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PubMed1991

Proximity relationships between sites on myosin and actin. Resonance energy transfer determination of the following distances, using a hybrid myosin: those between Cys-55 on the Mercenaria regulatory light chain, SH-1 on the Aequipecten myosin heavy chain, and Cys-374 of actin.

Resonance energy transfer measurements have been made on hybrid myosins in order to map distances between sites on the regulatory light chain, heavy chain, and actin as well as to assess potential conformational changes of functional importance. Using scallop (Aequipecten) myosin hybrid molecules possessing clam (Mercenaria) regulatory light chains, we have been able to map the distance between Cys-55 on the regulatory light chain and the fast-reacting thiol on the myosin heavy chain (SH-1). This distance is shown to be approximately 6.4 nm, and it is not altered by the presence or absence of Ca2+, MgATP, or actin. Experiments performed at low ionc strength on heavy meromyosin (HMM) derived from these hybrid myosins gave results similar to those performed on the soluble parent myosin preparations. The distances between Cys-374 on actin and each of the above sites were also measured. Mercenaria regulatory light-chain Cys-55, within the hybrid myosin molecule, was found to be greater than 8.0 nm away from actin Cys-374. Scallop heavy-chain SH-1 is shown to be approximately 4.5 nm away from actin Cys-374, in broad agreement with earlier measurements made by others in nonregulatory myosins. The significance of our results is discussed with respect to putative conformational changes within the region of the heavy chain connecting SH-1 to the N-terminal region of the light chain.

Actins↗
PubMed1988

Fluorescence properties of acrylodan-labeled tropomyosin and tropomyosin-actin: evidence for myosin subfragment 1 induced changes in geometry between tropomyosin and actin.

The Cys groups of rabbit skeletal tropomyosin (Tm) and rabbit skeletal alpha alpha Tm were specifically labeled with acrylodan (AC). The probe on Tm is quite immobile yet exposed to solvent as indicated by its limiting polarization (P0 = 0.38) and fluorescence emission spectrum (lambda max = 520 nm) and its accessibility to solute quenching. Changes in the shape of the excitation spectrum with temperature correlated with the helix thermal pretransition and main transition without much spectral change of the emission spectrum. The probe environment of ACTm did not significantly change on binding to F-actin, but fluorescence energy transfer between tryptophan in actin and AC on Tm was indicated by a 15-20% increase in AC fluorescence and a few percent decrease in tryptophan fluorescence. This energy transfer increased when myosin subfragment 1 (S1) was bound to the ACTm-actin filament, in quantitative agreement with the postulated shift in state of Tm associated with the cooperative binding of S1 to actin (Hill et al., 1980). The increase in energy transfer shows that there is a change in the spatial relationship between Tm and actin associated with the S1-induced change in state of Tm.

2-Naphthylamine↗
PubMed1999

Detection of nucleotide- and F-actin-induced movements in the switch II helix of the skeletal myosin using its differential oxidative cleavage mediated by an iron-EDTA complex disulfide-linked to the strong actin binding site.

We have synthesized the mixed disulfide, S-(2-nitro-5-thiobenzoic acid) cysteaminyl-EDTA, using a rapid procedure and water-soluble chemistry. Its disulfide-thiol exchange reaction with rabbit myosin subfragment-1 (S-1), analyzed by spectrophotometry, ATPase assays, and peptide mapping, led to the incorporation of the cysteaminyl-EDTA group into only Cys 540 on the heavy chain and into the unique cysteine on the alkali light chains. The former thiol, residing in the strong actin binding site, reacted at a much faster rate with a concomitant 3-fold decrease in the V(max) for acto-S-1 ATPase but without change in the essential enzymatic functions of S-1. Upon chelation of Fe(3+) ions to the Cys 540-bound EDTA and incubation of the S-1 derivative-Fe complex with ascorbic acid at pH 7.5, the 95 kDa heavy chain underwent a conformation-dependent, single-cut oxidative fragmentation within 5-15 A of Cys 540. Three pairs of fragments were formed which, after specific fluorescent labeling and SDS-PAGE, could be positioned along the heavy chain sequence as 68 kDa-26 kDa, 62 kDa-32 kDa, and 54 kDa-40 kDa. Densitometric measurements revealed that the yield of the 54 kDa-40 kDa pair of bands, but not that for the two other pairs, was very sensitive to S-1 binding to nucleotides or phosphate analogues as well as to F-actin. In binary complexes, all the former ligands specifically lowered the yield to 40% of S-1 alone, roughly in the following order: ADP = AMP-PNP > ATP = ADP.AlF(4) > ADP.BeF(x)() > PP(i). By contrast, rigor binding to F-actin increased the yield to 130%. In the ternary acto-S-1-ADP complex, the yield was again reduced to 80%, and it fell to 25% in acto-S-1-ADP.AlF(4), the putative transition state analogue complex of the acto-S-1 ATPase. These different quantitative changes reflect distinct ligand-induced conformations of the secondary structure element whose scission generates the 54 kDa-40 kDa species. According to the S-1 crystal structure, this element could be unambiguously assigned to the switch II helix (residues 475-507) whose N-terminus lies 14.2 A from Cys 540 and would include the ligand-responsive cleavage site. This motif is thought to be crucial for the transmission of sub-nanometer structural changes at the ATPase site to both the actin site and the lever arm domain during energy transduction. Our study illustrates this novel, actin site-specific chemical proteolysis of S-1 as a direct probe of the switch II helix conformational transitions in solution most likely associated with the skeletal cross-bridge cycle.

Actins↗
PubMedDate not supplied

Regulation of actin polymerization by non-polymerizable actin-like proteins.

Three functionally distinct actin-capping proteins from the slime mould Physarum are structurally closely related to actin itself. In Physarum, actin polymerization is regulated by a set of non-polymerizable actin-like proteins. It remains to be established whether these proteins and actin are each encoded by separate genes.

Actins↗
PubMed2005

Phospholipase D activity is essential for actin localization and actin-based motility in Dictyostelium.

PLD (phospholipase D) activity catalyses the generation of the lipid messenger phosphatidic acid, which has been implicated in a number of cellular processes, particularly the regulation of membrane traffic. In the present study, we report that disruption of PLD signalling causes unexpectedly profound effects on the actin-based motility of Dictyostelium. Cells in which PLD activity is inhibited by butan-1-ol show a complete loss of actin-based structures, accompanied by relocalization of F-actin into small clusters, and eventually the nucleus, without a visible fall in levels of F-actin. Addition of exogenous phosphatidic acid reverses the effects of butan-1-ol, confirming that these effects are caused by inhibition of PLD. Loss of motility correlates with complete inhibition of endocytosis and a reduction in phagocytosis. Inhibition of PLD caused a major decrease in the synthesis of PtdIns(4,5)P2, which could again be reversed by exogenously applied phosphatidic acid. Thus the essential role of PLD signalling in both motility and endocytosis appears to be mediated directly via regulation of PtdIns(4)P kinase activity. This implies that localized PLD-regulated synthesis of PtdIns(4,5)P2 is essential for Dictyostelium actin function.

Actins↗
PubMed1992

Thrombin promotes actin polymerization in U937 human monocyte-macrophage cells. Analysis of the signalling mechanisms mediating actin polymerization.

The U937 human monocyte-macrophage cell line was used to examine the effect of thrombin, an ill-defined chemoattractant, on the polymerization of actin, a process essential for cell motility. In differentiated macrophage-like U937 cells, thrombin (0.5-50 units/ml) caused a rapid dose-dependent increase in the formation of filamentous (F-) actin, detected by the staining of F-actin with the fluorescent toxin, 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. In contrast with other chemoattractants such as N-formylmethionyl-leucylphenylalanine or C5a, actin polymerization in response to thrombin occurred via a pertussis-toxin-insensitive G1-(inhibitory G-protein) independent signalling pathway. Further, this response was not affected by the Ca2+ chelator EGTA or by the specific protein kinase C (PKC) inhibitor RO-31-8220. The response to thrombin was not mimicked by the Ca2+ ionophore ionomycin or by the direct PKC activator phorbol 12-myristate 13-acetate. The thrombin response was, however, inhibited by the non-specific protein kinase inhibitor staurosporine. The present results suggest that in U937 cells thrombin stimulates the formation of F-actin via a signalling pathway independent of (i) the activation of PKC, (ii) the mobilization of intracellular Ca2+ and (iii) the activation of Ca(2+)-dependent protein kinases, but dependent on the activation of an undefined staurosporine-sensitive protein kinase.

Actins↗
PubMed1989

Coupling of nonpolymerizable monomeric actin to the F-actin binding region of the myosin head.

Polymerizations of skeletal G-actin induced by salt and myosin subfragment 1 (S-1) were suppressed by reaction of G-actin with m-maleimidobenzoyl-N-hydroxysuccinimide ester. The G-actin derivative, containing few intramolecular crosslinks and a free maleimide group, was covalently coupled in solution to the S-1 heavy chain. The resulting complex could no longer bind to F-actin. The SH-1 and SH-2 thiols of S-1 were not involved in the complexation and the covalent link was shown to be exclusively on the 50-kDa segment of the S-1 heavy chain. The specific conjugation of the two proteins followed formation of a reversibly associated pyrophosphate-sensitive binary complex which was characterized by different approaches. Potentially, these complexes may be useful in developing the crystallography of actin-bound S-1.

Actins↗
PubMed1999

The EVH2 domain of the vasodilator-stimulated phosphoprotein mediates tetramerization, F-actin binding, and actin bundle formation.

Vasodilator-stimulated phosphoprotein (VASP) is a member of the Ena/VASP family of proteins that are implicated in regulation of the actin cytoskeleton. All family members share a tripartite structural organization, comprising an N-terminal Ena/VASP homology (EVH) 1 domain, a more divergent proline-rich central part, and a common C-terminal EVH2 region of about 160-190 amino acids. Using chemical cross-linking, sucrose gradient sedimentation, and gel filtration analyses of different truncated VASP constructs, we demonstrate that the VASP EVH2 region is both necessary and sufficient for tetramerization. Moreover, co-sedimentation and fluorescent phalloidin staining showed that the EVH2 region binds and bundles F-actin in vitro and localizes to stress fibers in transfected cells. Analysis of the functional contribution of highly conserved blocks within this region indicated that residues 259-276 of human VASP are essential for the interaction with F-actin, whereas residues 343-380 are required for tetramerization, probably via coiled-coil formation. Interactions with F-actin are enhanced by VASP tetramerization. The results demonstrate that the C-terminal EVH2 segment is not only conserved in sequence but also forms a distinct functional entity. The data suggest that the EVH2 segment represents a novel oligomerization and F-actin binding domain.

Actins↗
PubMed2001

The rat homologue of Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) associates with actin filaments, recruits N-WASP from the nucleus, and mediates mobilization of actin from stress fibers in favor of filopodia formation.

We cloned and characterized the rat homologue of the Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP). Rat WIP shows 86% amino acid sequence identity to human WIP. Northern analyses revealed two major mRNA species of 5.0 and 3.8 kb, which were ubiquitously expressed, though predominantly in spleen and lung. Minor species of 2.4, 1.8, 1.4, and 1.1 kb were also detected in some tissues and cell lines. Thus, WIP is subject to tissue-specific alternative splicing. WIP bound to N-WASP in vivo, as revealed by co-immunoprecipitation. Expression of WIP in rat fibroblasts revealed a clear co-localization with actin stress fibers. However, expression in tumor cells lacking actin cables did not restore these structures. Interestingly, co-expression of WIP and N-WASP resulted in redistribution of N-WASP, abrogating its dominant nuclear expression and leading to co-localization with WIP in the perinuclear area and with actin in membrane protrusions. Moreover, stress fibers and, concomitantly, the associated WIP were largely dissolved. Very similar effects were seen upon epidermal growth factor stimulation of serum-starved cells. Our results suggest that WIP might be involved in transmitting mitogenic signals to cytoskeletal functions, perhaps by modulating the subcellular localization of N-WASP. Interaction of N-WASP with WIP may in turn lead to mobilization of actin from stress fibers and nucleation of new actin filaments in filopodia.

Actins↗
PubMed2002

Dynacortin is a novel actin bundling protein that localizes to dynamic actin structures.

Dynacortin is a novel protein that was discovered in a genetic suppressor screen of a Dictyostelium discoideum cytokinesis-deficient mutant cell line devoid of the cleavage furrow actin bundling protein, cortexillin I. While dynacortin is highly enriched in the cortex, particularly in cell-surface protrusions, it is excluded from the cleavage furrow cortex during cytokinesis. Here, we describe the biochemical characterization of this new protein. Purified dynacortin is an 80-kDa dimer with a large 5.7-nm Stokes radius. Dynacortin cross-links actin filaments into parallel arrays with a mole ratio of one dimer to 1.3 actin monomers and a 3.1 microm K(d). Using total internal reflection fluorescence microscopy, GFP-dynacortin and the actin bundling protein coronin-GFP are seen to concentrate in highly dynamic cortical structures with assembly and disassembly half-lives of about 15 s. These results indicate that cells have evolved different actin-filament cross-linking proteins with complementary cellular distributions that collaborate to orchestrate complex cell shape changes.

Actins↗
PubMed1999

Introduction of NAD decreases fMLP-induced actin polymerization in chicken polymorphonuclear leukocytes--the role of intracellular ADP-ribosylation of actin for cytoskeletal organization.

We reported previously that the arginine-specific ADP-ribosyltransferase in chicken polymorphonuclear leukocytes specifically modified actin, thereby inhibiting actin polymerization in vitro. In the present study, we investigated the effect of ADP-ribosylation on actin polymerization in situ. In the leukocytes, the introduction of NAD inhibited the increase in filamentous actin contents induced by a chemotactic peptide formyl-methionyl-leucyl-phenylalanine, while introduction of NAD together with novobiocin, a specific inhibitor for ADP-ribosyltransferase, did not. These results suggest that ADP-ribosylation regulates the formation of filamentous actin by the covalent modification of the protein in vivo.

Actins↗
PubMed1978

Polymerization of actin. IV. Role of Ca++ and H+ in the assembly of actin and in membrane fusion in the acrosomal reaction of echinoderm sperm.

When Pisaster, Asterias, or Thyone sperm are treated with the ionophore A23187 or X537A, an acrosomal reaction similar but not identical to a normal acrosomal reaction is induced in all the sperm. Based upon the response of the sperm, the acrosomal reaction consists of a series of temporally related steps. These include the fusion of the acrosomal vacuole with the cell surface, the polymerization of the actin, the alignment of the actin filaments, an increase in volume, an increase in the limiting membrane, and changes in the shape of the nucleus. In this report, we have concentrated on the first two steps in this sequence. Although fusion of the acrosomal vacuole with the cell surface requires Ca++, we found that the polymerization of actin instead appears to be dependent upon an increase in intracellular pH. This conclusion was reached by applying to sperm A23187, X537A, or nigericin, ionophores which all carry H+ at high affinity, yet vary in their affinity for other cations. When sperm are suspended in isotonic NaCl, isotonic KCl, calcium-free seawater, or seawater, all at pH 8.0, and the ionophore is added, the actin polymerizes explosively and an efflux of H+ from the cell occurs. However, if the pH, of the external medium is maintained at 6.5, the presumed intracellular pH, no effect is observed. And, finally, if egg jelly is added to sperm (the natural stimulus for the acrosomal reaction) at pH 8.0, H+ is also released. On the basis of these observations and those presented in earlier papers in this series, we conclude that a rise in intracellular pH induces the actin to disassociate from its binding proteins. Now it can polymerize.

Acrosome↗
PubMed1980

Association of actin with sperm centrioles: isolation of centriolar complexes and immunofluorescent localization of actin.

The centrioles of cnidarian sperm associate with striated specializations (pericentriolar processes) during spermiogenesis. Three functions have been proposed for the role of these structures: (a) an anchoring mechanism for the sperm flagellum, (b) a signal-transmitting mechanism for communication between sperm head and tal, and (c) a contractile mechanism involved in motor function of the sperm flagellum. To investigate these proposed functions, we developed a technique for the isolation and purification of Hydractinia sperm distal centriles with attached pericentriolar processes. SDS polyacrylamide electrophoretic profiles of whole sperm and pericentriolar process proteins revealed a prominent protein that comigrates with rabbit and penaeid shrimp muscle actin. To label and localize actin in hydroid spem, we produced in rabbits a highly specific antiserum to invertebrate actin that cross-reacts with both invertebrate and vertebrate muscle and nonmuscle actin. Immunofluorescent double antibody labeling of hydroid sperm with antiactin has demonstrated the presence of actin in the pericentriolar process region of the sperm. In earlier reports, it has been proposed that pericentriolar processes, if contractile, could alter the mid-piece asymmetry of hydroid sperm, facilitating the directional motility that these cells demonstrate in respone to egg-released chemoattractants. The present results support this hypothesis.

Actins↗
PubMed1983

A consistent picture of the actin filament related to the orientation of the actin molecule.

We show that freeze-dried actin filaments which have been rotary shadowed with a light coat of platinum appear very similar in morphology and width to negatively-stained filaments. The addition of a thicker coat of platinum to such preparations gives the actin filaments a different morphology and width, which are similar to those of the rotary-shadowed, quick-frozen filaments described by Heuser and Kirschner (J. Cell Biol. 1980, 86:212-234). The consistent view of the actin filament presented here, particularly its 7-8-nm width, can be interpreted in terms of the overall orientation of the actin subunit in the actin filament.

Actins↗
PubMed1984

A 45,000-mol-wt protein from unfertilized sea urchin eggs severs actin filaments in a calcium-dependent manner and increases the steady-state concentration of nonfilamentous actin.

A 45,000-mol-wt protein has been purified from unfertilized sea urchin (Strongylocentrotus purpuratus) eggs. The isolation scheme includes DEAE cellulose ion-exchange chromatography, gel filtration, and hydroxylapatite chromatography. The homogeneity of the isolated protein is greater than 90% by SDS PAGE. The 45,000-mol-wt protein reduces the viscosity of actin filaments in a Ca2+-dependent manner. The free calcium concentration required for the activity of this protein is in the micromolar range. Electron microscopic studies reveal that the formation of short filaments parallels the decrease in viscosity. Energy transfer and sedimentation experiments indicate a net disassembly of actin filaments and an increase in the steady-state nonfilamentous actin concentration in the presence of Ca2+ ions and the 45,000-mol-wt protein. The increase in the steady-state nonfilamentous actin concentration is proportional to the amount of 45,000-mol-wt protein added. The actin molecules disassembled by the addition of the 45,000-mol-wt protein are capable of polymerization.

Actins↗
PubMed2001

Profilin binding to poly-L-proline and actin monomers along with ability to catalyze actin nucleotide exchange is required for viability of fission yeast.

We tested the ability of 87 profilin point mutations to complement temperature-sensitive and null mutations of the single profilin gene of the fission yeast Schizosaccharomyces pombe. We compared the biochemical properties of 13 stable noncomplementing profilins with an equal number of complementing profilin mutants. A large quantitative database revealed the following: 1) in a profilin null background fission yeast grow normally with profilin mutations having >10% of wild-type affinity for actin or poly-L-proline, but lower affinity for either ligand is incompatible with life; 2) in the cdc3-124 profilin ts background, fission yeast function with profilin having only 2-5% wild-type affinity for actin or poly-L-proline; and 3) special mutations show that the ability of profilin to catalyze nucleotide exchange by actin is an essential function. Thus, poly-L-proline binding, actin binding, and actin nucleotide exchange are each independent requirements for profilin function in fission yeast.

Actins↗
PubMed1993

Genetic evidence for functional interactions between actin noncomplementing (Anc) gene products and actin cytoskeletal proteins in Saccharomyces cerevisiae.

We describe here genetic interactions between mutant alleles of Actin-NonComplementing (ANC) genes and actin (ACT1) or actin-binding protein (SAC6, ABP1, TPM1) genes. The anc mutations were found to exhibit allele-specific noncomplementing interactions with different act1 mutations. In addition, mutant alleles of four ANC genes (ANC1, ANC2, ANC3 and ANC4) were tested for interactions with null alleles of actin-binding protein genes. An anc1 mutant allele failed to complement null alleles of the SAC6 and TPM1 genes that encode yeast fimbrin and tropomyosin, respectively. Also, synthetic lethality between anc3 and sac6 mutations, and between anc4 and tpm1 mutations was observed. Taken together, the above results strongly suggest that the ANC gene products contribute to diverse aspects of actin function. Finally, we report the results of tests of two models previously proposed to explain extragenic noncomplementation.

Actins↗
PubMed1990

Actin with tumor-related mutation is antimorphic in Drosophila muscle: two distinct modes of myofibrillar disruption by antimorphic actins.

A mutant beta-actin with an amino acid substitution from Gly-245 to Asp has been shown to be related to tumorigenic transformation of a human fibroblast cell line (Leavitt, J. et al. (1987) Mol. Cell. Biol. 7, 2467-2476). To examine the effects of this mutation, we artificially introduced the same amino acid change into the Act88F actin gene of Drosophila melanogaster. The gene (Act88FGD245) was inserted in the Drosophila genome to make transgenic adult flies which synthesize the mutant actin in the indirect flight muscles. The mutant actin was found to be antimorphic with regard to flight and also to cause myofibrillar disruption in transformants even in the presence of two normal alleles. It was initially incorporated into myofibrils and later induced their degeneration from center to periphery. This mode of myofibrillar disruption is distinct from that of previously reported Act88F mutations, where defects are found only in the peripheral region of myofibrils. This indicates that actin functions are altered differently in the two classes of antimorphic mutations.

Actins↗