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PubMed1987

Isolation and characterization of actin from cultured BHK cells.

Cytoplasmic actin from cultured fibroblasts has been purified to homogeneity and characterized with respect to its polymerization and structure. It was found to be qualitatively similar to muscle actin in all respects, but significant quantitative differences in its properties were demonstrated. Although BHK actin did not polymerize in unfractionated cytoplasmic extracts, the purified BHK actin polymerized into filaments both in magnesium and calcium. The critical concentration, measured by the DNase I inhibition assay and by fluorimetry, was the same as that of muscle actin both in magnesium and calcium. Polymerization of pyrene-labelled BHK and muscle actin was followed by fluorimetry. Significant differences in kinetics were found under both ionic conditions tested. In the absence of Mg2+ ions (0.2 mM CaCl2, 85 mM KCl), BHK actin polymerized at a much slower rate than muscle actin. In the presence of magnesium and EGTA, the nucleation phase for BHK actin polymerization was shorter than that for muscle actin and the kinetics of polymerization was different. The structure of BHK actin filaments in the electron micrographs was very similar to that of muscle actin. In high concentrations of magnesium, BHK actin formed paracrystals which had the same appearance as muscle actin paracrystals. However, calcium-induced formation of actin paracrystals required higher concentration of Ca2+ ions for BHK actin than for muscle actin (12 mM and 8 mM respectively). These results suggest differences in divalent cation binding to both high- and low-affinity sites of the two actins.

Actins↗

PubMed1981

A radioimmunoassay for determination of anti-actin antibodies.

The reaction of spontaneously occurring human anti-human antibodies and experimentally produced rabbit anti-actin antibodies was investigated in a solid-phase radioimmunoassay (RIA). Three structurally different in vitro forms of actin monomeric G-actin filamentous F-actin and aggregated denatured actin were used as antigens. Human anti-actin antibodies reacted with F- and G-actin but not with aggregated actin, while rabbit anti-actin antibodies gave a strong reaction with all 3 forms of actin indicating differences in antibody specificities. The results of the anti-actin RIA were compared with those obtained by indirect immunofluorescence (IFL) on cryostat sections of rat stomach. The anti-actin RIA discriminated between patients' sera and control sera in most cases, although the indirect IFL test gave more conclusive results. The seemingly low sensitivity of the anti-actin RIA compared with that of indirect IFL test for detection of human anti-actin antibodies is probably due to favourable antigen distribution in tissue, not available in the solid phase. The anti-actin RIA was able to detect anti-actin antibodies in 8 out of 8 immunized rabbits although only two produced antibodies detectable by indirect IFL. The differences in reactivity between the two methods may depend on the presence of aggregated denatured actin in the antigen preparation used for immunization and exposure of the corresponding antigenic determinants of actin on the solid phase.

Actins↗

PubMed2000

Cross-linked dimers with nucleating activity in actin prepared from muscle acetone powder.

A covalently linked actin dimer is identified in solutions of actin prepared from an acetone powder from skeletal muscle. This actin dimer acts as an actin nucleating factor (ANF), decreasing the half-time for spontaneous actin polymerization. ANF reacts with antibodies to both the N- and C-terminal portions of actin on Western blots and migrates during reduced polyacrylamide gel electrophoresis like actin cross-linked with N, N'-p-phenylenebismaleimide. The origin of the cross-linked dimer appears to be related to the presence of carbonyl groups in purified actin. A large number of carbonyls (approximately 0.3/actin) are introduced into actin during the prolonged treatment with acetone in the preparation of the muscle acetone powder from which actin is extracted. Actin extracted from acetone powder prepared by a single acetone wash and actin prepared from bovine spleen, which is not washed with acetone, both contain fewer carbonyl groups (approximately 0.05 carbonyl/actin). ANF forms spontaneously in solutions of polymer actin containing 0.3 carbonyl/actin. We speculate that a reaction between a carbonyl on one actin polymer subunit and a lysine on a neighboring subunit is responsible for ANF formation. The presence of cross-linked actin dimers in commonly used skeletal muscle actin preparations could certainly affect studies of actin polymerization and, particularly, studies of the nucleation reaction. The physiological relevance of ANF is not clear, but given the large cellular concentration of actin, similar reactions yielding ANF could occur in vivo when increased levels of reactive oxygen species are present.

Acetone↗

PubMed2002

Decreased actin solubility observed during ATP-depletion is mimicked by severing agents but not depolymerizing agents in isolated and cultured proximal tubular cells.

The microvilli of the apical membrane of proximal tubule (PT) cells are supported by the underlying actin cytoskeleton. Ischaemic or anoxic ATP-depletion leads to the disruption of the actin cytoskeleton, resulting in microvillar retraction and loss of membrane polarity. Using isolated PT cells, we have previously demonstrated that actin filaments (F-actin) are likely severed during ATP-depletion. A sequential extraction protocol revealed a decrease in actin solubility, resulting in the sequestration of a distinct F-actin pool with the insoluble cellular complex in ATP-depleted PT cells. We demonstrate here that decreased actin solubility is not only a reliable end-marker of ATP-depletion induced injury in freshly isolated PT cells, but also serves as a biochemical marker in the cultured proximal tubular cell line LLC-PK1. In the present studies, we also investigated specific actin-binding drugs to determine if they mimic the effects observed during energy depletion. Jasplakinolide (JP), a compound which binds F-actin and prevents depolymerization, did not effect actin solubility during ATP-depletion. Furthermore, swinholide A (SA), an F-actin severing agent, resulted in decreased actin solubility, mimicking the effects of ATP-depletion. Interestingly, latrunculin A (LA), an agent which depolymerizes F-actin, did not reduce actin solubility, but rather resulted in an increase in digitonin-soluble actin. Taken collectively, our results support previous work and suggest that disruption of the actin cytoskeleton during ATP-depletion is mediated by F-actin severing/fragmentation and not depolymerization. The differential effects of F-actin disrupting agents and the consistencies observed in both models of ischaemic injury will provide a basis for a more detailed understanding of the pathological events of PT-cell dysfunction.

Actins↗

PubMed1991

Mechanism of the interaction of human platelet profilin with actin.

We have reexamined the interaction of purified platelet profilin with actin and present evidence that simple sequestration of actin monomers in a 1:1 complex with profilin cannot explain many of the effects of profilin on actin assembly. Three different methods to assess binding of profilin to actin show that the complex with platelet actin has a dissociation constant in the range of 1 to 5 microM. The value for muscle actin is similar. When bound to actin, profilin increases the rate constant for dissociation of ATP from actin by 1,000-fold and also increases the rate of dissociation of Ca2+ bound to actin. Kinetic simulation showed that the profilin exchanges between actin monomers on a subsecond time scale that allows it to catalyze nucleotide exchange. On the other hand, polymerization assays give disparate results that are inconsistent with the binding assays and each other: profilin has different effects on elongation at the two ends of actin filaments; profilin inhibits the elongation of platelet actin much more strongly than muscle actin; and simple formation of 1:1 complexes of actin with profilin cannot account for the strong inhibition of spontaneous polymerization. We suggest that the in vitro effects on actin polymerization may be explained by a complex mechanism that includes weak capping of filament ends and catalytic poisoning of nucleation. Although platelets contain only 1 profilin for every 5-10 actin molecules, these complex reactions may allow substoichiometric profilin to have an important influence on actin assembly. We also confirm the observation of I. Lassing and U. Lindberg (1985. Nature [Lond.] 318:472-474) that polyphosphoinositides inhibit the effects of profilin on actin polymerization, so lipid metabolism must also be taken into account when considering the functions of profilin in a cell.

Actins↗

PubMed1990

ADP-ribosylation of actin isoforms by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin.

The substrate specificities of the actin-ADP-ribosylating toxins, Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin were studied by using five different preparations of actin isoforms: alpha-skeletal muscle actin, alpha-cardiac muscle actin, gizzard gamma-smooth muscle actin, spleen beta- and gamma-cytoplasmic actin, and aortic smooth muscle actin containing alpha- and gamma-smooth muscle actin isoforms. C. perfringens iota toxin ADP-ribosylated all actin isoforms tested, whereas C. botulinum C2 toxin did not modify alpha-skeletal muscle actin or alpha-cardiac muscle actin. Spleen beta/gamma-cytoplasmic actin and gizzard gamma-smooth muscle actin were substrates of C. botulinum C2 toxin. In the aortic smooth muscle actin preparation, gamma-smooth muscle actin but not alpha-smooth muscle actin was ADP-ribosylated by C. botulinum C2 toxin. The data indicate that, in contrast to C. perfringens iota toxin, C. botulinum C2 toxin ADP-ribosylates only beta/gamma-cytoplasmic and gamma-smooth muscle actin and suggest that the N-terminal region of actin isoforms define the substrate specificity for ADP-ribosylation by C. botulinum C2 toxin.

ADP Ribose Transferases↗

PubMed1993

Vascular clearance and organ uptake of G- and F-actin in the rat.

This study comparatively evaluated the kinetics of removal and organ distribution of circulating G- and F-actin. Both F- and G-actin were cleared in two phases (fast component with a t1/2 of 3-5 min and a slow component with a t1/2 of hours). There was no effect of dose on either the fast- or slow-compartment clearance kinetics at the doses tested (5-100 micrograms/100 g body wt). However, at the same challenging dose of F- and G-actin, more F-actin was removed during the rapid phase. Although the time constants (Tfast) for F- and G-actin removal from the vasculature during the initial rapid phase were the same, during the slow phase the time constants (Tslow) for removal of F-actin were less (P < 0.001) than that of G-actin. The fraction of F-actin removed during the rapid phase ranged from 33 to 63% and was significantly greater (P < 0.01) than the fraction of G-actin removed during this phase (10-33%). The liver was the main organ of localization, and autoradiographic studies of liver tissue demonstrated that G-actin monomers were removed by Kupffer cells, whereas F-actin was predominantly removed by hepatic sinusoidal endothelial cells. In vivo endotoxin activation of Kupffer cells enhanced the rate of G-actin removal and increased liver localization of G-actin but had no effect on F-actin removal. This further supports a role for Kupffer cells in the clearance of G-actin. These studies therefore demonstrate that F- and G-actin clearance mechanisms are different. G-actin removal, presumably mediated by its binding to vitamin D binding protein, is accomplished by Kupffer cells, whereas F-actin removal at the same doses is due mainly to hepatic endothelial cell uptake.

Actins↗

PubMed2000

[The experimental study of intracellular actin in the scar fibroblasts].

OBJECTIVE: To study the relationship between intracellular actin and scar contracture. METHODS: Fibroblasts from 10 cases of hypertrophic scar and 5 cases of keloid were cultured in vitro. Total actin, filamentous actin(F actin), globular actin (G actin) and the ratio of F to G actin(F/G) were measured by densitometry after differential extraction and separation by polyacrylamide gel electrophoresis in the presence of sodium sulfate. RESULTS: Total actin, F actin, G actin and F/G in hypertrophic scar fibroblasts were 2.38 ng/10(4) cells, 0.98 ng/10(4) cells, 1.42 ng/10(4) cells and 0.68 respectively, while in keloid fibroblasts were 1.68 ng/10(4) cells. 0.46 ng/10(4) cells, 1.26 ng/10(4) cells, and 0.36 respectively. There was significant differences between two tissues fibroblasts in the items of total actin, F actin, G actin, and F/G (P < 0.01), while no significant difference in G actin (P > 0.05). CONCLUSION: Total intracellular actin, F actin, and F/G may play an important role in the scar contracture. The hypertrophic scar and keloid can be distinguished by the contents of total intracellular actin, F actin and F/G.

Actins↗

PubMed1989

Interactions among serum vitamin D binding protein, monomeric actin, profilin, and profilactin.

Human serum vitamin D binding protein (hDBP), a 58-kDa inter-alpha-globulin, is known to bind, monomeric actin (G-actin) in equimolar quantities. Using monoclonal and polyclonal anti-hDBP antibodies, hDBP, and radioiodinated actin, we developed a reliable saturation assay for actin bound to hDBP. By utilizing this assay, kinetic analysis, and ultracentrifugal sedimentation in sucrose gradients, these proteins' binding affinities (Kd = 10(-9) M) were demonstrated to be 10- to 100-fold greater than earlier estimates. At 4 degrees C, hDBP has an association rate constant of 2.2 x 10(4) M-1 s-1 and a rate of dissociation displaying a t1/2 of 22 h. This high affinity binding was largely unaffected by conditions favoring actin filament formation (1 mM MgCl2 and/or 50 mM KCl), by the range of pH from 6.8 to 8.6 or by temperatures from 4 to 37 degrees C. Compared with ATP-alpha-actin, a 2-fold decrease of binding affinity was observed for the nonmuscle isoactins (beta,gamma), ADP-G-alpha-actin, and N'-ethylmaleimide-modified G-alpha-actin. The 25-hydroxyvitamin D3 and 1 alpha,25-dihydroxyvitamin D3 holo-sterol forms of hDBP bound actin in a manner indistinguishable from the apo-sterol hDBP. The common polymorphisms of hDBP (DBP1 slow, DBP1 fast, and DBP2) were shown to have an equal avidity for G-actin binding. Human platelet profilin competed with hDBP for binding to G-actin, but was 1000-fold less potent (Ki = 1.9 microM). When platelet profilactin was incubated with hDBP, profilin was liberated and hDBP-actin complexes formed. DNase I, which forms a triprotein complex with hDBP-G actin, did not alter the affinity of binding of actin by hDBP. The very high affinity binding observed, which was largely unaffected by the state of G-actin, pH, and ionic conditions, appears to support a constitutive role for plasma DBP in the sequestration of actin monomers, as well as actin from actin-profilin complexes, that are liberated during cell injury.

Actins↗

PubMed1996

Adherence of human phagocytes results in characteristic reorganization and redistribution of distinct F-actin pools.

The F-actin based microfilamentous cytoskeleton (MFC) provides mobility for phagocytic immune cells including polymorphonuclear leukocytes (PMNs) and macrophages (MOs). In PMNs in suspension, the MFC is organized into two distinct F-actin pools [Triton Insoluble F-actin-(TIF), which form the sub-membranous, 3D actin meshwork and Triton Soluble F-actin (TSF), which exists as short oligomers] in equilibrium with G-actin. The structure of F-actin pools in adherent cells is unknown despite the fact that phagocytes are adherent in tissues in vivo. In order to determine the structure of F-actin pools in adherent phagocytes, human PMNs were isolated and allowed to adhere to plastic for 1 hour at 37 degrees C. Adherent cells were collected, actin pools separated and quantified by SDS-PAGE and compared to nonadherent PMNs in suspension. Likewise, the nonadherent human myeloid cell line U937 was induced to MO morphology and adherence by exposure to TPA (10(-6) M x 3 days) and similarly evaluated. Adherence of PMNs to plastic resulted in 75 +/- 15% adherence (n = 3). TPA differentiation of U937 cells resulted in 81 +/- 15% adherence (n = 10). In both cells, adherence resulted in a statistically significant increase in TIF, a decrease in TSF, and little to no change in G-actin. Basal, nonadherent PMNs in suspension contain TIF 40 +/- 0%, TSF 20 +/- 4%, and G-actin 40 +/- 4%, n = 3, whereas adherent PMNs contain TIF 61 +/- 3%, TSF 5 +/- 5%, G-actin 34 +/- 1%, n = 3. Basal U937 contain TIF 41 +/- 9%, TSF 17 +/- 6%, and G-actin 42 +/- 13%, n = 7. Adherent MO-like U937 contain TIF 53 +/- 4%, TSF 9 +/- 5%, and G-actin 38 +/- 4%. The results show that phagocyte adherence leads to a characteristic reorganization of actin pool structure that is remarkably quantitatively similar to, yet mechanistically distinct from, reorganization by chemotactic factor activation in suspension. Adherence-induced TIF-actin growth results exclusively from conversion of TSF-actin to TIF-actin.

Actins↗

PubMed1997

Reorganization of the cortical actin cytoskeleton during maturation division in the Tubifex egg: possible involvement of protein kinase C.

Tubifex eggs undergo a drastic reorganization of the cortical actin cytoskeleton during metaphase of the second meiosis. At the end of the first meiosis, the egg cortex displays only scattered actin filaments and tiny dots of F-actin; during the following 90 min, cortical F-actin gradually increases in amount, becomes organized into foci that are interlinked by actin bundles, and generates a geodesic dome-like organization. In this study, we have characterized this reorganization of the cortical actin cytoskeleton. In living eggs injected with rhodamine-phalloidin at the beginning of the second meiosis, cortical actin assembly (i.e., formation of actin foci and bundles) proceeds normally, but labeled F-actin is not found to be included significantly in the formed cortical actin network, suggesting that the increase in cortical F-actin is not simply ascribable to the recruitment of preexisting actin filaments. Cortical actin assembly can be induced precociously not only by calcium ionophore A23187 but also by a phorbol ester PMA, an agonist of protein kinase C (PKC). Conversely, the formation of actin foci and bundles is inhibited by PKC antagonists, although cortical F-actin increases to some extent in the presence of these inhibitors. Similar inhibition of the cortical reorganization is elicited in eggs whose intracellular free calcium level ([Ca2+]i) has been clamped low by microinjection of a calcium chelator BAPTA. The treatment of BAPTA-injected eggs with PMA results in the formation of actin foci and bundles. An experiment with eggs injected with fluo-3 shows that [Ca2+]i increases during metaphase of the second meiosis. These results suggest that the reorganization of cortical actin during metaphase of the second meiosis requires activation of PKC, which depends on increases in [Ca2+]i.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2005

Malaria parasite actin filaments are very short.

A novel form of actomyosin regulation has recently been proposed in which the polymerisation of new actin filaments regulates apicomplexan parasite motility. Here, we identified actin I in the merozoites of Plasmodium falciparum by mass spectrometry. The only post-translational modification is acetylation of the N terminus (acetyl-Gly-Glu-actin), while methylation of histidine 73, a common modification for actin, is absent. Results obtained with anti-actin antibodies suggest that, in contrast to a previous report, there is no actin-ubiquitin conjugate in merozoites. About half of the extracted monomeric actin polymerised and actin filaments could be sedimented at 500,000g. In contrast, centrifugation at 100,000g, conditions commonly used to sediment filamentous actin, yielded very little F-actin. In a functional characterisation using an in vitro motility assay, actin filaments moved over myosin at a velocity indistinguishable from that of rabbit skeletal actin. Filament length, however, was too short to be resolved by conventional fluorescence microscopy. On electron micrographs an average filament length of approximately 100nm was determined. We also identified by mass spectrometry proteins co-purifying with filamentous actin, which are potential actin-binding proteins. Our results demonstrate differences in actin filament dynamics for an apicomplexan parasite, which could be due to specific properties of the actin and/or actin-regulatory proteins.

Actin Cytoskeleton↗

PubMed2002

Structural effects of cofilin on longitudinal contacts in F-actin.

Structural effects of yeast cofilin on skeletal muscle and yeast actin were examined in solution. Cofilin binding to native actin was non-cooperative and saturated at a 1:1 molar ratio, with K(d)<or=0.05 microM for both CaATP-G-actin and F-actin. Cofilin binding enhanced the fluorescence of dansyl ethylenediamine (DED) attached to Gln41 on the DNase I binding loop of skeletal muscle F-actin and decreased the fluorescence of AEDANS at Cys41 on yeast Q41C/C374S mutant F-actin. However, cofilin had no effect on the spectral properties of DED or AEDANS on CaATP-G-actin. Fluorescence energy transfer (FRET) from tryptophan residues to DED at Gln41 on skeletal muscle actin and to AEDANS at Cys41 on yeast Q41C/C374S actin was decreased by cofilin binding to F- but not to G-actin. Cofilin inhibited strongly the rate of interprotomer disulfide cross-linking of Cys41 to Cys374 on yeast Q41C mutant F-actin. Binding of cofilin enhanced excimer formation between pyrene probes attached to Cys41 and Cys374 on Q41C F-actin. These results indicate that cofilin alters the interface between subdomains 1 and 2 and shifts the DNase I binding loop away from subdomain 1 of an adjacent actin protomer. Cofilin reduced FRET from tryptophan residues to 4-azido-2-nitrophenyl-putrescine (ANP) at Gln41 in skeletal muscle F-but not in G-actin. However, following the interprotomer cross-linking of Gln41 to Cys374 in F-actin by ANP, cofilin binding did not change FRET from the tryptophan residues to ANP. This suggests that cofilin binding and the conformational effect on F-actin are not coupled tightly. Overall, this study provides solution evidence for the weakening of longitudinal, subdomain 2/1 contacts in F-actin by cofilin.

Actin Depolymerizing Factors↗

PubMed1984

Cofilin, a protein in porcine brain that binds to actin filaments and inhibits their interactions with myosin and tropomyosin.

Cofilin, a 21 000 molecular weight protein of porcine brain, reacts stoichiometrically with actin in a 1:1 molar ratio. Upon binding of cofilin, the fluorescence of pyrene-labeled actin under polymerizing conditions is changed into the monomer form, irrespective of whether cofilin is added to actin before or after polymerization. Cofilin decreases the viscosity of actin filaments but increases the light-scattering intensity of the filaments. The centrifugation assay and the DNase I inhibition assay demonstrate that cofilin binds to actin filaments in a 1:1 molar ratio of cofilin to actin monomer in the filament and that cofilin increases the monomeric actin to a limited extent (up to 1.1-1.5 microM monomer) in the presence of physiological concentrations of Mg2+ and KCl. Cofilin is also able to bind to monomeric actin, as demonstrated by gel filtration. Electron microscopy showed that actin filaments are shortened and slightly thickened in the presence of cofilin. No bundle formation was observed in the presence of various concentrations of cofilin. The gel point assay using an actin cross-linking protein and the nucleation assay also suggested that cofilin shortens the actin filaments and hence increases the filament number. Cofilin blocks the binding of tropomyosin to actin filaments. Tropomyosin is dissociated from actin filaments by the binding of cofilin to actin filaments. Cofilin was found to inhibit the superprecipitation of actin-myosin mixtures as well as the actin-activated myosin ATPase. All these results suggest that cofilin is a new type of actin-associated protein.

Actin Depolymerizing Factors↗

PubMed2007

Mapping the interaction of cofilin with subdomain 2 on actin.

Cofilin, a member of the actin-depolymerizing factor (ADF)/cofilin family of proteins, is a key regulator of actin dynamics. Cofilin binds to monomer (G-) and filamentous (F-) actin, severs the filaments, and increases their turnover rate. Electron microscopy studies suggested cofilin interactions with subdomains 2 and 1/3 on adjacent actin protomers in F-actin. To probe for the presence of a cryptic cofilin binding site in subdomain 2 in G-actin, we used transglutaminase-mediated cross-linking, which targets Gln41 in subdomain 2. The cross-linking proceeded with up to 85% efficiency with skeletal alpha-actin and WT yeast actin, yielding a single product corresponding to a 1:1 actin-cofilin complex but was strongly inhibited in Q41C yeast actin (in which Q41 was substituted with cysteine). LC-MS/MS analysis of the proteolytic fragments of this complex mapped the cross-linking to Gln41 on actin and Gly1 on recombinant yeast cofilin. The actin-cofilin (AC) heterodimer was purified on FPLC for analytical ultracentrifugation and electron microscopy analysis. Sedimentation equilibrium and velocity runs revealed oligomers of AC in G-actin buffer. In the presence of excess cofilin, the covalent AC heterodimer bound a second cofilin, forming a 2:1 cofilin/actin complex, as revealed by sedimentation results. Under polymerizing conditions the cross-linked AC formed mostly short filaments, which according to image reconstruction were similar to uncross-linked actin-cofilin filaments. Although a majority of the cross-linking occurs at Gln41, a small fraction of the AC cross-linked complex forms in the Q41C yeast actin mutant. This secondary cross-linking site was sequenced by MALDI-MS/MS as linking Gln360 in actin to Lys98 on cofilin. Overall, these results demonstrate that the region around Gln41 (subdomain 2) is involved in a weak binding of cofilin to G-actin.

Actin Depolymerizing Factors↗

PubMed1992

High level expression of transfected beta- and gamma-actin genes differentially impacts on myoblast cytoarchitecture.

The impact of the human beta- and gamma-actin genes on myoblast cytoarchitecture was examined by their stable transfection into mouse C2 myoblasts. Transfectant C2 clones expressing high levels of human beta-actin displayed increases in cell surface area. In contrast, C2 clones with high levels of human gamma-actin expression showed decreases in cell surface area. The changes in cell morphology were accompanied by changes in actin stress-fiber organization. The beta-actin transfectants displayed well-defined filamentous organization of actin; whereas the gamma-actin transfectants displayed a more diffuse organization of the actin cables. The role of the beta-actin protein in generating the enlarged cell phenotype was examined by transfecting a mutant form of the human beta-actin gene. Transfectant cells were shown to incorporate the aberrant actin protein into stress-fiber-like structures. High level expression of the mutant beta-actin produced decreases in cell surface area and disruption of the actin microfilament network similar to that seen with transfection of the gamma-actin gene. In contrast, transfection of another mutant form of the beta-actin gene which encodes an unstable protein had no impact on cell morphology or cytoarchitecture. These results strongly suggest that it is the nature of the encoded protein that determines the morphological response of the cell. We conclude that the relative gene expression of beta- and gamma-actin is of relevance to the control of myoblast cytoarchitecture. In particular, we conclude that the beta- and gamma-actin genes encode functionally distinct cytoarchitectural information.

Actin Cytoskeleton↗

PubMed1992

Actin dynamics during the cell cycle in Chlamydomonas reinhardtii.

We have used two monoclonal antibodies to demonstrate the presence and localization of actin in interphase and mitotic vegetative cells of the green alga Chlamydomonas reinhardtii. Commercially available monoclonal antibodies raised against smooth muscle actin (Lessard: Cell Motil. Cytoskeleton 10:349-362, 1988; Lin: Proc. Natl. Acad. Sci. USA 78:2335-2339, 1981) identify Chlamydomonas actin as a approximately 43,000-M(r) protein by Western immunoblot procedures. In an earlier study, Detmers and coworkers (Cell Motil. 5:415-430, 1985) first identified Chlamydomonas actin using NBD-phallacidin and an antibody raised against Dictyostelium actin; they demonstrated that F-actin is localized in the fertilization tubule of mating gametes. Here, we show by immunofluorescence that vegetative Chlamydomonas cells have an array of actin that surrounds the nucleus in interphase cells and undergoes dramatic reorganization during mitosis and cytokinesis. This includes the following: reorganization of actin to the anterior of the cell during preprophase; the formation of a cruciate actin band in prophase; reorganization to a single anterior actin band in metaphase; rearrangement forming a focus of actin anterior to the metaphase plate; reextension of the actin band in anaphase; presence of actin in the forming cleavage furrow during telophase and cytokinesis; and finally reestablishment of the interphase actin array. The studies presented here do not allow us to discriminate between G and F-actin. None the less, our observations, demonstrating dynamic reorganization of actin during the cell cycle, suggest a role for actin that may include the movement of basal bodies toward the spindle poles in mitosis and the formation of the cleavage furrow during cytokinesis.

Actins↗

PubMed2002

Developmentally regulated changes in cellular compartmentation and synaptic distribution of actin in hippocampal neurons.

Actin dynamics and actin-based motility are important for neurite outgrowth and synapse plasticity. Recent work implicates actin in synapse assembly, but the morphological relationship between actin and synapses during development is unclear. Here we used developing hippocampal neurons grown in culture to examine the relationship between F- and G-actin and clusters of synaptic proteins. Both F- and G-actin are most enriched in dendritic and axonal growth cones, but only G-actin is present within the distal tips of filopodia. Outside of growth cones, F-actin levels are greater in dendrites than in axons, whereas G-actin levels are slightly greater in axons than in dendrites. The distribution of both F- and G-actin is consistent with their presence at synapses, but only F-actin levels become detectably enhanced at synaptic sites. Quantitative analyses suggest that first-forming synapses are associated with enhanced levels of pre- and postsynaptic F-actin that do not necessarily remain elevated during synapse maturation. However, nearly all mature excitatory synapses become associated with high, mostly postsynaptic concentrations of F-actin contained principally within dendritic spines. Mature shaft and GABAergic synapses are also associated with enhanced levels of F-actin, but to a lesser degree. Thus, although F-actin is essential for function and maintenance of young synapses, it need not be highly concentrated at every site. The large increase in postsynaptic F-actin concentration observed in mature neurons is likely to reflect actin's role in dendritic spine morphology and in synapse plasticity.

Actins↗