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PubMed1987

Actin typing of rhabdomyosarcomas shows the presence of the fetal and adult forms of sarcomeric muscle actin.

We analyzed actin expression in two human rhabdomyosarcomas as well as in three rhabdomyosarcomas induced in rats by the injection of nickel sulfide. All five tumors exhibited appreciable amounts of the sarcomeric alpha-actin types, in line with their myogenic differentiation. The level of these actins was particularly high in the rat tumors, which according to morphological criteria, all showed a higher degree of differentiation than the human tumors. Interestingly, in both human tumors and in two of the three rat tumors, the level of the cardiac alpha-actin type was significantly higher than that of adult skeletal muscle alpha-actin. Taken together with the results of recent reports indicating that the cardiac alpha-actin type is a marker of embryonic and fetal skeletal muscle, our findings indicate that rhabdomyosarcomas express the embryonic sarcomeric actin isoform.

Actins↗
PubMed1981

Actin typing on total cellular extracts: a highly sensitive protein-chemical procedure able to distinguish different actins.

Based on the finding that the amino-terminal tryptic peptide of actin is a reliable marker for actin divergence, we describe in detail a highly sensitive protein-chemical procedure for actin typing. The method is performed on non-radioactivity labeled cells and tissues and six actins can be identified unambiguously in warm-blooded vertebrates. The method is quantitative and gives directly the ratio of the different actions in the specimens. It does not require previous purification of actin and can be used on total cellular extracts without any prior fractionation. The procedure can be extended to actins not previously characterized by amino acid sequence analysis and makes certain predictions possible about the partial amino acid sequences of the amino-terminal tryptic peptides, mostly sufficient for a correlation with DNA sequences derived from cloned actin genes. This is done as an example for the cytoplasmic action present in Schneider L-2 Drosophila melanogaster cells. Although the method is currently used routinely on 10(5) cells, modifications are discussed, which should allow the analysis to be performed with even higher sensitivity.

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PubMed1986

Isolation of a domain of villin retaining calcium-dependent interaction with G-actin, but devoid of F-actin fragmenting activity.

Villin is an F-actin binding protein located in the microfilament bundle of intestinal epithelial cell microvilli. Extensive in vitro proteolysis with Staphylococcus aureus V8 protease results in the production of a stable domain (apparent Mr 44000) which can be isolated due to its Ca2+-dependent interaction with G-actin bound to immobilized DNase-I, the standard procedure for the purification of villin. This 44-kDa fragment retains a single Ca2+ binding site with an apparent Kd = 2 X 10(-6) M, binds to G-actin, and inhibits the rate of actin polymerization. However, the 44-kDa domain does not shown any Ca2+-activated severing activity nor does it compete with villin for F-actin binding. These results suggest that villin contains three domains: headpiece containing an F-actin binding site, 44-kDa fragment containing a G-actin binding site, and an amino-terminal fragment responsible for the Ca2+-dependent severing activity.

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PubMed1987

pH-dependent rate of formation of the gelsolin-actin complex from gelsolin and monomeric actin.

The assembly of gelsolin with actin was followed by the increase of the fluorescence intensity of a fluorescence label bound to actin. The time course of the formation of the gelsolin-actin complex in the presence of micromolar [Ca2+] could be quantitatively interpreted by a model in which one actin molecule binds slowly to gelsolin in a rate-determining step and subsequently a second actin molecule is bound at least 40 times more rapidly. The rate of binding of the first actin molecule to gelsolin was found to be remarkably slow and to depend on the pH. The rate constants of formation of the gelsolin-actin complex range from 1.5 X 10(4) M-1 s-1 at pH 8 to 7 X 10(4) M-1 s-1 at pH 6.

Actins↗
PubMed1990

Structural study of gizzard caldesmon and its interaction with actin. Binding involves residues of actin also recognised by myosin subfragment 1.

The interaction between actin and caldesmon that is associated with the inhibition of actomyosin ATPase activity in smooth muscle has been studied using 1H-NMR spectroscopy. Binding studies using the intact molecules were complemented by the use of thrombic cleavage fragments of both turkey and chicken gizzard caldesmon as well as defined peptides of actin, in order to investigate the conformational properties of caldesmon and to localise regions of the primary structures that participate in protein-protein contacts. The binding of caldesmon is shown to involve distinct segments on the N-terminal region (residues 1-44) of actin, as previously observed for the inhibitory component of the thin filament of striated muscle, troponin I [Levine et al. (1988) Eur. J. Biochem. 153, 389-397]. The comparable structural properties of these tissue-specific inhibitors of actomyosin ATPase and the similarities in their mode of interaction at the N-terminal region of actin suggest common aspects to the structural mechanism for thin-filament regulation in smooth and striated muscle. Unlike the inhibitory interaction of troponin I, however, the binding of caldesmon to the N-terminal region of actin directly involves groups within residues 20-41 of actin that are also recognised by myosin subfragment 1. The complementary segment of caldesmon has been localised to a 15-kDa thrombic fragment (residues 483-578) derived from the N-terminal portion of a 35-kDa proteolytic cleavage product from the C-terminal of caldesmon whose interaction with actin is modulated by calmodulin. The results are discussed in relation to the calcium-mediated mechanism for thin-filament regulation in smooth and striated muscle.

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PubMed1991

Actin-caldesmon-myosin-subfragment-1 ternary complex viewed by electron microscopy. Competitive actin binding region for caldesmon and myosin subfragment-1.

An earlier electron microscopic study using different caldesmon forms complexed with actin revealed that the aggregates produced display regular periodic striation after antibody labeling of the 35-kDa caldesmon fragment. This approach provides further evidence that a caldesmon fragment, even as small as 15 kDa, can induce actin filaments to assemble into bundles. The observed difference in the compactness of these structures, depending on the use of the 15-kDa fragment instead of the 35-kDa fragment, suggests the existence of more than one actin-binding site in the caldesmon molecule. In this study, the caldesmon-induced process of F-actin association was investigated in the presence of skeletal myosin subfragment-1, using light-scattering methods, cosedimentation experiment and electron microscopic techniques. We show that the actin-caldesmon association is partially destabilized in the presence of subfragment-1 and this leads to a ternary complex formation. Immunogold labelling of the actin filaments still reveals the presence of caldesmon within this structure. This latter result strengthens the hypothesis that actin has a site(s) able to bind both caldesmon and myosin subfragment-1, as detected by recent NMR observations. This evidence is discussed with respect to the regulatory function of caldesmon during smooth muscle contraction.

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PubMed2006

Rotavirus spike protein VP4 binds to and remodels actin bundles of the epithelial brush border into actin bodies.

We demonstrate here that VP4, a rotaviral protein, is able to specifically bind to bundled actin microfilaments that are subsequently profoundly remodeled into actin bodies. These cytoplasmic actin bodies do not localize within identified intracellular compartments. VP4-induced actin remodeling is similar to cytochalasin D effects with kinetics compatible with that of rotavirus infection. Actin bundles' remodeling occurs both in infected and in VP4-transfected cells and in various cell lines, indicating that this is a general property of the viral protein itself. Interestingly, in intestinal epithelial cells, which represent the natural target of rotavirus, VP4 is addressed to the apical membrane where it binds specifically to brush border actin bundles and elicits its remodeling, whereas cytochalasin D impaired all the filamentous actin. These observations indicate that these original properties of VP4 likely explain the previously described brush border alterations that follow rotavirus infection of enterocytes and may also participate to the mechanism of rotavirus final assembly.

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PubMed2000

Mouse A6/twinfilin is an actin monomer-binding protein that localizes to the regions of rapid actin dynamics.

In our database searches, we have identified mammalian homologues of yeast actin-binding protein, twinfilin. Previous studies suggested that these mammalian proteins were tyrosine kinases, and therefore they were named A6 protein tyrosine kinase. In contrast to these earlier studies, we did not find any tyrosine kinase activity in our recombinant protein. However, biochemical analysis showed that mouse A6/twinfilin forms a complex with actin monomer and prevents actin filament assembly in vitro. A6/twinfilin mRNA is expressed in most adult tissues but not in skeletal muscle and spleen. In mouse cells, A6/twinfilin protein is concentrated to the areas at the cell cortex which overlap with G-actin-rich actin structures. A6/twinfilin also colocalizes with the activated forms of small GTPases Rac1 and Cdc42 to membrane ruffles and to cell-cell contacts, respectively. Furthermore, expression of the activated Rac1(V12) in NIH 3T3 cells leads to an increased A6/twinfilin localization to nucleus and cell cortex, whereas a dominant negative form of Rac1(V12,N17) induces A6/twinfilin localization to cytoplasm. Taken together, these studies show that mouse A6/twinfilin is an actin monomer-binding protein whose localization to cortical G-actin-rich structures may be regulated by the small GTPase Rac1.

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PubMed1996

Identification and characterization of espin, an actin-binding protein localized to the F-actin-rich junctional plaques of Sertoli cell ectoplasmic specializations.

Ectoplasmic specializations are membrane-cytoskeletal assemblages found in Sertoli cells at sites of attachment to elongate spermatids or neighboring Sertoli cells. They are characterized in part by the presence of a unique junctional plaque which contains a narrow layer of parallel actin bundles sandwiched between the Sertoli cell plasma membrane and an affiliated cistern of endoplasmic reticulum. Using a monoclonal antibody, we have identified 'espin,' a novel actin-binding protein localized to ectoplasmic specializations. By immunogold electron microscopy, espin was localized to the parallel actin bundles of ectoplasmic specializations at sites where Sertoli cells contacted the heads of elongate spermatids. The protein was also detected at the sites of ectoplasmic specializations between neighboring Sertoli cells. Espin exhibits an apparent molecular mass of approximately 110 kDa in SDS gels. It is encoded by an approximately 2.9 kb mRNA, which was found to be specific to testis among the 11 rat organs and tissues examined. On the basis of cDNA sequence, espin is predicted to be an 836 amino acid protein which contains 8 ankyrin-like repeats in its N-terminal third, a potential P-loop, two proline-rich peptides and two peptides which contain clusters of multiple glutamates bracketed by arginines, lysines and glutamines in a pattern reminiscent of the repetitive motif found in the protein trichohyalin. The ankyrin-like repeats and a 66 amino acid peptide in the C terminus show significant sequence similarity to proteins encoded by the forked gene of Drosophila. A fusion protein containing the C-terminal 378 amino acids of espin was found to bind with high affinity (Kd = approximately 10 nM) to F-actin in vitro with a stoichiometry of approximately 1 espin per 6 actin monomers. When expressed by transfected NRK fibroblasts, the same C-terminal fragment of espin was observed to decorate actin fibers or cables. On the basis of its structure, localization and properties, we hypothesize that espin is involved in linking actin filaments to each other or to membranes, thereby potentially playing a key role in the organization and function of the ectoplasmic specialization.

Actins↗
PubMed2004

Mobile actin clusters and traveling waves in cells recovering from actin depolymerization.

At the leading edge of a motile cell, actin polymerizes in close apposition to the plasma membrane. Here we ask how the machinery for force generation at a leading edge is established de novo after the global depolymerization of actin. The depolymerization is accomplished by latrunculin A, and the reorganization of actin upon removal of the drug is visualized in Dictyostelium cells by total internal reflection fluorescence microscopy. The actin filament system is reorganized in three steps. First, F-actin assembles into globular complexes that move along the bottom surface of the cells at velocities up to 10 microm/min. These clusters are transient structures that eventually disassemble, fuse, or divide. In a second step, clusters merge into a contiguous zone at the cell border that spreads and gives rise to actin waves traveling on a planar membrane. Finally, normal cell shape and motility are resumed. These data show that the initiation of actin polymerization is separated in Dictyostelium from front protrusion, and that the coupling of polymerization to protrusion is a later step in the reconstitution of a leading edge.

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PubMed2006

Inflammation in cardiovascular disease and regulation of the actin cytoskeleton in inflammatory cells: the actin cytoskeleton as a target.

Cardiovascular disease is prevalent in developed countries causing very large burdens to health services. The underlying pathology is atheromatous plaque in the sub-endothelial region of the vascular wall. High levels of low density lipoprotein cholesterol and high blood pressure cause endothelial damage. Atheroma develop from a response to this injury that is perpetuated to chronic inflammation. The invasion of inflammatory leukocytes into atheroma during its development and in the precipitation of acute thrombotic events is mediated by adhesion molecules on the cell surface. These are regulated by the actin filament cytoskeleton which also mediates intracellular signalling from them. The actin cytoskeleton is central to NADPH oxidase activation that produces superoxide which is an intracellular signalling molecule for the hypertensive and inflammatory actions of angiotensin II. There are polymorphisms in actin filament proteins such as adducin and caldesmon and in the promoter regions of tropomyosins that may cause individual variation in these processes. Many signalling molecules in the actin filament response to inflammatory stimuli and in signalling downstream from actin filaments are small G-proteins that require post-transcriptional modification by isoprenoids from the cholesterol synthetic pathway. Statins deplete the isoprenoids and so down regulate G-proteins that mediate the inflammatory response. Angiotensin converting enzyme inhibitors and angiotensin II receptor type 1 antagonists decrease angiotensin II stimulated superoxide production thus decreasing not only blood pressure but also inflammation. The anti-inflammatory effects of these drugs, involving altered actin filament function, are a major contributor to their benefits in the treatment of cardiovascular disease. The feasibility of modifying the behaviour of actin filament proteins as a therapeutic approach for cardiovascular disease is considered.

Actins↗
PubMed1999

Specific inhibition of skeletal alpha-actin gene transcription by applied mechanical forces through integrins and actin.

Skeletal alpha-actin (skA), a prominent fetal actin isoform that is re-expressed by adult cardiac myocytes after chronic overload in vivo, provides a model for studying cytoskeletal gene regulation by mechanical forces in vitro. We have determined the mechanisms by which perpendicular applied forces acting through integrins and the actin cytoskeleton regulate the expression of skA. Rat-2 fibroblasts were transiently transfected with plasmids containing 5'-regulatory regions of the skA gene fused to luciferase coding sequences. A constant, perpendicular force (0.2 pN/micrometer(2)) was applied by using a collagen-magnetic bead model; a 25% deformation was obtained on the dorsal cell surface. In this system, force is applied through focal adhesion integrins and strongly induces actin assembly [Glogauer, Arora, Yao, Sokholov, Ferrier and McCulloch (1997) J. Cell Sci. 110, 11-21]. skA promoter activity was inhibited by 68% in cells subjected to 4 h of applied force, whereas Rous sarcoma virus promoter activity was unaffected. In cells transiently transfected with a skA expression vector there was also a parallel 40% decrease in skA protein levels by force, as shown by Western blotting. In L8 cells, constitutive skA expression was decreased by more than 50%. Analyses of specific motifs in the skA promoter revealed that transcriptional enhancer factor 1 and Yin and Yang 1 sites, but not serum response factor and Sp1 sites, mediated inhibitory responses to force. In cells treated with cycloheximide the force-induced inhibition was abrogated, indicating a dependence on new protein synthesis. Inhibition of actin filament assembly with either cytochalasin D or Ca(2+)-depleted medium blocked the inhibitory effect induced by the applied force, suggesting that actin filaments are required for the regulation of skA promoter activity. Western blot analysis showed that p38 kinase, but not Jun N-terminal kinase or extracellular signal-regulated protein kinase 1/2, was activated by force; indeed, the p38 kinase inhibitor SB203580 relieved the force-induced inhibition of skA. We conclude that the force-induced inhibition of skA promoter activity requires an intact actin cytoskeleton and can be mapped to two different response elements. This inhibition might be mediated through the p38 kinase.

Actins↗
PubMed1986

Simultaneous expression of skeletal muscle and heart actin proteins in various striated muscle tissues and cells. A quantitative determination of the two actin isoforms.

A procedure was developed to determine the percentage of skeletal muscle actin and cardiac actin present in different striated muscle tissues. The method was applied to 2 mg of actin mixtures isolated from various origins. All samples show simultaneous expression of both striated muscle isoactins, with the cardiac actin being the major form (congruent to 80%) in 11-day-old chick embryonic leg muscle, decreasing to approximately 50% values in the late fetal stage of chicken, mouse, and in fused mouse muscle cell cultures and becoming the minor species (less than 5%) in adult skeletal muscle tissues. We also find a significant amount (up to 20%) of the skeletal muscle isoform in adult heart (ventricle) of porcine, bovine, and human origin and no differences in muscle actin ratios in human atrium and ventriculum cells. Similarly, no significant variation in the actin ratios was observed between a normal heart and a heart from a patient with hereditary obstructive myopathy. For those cells and tissues where comparison with levels of mRNA was possible we mostly find a good correlation between the relative ratios of expression of cardiac and skeletal actin proteins and mRNAs.

Actins↗
PubMed1986

Anthracycline effects on actin and actin-containing thin filaments in cultured neonatal rat myocardial cells.

Adriamycin (ADR, Doxorubicin) effects on actin and other proteins in cultured neonatal rat cardiac myocytes were investigated. Heart cells were exposed to ADR in doses of 10(-8) M to 10(-5) M for 24 hours. Cells were harvested in 2 mM of Tris buffer containing Triton X-100, homogenized and centrifuged in a microfuge. Parallel dishes of cultured cardiac myocytes were washed in buffered saline and were fixed at 4 degrees C in Karnovsky's fixative. The supernatant solutions were dialyzed and then incubated with pancreatic DNAase I to quantify actin by enzyme inhibition. In parallel studies, both cell supernatant solutions and pellets were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and the stained polypeptide bands were quantified by densitometry. Results showed that heart cells exposed to 10(-6) M of ADR for 24 hours had unpolymerized actin levels reduced to 7.7 micrograms/10(6) cells (as measured by DNAase I inhibition or by sodium dodecyl sulfate polyacrylamide gel electrophoresis along with densitometry) compared to 11.0 micrograms/10(6) cells in untreated culture heart cells. When ADR concentration was 10(-7) or 10(-8) M, unpolymerized actin levels were similar to the levels of untreated heart cells. Protein content of extract solutions of untreated and ADR-treated myocytes were 1.2 mg/ml and 0.8 mg/ml, respectively. Gel densitometry of electrophoretograms showed actin to account for 12 to 16% of total density of bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Comparative densitometry of ADR-treated cells treated with 10(-6) M of ADR show depolymerized actin to account for 77% of total actin. Ultrastructural results show a large clear cytoplasmic zone of disorganized 12 to 14-nm filaments in cultured myocytes exposed to 10(-6) M ADR. Little change in myocyte ultrastructure was seen at 10(-7) M or 10(-8) M ADR exposure. Data support ADR as a cellular disruptor with toxic effects on cardiac cytoplasmic and contractile proteins and filaments. This ADR effect on heart cells in culture is dose-related.

Actins↗
PubMed1983

Rabbit autoantibodies to actin induced by immunization with heterologous actins; a possible mechanism of smooth muscle antibody production.

Antisera against actins from chicken gizzard smooth muscle and ascaris body wall were prepared in rabbits. Immunological cross-reactivity of the antisera with actins of several different species was demonstrated by precipitation reactions in agarose gels and by immunofluorescence studies. The antisera were also reactive with actin of rabbits, the homologous animal used for immunization. The latter finding indicated termination of natural tolerance to actin by immunization of cross-reactive actins. A possible mechanism operating in the induction of smooth muscle antibodies, autoantibodies which have a similar nature to the anti-actin antibody is discussed in relation to the termination of natural tolerance to actin.

Actins↗
PubMed1980

Cytochalasin B and the structure of actin gels. II. Further evidence for the splitting of F-actin by cytochalasin B.

Cytochalasin B decreased the flow birefringence and s20,w and increased the extinction angle of actin filaments in salt solutions favoring polymerization of the protein. These changes occurred without a detectable increase in the equilibrium actin monomer concentration determined by a radioassay. These results complement earlier observations indicating that cytochalasin B shortens actin filaments without net depolymerization. Analyzed in terms of Flory's classical network theory, this shortening accounts for the marked effect of cytochalasin B in dissolving the gel structure of F-actin crosslinked by actin-binding protein concentrations near the critical concentration for incipient gelation. Cytochalasin B decreased the annealing rate of low concentrations of actin filament fragments prepared by sonic disruption. The result is consistent with the idea that cytochalasin B binds to the ends of actin filaments, and may explain how cytochalasin B causes filament shortening.

Actins↗
PubMed1994

Chicken antibodies to rabbit muscle actin with a restricted repertoire of F-actin recognition.

This report describes a polyclonal antibody against actin with unexpected and unusual properties. The antibody was raised in chicken immunized with a complex of DNase I and rabbit skeletal muscle actin, and purified from egg yolk by affinity chromatography. In Western blots, it reacted with alpha, beta and gamma isoforms of actin. In immunofluorescence and dot blot assays, however, it recognized selectively actin filaments in myofibrils, microvilli of brush border-type epithelium and the "comet tails" of the intracellular parasite Listeria monocytogenes, while it did not react with stress fibers and peripheral belts of fibroblasts and epithelial cells, respectively. This reactivity pattern is reminiscent of that previously described for a monoclonal mouse antibody raised against smooth muscle actin (Sawtell et al., Cell Motil. Cytoskel. 11, 318, 1988). The data presented in this study are consistent with the hypothesis that the chicken antibody recognizes an actin epitope/actin epitopes either accessible in only a subpopulation of microfilaments, or expressed only in a particular conformation of F-actin.

Actins↗
PubMed1994

Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin.

Jasplakinolide, a naturally occurring cyclic peptide from the marine sponge, Jaspis johnstoni, has both fungicidal and antiproliferative activity. We now report that this peptide is a potent inducer of actin polymerization in vitro. The peptide has a much greater effect on Mg(2+)-actin than on Ca(2+)-actin. Competitive binding studies using rhodamine-phalloidin suggest that jasplakinolide binds to F-actin competitively with phalloidin with a dissociation constant of approximately 15 nM. This compares favorably to the previously reported IC50 of 35 nM for the antiproliferative effect of jasplakinolide on PC3 prostate carcinoma cells. The binding curve suggests that nearest neighbor positive cooperativity influences the binding of jasplakinolide (and perhaps also phalloidin) to F-actin. These results imply that jasplakinolide may exert its cytotoxic effect in vivo by inducing actin polymerization and/or stabilizing pre-existing actin filaments.

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