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PubMed2004

Release of soluble CD40L from platelets is regulated by glycoprotein IIb/IIIa and actin polymerization.

OBJECTIVES: The purpose of this study was to examine the effects of glycoprotein (GP) IIb/IIIa antagonists (abciximab, eptifibatide, and tirofiban) and other inhibitors on translocation of CD40L from intraplatelet stores to the platelet surface and on the release of soluble CD40L (sCD40L) from platelets. BACKGROUND: CD40L is a proinflammatory and prothrombotic ligand in the tumor necrosis factor family. METHODS: Platelet surface CD40L was measured by flow cytometry, and sCD40L was measured by enzyme-linked immunosorbent assay. RESULTS: Translocation of CD40L from intraplatelet stores to the platelet surface was not inhibited by GP IIb/IIIa antagonists. However, release of sCD40L from the surface of activated platelets was inhibited by GP IIb/IIIa antagonists in a dose-dependent manner, in concert with inhibition of PAC1 binding to platelets (a surrogate marker for fibrinogen binding). Release of sCD40L from activated platelets was also markedly reduced in Glanzmann platelets (deficient in GP IIb/IIIa). Ethylenediaminetetraacetic acid was an effective inhibitor of sCD40L release, but only when added before platelet activation. Both cytochalasin D (an inhibitor of actin polymerization) and GM6001 (an inhibitor of matrix metalloproteinases [MMPs]) inhibited the release of sCD40L from platelets when added before, as well as 3 min after, platelet activation. However, neither cytochalasin D nor GM6001 affected translocation of CD40L to the platelet surface. CONCLUSIONS: The GP IIb/IIIa antagonists inhibit release of sCD40L from activated platelets. Release of sCD40L from platelets is regulated, at least in part, by GP IIb/IIIa, actin polymerization, and an MMP inhibitor-sensitive pathway. In addition to their well-characterized inhibition of platelet aggregation, GP IIb/IIIa antagonists may obviate the proinflammatory and prothrombotic effects of sCD40L.

Actin Depolymerizing Factors↗
PubMed1984

Calorimetric studies on monomeric and polymeric actin.

Differential scanning calorimetry of polymeric F-actin at pH 8.0 showed that the polymer had a concentration-independent thermal profile with a single transition temperature of 81 degrees C. In contrast, the thermal profile of G-actin was concentration-dependent, and although it resembled the F-actin profile at lower concentrations, it was found to have a more complex profile at higher protein concentrations.

Actins↗
PubMed1998

A six-module human nebulin fragment bundles actin filaments and induces actin polymerization.

We have investigated the interaction of a 6-repeat recombinant human nebulin fragment (S6R2R7) with F-actin, with Mg2+-induced actin paracrystals, and G-actin, respectively. This fragment corresponds to super-repeat 6, repeat 2 to 7 of human nebulin, and is located in the N-terminal part of the super-repeat region of the nebulin molecule. The S6R2R7 fragment included an immuno-tag of three amino-acid residues (EEF) at one end which was detectable by a monoclonal anti-tubulin YL1/2. By a cosedimentation assay, interaction between F-actin and S6R2R7 was observed. Electron microscopy revealed the formation of large bundle-like aggregates containing highly parallelized actin filaments, apparently caused by actin bundling of the nebulin fragment. Compared with Mg2+-induced actin paracrystals where the helices of the actin filaments are arranged in register, the filaments in the actin-nebulin bundles seem to be packed in a different way and show no obvious periodicity. The bundles were also visible in the light microscope, and immunofluorescence microscopy revealed binding of the nebulin fragment S6R2R7 to both preformed Mg2+ paracrystals and to F-actin. We also analyzed the effect of S6R2R7 on actin under non-polymerizing conditions by cosedimentation assays and pyrene actin fluorimetry, as well as fluorescence microscopy and electron microscopy. Nebulin-induced actin polymerization was observed with an enhancement of the nucleation step indicating a stabilization of actin nuclei by S6R2R7. Light and electron microscopy revealed bundle-like actin-nebulin aggregates similar to those formed by pre-assembled F-actin and S6R2R7. Thus, even in the absence of salt, S6R2R7 promotes actin polymerization and induces formation of tightly packed actin filament bundles. We assume that the actin filaments are crosslinked by the nebulin fragments, indicating a rather low cooperativity of binding to a single filament.

Actins↗
PubMed1984

Rate constants for actin polymerization in ATP determined using cross-linked actin trimers as nuclei.

Chemically cross-linked actin trimer, prepared from F-actin covalently cross-linked by N-N'-p-phenylenebismaleimide, was used to nucleate the assembly of actin polymers under a variety of ionic conditions at 25 degrees C. Gel-filtered G-actin (5% labeled with N-pyrenyl iodoacetamide) was prepared in buffers containing 5 mM Tris X HCl, 0.2 mM ATP, 0.05 mM MgCl2 or 0.1 mM CaCl2, 0.2 mM dithiothreitol, and 0.01% sodium azide, pH 7.5. Polymerization was initiated by adding salt to a solution of actin monomer (2-25 microM) mixed with actin trimer (0-24 nM). Pseudo-first-order polymerization kinetics were observed in all cases following a lag phase of 30-75 s. The length of the lag phase was independent of trimer concentration, monomer concentration, or preincubation of monomer or trimer in polymerizing buffer. A simple model is presented which explains all of the observed features of the lag phase. Lower limits for the sums of the elongation rate constants at the two filament ends, calculated assuming each trimer acts as a seed, were in the range 1.4-5.2 microM-1 s-1, and lower limits for the polymer dissociation rate constants (calculated from the elongation rate constants and the critical actin concentration) were 0.4-1.9 s-1, depending on ionic conditions. The observed rate constants were independent of actin concentration or trimer concentration under a given ionic condition. These polymerization rate constants are in good agreement with the values others have obtained by measuring the growth of actin filaments by electron microscopy.

Actins↗
PubMed2002

Eukaryotic chaperonin containing T-complex polypeptide 1 interacts with filamentous actin and reduces the initial rate of actin polymerization in vitro.

We have previously observed that subunits of the chaperonin required for actin production (type-II chaperonin containing T-complex polypeptide 1 [CCT]) localize at sites of microfilament assembly. In this article we extend this observation by showing that substantially substoichiometric CCT reduces the initial rate of pyrene-labeled actin polymerization in vitro where eubacterial chaperonin GroEL had no such effect. CCT subunits bound selectively to F-actin in cosedimentation assays, and CCT reduced elongation rates from both purified actin filament "seeds" and the short and stabilized, minus-end blocked filaments in erythrocyte membrane cytoskeletons. These observations suggest CCT might remain involved in biogenesis of the actin cytoskeleton, by acting at filament (+) ends, beyond its already well-established role in producing new actin monomers.

Actin Cytoskeleton↗
PubMed1979

Complex influence of cytochalasin B on actin polymerization.

In the presence of very low concentrations (about 2X10(-7) M) of cytochalasin B (CB) the time course of actin polymerization is much more sigmoidal when followed by viscosity measurements than when followed by light scattering measurements. This suggests that under these conditions actin polymers do not immediately reach their final length but only via short "bent" polymers which can be detected only by light scattering but not by viscosity measurements. At higher CB concentrations (about equimolar to those of actin) CB reduces the average degree of polymerization and favors the nucleation step necessary for polymerization.

Actins↗
PubMed1996

Cellular changes of rat embryonic fibroblasts by an actin-polymerization inhibitor, bistheonellide A, from a marine sponge.

Bistheonellide A, an inhibitor of actin polymerization from the marine sponge Theonella sp., was introduced at a concentration of 100 nM into rat fibroblast of 2.4 x 10(4) cells/ml. Within 1 h, it disrupted stress fibers, accompanied by a marked change of the cell morphology, resulting in the formation of processes from the cell surface. Further incubation for 24 h in the presence of 100 nM bistheonellide A led to binucleation in most cells and subsequent inhibition of cell cycle progression. When bistheonellide A was withdrawn from the culture medium, binuclear cells began to grow again within 20 h and reverted to mononuclear morphology. Flow cytometric analysis fluorescence-activated cell sorting showed that 2C diploid DNA content in G1 phase was changed into 4C content of tetraploid for the bistheonellide A treated-cells in G1 phase and into 8C content during G2 and M phase. Therefore, we suggested that the bistheonellide A treatment inhibited cytokinesis, but not mitosis in M phase, and that treated cells were arrested at the early G1 phase. These effects of bistheonellide A on the cell cycle progression of 3Y1 fibroblast were also observed more prominently in cells synchronized in S phase with hydroxyurea. Cells in G0 phase were then activated by the addition of fetal calf serum in the presence of 100 nM bistheonellide A. Cell cycle progression of the bistheonellide A-treated cells was obviously slowed down or completely inhibited during G1 phase. These results reveal that actin filaments are not only essential to cytokinesis but also for promoting the progression of cell cycle G1 to S phase.

Actins↗
PubMed2002

An unexplained sequestration of latrunculin A is required in neutrophils for inhibition of actin polymerization.

Latrunculin A (LatA) is a toxic natural product that causes disruption of the actin cytoskeleton in many eukaryotic cells at submicromolar concentrations. LatA has been found to bind G-actin with a dissociation constant of 0.2 microM, and more recently to bind profilin-G-actin and, weakly, thymosin beta4-G-actin. A number of investigators have used LatA as a G-actin sequestering agent. Thus, we studied neutrophil chemotaxis and its requisite conversion of G-actin to F-actin, supported by an extensive pool of G-actin, mainly bound to thymosin beta4. Calculations suggest that the affinity of LatA is insufficient to cause significant sequestration of this pool, and the pool's buffering action should protect neutrophils from depletion of productive G-actin species by submicromolar LatA. Nonetheless, we found that both chemoattractant stimulated migration and F-actin polymerization in neutrophils were inhibited by LatA at these concentrations. The latter effect was accompanied by sequestration of LatA and showed a cell density dependence that was consistent with G-actin sequestration. The apparent contradiction between the calculations and the experimental observations could be reconciled by assuming the presence of an accessory species, of unknown normal function, which forms a high affinity ternary complex with LatA and G-actin, thus causing the cells to concentrate LatA. Other models that could not be ruled out also invoke new actions of LatA, suggesting caution in the interpretation of its effects on cells.

Actin Cytoskeleton↗
PubMed1993

Use of bimanyl actin derivative (TMB-actin) for studying complexation of beta-thymosins. Inhibition of actin polymerization by thymosin beta 9.

By reacting trimethylammoniobromobimane bromide (TMB bromide) with rabbit muscle actin, a fluorescent reporter group was linked to cysteine at position 374. Fluorescence of TMB-actin decreased significantly on addition of thymosin beta 4 (T beta 4), a peptide of 43 amino acid residues reported to bind to monomeric actin and to prevent filament formation. Based on this effect, we determined the KD value of the thymosin beta 4 complex as 0.8 microM, a value that is in agreement with previous determinations. In addition to the main compound thymosin beta 4, bovine tissue contains a related peptide, thymosin beta 9 (T beta 9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that T beta 9, similar to T beta 4, forms a 1:1 complex with monomeric actin, and hereby inhibits actin polymerization. With a KD value of 1.1 microM the affinity of T beta 9 is in the same range as that of T beta 4, suggesting that T beta 9, like T beta 4, contributes to maintaining the pool of monomeric actin in bovine non-muscle cells. Further proof of the interaction of T beta 9 with actin was provided by native PAGE, where the complex showed the reported higher mobility, as well as by crosslinking experiments. Using different crosslinking reagents, like water-soluble carbodiimide (EDC), m-maleimidobenzoyl-N-hydroxysuccinimidate (MBS), and disuccinimidylsuberate (DSS), we were able to produce conjugates of 47 kDa. In one of these (from MBS) both actin and T beta 9 could be identified by immunoblotting. When, in the MBS crosslinking experiments, native actin was replaced with (374-NEM)-actin, the 47 kDa band was not seen, indicating that Cys-374 takes part in the thiol-specific crosslinking reaction. This suggests that part of the binding site of T beta 9 must be located close to the carboxy-terminus.

Actins↗
PubMed2004

Activation of Arp2/3 complex-dependent actin polymerization by plant proteins distantly related to Scar/WAVE.

The Arp2/3 complex, a highly conserved nucleator of F-actin polymerization, plays a key role in the regulation of actin dynamics eukaryotic cells. In animal cells and yeasts, Wiskott-Aldrich Syndrome protein (WASP)/suppressor of cAMP receptor (Scar)/WASP family verprolin homologous (WAVE) family proteins activate the Arp2/3 complex in response to localized cues. Like other eukaryotes, plants have an Arp2/3 complex, which has recently been shown to play an important role in F-actin organization and cell morphogenesis. However, no activators of the Arp2/3 complex have been identified in plants, which lack obvious homologs of WASP/Scar/WAVE family proteins. Here, we identify a family of Scar/WAVE-related plant Arp2/3 activators. Like Scar/WAVE proteins, four proteins identified in Arabidopsis thaliana (AtSCAR1 to AtSCAR4) and one in maize (ZmSCAR1) have a C-terminal WASP homology 2 (WH2)/acidic (WA)-verprolin homology/cofilin homology/acidic (VCA)-like domain, which we show can activate the bovine Arp2/3 complex. At their N termini, AtSCAR1 to ATSCAR4, along with a fifth protein lacking a VCA/WA-like domain at its C terminus (At4g18600), are related to the N-terminal Scar homology domains of Scar/WAVE family proteins. Analysis of gene expression patterns suggests functional redundancy among members of the AtSCAR family. Full-length AtSCAR1 and ATSCAR3 proteins and their Scar homology domains bind in vitro to AtBRICK 1 (AtBRK1), the Arabidopsis homolog of HSPC300, a WAVE-binding protein recently identified as a component of a complex implicated in the regulation of Scar/WAVE activity. Thus, AtSCAR proteins are likely to function in association with AtBRK1, and perhaps other Arabidopsis homologs of WAVE complex components, to regulate activation of the Arp2,3 complex in vivo.

Actin-Related Protein 2↗
PubMed1990

Actin polymerization and ATP hydrolysis.

This paper surveys several aspects of the consequences of ATP hydrolysis associated with actin polymerization, and their physiological implications. ATP hydrolysis occurs on F-actin in two subsequent reactions, cleavage of ATP followed by the slower release of Pi. The latter reaction is linked to a conformation change of the actin subunit that causes a destabilization of the actin-actin interactions in the filament, i.e., a structural change of the filament. The nature of the nucleotide bound to terminal subunits therefore affects the dynamics of actin filaments. It is shown that this regulation is different at the two ends, terminal F-ADP-Pi subunits being present at steady state at the barbed end, while F-ADP-subunits are present at the pointed end. While cleavage of ATP on F-actin is irreversible, Pi release is reversible, which allows the regulation of filament dynamics by cellular Pi. The nature of the divalent metal ion--Ca2+ or Mg2(+)--tightly bound to actin, in direct interaction with ATP, also affects the conformation of actin and the rate of ATP hydrolysis, therefore regulating actin dynamics. Finally, the rate of nucleotide exchange on G-actin is relatively slow, which allows the critical concentration to increase with the number of filaments in ATP, a property largely used by the cell via the action of severing proteins.

Actin Cytoskeleton↗
PubMed1984

Mechanism of regulation of actin polymerization by Physarum profilin.

Physarum profilin reduces the rates of nucleation and elongation of F-actin and also reduces the extent of polymerization of actin at the steady state in a concentration-dependent fashion. The apparent critical concentration for polymerization of actin is increased by the addition of profilin. These results can be explained by the idea that Physarum profilin forms a 1:1 complex with G-actin and decreases the concentration of actin available for polymerization. The dissociation constant for binding of profilin to G-actin is estimated from the kinetics of polymerization of G-actin and elongation of F-actin nuclei and from the increase of apparent critical concentration in the presence of profilin. The dissociation constants for binding of Physarum profilin to Physarum and muscle actins under physiological ionic conditions are in the ranges of 1.4-3.7 microM and 11.3-28.5 microM, respectively. When profilin is added to an F-actin solution, profilin binds to G-actin which co-exists with F-actin, and then G-actin is dissociated from F-actin to compensate for the decrease of the concentration of free G-actin and to keep it constant at the critical concentration. At the steady state, free G-actin of the critical concentration is in equilibrium not only with F-actin but also with profilin-G-actin complex. The stoichiometry of 1:1 for the formation of complex between profilin and G-actin is directly shown by means of chemical cross-linking.

Actins↗
PubMed1991

A 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein.

The 25-kD inhibitor of actin polymerization (25-kD IAP), isolated from turkey smooth muscle (Miron, T., M. Wilchek, and B. Geiger, 1988. Eur. J. Biochem. 178:543-553), is shown here to be a low molecular mass heat shock protein (HSP). Direct sequence analysis of the purified protein, as well as cloning and sequencing of the respective cDNA, disclosed a high degree of homology (67% identity, 80% similarity) to the human 27-kD HSP. Southern blot of chicken genomic DNA disclosed one band, suggesting the presence of a single gene, and Northern blot analysis revealed abundant transcript of approximately 1 kb in gizzard and heart tissues and lower amounts in total 18-d chick embryo RNA and in cultured fibroblasts. Exposure of the latter cells to 45 degrees C resulted in over 15-fold increase in the apparent level of the 25-kD IAP protein, confirming that its expression is regulated by heat shock. Immunofluorescent microscopic localization indicated that after heat treatment, the levels of the 25-kD IAP were markedly increased and the protein was apparently associated with cytoplasmic granules. Heat shock also had a transient, yet prominent, effect on the microfilament system in cultured fibroblasts: stress fibers disintegrated within 10-15 min after incubation at 45 degrees C, yet upon further incubation at the elevated temperature, conspicuous actin bundles were apparently reformed.

Actin Cytoskeleton↗
PubMed2006

Optimization of WAVE2 complex-induced actin polymerization by membrane-bound IRSp53, PIP(3), and Rac.

WAVE2 activates the actin-related protein (Arp) 2/3 complex for Rac-induced actin polymerization during lamellipodium formation and exists as a large WAVE2 protein complex with Sra1/PIR121, Nap1, Abi1, and HSPC300. IRSp53 binds to both Rac and Cdc42 and is proposed to link Rac to WAVE2. We found that the knockdown of IRSp53 by RNA interference decreased lamellipodium formation without a decrease in the amount of WAVE2 complex. Localization of WAVE2 at the cell periphery was retained in IRSp53 knockdown cells. Moreover, activated Cdc42 but not Rac weakened the association between WAVE2 and IRSp53. When we measured Arp2/3 activation in vitro, the WAVE2 complex isolated from the membrane fraction of cells was fully active in an IRSp53-dependent manner but WAVE2 isolated from the cytosol was not. Purified WAVE2 and purified WAVE2 complex were activated by IRSp53 in a Rac-dependent manner with PIP(3)-containing liposomes. Therefore, IRSp53 optimizes the activity of the WAVE2 complex in the presence of activated Rac and PIP(3).

Actin-Related Protein 2-3 Complex↗
PubMed2005

Polycomb group protein ezh2 controls actin polymerization and cell signaling.

Polycomb group protein Ezh2, one of the key regulators of development in organisms from flies to mice, exerts its epigenetic function through regulation of histone methylation. Here, we report the existence of the cytosolic Ezh2-containing methyltransferase complex and tie the function of this complex to regulation of actin polymerization in various cell types. Genetic evidence supports the essential role of cytosolic Ezh2 in actin polymerization-dependent processes such as antigen receptor signaling in T cells and PDGF-induced dorsal circular ruffle formation in fibroblasts. Revealed function of Ezh2 points to a broader usage of lysine methylation in regulation of both nuclear and extra-nuclear signaling processes.

Actins↗
PubMed2005

Actin polymerization machinery: the finish line of signaling networks, the starting point of cellular movement.

Dynamic assembly of actin filaments generates the forces supporting cell motility. Several recent biochemical and genetic studies have revealed a plethora of different actin binding proteins whose coordinated activity regulates the turnover of actin filaments, thus controlling a variety of actin-based processes, including cell migration. Additionally, emerging evidence is highlighting a scenario whereby the same basic set of actin regulatory proteins is also the convergent node of different signaling pathways emanating from extracellular stimuli, like those from receptor tyrosine kinases. Here, we will focus on the molecular mechanisms of how the machinery of actin polymerization functions and is regulated, in a signaling-dependent mode, to generate site-directed actin assembly leading to cell motility.

Actin Cytoskeleton↗