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The dynamic nature of actin polymers is modulated to facilitate a diverse\ud range of cellular processes. These dynamic properties are modulated by\ud different isoforms of Tropomyosin, which are recruited to distinct subpopulations\ud of actin polymers to differentially modulate their functional\ud properties. This makes them an attractive target for labelling discrete actin\ud populations. We have assessed the effect of different fluorescent labelling\ud strategies for this protein. Although tropomyosin fluorescent fusions\ud decorate actin in vivo, they are either non-functional or perturb regulation\ud of actin nucleation and cell cycle timings. Thus conclusions and\ud physiological relevance should be carefully evaluated when using\ud tropomyosin fusions.

Tropomyosin plays an important role in regulating the organization and functions of the actin cytoskeleton. Here, we describe a new approach to analyze the effects of tropomyosin on actin dynamics. Using F-actin proteolytically modified within the DNase-binding loop [ECP-actin], we show that tropomyosin binding almost completely suppresses the increased subunit exchange intrinsic for this F-actin. The effect is both concentration-dependent and cooperative, with half-maximal inhibition observed at about a 1:50 tropomyosin:actin ratio. Tropomyosin decreases not only the number concentration of ECP-actin filaments, but also the rate of the filament subunit exchange. Our data suggest that tropomyosin regulates the dynamics of actin filaments by an allosteric strengthening of intermonomer contacts in the actin filament, and that this mechanism may be involved in the modulation of cytoskeletal dynamics. This article is protected by copyright. All rights reserved.

We examined the hypothesis that certain actin binding proteins might be upregulated by laminar shear stress (LSS) and could contribute to endothelial wound healing. Analysis of mRNA expression profiles of human umbilical vein endothelial cells under static and LSS-exposed conditions provided a list of LSS-induced actin binding proteins including synaptopodin (SYNPO) whose endothelial expression has not been previously reported. Additional studies demonstrated that SYNPO is a key mediator of endothelial wound healing because small interfering RNA-mediated suppression of SYNPO attenuated wound closure under LSS whereas overexpression of exogenous SYNPO enhanced endothelial wound closure in the absence of LSS. This study suggests that LSS-induced actin binding proteins including SYNPO may play a critical role in the endothelial wound healing stimulated by LSS.

International audience; Understanding the structural basis of actin cytoskeleton remodeling requires stabilization of actin monomers, oligomers, and filaments in complex with partner proteins, using various biochemical strategies. Here, we report a dramatic destabilization of the dynamic interaction with a model β-thymosin/WH2 domain induced by mutations in actin. This result underlines that mutant actins should be used with prudence to characterize interactions with intrinsically disordered partners as destabilization of dynamic interactions, although identifiable by NMR, may be invisible to other structural techniques. It also highlights how both β-thymosin/WH2 domains and actin tune local structure and dynamics in regulatory processes involving intrinsically disordered domains.

Phenotype-based chemical screening is an attractive strategy for the identification of bioactive compounds. We searched for a compound that induces cellular morphological change and identified a novel compound that we named seco-clavilactone B (Seco-CB). Treatment with Seco-CB decreased the ratio of filament actin (F-actin) to globular actin (G-actin). An in vitro actin polymerization assay revealed that Seco-CB inhibited actin polymerization directly. Further analysis demonstrated that the inhibitory effect of Seco-CB on actin polymerization was associated with Seco-CB binding to either Thr5 or Cys285 of actin. These data indicate that Seco-CB is a novel actin polymerization inhibitor.

Most active processes by immune cells including adhesion, migration, and phagocytosis require the coordinated polymerization and depolymerization of filamentous actin (F-actin), which is an essential component of the actin cytoskeleton. This review focuses on a newly characterized hematopoietic cell-specific actin regulatory protein called hematopoietic protein-1 [Hem-1, also known as Nck-associated protein 1-like (Nckap1l or Nap1l)]. Hem-1 is a component of the “WAVE [WASP (Wiskott-Aldrich syndrome protein)-family verprolin homologous protein]” complex, which signals downstream of activated Rac to stimulate F-actin polymerization in response to immuno-receptor signaling. Genetic studies in cell lines and in mice suggest that Hem-1 regulates F-actin polymerization in hematopoietic cells, and may be essential for most active processes dependent on reorganization of the actin cytoskeleton in immune cells.

Cell biological functions of actin have recently expanded from cytoplasm to nucleus, with actin implicated in such diverse processes as gene expression, transcription factor regulation and intranuclear motility. Actin in the nucleus seems to behave differently than in the cytoplasm, raising new questions regarding the molecular mechanisms by which actin functions in cells. In this review, I will discuss dynamic properties of nuclear actin that are related to its polymerization cycle and nucleocytoplasmic shuttling. By comparing the behaviour of nuclear and cytoplasmic actin and their regulators, I try to dissect the underlying differences of these equally important cellular actin pools.

Shigella, the causative agent of bacillary dysentery, invades epithelial cells. Upon bacterial–cell contact, the type III bacterial effector IpaA binds to the cytoskeletal protein vinculin to promote actin reorganization required for efficient bacterial uptake. We show that the last 74 C-terminal residues of IpaA (A559) bind to human vinculin (HV) and promotes its association with actin filaments. Polymerisation experiments demonstrated that A559 was sufficient to induce HV-dependent partial capping of the barbed ends of actin filaments. These results suggest that IpaA regulates actin polymerisation/depolymerisation at sites of Shigella invasion by modulating the barbed end capping activity of vinculin.

Sjl2p is one of three yeast phosphoinositide 5′-phosphatases that belong to the conserved family of synaptojanins. Here, we show that Sjl2p is specifically associated with cortical actin patches which aggregate upon loss of the actin-regulating kinases Ark1p and Prk1p. The Sjl2p-containing clumps overlap with clathrin and early endocytic structures generated independently of NSF/Sec18p, but not with endosome- and trans Golgi network-derived membranes. Consistent with the finding that Sjl2p can bind to clathrin heavy chain in vitro, our results suggest that Sjl2p localizes to smooth endocytic vesicles that may be derived from clathrin-coated structures.

In a yeast two-hybrid system screen for Cdc42-interacting proteins, we identified a protein with similarity to the CrkII-binding protein DOCK180. A cDNA clone of this protein, designated DOCK8, encoded a gene-product of 1701 amino acid residues with a molecular mass of 190 kDa. Immunofluorescence staining showed that transiently transfected HA-tagged DOCK8, as well as endogenous DOCK8, was present at the cell edges in areas undergoing lamellipodia formation. Transient transfection of a C-terminal fragment of DOCK8 resulted in the formation of vesicular structures. Interestingly, these vesicles also contained filamentous actin. These data suggest an involvement of DOCK8 in processes that affect the organisation of filamentous actin.

The actin cytoskeleton has been implicated in protein trafficking at the Golgi apparatus and in Golgi orientation and morphology. Actin dynamics at the Golgi are regulated in part by recruiting Cdc42 or Rac to the membrane through a binding interaction with the coatomer-coated (COPI)-vesicle coat protein, coatomer. This leads to actin polymerization through the effector, N-WASP and the Arp2/3 complex. Here, we have used reconstitution of vesicle budding to test whether Arp2/3 is recruited to membranes during the formation of COPI vesicles. Our results revealed that ARF1 activation leads to greatly increased Arp3 levels on the membranes. Coatomer-bound Cdc42 and pre-existing F-actin are important for Arp2/3 binding. ARF1-dependent Arp2/3 recruitment and actin polymerization can be reconstituted on liposomal membranes, indicating that no membrane proteins are necessary. These results show that activated ARF1 can stimulate Arp2/3 recruitment to Golgi membranes through coatomer, Cdc42 or Rac, and N-WASP.

Thymosin β-10 (TB10) is a small G-actin binding protein that induces depolymerization of intracellular F-actin pools by sequestering actin monomers. Previously, we demonstrated that overexpression of TB10 in ovarian tumor cells increased the rate of cell death. As an initial step to define molecular mechanism of TB10-dependent apoptotic process in ovarian tumor cells, we searched a human ovary cDNA library for a novel TB10 binding protein using a yeast two-hybrid system. The selected protein was human E-tropomodulin (E-Tmod), another component of the actin binding proteins. Subsequently, two interacting protein components were determined quantitatively. Results showed that the full-length TB10 is required to bind with E-Tmod, and the TB10 binding site on E-Tmod partially overlaps with the actin binding site on E-Tmod. Moreover, introduction of E-Tmod cDNA into a tumor cell line reversed TB10 mediated apoptosis and restored actin architectures. These results may suggest that TB10 regulates apoptotic homeostasis by not only just binding to actin but also competing or blocking the protein complex formation of E-Tmod with actin.

To detect the posttranslational N-myristoylation of caspase substrates, the susceptibility of the newly exposed N-terminus of known caspase substrates to protein N-myristoylation was evaluated by in vivo metabolic labeling with [3H]myristic acid in transfected cells using a fusion protein in which the query sequence was fused to a model protein. As a result, it was found that the N-terminal nine residues of the newly exposed N-terminus of the caspase-cleavage product of cytoskeletal actin efficiently direct the protein N-myristoylation. Metabolic labeling of COS-1 cells transiently transfected with cDNA coding for full-length truncated actin (tActin) revealed the efficient incorporation of [3H]myristic acid into this molecule. When COS-1 cells transiently transfected with cDNA coding for full-length actin were treated with staurosporine, an apoptosis-inducing agent, an N-myristoylated tActin was generated. Immunofluorescence staining coupled with MitoTracker or fluorescence tagged-phalloidin staining revealed that exogenously expressed tActin colocalized with mitochondria without affecting cellular and actin morphology. Taken together, these results demonstrate that the C-terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage during apoptosis and targeted to mitochondria.

The vasodilator-stimulated phosphoprotein (VASP) functions as a cellular regulator of actin dynamics. VASP may initialise actin polymerisation, suggesting a direct interaction with monomeric actin. The present study demonstrates that VASP directly binds to actin monomers and that complex formation depends on a conserved four amino acid motif in the EVH2 domain. Point mutations within this motif drastically weaken VASP/G-actin interactions, thereby abolishing any actin-nucleating activity of VASP. Additionally, actin nucleation was found to depend on VASP oligomerisation since VASP monomers fail to induce the formation of actin filaments. Phosphorylation negatively affects VASP/G-actin interactions preventing VASP-induced actin filament formation. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Ca2+ of 0.3-1.0 muM induces both the exposure of tryptic cleavage sites within the gelsolin molecule inaccessible in the Ca-free conformation, and binding of one actin monomer to the N-terminal half of gelsolin. On the other hand, gelsolin-induced enhancement of pyrene actin fluorescence was observed only above 50 muM Ca2+, and a ternary actin/gelsolin complex preformed in 200 muM Ca2+ was stable only above 30 muM Ca2+. These results provide direct evidence for a Ca-induced transitions from closed to open conformation of the gelsolin molecule in the range of 3 x 10(-7) to 10(-6) M Ca2+. They also suggest that Ca2+ > 10(-5) M is required to stabilize actin-actin contacts in the 2:1 actin/gelsolin complex. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Actin ADP-ribosylated at Arg177 was previously shown not to polymerise after increasing the ionic strength, but to cap the barbed ends of filaments. Here we confirm that the polymerisation of ADP-ribosylated actin is inhibited, however, under specific conditions the modified actin copolymerises with native actin, indicating that its ability to take part in normal subunit interactions within filaments is not fully eliminated. We also show that ADP-ribosylated actin forms antiparallel but not parallel dimers: the former are not able to form filaments. ADP-ribosylated actin interacts with deoxyribonuclease I, vitamin D binding protein, thymosin β4, cofilin and gelsolin segment 1 like native actin. Interaction with myosin subfragment 1 revealed that the potential of the modified actin to aggregate into oligomers or short filaments is not fully eliminated.

Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

In vitro actin based motility assays with bacterial pathogens have provided powerful systems to both understand and dissect actin dynamics as well as cell motility. Taking advantage of endogenous membrane vesicles in Xenopus extracts we have developed an in vitro assay to study membrane dependent actin polymerization. Our results demonstrate that membrane dependent actin polymerization, in contrast to Listeria stimulated actin filament assembly, is dependent on small GTPases of the Rho family. Using a combination of depletion and reconstitution experiments we have shown that Cdc42 but not Rac or Rho is required to stimulate actin polymerization from membranes. The in vitro system we have described here is amenable to identification of the downstream effectors of Cdc42 required for membrane dependent actin polymerization.

Recombinant plant (birch) profilin was analyzed for its ability to promote actin polymerization from the actin:thymosin beta4 and beta9 complex. Depending on the nature of the divalent cation, recombinant plant (birch) profilin exhibited two different modes of interaction with actin, like mammalian profilin. In the presence of magnesium ions birch profilin promoted the polymerization of actin from A:Tbeta4. In contrast, in the presence of calcium but absence of magnesium ions birch profilin was unable to initiate the polymerization of actin from the complex with Tbeta4. However, under these conditions profilin formed a stable stoichiometric complex with skeletal muscle alpha-actin, as verified by its ability to increase the critical concentration of actin polymerization. Chemical cross-linking indicated that birch profilin competes with Tbeta4 for actin binding. Ternary complex formation of birch profilin with actin:DNase I complex was suggested by chemical cross-linking. However, the determination of the critical concentrations of actin polymerization in the simultaneous presence of birch profilin and DNase I indicated that profilin and DNase I did not form a ternary complex. These data indicated a negative co-operativity between the profilin and DNase I binding sites on actin.

We have studied the intracellular localization of glycogenin by fusing green fluorescent protein (GFP) to the N-terminus of rabbit muscle glycogenin and expressing the chimeric protein in C2C12, COS-1 and rat hepatic cells. The fusion protein showed a nuclear and cytosolic distribution and partially co-localized with actin in the cytosol. Disruption of the actin cytoskeleton with cytochalasin D led to a change in the pattern of green fluorescence, which coincided with that observed for the remaining non-depolymerized actin. The distribution of the single point mutant K324A was completely uniform and was not affected by this drug. These findings indicate that rabbit muscle glycogenin binds to actin through the heptapeptide 321DNIKKKL327, a common motif found in other actin-binding proteins, which is located at the C-terminal end of this protein, and suggest that the actin cytoskeleton plays an important role in glycogen metabolism.

C- and N-terminally truncated fragments of earthworm gelsolin were constructed, cloned and expressed in Escherichia coli. G-actin-binding properties of these fragments and their influences on the polymeric state of actin were investigated. A construct lacking a large part of the third segment [E(1–295)] supports actin nucleation similar to the complete protein and shows reduced actin fragmentation property, but is no longer Ca2+-sensitive in its activity. The first and the second segments (E1 and E2) each contain one actin-binding site. In contrast to human gelsolin, E1 in combination with a short N-terminal region of E2 is not sufficient for the F-actin-severing activity of the protein.

Smooth muscle cell calponin (h1 or basic isoform) is an actin-binding protein that inhibits actomyosin MgATPase activity and is abundantly expressed in differentiated smooth muscle. Western blots showed bovine tracheal (BT) smooth muscle cells in culture expressed only 2 +/- 1% (n = 8) of the amount of calponin in tissues, while NIH-3T3 fibroblasts expressed none. We tested the hypothesis that introduction of calponin to cultured BT and 3T3 cells would inhibit cytoskeletal activities associated with cell proliferation. To achieve high-efficiency expression, an adenovirus encoding the CMV-calponin construct (Adv-CaP) was generated by homologous recombination in 293 cells. With greater than 90% of BT and 3T3 cells infected with Adv-CaP, calponin expression (32 and 11 microg/mg total protein, respectively) was similar to that in smooth muscle tissues (51 microg/mg). Cells were infected with Adv-CaP for 48 h, replated at low density and proliferation rates were assessed by cell density and [3H]thymidine incorporation. Cell growth and DNA synthesis by Adv-CaP-infected cells were inhibited to one-third control values for both BT and 3T3 cells. Expressed calponin was localized primarily on stress fibers in both cell types. Calponin may act at the cytoskeletal level to retard signaling pathways that normally lead to tight coupling between cell shape and DNA synthesis.

The small GTPase Rho regulates several actomyosin-based cellular processes such as cell adhesion, cytokinesis and contraction. The biochemical mechanisms of these actions remain unknown. Recently, several GTP-Rho binding proteins were isolated. Among them, p140mDia and p160ROCK appear to work as Rho effectors mediating its action on the cytoskeleton. p140mDia induces actin polymerization by recruiting an actin binding protein, profilin, to the site of Rho action. p160ROCK induces focal adhesions and stress fibers by activating integrin and clustering them by the use of myosin-based contractility.

A cDNA corresponding to a putative actin-binding protein was cloned from a cold-acclimated wheat cDNA library. The cDNA, designated Wcor719, encodes a polypeptide of 142 amino acids with a calculated molecular mass of 15.8 kDa and a pI of 4.27. The protein has the two conserved domains identified as actin and phosphatidylinositol 4,5-biphosphate (PIP2) binding sites found in members of the cofilin family. Northern analyses revealed that Wcor719 transcript accumulation is rapid and strongly up-regulated by low temperature. This accumulation was greater in the tolerant winter wheat and rye species compared to the less tolerant ones. The rapidity of transcript induction and the significant homology with actin-binding proteins (ABP) from different organisms suggest that the product of this gene might be involved in the dynamic reorganization of the cytoskeleton during low temperature acclimation. It may also serve as a key factor in the signal transduction pathway during cold acclimation.

We have recently described a method for identifying contact sites between actin and thymosin beta4 (Tbeta4) by following spectrophotometrically the extent and kinetics of distinct, thiol-specific crosslinking reactions between appropriate derivatives of the two proteins [Reichert et a]. (1996) J. Biol. Chem. 271, 1301-1308]. In the present study this method was used to show that such crosslinking, which is indicative of complex formation, occurs to the same extent with the actin-DNase I complex as with pure actin, although at a somewhat lower rate. Further evidence for the formation of the ternary complex was given by gel electrophoresis. From fluorescence spectroscopy the KD value of Tbeta4 from the actin-DNase I complex was found to be identical to that from pure actin. In line with these data, the capacity of actin for inhibiting DNase I was not affected by the addition of Tbeta4. In conclusion, DNase I and Tbeta4 are independent of each other in their interaction with actin, suggesting that the binding sites of thymosin beta4 and DNase I on actin do not overlap. A ternary complex of DNase I, actin and Tbeta4, if obtained in crystalline form, could thus provide an approach for studying the interface of Tbeta4 and actin by X-ray analysis.

Two-dimensional hydrophobic clusters analysis (HCA) was used to compare the distribution of hydrophobic clusters along various actin sequence. HCA-deduced patterns were not altered by amino-acid variations throughout the evolution of actin and we observed similar hydrophobic motifs comprising myosin subfragment-1 ATP-independent binding sites. HCA suggested the presence of two groups of identical hydrophobic motifs (A1 and A2) which bound on each side of the S1 (63 kDa-31 kDa) connecting segment in relation with two actin monomers. This connection is important in communications between actin- and nucleotide-binding sites. We postulate that some relation and message between the two motifs A1 and A2 take place through myosin subfragment-1 (63 kDa-31 kDa) connecting segment.

Actin-crosslinking proteins link F-actin into the bundles and networks that constitute the cytoskeleton. Dystrophin, β-spectrin, α-actinin, ABP-120, ABP-280, and fimbrin share homologous actin-binding domains and comprise an actin crosslinker superfamily. We have identified a novel member of this superfamily (ACF7) using a degenerate primer-mediated PCR strategy that was optimized to resolve less-abundant superfamily sequences. The ACF7 gene is on human chromosome 1 and hybridizes to high molecular weight bands on northern blots. Sequence comparisons argue that ACF7 does not fit into one of the existing families, but represents a new class within the superfamily.

A 57 kDa protein (p57) was obtained during the study on phosphatidylinositol-specific phospholipase C. Its cDNA was isolated from calf spleen and human leukemia cell line HL60 libraries and cloned. In the primary structures of p57, they have two unique amino acid sequence motifs, a WD repeat and a leucine zipper motif. Furthermore, p57 shared sequence similarity (40%) with coronin, an actin-binding protein responsible for chemotaxis, cell motility, and cytokinesis of Dictyostelium discoideum, which has only the WD repeat. p57 also showed an actin-binding activity and was mainly expressed in immune tissues. From these results, we conclude that p57 is a coronin-like novel actin-binding protein in mammalian cells but may also have a different function from coronin.

The F-actin binding properties of chicken villin, its headpiece and domains 2-3 (V2-3) have been analysed to identify sites involved in bundle formation. Headpiece and V2-3 bind actin with Kd values of approximately 7 microM and approximately 0.3 microM, respectively, at low ionic strength. V2-3 binding, like that of villin, is weakened with increasing salt concentration; headpiece binding is not. Competition experiments show that headpiece and V2-3 bind to different sites on actin, forming the two cross-linking sites of villin. Headpiece does not compete with the F-actin binding domains of gelsolin or alpha-actinin, but it dissociates actin depolymerizing factor. We suggest that the F-actin binding domains of actin severing, crosslinking and capping proteins can be organized into two classes.

Evolutionary conservation for structure function relations is commonly accepted. Here we hypothesize that closely related single domain paralogous proteins, having similar expression profiles and redundant biochemical core functions, additionally evolved to allow and maintain isoform specific differential regulation by single conserved amino acid substitutions. To substantiate this, we considered two families of closely related actin binding proteins combined with data mining of phosphorylated residues in human and mouse proteins. We show that such residues are identical in other orthologs whereas paralogs have a different, but also conserved, non-phosphorylatable residue at the equivalent positions.

RUN domain is present in several proteins related to the functions of Rap and Rab family GTPases. Accumulating evidence supports the hypothesis that RUN domain-containing proteins act as a component of vesicle traffic and might be responsible for an interaction with a filamentous network linked to actin cytoskeleton or microtubules. That is to say, on one hand, RUN domains associate with Rab or Rap family proteins, on the other hand, they also might interact with motor proteins such as kinesin or myosin via intervention molecules. In this review, we summarize the background and current status of RUN domain research with an emphasis on the interaction between RUN domain and motor proteins with respect to the vesicle traffic on filamentous network.

We provide evidence that one of the 11 Arabidopsis actin-depolymerizing factors (ADFs), namely ADF9, does not display typical F-actin depolymerizing activity. Instead, ADF9 effectively stabilizes actin filaments in vitro and concomitantly bundles actin filaments with the highest efficiency under acidic conditions. Competition experiments show that ADF9 antagonizes ADF1 activity by reducing its ability to potentiate F-actin depolymerization. Accordingly, ectopic expression of ADF1 and ADF9 in tobacco cells has opposite effects. ADF1 severs actin filaments/bundles and promotes actin cytoskeleton disassembly, whereas ADF9 induces the formation of long bundles. Together these data reveal an additional degree of complexity in the comprehension of the biological functions of the ADF family and illustrate that antagonist activities can be displayed by seemingly equivalent actin-binding proteins.

The 100 kDa actin-binding protein from Dictyostelium amoebae is an F-actin capping protein that displays neither severing nor crosslinking nor nucleating activities [Hofmann et al. (1992) Cell Motil. Cytoskel. 23,133-144]. Cloning and sequencing of the gene revealed that the protein is highly homologous to vertebrate villin, a unique component of brush border microvilli and contains six domains fused to a villin-like headpiece domain via a threonine/proline rich neck region. The functional differences and similarities between the 100 kDa protein and villin are reflected in the amino acid sequences. We draw from the data the following conclusions, (i) The presence of a six domain protein in Dictyostelium suggests that in contrast to the current view gene duplications must have happened before Dictyostelium branched off during evolution, (ii) The villin-like molecule in Dictyostelium appears to be a premature villin (‘protovillin’) which is able to cap actin filaments but still lacks the other villin-type actin-binding activities. This renders capping of actin filaments as the evolutionarily oldest function of an F-actin binding protein.

It has been shown that the EGTA-resistant actin, one of the two actin molecules associated to gelsolin, can be predominantly cross-linked to gelsolin by benzophenone-4-maleimide (BPM), a photoaffinity-labeling reagent, which was conjugated to Cys-374 of actin prior to cross-linking (Doi, Y., Banba, M. and Vertut-Doï, A. (1991) Biochemistry 30, 5769–5777). When a chymotryptic digest of gelsolin containing the amino-terminal 15-kDa fragment was mixed with BPM-actin (42 kDa) and irradiated for cross-linking, a band of 58 kDa appeared on SDS-PAGE which was shown to contain actin molecule by using fluorescently labeled actin. The amino-terminal sequence of the 58-kDa complex was identical to that of gelsolin, confirming that the amino-terminal segment (residues 1–133) of pig plasma gelsolin lies closely to Cys-374 of actin in the EGTA-resistant complex.

The amino-terminal region of actin participates in the binding of myosin subfragment 1 (S1) during cross-bridge cycling, thereby assisting in the activation of the magnesium-dependent myosin ATPase. Effects of three actin fragments on the magnesium-dependent S1 and acto-S1 ATPase activities in solution were studied. One of the peptides, containing residues actin 1–44, mimicked the S1 ATPase-activating properties of actin and in turn inhibited acto-S1 ATPase both in a concentration-dependent manner. This suggests peptide competition for the actin binding site on myosin. The other fragments, residues actin 1–18 and 82–119, respectively, had no detectable effect on S1- and acto-S1 ATPase activity.

Platelet talin binds to actin in vitro and hence is an actin binding protein. By four different non-interfering assay conditions (fluorescence, fluorescence recovery after photobleaching, (FRAP), dynamic light scattering and DNase-I inhibition) we show that talin promotes filament nucleation, raises the filament number concentration and increases the net rate of actin polymerization but has no inhibitory effect on filament elongation. Binding of talin to actin occurs at a maximal molar ratio of 1:3 as determined by fluorescencetitration under G-buffer conditions. The overall binding constant was approximately 0.25 microM.

The effect of troponin I and caldesmon on phalloidin-rhodamine- and 1,5-IAEDANS-labelled actin in skeletal muscle ghost fibers was investigated by polarized fluorescence. Both these proteins inhibited the structural alterations in the actin monomer and the increase of flexibility of actin filaments occurring on binding of myosin heads, and their effects were potentiated by tropomyosin. This immobilization of the actin filament through troponin I and caldesmon seems to originate from restriction of the relative motions of the two domains within the monomer.

Microfilaments exist in a dynamic equilibrium between monomeric and polymerized actin and the ratio of monomers to polymeric forms is influenced by a variety of extracellular stimuli. The polymerization, depolymerization and redistribution of actin filaments are modulated by several actin-binding proteins, which are regulated by upstream signalling molecules. Actin cytoskeleton is involved in diverse cellular functions including migration, ion channels activity, secretion, apoptosis and cell survival. In this review we have outlined the role of actin dynamics in representative cell functions induced by the early response to extracellular stimuli.

Villin is a tissue-specific actin modifying protein that is associated with actin filaments in the microvilli and terminal web of epithelial cells. It belongs to a large family of actin-binding proteins which includes actin-capping, -nucleating and/or -severing proteins such as gelsolin, severin, fragmin, adseverin/scinderin and actin crosslinking proteins such as dematin and supervillin. Studies done in epithelial cell lines and villin knock-out mice have demonstrated the function of villin in regulating actin dynamics, cell morphology, epithelial-to-mesenchymal transition, cell migration and cell survival. In addition, the ligand-binding properties of villin (F-actin, G-actin, calcium, phospholipids and phospholipase C-γ1) are mechanistically important for the crosstalk between signaling pathways and actin reorganization in epithelial cells.

Pan1 is an actin patch-associated protein involved in endocytosis. Our studies revealed that in oleate-grown cells Pan1 is located in the nucleus as well as in patches. One of three putative nuclear localization signals (NLS) of Pan1, NLS2, directed beta-galactosidase (beta-gal) to the nucleus. However, GFP-Pan1(886-1219), containing NLS2, was found in the cytoplasm indicating that it may contain a nuclear export signal (NES). A putative Pan1 NES, overlapping with NLS3, re-addressed NLS(H2B)-NES/NLS3-beta-gal from the nucleus to the cytoplasm. Inactivation of the NES allowed NLS3 to be effective. Thus, Pan1 contains functional NLSs and a NES and appears to shuttle in certain circumstances.

Actin has been reported to enhance the superoxide-generating activity of neutrophil NADPH oxidase in a cell-free system and to interact with p47phox, a regulatory subunit of the oxidase. In the present study, we searched for an actin-binding site in p47phox by far-western blotting and blot-binding assays using truncated forms of p47phox. The amino-acid sequence 319–337 was identified as an actin-binding site, and a synthetic peptide of this sequence bound to actin. The sequence shows no homology to other known actin-binding motifs. It is located in the autoinhibitory region of p47phox and includes Ser-328, a phosphorylation site essential for unmasking. Although a phosphorylation-mimetic p47phox mutant bound to actin with a lower affinity than the wild type, the same mutant interacted with filamentous actin more efficiently than the wild type. A mutant peptide p47phox (319–337, Ser328Glu) bound to filamentous actin more tightly than to monomer actin. These results suggest that p47phox moves to cortical actin when it becomes unmasked in the cells.

p116Rip, originally identified as a binding partner of activated RhoA, is an actin-binding protein that interacts with the regulatory myosin-binding subunit (MBS) of myosin-II phosphatase and is essential for Rho-regulated cytoskeletal contractility. Here, we have examined the role of p116Rip in RhoA-mediated activation of the transcription factor SRF. We show that p116Rip oligomerizes via its C-terminal coiled-coil domain and, when overexpressed, inhibits RhoA-induced SRF activation without affecting RhoA-GTP levels. Mutant forms of p116Rip that fail to oligomerize or bind to MBS are still capable of inhibiting SRF activity. Our results suggest that p116Rip interferes with RhoA-mediated transcription through its ability to disassemble the actomyosin cytoskeleton downstream of RhoA.

The immune cell specific protein Fyn-T binding protein (Fyb) has been identified as a target of the Yersinia antiphagocytic effector Yersinia outer protein H (YopH), but its role in macrophages is unknown. By using Fyb domains as bait to screen a mouse lymphoma cDNA library, we identified a novel interaction partner, mammalian actin binding protein 1 (mAbp1). We show that mAbp1 binds the Fyb N-terminal via its C-terminally located src homology 3 domain. The interaction between Fyb and mAbp1 is detected in macrophage lysates and the proteins co-localize with F-actin in the leading edge. Hence, mAbp1 is likely to constitute a downstream effector of Fyb involved in F-actin dynamics.

Cysteine-rich proteins (CRPs) have been shown to be involved in cell differentiation, transcriptional regulation and the organisation of the actin cytoskeleton. Thus far, the latter function has been inferred solely from the in vitro interaction of CRP1, CRP2, and CRP3 with alpha-actinin and zyxin. We show here that purified, recombinant CRP2 binds directly to F-actin in vitro in co-sedimentation assays. Using a green fluorescent protein (GFP)-tagged construct of CRP2 we analysed its localisation and dynamics in A7r5 rat smooth muscle cells. CRP2 was associated with the actin cytoskeleton and decorated actin stress fibres in a continuous fashion, unlike the periodic labelling pattern observed for alpha-actinin and zyxin, which also accumulate in focal adhesions. Using live video fluorescence microscopy we observed the behaviour of GFP-CRP2 during the dynamic rearrangement of the actin cytoskeleton in phorbol 12,13-dibutyrate-treated A7r5 cells. In contrast to the actin-binding proteins SM22alpha and alpha-actinin, GFP-CRP2 did not translocate into the podosomes induced by this treatment, but remained preferentially bound to the stress fibres, suggesting an actin filament-stabilising role for CRP2. When fused to the mitochondrial targeting sequence from the Listeria protein ActA, GFP-CRP2 was almost completely localised to mitochondria, but no significant recruitment of either alpha-actinin or zyxin could be observed. Taken together, our results demonstrate that CRP2 can bind to F-actin directly and that the association with the actin cytoskeleton is independent of alpha-actinin or zyxin localisation in the cell, thus questioning the role of CRP2 as a regulator of alpha-actinin function in vivo.

An actin-binding protein domain we call here ‘calponin-homology’ or CH is present in signalling proteins such as Vav which are involved in activation and inactivation of small G-proteins. Using profile methods, we have detected two repeats of this domain in the actin-binding region of α-actinin and related proteins. Based on this, we propose that CH domain in Vav and other signalling proteins is employed for association with filamentous actin, and that this function correlates with their control on the G-proteins Rac and Rho which are involved in the organization of cytoskeleton.