilobases. Southern blots suggest that the ADF gene is present in a single copy within the chicken genome. ADF contains regions of homology with other actin binding proteins including tropomyosin, gelsolin, and depactin.

• Hartmann H, Noegel AA, Eckerskorn C, Rapp S, Schleicher M
• Ca2+-independent F-actin capping proteins. Cap 32/34, a capping protein from Dictyostelium discoideum, does not share sequence homologies with known actin-binding proteins.
• J Biol Chem. 1989; 264: 12639-47
• Display abstract

• Polyclonal and monoclonal antibodies against purified 32/34-kDa F-actin-capping protein from Dictyostelium discoideum were generated and used to isolate clones coding for both subunits from a lambda gt11 expression library. In addition genomic clones were isolated for the 34-kDa subunit. The sequences of the corresponding inserts were determined and the amino acid sequences of the proteins deduced. The amino acid sequences have been confirmed by sequencing tryptic peptides of both subunits. As judged from immunological data and sequence comparison, the subunits are completely different. Each of them is encoded by a single copy gene. The mRNAs are present throughout all stages of development. Severing proteins, which exert also capping activity, show no sequence similarity with the cap 32/34-kDa protein. However, a pronounced homology between the 32-kDa subunit and a hypothetical yeast protein was found indicating the presence of a protein homologous to the 32-kDa subunit of the D. discoideum capping protein in yeast.

• Adams AE, Botstein D, Drubin DG
• A yeast actin-binding protein is encoded by SAC6, a gene found by suppression of an actin mutation.
• Science. 1989; 243: 231-3
• Display abstract

• The protein encoded by SAC6, a gene that can mutate to suppress a temperature-sensitive defect in the yeast actin gene, has been identified as a 67-kilodalton actin-binding protein (ABP 67) that associates with all identifiable actin structures. This finding demonstrates the in vivo functional importance of the actin-ABP 67 interaction previously established in vitro and illustrates the use of suppressor analysis to identify physically interacting proteins.

• Abe H, Obinata T
• An actin-depolymerizing protein in embryonic chicken skeletal muscle: purification and characterization.
• J Biochem (Tokyo). 1989; 106: 172-80
• Display abstract

• In embryonic skeletal muscle, a large amount of non-polymerized actin exists in the cytoplasm (Shimizu and Obinata [1986] J. Biochem. 99, 751-759). A 19-kDa protein (called 19K protein) which binds to G-actin was purified by sequential chromatography on DNase I-agarose, hydroxylapatite, SP-Sephadex, and Sephadex G-75, from the sarcoplasmic fraction of embryonic chicken skeletal muscle. This protein decreased the extent of actin polymerization at a steady state and increased the monomeric actin in a concentration-dependent fashion; it also caused quick depolymerization of F-actin, as determined by spectrophotometry at 237 nm, viscometry, DNase I inhibition assay, and electron microscopy. The molar ratio of 19K protein and actin interacting with each other was estimated to be 1:1. From these results, 19K protein was regarded as being actin depolymerizing protein. The amount of 19K protein in muscle decreased during development. The inhibitory action of 19K protein was removed by myosin or heavy meromyosin, and actin filaments were formed on the surface of myosin filaments when myosin filaments were added to a mixture of actin and 19K protein in a physiological salt solution. We propose that actin assembly is dually controlled in the developing muscle by the inhibitor(s) and an accelerator (myosin); this mechanism may enable the ordered assembly of actin and myosin in the early phase of myofibrillogenesis.

• Yonezawa N, Nishida E, Maekawa S, Sakai H
• Studies on the interaction between actin and cofilin purified by a new method.
• Biochem J. 1988; 251: 121-7
• Display abstract

• Cofilin is a 21,000-Mr actin-binding protein that widely exists in mammalian tissues. (1) A new purification procedure for porcine brain cofilin has been developed that involves (NH4)2SO4 fractionation and sequential chromatographies on Toyo Pearl and butyl-Toyo Pearl hydrophobic columns, hydroxyapatite, phosphocellulose and Sephadex G-75 gel-filtration columns. The purified cofilin bound to F-actin and increased the amount of G-actin to a limited extent, as previously reported [Nishida, Maekawa & Sakai (1984) Biochemistry 23, 5307-5313]. (2) The binding of cofilin to F-actin was scarcely affected by Mg2+, Ca2+ or by calmodulin. However, the binding was diminished by increasing concentrations of KCl, but was only slightly affected by temperature. (3) Cofilin and either alpha-actinin or filamin could bind to F-actin simultaneously with some competition, but the binding of caldesmon to F-actin was markedly inhibited by cofilin. Phalloidin inhibited the binding of cofilin to F-actin, and protected F-actin from depolymerization by cofilin.

• Matsuzaki F, Matsumoto S, Yahara I, Yonezawa N, Nishida E, Sakai H
• Cloning and characterization of porcine brain cofilin cDNA. Cofilin contains the nuclear transport signal sequence.
• J Biol Chem. 1988; 263: 11564-8
• Display abstract

• Cofilin is a widely distributed, pH-sensitive, actin-modulating protein with an apparent molecular mass of 21 kDa, which forms intranuclear and/or cytoplasmic actin/cofilin rods in cultured fibroblastic cells under specific conditions. In this study, a cDNA library from porcine brain mRNA was constructed, and full-length brain cofilin cDNA clones were isolated by screening with oligonucleotide probes. The deduced amino acid sequence of cofilin is 166 residues long and contains a sequence of Lys-Lys-Arg-Lys-Lys which is very similar to the nuclear transport signal sequence (Pro-Lys-Lys-Lys-Arg-Lys-Val) of SV40 large T antigen. The sequence may act as a signal capable of inducing nuclear accumulation of cofilin in cells exposed to heat shock or dimethyl sulfoxide. The cofilin sequence contains a hexapeptide (Asp-Ala-Ile-Lys-Lys-Lys) identical to the amino-terminal sequence (residues 2-7) of muscle and nonmuscle tropomyosin. Cofilin also has in the carboxyl-terminal portion a region homologous to the sequence shared by gelsolin, fragmin, and Acanthamoeba profilin. Furthermore, the overall amino acid sequence of cofilin shows weak homology with the rod portion of myosin and suggests a high alpha-helical content.

• Koffer A, Edgar AJ, Bamburg JR
• Identification of two species of actin depolymerizing factor in cultures of BHK cells.
• J Muscle Res Cell Motil. 1988; 9: 320-8
• Display abstract

• High-speed supernatant obtained from the lysate of cultured BHK cells has been chromatographed on Sepharose-4B, DEAE-cellulose and hydroxyapatite columns, and a fraction has been identified with characteristics similar to an actin depolymerizing factor (ADF), a small protein previously isolated from embryonic chick brain. Using a rabbit antibody against the chick brain protein, two immunoreactive forms were identified: a 19 kDa form co-migrating in SDS-polyacrylamide gels with embryonic chick brain ADF, and a 20 kDa form. The two species could be separated on a hydroxyapatite or green A dye matrix columns and only the 20 kDa protein was active when assayed for effects on pyrene-G-actin assembly. It enhanced the rate of F-actin assembly, but only after an initial lag phase, and decreased the final proportion of actin in filamentous form. These effects were calcium-independent. Actin depolymerizing factor constituted at least 0.5% of the total protein in the cytoplasmic fraction. A Triton extract of plasma membrane-enriched fraction from BHK cells was fractionated on a Sepharose-4B column and again, a fraction was found which had an ADF-like activity and also contained the two immuno-cross-reactive forms, 19 kDa and 20 kDa. These results suggest a novel regulation of the microfilament system in eukaryotic cells via the control of the ADF activity.

• Drubin DG, Miller KG, Botstein D
• Yeast actin-binding proteins: evidence for a role in morphogenesis.
• J Cell Biol. 1988; 107: 2551-61
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• Three yeast actin-binding proteins were identified using yeast actin filaments as an affinity matrix. One protein appears to be a yeast myosin heavy chain; it is dissociated from actin filaments by ATP, it is similar in size (200 kD) to other myosins, and antibodies directed against Dictyostelium myosin heavy chain bind to it. Immunofluorescence experiments show that a second actin-binding protein (67 kD) colocalizes in vivo with both cytoplasmic actin cables and cortical actin patches, the only identifiable actin structures in yeast. The cortical actin patches are concentrated at growing surfaces of the yeast cell where they might play a role in membrane and cell wall insertion, and the third actin-binding protein (85 kD) is only detected in association with these structures. This 85-kD protein is therefore a candidate for a determinant of growth sites. The in vivo role of this protein was tested by overproduction; this overproduction causes a reorganization of the actin cytoskeleton which in turn dramatically affects the budding pattern and spatial growth organization of the yeast cell.

• Selve N, Wegner A
• Rate of treadmilling of actin filaments in vitro.
• J Mol Biol. 1986; 187: 627-31
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• Actin filaments capped at the barbed ends were formed by polymerizing monomeric actin onto a gelsolin-actin complex. The rate of depolymerization and polymerization of the pointed ends was determined by diluting gelsolin-capped actin filaments into various concentrations of monomeric actin. Under the conditions of the experiments (100 mM-KCl, 2 mM-MgCl2 at 37 degrees C) the rate constant of dissociation of subunits both from a shortening and a lengthening filament was found to be 0.21 s-1. As the rate of dissociation of subunits from the slow pointed end determines the rate of treadmilling, it is concluded that actin filaments treadmill with a rate of about 2 micron/h.

• Yonezawa N, Nishida E, Sakai H
• pH control of actin polymerization by cofilin.
• J Biol Chem. 1985; 260: 14410-2
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• Cofilin, a 21,000 molecular weight actin-regulatory protein (Nishida, E., Maekawa, S., and Sakai, H. (1984) Biochemistry 23, 5307-5313), was here shown to be capable of reversibly controlling actin polymerization and depolymerization in a pH-sensitive manner. When cofilin was reacted with F-actin at different pH, the depolymerized actin concentration (= monomeric actin concentration) was higher at elevated pH. At pH less than 7.3, the monomeric actin concentrations did not exceed approximately 1 microM even in the presence of excess amounts of cofilin, whereas at pH greater than 7.3 it increased in proportion to the concentration of cofilin added, and complete depolymerization of F-actin occurred by the addition of an excess amount of cofilin. Moreover, in the presence of cofilin, rapid interconversion of monomeric and polymeric forms of actin can be induced by simply changing the pH of the medium. Thus, this study provides a new possible mechanism regulating actin polymerization, pH control.

• Hartwig JH, Niederman R, Lind SE
• Cortical actin structures and their relationship to mammalian cell movements.
• Subcell Biochem. 1985; 11: 1-49

• Bernstein BW, Bamburg JR
• Tropomyosin binding to F-actin protects the F-actin from disassembly by brain actin-depolymerizing factor (ADF).
• Cell Motil. 1982; 2: 1-8
• Display abstract

• Brain or muscle F-actin is rapidly depolymerized to monomeric actin in vitro by actin-depolymerizing factor, a protein isolated from chick embryo brain. Binding of muscle tropomyosin to muscle F-actin protects the F-actin from depolymerization by this factor. A 8.4/1.0 molar ratio of actin subunits to tropomyosin, achieved by incubation of the F-actin with excess tropomyosin, protects 58% of the F-actin from depolymerization by excess actin-depolymerizing factor for at least 3 hr at 25 degrees C. Thus, actin-depolymerizing factor seems to be specifically directed toward actin filaments lacking tropomyosin.

• Bamburg JR, Harris HE, Weeds AG
• Partial purification and characterization of an actin depolymerizing factor from brain.
• FEBS Lett. 1980; 121: 178-82

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