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At least 37 records · Page 2Linked to original sources
PubMed1998

Disruption of the actin cytoskeleton of mammalian cells by the capping complex actin-fragmin is inhibited by actin phosphorylation and regulated by Ca2+ ions.

Fragmin from Physarum polycephalum is a gelsolin-like actin-binding protein and interferes with the growth of actin filaments in vitro by severing actin filaments and capping their barbed ends through formation of an actin-fragmin dimer in a Ca2+-dependent manner. The actin-fragmin dimer is phosphorylated in vivo and in vitro on the actin subunit by the actin-fragmin kinase. We have studied the properties of these capping proteins and their regulation by actin phosphorylation and Ca2+ ions in living PtK2, CV1 and NIH3T3 cultured cells by microinjection or by expression in conjunction with immunostaining and fluorescence microscopy. Microinjection of the actin-fragmin dimer disintegrated the actin cytoskeleton and altered cell morphology. This in vivo effect could be blocked by phosphorylation of the actin subunit by the actin-fragmin kinase in low Ca2+ conditions, and the capping activity could be recovered by high Ca2+ concentration, probably through activation of the second actin-binding site in fragmin. This suggests that in Physarum microplasmodia, actin polymerization can be controlled in a Ca2+-dependent manner through the phosphorylation of actin. Microinjected or overexpressed recombinant fragmin did not affect the actin-based cytoskeleton or cell morphology of resting cells, unless the cytosolic free Ca2+ concentration was increased by microinjection of a Ca2+-containing buffer. The cells were able to revert to their normal phenotype which indicates that endogenous regulatory mechanisms counteracted fragmin activity, probably by uncapping fragmin from the barbed ends of filaments. Fragmin also antagonized formation of stress fibers induced by lysophosphatidic acid. Our findings demonstrate that the interactions between actin and fragmin are tightly regulated by the cytosolic Ca2+ concentration and this provides a basis for a more general mechanism in higher organisms to regulate microfilament organization.

3T3 Cells↗
PubMed1997

F-actin and G-actin binding are uncoupled by mutation of conserved tyrosine residues in maize actin depolymerizing factor (ZmADF).

Actin depolymerizing factors (ADF) are stimulus responsive actin cytoskeleton modulating proteins. They bind both monomeric actin (G-actin) and filamentous actin (F-actin) and, under certain conditions, F-actin binding is followed by filament severing. In this paper, using mutant maize ADF3 proteins, we demonstrate that the maize ADF3 binding of F-actin can be spatially distinguished from that of G-actin. One mutant, zmadf3-1, in which Tyr-103 and Ala-104 (equivalent to destrin Tyr-117 and Ala-118) have been replaced by phenylalanine and glycine, respectively, binds more weakly to both G-actin and F-actin compared with maize ADF3. A second mutant, zmadf3-2, in which both Tyr-67 and Tyr-70 are replaced by phenylalanine, shows an affinity for G-actin similar to maize ADF3, but F-actin binding is abolished. The two tyrosines, Tyr-67 and Tyr-70, are in the equivalent position to Tyr-82 and Tyr-85 of destrin, respectively. Using the tertiary structure of destrin, yeast cofilin, and Acanthamoeba actophorin, we discuss the implications of removing the aromatic hydroxyls of Tyr-82 and Tyr-85 (i.e., the effect of substituting phenylalanine for tyrosine) and conclude that Tyr-82 plays a critical role in stabilizing the tertiary structure that is essential for F-actin binding. We propose that this tertiary structure is maintained as a result of a hydrogen bond between the hydroxyl of Tyr-82 and the carbonyl of Tyr-117, which is located in the long alpha-helix; amino acid components of this helix (Leu-111 to Phe-128) have been implicated in G-actin and F-actin binding. The structures of human destrin and yeast cofilin indicate a hydrogen distance of 2.61 and 2.77 A, respectively, with corresponding bond angles of 99.5 degrees and 113 degrees, close to the optimum for a strong hydrogen bond.

Actin Depolymerizing Factors↗
PubMed1997

Mapping the binding site of thymosin beta4 on actin by competition with G-actin binding proteins indicates negative co-operativity between binding sites located on opposite subdomains of actin.

The beta-thymosins are small monomeric (G-)actin-binding proteins of 5 kDa that are supposed to act intracellularly as actin-sequestering factors stabilizing the cytoplasmic monomeric pool of actin. The binding region of thymosin beta4 was determined by analysing the binding of thymosin beta4 to actin complexed with DNase I, gelsolin or gelsolin segment 1. Binding was analysed by determining the increase in the critical concentration of actin polymerization by native gel electrophoresis or chemical cross-linking. The formation of a ternary complex including thymosin beta4 should indicate that the actin-binding proteins attach to different sites on actin. Competition would be indicative of binding to identical or overlapping sites on actin or of a negative co-operative linkage between the two binding sites. Competition of thymosin beta4 for actin binding was observed in the presence of intact gelsolin or the N-terminal gelsolin fragment, segment 1, indicating that thymosin beta4 binds to a site close to or identical with the gelsolin segment 1-binding site. The ternary complex of actin-DNase I-thymosin beta4 was obtained only when using the chemically cross-linked actin-thymosin beta4 complex, indicating that thymosin beta4 is dissociated by the binding of DNase I to actin. It is suggested that the dissociation of thymosin beta4 by DNase I binding to actin is caused by negative co-operativity between their spatially separated binding sites on actin. A similar negative co-operativity was observed between DNase I and gelsolin segment 1 binding to actin. The results therefore indicate that the respective binding sites for DNase I and segment 1 on subdomains 1 and 2 of actin are linked in a negative co-operative manner.

Actins↗
PubMed1986

The effect of the 540-kilodalton actin cross-linking protein, actin-binding protein, on the mechanical properties of F-actin.

This study describes the effect of actin-binding protein derived from rabbit lung macrophages on the mechanical properties of F-actin. The dynamic storage modulus, G'(omega), and loss modulus, G"(omega) of F-actin, at concentrations from 1 to 4 mg/ml, in the absence or presence of actin-binding protein at molar ratios to actin of 1:1000 to 1:125, were measured at frequencies ranging from 3 X 10(-3) to 0.5 Hz. Actin-binding protein increased the dynamic moduli of F-actin, but this increase was much greater as either the actin-binding protein/actin ratio or the total protein concentration increased. Moreover, there was a convergence of the values of G' and G" at high frequencies for F-actin which became more prominent upon the addition of actin-binding protein. The value of the modulus obtained by an extrapolation of these data to actin concentrations similar to that found in the cell cortex was close to values which have been obtained by direct measurements. The addition of actin-binding protein to an F-actin solution enabled it to reach an equilibrium strain following the application of a stress, in contrast to pure F-actin. These data allow a more rigorous definition of the "sol" to "gel" transition and suggest that the cross-linking of actin filaments by actin-binding protein leads to the formation of a network structure whose underlying mechanism of mechanical behavior is short range intrafilament bending in contrast to the classical rubber network.

Actins↗
PubMed1978

Actin amino-acid sequences. Comparison of actins from calf thymus, bovine brain, and SV40-transformed mouse 3T3 cells with rabbit skeletal muscle actin.

Actin was purified from calf thymus, bovine brain and SV40-transformed mouse 3T3 cells grown in tissue culture. Isoelectric focusing analysis showed the presence of the two actin polypeptides beta and gamma typical for non-muscle actins in all three actins. Tryptic and thermolytic peptides accounting for the complete amino-acid sequence of the cytoplasmic actins were separated and isolated by preparative fingerprint techniques. All peptides were characterized by amino-acid analysis and compared with the corresponding peptides from rabbit skeletal muscle actin. Peptides which differed in amino-acid composition from the corresponding skeletal muscle actin peptides were subjected to sequence analysis in order to localize the amino-acid replacement. The results obtained show that all three mammalian cytoplasmic actins studied contain the same amino-acid exchanges indicating that mammalian cytoplasmic actins are very similar if not identical in amino-acid sequence. The presence of two different isoelectric species beta and gamma in cytoplasmic actins from higher vertebrates is acccounted for by the isolation of two very similar but not identical amino-terminal peptides in all three actin preparations. The nature of the amino-acid replacements in these two peptides not only accounts for the different isoelectric forms but also shows that beta and gamma cytoplasmic actins are the products of two different structural genes expressed in the same cell. The total number of amino-acid replacements so far detected in the comparison of these cytoplasmic actins and skeletal muscle actin is 25 for the beta chain and 24 for the gamma chain. With the exception of the amino-terminal three or four residues, which are responsible for the isoelectric differences, the replacements do not involve charged amino acids. The exchanges are not randomly distributed. No replacements were detected in regions 18--75 and 299--356 while the regions between residues 2--17 and 259--298 show a high number of replacements. In addition documentation for a few minor revisions of the amino acid sequence of rabbit skeletal muscle actin is provided.

Actins↗
PubMed2001

Role of actin polymerization and actin cables in actin-patch movement in Schizosaccharomyces pombe.

Factors that are involved in actin polymerization, such as the Arp2/3 complex, have been found to be packaged into discrete, motile, actin-rich foci. Here we investigate the mechanism of actin-patch motility in S. pombe using a fusion of green fluorescent protein (GFP) to a coronin homologue, Crn1p. Actin patches are associated with cables and move with rates of 0.32 microm s(-1) primarily in an undirected manner at cell tips and also in a directed manner along actin cables, often away from cell tips. Patches move more slowly or stop when actin polymerization is attenuated by Latrunculin A or in arp3 and cdc3 (profilin) mutants. In a cdc8 (tropomyosin) mutant, actin cables are absent, and patches move with similar speed but in a non-directed manner. Patches are sites of Arp3-dependent F-actin polymerization in vitro. Rapid F-actin turnover rates in vivo indicate that patches and cables are maintained continuously by actin polymerization. Our studies give rise to a model in which actin patches are centres for actin polymerization that drive their own movement on actin cables using Arp2/3-based actin polymerization.

Actin-Related Protein 2↗
PubMed2002

Actin filament barbed end elongation with nonmuscle MgATP-actin and MgADP-actin in the presence of profilin.

We have quantitated the in vitro interactions of profilin and the profilin-actin complex (PA) with the actin filament barbed end using profilin and nonmuscle beta,gamma-actin prepared from bovine spleen. Actin filament barbed end elongation was initiated from spectrin seeds in the presence of varying profilin concentrations and followed by light scattering. We find that profilin inhibits actin polymerization and that this effect is much more pronounced for beta,gamma-actin than for alpha-skeletal muscle actin. Profilin binds to beta,gamma-actin filament barbed ends with an equilibrium constant of 20 microM, decreases the filament elongation rate by blocking addition of actin monomers, and increases the dissociation rate of actin monomers from the filament end. PA containing bound MgADP supports elongation of the actin filament barbed end, indicating that ATP hydrolysis is not necessary for PA elongation of filaments. Initial analysis of the energetics for these reactions suggested an apparent greater negative free energy change for actin filament elongation from PA than elongation from monomeric actin. However, we calculate that the free energy changes for the two elongation pathways are equal if the profilin-induced weakening of nucleotide binding to actin is taken into consideration.

Actin Cytoskeleton↗
PubMed1998

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.

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.

Acanthamoeba↗
PubMed1996

Slow axonal transport of soluble actin with actin depolymerizing factor, cofilin, and profilin suggests actin moves in an unassembled form.

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.

Actin Depolymerizing Factors↗
PubMed2004

Anti-actin antibodies generated against profilin:actin distinguish between non-filamentous and filamentous actin, and label cultured cells in a dotted pattern.

Actin polymerization is a prominent feature of migrating cells, where it powers the protrusion of the leading edge. Many studies have characterized the well-ordered and dynamic arrangement of filamentous actin in this submembraneous space. However, less is known about the organization of unpolymerized actin. Previously, we reported on the use of covalently coupled profilin:actin to study actin dynamics and presented evidence that profilin-bound actin is a major source of actin for filament growth. To locate profilin:actin in the cell we have now used this non-dissociable complex for antibody generation, and obtained monospecific anti-actin and anti-profilin antibodies from two separate immunizations. Fluorescence microscopy revealed drastic differences in the staining pattern generated by the anti-actin antibody preparations. With one, distinct puncta appeared at the actin-rich leading edge and sometimes aligned with microtubules in the interior of the lamella, while the other displayed typical actin filament staining. Labelling experiments in vitro demonstrated failure of the first antibody to recognize filamentous actin and none of the two bound microtubules. The two anti-profilin antibodies purified in parallel generated a punctated pattern similar to that seen with the first anti-actin antibody. All antibody preparations labelled the nuclei.

Actin Cytoskeleton↗
PubMed1999

Autoregulation of actin synthesis requires the 3'-UTR of actin mRNA and protects cells from actin overproduction.

Monomeric (G) actin was shown to be involved in inhibiting its own synthesis by an autoregulatory mechanism that includes enhanced degradation of the actin mRNA [Bershadsky et al., 1995; Lyubimova et al., 1997]. We show that the 3'-untranslated region (3'-UTR) of beta-actin mRNA, but not its 5'-untranslated region, is important for this regulation. The level of full-length beta-actin mRNA in cells was reduced when actin filaments were depolymerized by treatment with latrunculin A and elevated when actin polymerization was induced by jasplakinolide. By contrast, the level of actin mRNA lacking the 3'-UTR remained unchanged when these drugs modulated the dynamics of actin assembly in the cell. Moreover, the transfection of cells with a construct encoding the autoregulation-deficient form of beta-actin mRNA led to very high levels of actin expression compared with transfection with the control actin construct and was accompanied by characteristic changes in cell morphology and the structure of the actin cytoskeleton. These results suggest that the autoregulatory mechanism working via the 3'-UTR of actin mRNA is involved in controlling the maintenance of a defined pool of actin monomers that could be necessary for the proper organization of the microfilament system and the cytoskeleton-mediated signaling.

3' Untranslated Regions↗
PubMed1993

Bacterial lipopolysaccharide induces actin reorganization, intercellular gap formation, and endothelial barrier dysfunction in pulmonary vascular endothelial cells: concurrent F-actin depolymerization and new actin synthesis.

Bacterial lipopolysaccharide (LPS) influences pulmonary vascular endothelial barrier function in vitro. We studied whether LPS regulates endothelial barrier function through actin reorganization. Postconfluent bovine pulmonary artery endothelial cell monolayers were exposed to Escherichia coli 0111:B4 LPS 10 ng/ml or media for up to 6 h and evaluated for: 1) transendothelial 14C-albumin flux, 2) F-actin organization with fluorescence microscopy, 3) F-actin quantitation by spectrofluorometry, and 4) monomeric G-actin levels by the DNAse 1 inhibition assay. LPS induced increments in 14C-albumin flux (P < 0.001) and intercellular gap formation at > or = 2-6 h. During this same time period the endothelial F-actin pool was not significantly changed compared to simultaneous media controls. Mean (+/- SE) G-actin (micrograms/mg total protein) was significantly (P < 0.002) increased compared to simultaneous media controls at 2, 4, and 6 h but not at 0.5 or 1 h. Prior F-actin stabilization with phallicidin protected against the LPS-induced increments in G-actin (P = 0.040) as well as changes in barrier function (P < 0.0001). Prior protein synthesis inhibition unmasked an LPS-induced decrement in F-actin (P = 0.0044), blunted the G-actin increment (P = 0.010), and increased LPS-induced changes in endothelial barrier function (P < 0.0001). Therefore, LPS induces pulmonary vascular endothelial F-actin depolymerization, intercellular gap formation, and barrier dysfunction. Over the same time period, LPS increased total actin (P < 0.0001) and new actin synthesis (P = 0.0063) which may be a compensatory endothelial cell response to LPS-induced F-actin depolymerization.

Actins↗
PubMed1992

Actin kinase: a protein kinase that phosphorylates actin of fragmin-actin complex.

Actin of fragmin-actin complex is phosphorylated by an endogenous kinase from plasmodium of Physarum polycephalum. The phosphorylation abolishes the nucleation and capping activities of fragmin-actin complex. The kinase has been purified and termed actin kinase [Furuhashi, K. & Hatano, S. (1990) J. Cell Biol. 111, 1081-1087]. Enzymatic properties of the purified actin kinase were studied in detail. Actin kinase exhibited the highest activity under conditions physiological for the plasmodium (30 mM KCl, 6 mM MgCl2, pH 7.0). The Vmax and the Km of the enzyme for ATP were about 83 mumol/min/mg and 25 microM, respectively. The Km for fragmin-actin complex was 190 nM. The purified actin kinase phosphorylated actin of fragmin-actin complex at a constant rate regardless of Ca2+ concentration. Similarly, 2 microM cAMP, 2 microM cGMP, 2 micrograms/ml calmodulin in the presence of Ca2+ or 1 mM GTP showed no effect on the activity of the purified enzyme. Actin kinase did not phosphorylate histone H1, H2B, alpha-casein, or beta-casein, suggesting that actin kinase is a new kind of protein kinase which specifically phosphorylates actin of the fragmin-actin complex.

Actins↗
PubMed1992

Actin-actin contact: inhibition of actin-polymerization by subdomain 4 peptide fragments.

F-Actin was digested with alpha-chymotrypsin in 6 M urea, and two peptide fragments from subdomain 4 of actin molecule [Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F., & Holmes K.C. (1990) Nature 347, 37-44] were purified by reverse-phase HPLC and Sephadex G-50 gel filtration. The peptide fragments were identified as segments from Arg-177 to Tyr-198 (2.6-kDa peptide) and from Ser-199 to Tyr-279 (9.1-kDa peptide). Their effects on actin polymerization induced by 50 or 100 mM KCl were studied by measuring the increase in viscosity by the falling ball method. The 2.6-kDa peptide decreased the rate of actin polymerization and increased the critical concentration for the polymerization. Based on the atomic model of the actin filament [Holmes, K.C., Popp, D., Gebhard, W., & Kabsch, W. (1990) Nature 347, 44-49], the peptide is presumed to bind to the barbed end of the actin filament and inhibit the polymerization. By assuming that the peptide affected the rate of association of the actin monomer to the end of the actin filament, well-fitting curves for the polymerization kinetics were calculated. Computer-assisted results indicated that the dissociation constant of the 2.6-kDa peptide for F-actin is 200 to 260 microM. In contrast, the 9.1-kDa peptide only slightly inhibited actin polymerization. These results suggest that the actin-actin interface in the region between Arg-177 and Tyr-198 has a stronger interaction than those between Ser-199 and Tyr-279. The amino acid sequence L-T-D-Y-L present in the 2.6-kDa segment is homologous to a common sequence in the F-actin capping domain of various actin-binding proteins.

Actins↗
PubMed1983

Immunological characterization of an anti-actin antibody specific for cytoplasmic actins and its use for the immunocytological localization of actin in Aplysia nervous tissue.

Previous immunochemical and immunocytochemical studies have shown that an antibody to actin prepared from body wall muscle of the marine mollusc Aplysia californica is specific for vertebrate cytoplasmic actins. The ability of this anti-actin to distinguish between different forms of actin most likely reflects the recognition of amino acid sequences unique to cytoplasmic actins. We have confirmed the specificity of this antibody for cytoplasmic actins using nervous tissue as a source of cytoplasmic actin in further immunochemical studies. In addition to binding cytoplasmic actin in purified preparations, the antibody removed actin selectively from crude extracts of nervous tissue of some but not all of the species tested. Our results also suggest that tissue-specific differences in the distribution of cytoplasmic actins may exist. Immunofluorescence studies of Aplysia nervous tissue stained with anti-actin revealed that actin is present in the cell body and axonal processes of Aplysia neurons. Although the function of actin in nerve cells is not understood, the observed pattern of immunofluorescence staining is consistent with the idea that actin may be involved in movement within the axoplasm.

Actins↗
PubMed1988

Binding of phosphate to F-ADP-actin and role of F-ADP-Pi-actin in ATP-actin polymerization.

Our previous work (Carlier, M.-F., and Pantaloni, D. (1986) Biochemistry 25, 7789-7792) had shown that F-ADP-Pi-actin is a major intermediate in ATP-actin polymerization, due to the slow rate of Pi release following ATP cleavage on filaments. To understand the mechanism of ATP-actin polymerization, we have prepared F-ADP-Pi-actin and characterized its kinetic parameters. 32Pi binds to F-ADP-actin with a stoichiometry of 1 mol/mol of F-actin subunit and an equilibrium dissociation constant Kpi of 1.5 mM at pH 7.0 Kpi increases with pH, indicating that the H2PO-4 species binds to F-actin. ADP-Pi-actin subunits dissociate much more slowly from filament ends than ADP-actin subunits; therefore, the stability of filaments in ATP is due to terminal ADP-Pi subunits. The slow rate of dissociation of ADP-Pi-actin also explains the decrease in critical concentration of ADP-actin in the presence of Pi reported by Rickard and Sheterline (Richard, J. E., and Sheterline, P. (1986) J. Mol. Biol. 191, 273-280). The effect of Pi on the rate of actin dissociation from filaments is much more pronounced at the barbed end than at the pointed end. Using gelsolin to block the barbed end, we have shown that the two ends are energetically different in the presence of ATP and saturating Pi, but less different than in the absence of Pi. The results are interpreted within a new model for actin polymerization. It is possible that phosphate binding to F-actin can regulate motile events in muscle and nonmuscle cells.

Actins↗
PubMed1985

Polymerization of ADP-actin and ATP-actin under sonication and characteristics of the ATP-actin equilibrium polymer.

Polymerization under sonication has been developed as a new method to study the rapid polymerization of actin with a large number of elongating sites. The theory proposed assumes that filaments under sonication are maintained at a constant length by the constant input of energy. The data obtained for the reversible polymerization of ADP-actin under sonication have been successfully analyzed according to the proposed model and, therefore, validate the model. The results obtained for the polymerization of ATP-actin under sonication demonstrate the involvement of ATP hydrolysis in the polymerization process. At high actin concentration, polymerization was fast enough, as compared to ATP hydrolysis on the F-actin, to obtain completion of the reversible polymerization of ATP-actin before significant hydrolysis of ATP occurred. A critical concentration of 3 microM was determined as the ratio of the dissociation and association rate constants for the interaction of ATP-actin with the ATP filament ends in 1 mM MgCl2, 0.2 mM ATP. The plot of the rate of elongation of filaments versus actin monomer concentration exhibited an upward deviation at high actin concentration that is consistent with this result. The fact that F-actin at steady state is more stable than the ATP-F-actin polymer at equilibrium suggests that the interaction between ADP-actin and ATP-actin subunits at the end of the ATP-capped filament is much stronger than the interaction between two ATP-actin subunits.

Actins↗
PubMed2000

The C-terminal tail of UNC-60B (actin depolymerizing factor/cofilin) is critical for maintaining its stable association with F-actin and is implicated in the second actin-binding site.

Actin depolymerizing factor (ADF)/cofilin changes the twist of actin filaments by binding two longitudinally associated actin subunits. In the absence of an atomic model of the ADF/cofilin-F-actin complex, we have identified residues in ADF/cofilin that are essential for filament binding. Here, we have characterized the C-terminal tail of UNC-60B (a nematode ADF/cofilin isoform) as a novel determinant for its association with F-actin. Removal of the C-terminal isoleucine (Ile152) by carboxypeptidase A or truncation by mutagenesis eliminated F-actin binding activity but strongly enhanced actin depolymerizing activity. Replacement of Ile152 by Ala had a similar but less marked effect; F-actin binding was weakened and depolymerizing activity slightly enhanced. Truncation of both Arg151 and Ile152 or replacement of Arg151 with Ala also abolished F-actin binding and enhanced depolymerizing activity. Loss of F-actin binding in these mutants was accompanied by loss or greatly decreased severing activity. All of the variants of UNC-60B interacted with G-actin in an indistinguishable manner from wild type. Cryoelectron microscopy showed that UNC-60B changed the twist of F-actin to a similar extent to vertebrate ADF/cofilins. Helical reconstruction and structural modeling of UNC-60B-F-actin complex reveal how the C terminus of UNC-60B might be involved in one of the two actin-binding sites.

Actin Depolymerizing Factors↗