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PubMed1999

Computer simulations of actin polymerization can explain the barbed-pointed end asymmetry.

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

Actin Cytoskeleton↗
PubMed1994

[The effect of different antitubercular agents on phagosome fusion with lysosomes, on the F-actin content in mouse peritoneal macrophages and on G-actin polymerization in vitro].

Effects of various antituberculosis remedies (ATR)--isoniazid (INA), saluzid (SA), streptosaluzid (SS), ethambutol (EB), sodium para-aminosalicylate (SPAS) on phagosome-lysosome (PL) fusion, on F-actin content in mouse macrophages and on G-actin polymerization in vitro were studied. The ATR of choice have been shown to stimulate the PL fusion. INA (0.2 mM), SA (0.02 mM), SS (0.05 mM), EB (0.08 mM) and SPAS (0.5 mM) increased the F-actin content and changed its localization within macrophages. ATR changed the character of G-actin polymerization in vitro. Possible mechanisms of interrelation between cytoskeleton (actin part) changes and PL fusion are discussed. The results obtained suggest to use the ATR-induced changes in PL fusion and F-actin content in the cells for estimating therapeutic effects of respective antituberculosis remedies.

Actins↗
PubMed1989

Polymorphonuclear leukocyte adherence induces actin polymerization by a transduction pathway which differs from that used by chemoattractants.

Nitrobenzoxadiazole-phallacidin in combination with quantitative fluorescent microscopy have been used to measure F-actin concentrations in human polymorphonuclear leukocytes (PMN) as they adhere to a plastic surface. Like stimulation with chemoattractants, adherence is associated with a twofold rise in F-actin content. However unlike the rapid rise in F-actin induced by chemoattractants which peaks within 30 s, actin assembly induced by adherence is slower, maximum F-actin values not being observed until 10 min. Furthermore the rise in F-actin induced by adherence is persistent, remaining constant over 60 min while F-actin returns to near basal levels after 20 min exposure to chemoattractant. The combination of adherence (5 min) followed by chemoattractant (FMLP 5 x 10(-8) M for 40 s) resulted in an additive rise in F-actin content to greater than threefold over unstimulated values. Unlike chemoattractant induced actin assembly, adherence-associated PMN actin polymerization was not inhibited by pertussis toxin, but was markedly reduced by lowering extracellular Ca2+. Fluorescent micrographs of adherent PMN stained with nitrobenzoxadiazole-phallacidin revealed F-actin in the lamellipodia and in small foci on the adherent surface. These findings suggest that the transduction mechanisms by which adherence induces PMN actin polymerization differ from those used by chemoattractant receptors.

Actins↗
PubMed2006

CFTR surface expression and chloride currents are decreased by inhibitors of N-WASP and actin polymerization.

The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid turnover at the plasma membrane in various cell types. The ubiquitously expressed N-WASP promotes actin polymerization and regulates endocytic trafficking of other proteins in response to signaling molecules such as Rho-GTPases. In the present study we investigated the effects of wiskostatin, an N-WASP inhibitor, on the surface expression and activity of CFTR. We demonstrate, using surface biotinylation methods, that the steady-state surface CFTR pool in stably transfected BHK cells was dramatically decreased following wiskostatin treatment with a corresponding increase in the amount of intracellular CFTR. Similar effects were observed for latrunculin B, a specific actin-disrupting reagent. Both reagents strongly inhibited macroscopic CFTR-mediated Cl(-) currents in two cell types including HT29-Cl19A colonic epithelial cells. As previously reported, CFTR internalization from the cell surface was strongly inhibited by a cyclic-AMP cocktail. This effect of cyclic-AMP was only partially blunted in the presence of wiskostatin, which raises the possibility that these two factors modulate different steps in CFTR traffic. In kinetic studies wiskostatin appeared to accelerate the initial rate of CFTR endocytosis as well as inhibit its recycling back to the cell surface over longer time periods. Our studies implicate a role for N-WASP-mediated actin polymerization in regulating CFTR surface expression and channel activity.

Actins↗
PubMed1990

Effects of bile acids on actin polymerization in vitro.

Bile acids are major determinants of canalicular bile secretion, and there are indications that choleretic bile acids increase bile canalicular contractions, in isolated rat hepatocytes. Therefore, we examined the influence of various bile acids on the rate of actin polymerization in vitro. The free forms of cholic acid, ursodeoxycholic acid, and chenodeoxycholic acid, as well as their taurine and glycine conjugates, were incubated with purified muscle actin, at a concentration of 100-300 nmoles/mg actin. The rate of actin polymerization was measured by viscometry and the fluorescence of the pyrene probe, linked to actin. Results showed that all bile acids slow the rate of polymerization, and that the effect was dose-dependent. However, the reduction by chenodeoxycholic acid was greater than that caused by the other bile acids. The results indicate that bile acids, particularly in high concentrations interact with actin, a finding that may be related to the increased bile canalicular contractility, and altered canalicular membrane morphology, induced by choleretic bile acids.

Actins↗
PubMed1989

Diacylglycerols and PMA induce actin polymerization and distinct shape changes in lymphocytes: relation to fluid pinocytosis and locomotion.

Shape changes have been determined in human blood lymphocytes stimulated with OAG, diC8, PMA, colchicine or the hexapeptide fNLPNTL in short-term assays (30 min). Distinct types of shape-change responses were observed. Colchicine was active in generating a relatively small proportion of polarized lymphocytes (front-tail polarity). OAG, diC8 and PMA produced different types of shape change (non-polar cells with surface projections), and these were closely associated with an increase in actin polymerization and a shift of F-actin into the projections at the cell periphery. The diacylglycerols OAG and diC8 produced biphasic dose-response curves leading to rounding up of cells at very high stimulant concentrations. PMA produced no comparable biphasic response when tested over a much wider concentration range. Though the nonpolar cells with surface projections generated by OAG, diC8 or PMA showed vigorous shape changes, they lacked significant locomotor activity. alpha-Phorbol, 4 alpha-PDD, lumicolchicine or fNLPNTL were inactive. Small blood lymphocytes stimulated by OAG, diC8 or PMA showed a very small increase in the net uptake of FITC-dextran by fluid pinocytosis. Unlike neutrophils, which show a high net uptake, lymphocytes did not concentrate FITC-dextran in large granules, indicating that they do not develop a 'storage' compartment in the form of large vesicles. However, small fluorescent spots were consistently found in at least a fraction of blood lymphocytes. The results indicate that stimulated surface movement may be instrumental in fluid pinocytosis. Diacylglycerols may act as second messengers to induce pinocytosis, shape changes and altered actin polymerization in lymphocytes.

Actins↗
PubMed2000

The novel acute phase protein, IHRP, inhibits actin polymerization and phagocytosis of polymorphonuclear cells.

OBJECTIVE AND DESIGN: In the present study, the involvement of the binding of IHRP (inter-alpha-trypsin inhibitor family heavy chain-related protein) and actin in phagocyte activity was investigated. MATERIALS AND METHODS: The actin polymerization and the phagocytic activity of the polymorphonuclear (PMN) cells were studied in the presence of IHRP. RESULTS: IHRP inhibited the polymerization of actin and the phagocytic activity of the PMN cells. CONCLUSION: 1) IHRP may bind to actin released from the damaged cells and suppress its toxic action by preventing the formation of actin fibril. 2) IHRP may bind to cell surface actin on PMN cells and inhibit their phagocytic activities. 3) From these results, IHRP may act as an anti-inflammatory protein.

Actins↗
PubMed2002

The role of MeH73 in actin polymerization and ATP hydrolysis.

In actin from many species H73 is methylated, but the function of this rare post-translational modification is unknown. Although not within bonding distance, it is located close to the gamma-phosphate of the actin-bound ATP. In most crystal structures of actin, the delta1-nitrogen of the methylated H73 forms a hydrogen bond with the carbonyl of G158. This hydrogen bond spans the gap separating subdomains 2 and 4, thereby contributing to the forces that close the interdomain cleft around the ATP polyphosphate tail. A second hydrogen bond stabilizing interdomain closure exists between R183 and Y69. In the closed-to-open transition in beta-actin, both of these hydrogen bonds are broken as the phosphate tail is exposed to solvent. Here we describe the isolation and characterization of a mutant beta-actin (H73A) expressed in the yeast Saccharomyces cerevisiae. The properties of the mutant are compared to those of wild-type beta-actin, also expressed in yeast. Yeast does not have the methyl transferase necessary to methylate recombinant beta-actin. Thus, the polymerization properties of yeast-expressed wild-type beta-actin can be compared with normally methylated beta-actin isolated from calf thymus. Since earlier studies of the actin ATPase almost invariably employed rabbit skeletal alpha-actin, this isoform was included in these comparative studies on the polymerization, ATP hydrolysis, and phosphate release of actin. It was found that H73A-actin exchanged ATP at an increased rate, and was less stable than yeast-expressed wild-type actin, indicating that the mutation affects the spatial relationship between the two domains of actin which embrace the nucleotide. At physiological concentrations of Mg(2+), the kinetics of ATP hydrolysis of the mutant actin were unaffected, but polymer formation was delayed. The comparison of methylated and unmethylated beta-actin revealed that in the absence of a methyl group on H73, ATP hydrolysis and phosphate release occurred prior to, and seemingly independently of, filament formation. The comparison of beta and alpha-actin revealed differences in the timing and relative rates of ATP hydrolysis and P(i)-release.

Actins↗
PubMed2000

Two tandem verprolin homology domains are necessary for a strong activation of Arp2/3 complex-induced actin polymerization and induction of microspike formation by N-WASP.

All WASP family proteins share a common C terminus that consists of the verprolin homology domain (V), cofilin homology domain (C), and acidic region (A), through which they activate Arp2/3 complex-induced actin polymerization. In this study, we characterized the Arp2/3 complex-mediated actin polymerization activity of VCA fragments of all of the WASP family proteins: WASP, N-WASP, WAVE1, WAVE2, and WAVE3. All of the VCA fragments stimulated the nucleating activity of Arp2/3 complex. Among them, N-WASP VCA, which possesses two tandem V motifs, had a more potent activity than other VCA proteins. The chimeric protein experiments revealed that the V motif was more important to the activation potency than the CA region; two V motifs were required for full activity of N-WASP. COS7 cells overexpressing N-WASP form microspikes in response to epidermal growth factor. However, when a chimeric protein in which the VCA region of N-WASP is replaced with WAVE1 VCA was overexpressed, microspike formation was suppressed. Interestingly, when the N-WASP VCA region was replaced with WAVE1 VCA, having two V motifs, this chimeric protein could induce microspike formation. These results indicate that strong activation of Arp2/3 complex by N-WASP is mainly caused by its two tandem V motifs, which are essential for actin microspike formation.

Actin-Related Protein 2↗
PubMed1983

Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization.

The fluorescence of pyrene-labelled actin is much higher after polymerization. We have characterized in detail the polymerization properties of pyrene actin and report that native and pyrene actin are identical using the following criteria: (1) the time course of polymerization; (2) the elongation rate constants; (3) the intrinsic viscosity; and (4) the critical concentration. Native and pyrene actin copolymerize. Fluorescence of polymerized pyrene actin is 7-10 times higher than monomer. The fluorescent signal is proportional to polymer weight concentration and is insensitive to filament length distribution. Bleaching can be minimized by appropriate filters to allow continuous monitoring of signal. Measurements do not influence polymerization kinetics. This establishes that pyrene actin fluorescence is a valid assay for actin polymerization that is more sensitive than any other current assay.

Actins↗
PubMed2005

Differential roles for actin polymerization and a myosin II motor in assembly of the epithelial apical junctional complex.

Differentiation and polarization of epithelial cells depends on the formation of the apical junctional complex (AJC), which is composed of the tight junction (TJ) and the adherens junction (AJ). In this study, we investigated mechanisms of actin reorganization that drive the establishment of AJC. Using a calcium switch model, we observed that formation of the AJC in T84 intestinal epithelial cells began with the assembly of adherens-like junctions followed by the formation of TJs. Early adherens-like junctions and TJs readily incorporated exogenous G-actin and were disassembled by latrunculin B, thus indicating dependence on continuous actin polymerization. Both adherens-like junctions and TJs were enriched in actin-related protein 3 and neuronal Wiskott-Aldrich syndrome protein (N-WASP), and their assembly was prevented by the N-WASP inhibitor wiskostatin. In contrast, the formation of TJs, but not adherens-like junctions, was accompanied by recruitment of myosin II and was blocked by inhibition of myosin II with blebbistatin. In addition, blebbistatin inhibited the ability of epithelial cells to establish a columnar phenotype with proper apico-basal polarity. These findings suggest that actin polymerization directly mediates recruitment and maintenance of AJ/TJ proteins at intercellular contacts, whereas myosin II regulates cell polarization and correct positioning of the AJC within the plasma membrane.

Actins↗
PubMed1981

7-Chloro-4-nitrobenzeno-2-oxa-1,3-diazole actin as a probe for actin polymerization.

Lysine 372 of N-ethylmaleimide actin was specifically (60%) labeled by 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole chloride (NBD-Cl), which also reacted with lysines on cyanogen bromide fragment 17 (20%) and other undetermined residues (20%). Isolation of N-ethylmaleimide peptides and two-dimensional peptide mapping demonstrated that 90% of bound N-ethylmaleimide was attached to an adjacent residue, cysteine 373, independent of the polymerization state of actin during the labeling reaction. Formation of NBD cysteine severely inhibited lysine modification. After N-ethylmaleimide blockage of cysteine 373, lysine labeling with NBD was greatly accelerated. The kinetics of formation of fluorescent compounds were biphasic, with fluorescence decreasing upon prolonged incubation of actin in NBD-Cl. Lysine 372 of purified NBD actin reproducibly responded to polymerization by a 2.2- to 2.3-fold enhancement of fluorescence. By contrast, interaction of NBD actin with several actin-binding proteins caused only very small or undetectable changes in fluorescence intensity: 10% enhancement on myosin subfragment 1 binding, about 6% quenching by DNase I, and no change at all by tropomyosin-troponin. Despite its sensitivity to polymerization the probe did not affect it. Native and modified actin polymerized randomly indicating that the rate constants for polymerization remained the same. Labeling actin with NBD did not diminish its cofactor activity for myosin ATPase activity. Contrary to previous reports we observed that myosin subfragment 1 (single myosin heads) caused actin polymerization in the absence of salt.

4-Chloro-7-nitrobenzofurazan↗
PubMed2006

Interaction between Tiam1 and the Arp2/3 complex links activation of Rac to actin polymerization.

The Rac-specific GEF (guanine-nucleotide exchange factor) Tiam1 (T-lymphoma invasion and metastasis 1) regulates migration, cell-matrix and cell-cell adhesion by modulating the actin cytoskeleton through the GTPase, Rac1. Using yeast two-hybrid screening and biochemical assays, we found that Tiam1 interacts with the p21-Arc [Arp (actin-related protein) complex] subunit of the Arp2/3 complex. Association occurred through the N-terminal pleckstrin homology domain and the adjacent coiled-coil region of Tiam1. As a result, Tiam1 co-localizes with the Arp2/3 complex at sites of actin polymerization, such as epithelial cell-cell contacts and membrane ruffles. Deletion of the p21-Arc-binding domain in Tiam1 impairs its subcellular localization and capacity to activate Rac1, suggesting that binding to the Arp2/3 complex is important for the function of Tiam1. Indeed, blocking Arp2/3 activation with a WASP (Wiskott-Aldrich syndrome protein) inhibitor leads to subcellular relocalization of Tiam1 and decreased Rac activation. Conversely, functionally active Tiam1, but not a GEF-deficient mutant, promotes activation of the Arp2/3 complex and its association with cytoskeletal components, indicating that Tiam1 and Arp2/3 are mutually dependent for their correct localization and signalling. Our data suggests a model in which the Arp2/3 complex acts as a scaffold to localize Tiam1, and thereby Rac activity, which are both required for activation of the Arp2/3 complex and further Arp2/3 recruitment. This 'self-amplifying' signalling module involving Tiam1, Rac and the Arp2/3 complex could thus drive actin polymerization at specific sites in cells that are required for dynamic morphological changes.

Actin Cytoskeleton↗
PubMed1986

Effect of toxin from the cyanobacterium Microcystis aeruginosa on ultrastructural morphology and actin polymerization in isolated hepatocytes.

Freshly isolated rat hepatocytes incubated with the hepatotoxin from the cyanobacterium Microcystis aeruginosa are rapidly deformed (blebbed). Transmission electron microscopy shows the appearance of unusual intracellular structures and rearrangement of cellular organelles, without any change in the polymerization state of actin. Cytochalasin E (20 microM), a fungal metabolite that causes blebbing of hepatocytes, had no significant effect on the polymerization state of cellular actin, but if Microcystis toxin (10 microM) was added together with cytochalasin E (20 microM), there was a significant increase (from 30% to 44%) in the proportion of unpolymerized G-actin in hepatocytes. These findings are in contrast to the effect of phalloidin (12.5 - 37.5 microM), a peptide hepatotoxin from the poisonous mushroom Amanita phalloides, which also causes blebbing of hepatocytes, and was shown in this study to decrease the level of unpolymerized G-actin in the cells to below measurable levels when added by itself or together with Microcystis toxin or cytochalasin E.

Actins↗
PubMed2003

Regulation of the mesangial cell myofibroblast phenotype by actin polymerization.

Mesangial cells in diverse glomerular diseases become myofibroblast-like, characterized by activation of smooth muscle alpha-actin (alpha-SMA) expression. In cultured mesangial cells, serum-deprivation markedly increases alpha-SMA expression, cell size, and stress fiber formation. Since stress fibers are assembled from actin monomers, we investigated the hypothesis that alterations in stress fiber formation regulate alpha-SMA expression and hypertrophy. Human mesangial cells were treated with agents that disrupt or stabilize actin stress fibers. Depolymerization of actin stress fibers in serum-deprived cells with actin-depolymerizing agents, cytochalasin B (CytB) and latrunculin B (LatB), or with inhibitors of Rho-kinase, Y-27632 and HA-1077 decreased alpha-SMA mRNA as judged by Northern blot analysis. Western blot analysis showed that CytB also reduced alpha-SMA protein levels. In serum-fed cells, agents that stabilized actin stress fibers, jasplakinolide (Jas) and phalloidin, increased alpha-SMA mRNA and protein. Treatment of human or rat mesangial cells with CytB, LatB, or Y-27632 decreased alpha-SMA promoter activity. In contrast, Jas increased promoter activity 5.6-fold in rat mesangial cells. The presence of an RNA polymerase inhibitor blocked degradation of alpha-SMA mRNA in cells treated with CytB suggesting that destabilization of this message is dependent on a newly transcribed or rapidly degraded factor. Inhibition of actin polymerization by CytB, LatB, Y-27623, and HA-1077 inhibited incorporation of (3)[H]-leucine into newly synthesized protein. Additionally, CytB and LatB decreased cell volume as determined by flow cytometry. Collectively, these results indicate that the state of polymerization of the actin cytoskeleton regulates alpha-SMA expression, hypertrophy, and myofibroblast differentiation in mesangial cells.

Actin Cytoskeleton↗
PubMed2006

Differential requirements for actin polymerization, calmodulin, and Ca2+ define distinct stages of lysosome/phagosome targeting.

Fusion of phagosomes with late endocytic organelles is essential for cellular digestion of microbial pathogens, senescent cells, apoptotic bodies, and retinal outer segment fragments. To further elucidate the biochemistry of the targeting process, we developed a scintillation proximity assay to study the stepwise association of lysosomes and phagosomes in vitro. Incubation of tritium-labeled lysosomes with phagosomes containing scintillant latex beads led to light emission in a reaction requiring cytosol, ATP, and low Ca(2+) concentrations. The nascent complex was sensitive to disruption by alkaline carbonate, indicating that the organelles had "docked" but not fused. Through inhibitor studies and fluorescence microscopy we show that docking is preceded by a tethering step that requires actin polymerization and calmodulin. In the docked state ongoing actin polymerization and calmodulin are no longer necessary. The tethering/docking activity was purified to near homogeneity from rat liver cytosol. Major proteins in the active fractions included actin, calmodulin and IQGAP2. IQGAPs are known to bind calmodulin and cross-link F-actin, suggesting a key coordinating role during lysosome/phagosome attachment. The current results support the conclusion that lysosome/phagosome interactions proceed through distinct stages and provide a useful new approach for further experimental dissection.

Actins↗
PubMed2003

Cofilin phosphorylation and actin polymerization by NRK/NESK, a member of the germinal center kinase family.

Nck-interacting kinase (NIK)-related kinase (NRK)/NIK-like embryo-specific kinase (NESK) is a protein kinase that belongs to the germinal center kinase family, and activates the c-Jun N-terminal kinase (JNK) signaling pathway. In this study, we examined the effect of NRK/NESK on actin cytoskeletal organization. Overexpression of NRK/NESK in COS7 cells induced accumulation of polymerized actin at the perinuclear. Phosphorylation of cofilin, an actin-depolymerizing factor, was increased in NRK/NESK-expressing HEK 293T cells. In addition, in vitro phosphorylation of cofilin was observed on NRK/NESK immunoprecipitates from HEK 293T cells expressing the kinase domain of NRK/NESK. The cofilin phosphorylation occurred at the serine residue of position 3 (Ser-3). Since the phosphorylation at Ser-3 inactivates the actin-depolymerizing activity of cofilin, these results suggest that NRK/NESK induces actin polymerization through cofilin phosphorylation. The cofilin phosphorylation did not appear to be mediated through activation of LIM-kinasel, a cofilin-phosphorylating kinase, or through the activation of JNK. Thus, cofilin is likely to be a direct substrate of NRK/NESK. NRK/NESK is predominantly expressed in skeletal muscle during the late stages of mouse embryogenesis. Thus, NRK/NESK may be involved in the regulation of actin cytoskeletal organization in skeletal muscle cells through cofilin phosphorylation.

Actin Depolymerizing Factors↗