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At least 235 records · Page 13Linked to original sources
PubMed2001

Interactions among a fimbrin, a capping protein, and an actin-depolymerizing factor in organization of the fission yeast actin cytoskeleton.

We report studies of the fission yeast fimbrin-like protein Fim1, which contains two EF-hand domains and two actin-binding domains (ABD1 and ABD2). Fim1 is a component of both F-actin patches and the F-actin ring, but not of F-actin cables. Fim1 cross-links F-actin in vitro, but a Fim1 protein lacking either EF-hand domains (Fim1A12) or both the EF-hand domains and ABD1 (Fim1A2) has no actin cross-linking activity. Overexpression of Fim1 induced the formation of F-actin patches throughout the cell cortex, whereas the F-actin patches disappear in cells overexpressing Fim1A12 or Fim1A2. Thus, the actin cross-linking activity of Fim1 is probably important for the formation of F-actin patches. The overexpression of Fim1 also excluded the actin-depolymerizing factor Adf1 from the F-actin patches and inhibited the turnover of actin in these structures. Thus, Fim1 may function in stabilizing the F-actin patches. We also isolated the gene encoding Acp1, a subunit of the heterodimeric F-actin capping protein. fim1 acp1 double null cells showed more severe defects in the organization of the actin cytoskeleton than those seen in each single mutant. Thus, Fim1 and Acp1 may function in a similar manner in the organization of the actin cytoskeleton. Finally, genetic studies suggested that Fim1 may function in cytokinesis in cooperation with Cdc15 (PSTPIP) and Rng2 (IQGAP), respectively.

Actin Depolymerizing Factors↗
PubMed2001

Cofilin phosphorylation by protein kinase testicular protein kinase 1 and its role in integrin-mediated actin reorganization and focal adhesion formation.

Testicular protein kinase 1 (TESK1) is a serine/threonine kinase with a structure composed of a kinase domain related to those of LIM-kinases and a unique C-terminal proline-rich domain. Like LIM-kinases, TESK1 phosphorylated cofilin specifically at Ser-3, both in vitro and in vivo. When expressed in HeLa cells, TESK1 stimulated the formation of actin stress fibers and focal adhesions. In contrast to LIM-kinases, the kinase activity of TESK1 was not enhanced by Rho-associated kinase (ROCK) or p21-activated kinase, indicating that TESK1 is not their downstream effector. Both the kinase activity of TESK1 and the level of cofilin phosphorylation increased by plating cells on fibronectin. Y-27632, a specific inhibitor of ROCK, inhibited LIM-kinase-induced cofilin phosphorylation but did not affect fibronectin-induced or TESK1-induced cofilin phosphorylation in HeLa cells. Expression of a kinase-negative TESK1 suppressed cofilin phosphorylation and formation of stress fibers and focal adhesions induced in cells plated on fibronectin. These results suggest that TESK1 functions downstream of integrins and plays a key role in integrin-mediated actin reorganization, presumably through phosphorylating and inactivating cofilin. We propose that TESK1 and LIM-kinases commonly phosphorylate cofilin but are regulated in different ways and play distinct roles in actin reorganization in living cells.

Actin Depolymerizing Factors↗
PubMed1993

Unexpected combinations of null mutations in genes encoding the actin cytoskeleton are lethal in yeast.

To understand the role of the actin cytoskeleton in cell physiology, and how actin-binding proteins regulate the actin cytoskeleton in vivo, we and others previously identified actin-binding proteins in Saccharomyces cerevisiae and studied the effect of null mutations in the genes for these proteins. A null mutation of the actin gene (ACT1) is lethal, but null mutations in the tropomyosin (TPM1), fimbrin (SAC6), Abp1p (ABP1), and capping protein (CAP1 and CAP2) genes have relatively mild or no effects. We have now constructed double and triple mutants lacking 2 or 3 of these actin-binding proteins, and studied the effect of the combined mutations on cell growth, morphology, and organization of the actin cytoskeleton. Double mutants lacking fimbrin and either Abp1p or capping protein show negative synthetic effects on growth, in the most extreme case resulting in lethality. All other combinations of double mutations and the triple mutant lacking tropomyosin, Abp1p, and capping protein, are viable and their phenotypes are similar to or only slightly more severe than those of the single mutants. Therefore, the synthetic phenotypes are highly specific. We confirmed this specificity by overexpression of capping protein and Abp1p in strains lacking fimbrin. Thus, while overexpression of these proteins has deleterious effects on actin organization in wild-type strains, no synthetic phenotype was observed in the absence of fimbrin. We draw two important conclusions from these results. First, since mutations in pairs of actin-binding protein genes cause inviability, the actin cytoskeleton of yeast does not contain a high degree of redundancy. Second, the lack of structural and functional homology among these genetically redundant proteins (fimbrin and capping protein or Abp1p) indicates that they regulate the actin cytoskeleton by different mechanisms. Determination of the molecular basis for this surprising conclusion will provide unique insights into the essential mechanisms that regulate the actin cytoskeleton.

Actin Depolymerizing Factors↗
PubMed1993

The alpha and beta subunits of nematode actin capping protein function in yeast.

We cloned and analyzed two genes, cap-1 and cap-2, which encode the alpha and beta subunits of Caenorhabditis elegans capping protein (CP). The nematode CP subunits are 55% (cap-1) and 66% (cap-2) identical to the chicken CP subunits and 32% (cap-1) and 48% (cap-2) identical to the yeast CP subunits. Purified nematode CP made by expression of both subunits in yeast is functionally similar to chicken skeletal muscle CP in two different actin polymerization assays. The abnormal cell morphology and disorganized actin cytoskeleton of yeast CP null mutants are restored to wild-type by expression of the nematode CP subunits. Expression of the nematode CP alpha or beta subunit is sufficient to restore viability to yeast cap1 sac6 or cap2 sac6 double mutants, respectively. Therefore, despite evolution of the nematode actin cytoskeleton to a state far more complex than that of yeast, one important component can function in both organisms.

Actin Capping Proteins↗
PubMed1995

Actin filament barbed-end capping activity in neutrophil lysates: the role of capping protein-beta 2.

A barbed-end capping activity was found in high speed supernates of neutrophils lysed in submicromolar calcium. In dilute supernate (> or = 100-fold dilution of cytoplasm), this activity accounted for most of the inhibition of barbed-end elongation of pyrenyl-G-actin from spectrin-F-actin seeds. Pointed-end elongation from gelsolin-capped F-actin seeds was not inhibited at comparable concentrations of supernate, thus excluding actin monomer sequestration as a cause of the observed inhibition. Most of the capping activity was due to capping protein-beta 2 (a homologue of cap Z). Thus, while immunoadsorption of > or = 95% of the gelsolin in the supernate did not decrease capping activity, immunoadsorption of capping protein-beta 2 reduced capping activity proportionally to the amount of capping protein-beta 2 adsorbed. Depletion of > 90% of capping protein-beta 2 from the supernate removed 90% of its capping activity. The functional properties of the capping activity were defined. The dissociation constant for binding to barbed ends (determined by steady state and kinetic analyses) was approximately 1-2 nM; the on-rate of capping was between 7 x 10(5) and 5 x 10(6) M-1 s-1; and the off-rate was approximately 2 x 10(-3) s-1. The concentration of capper free in the intact cell (determined by adsorption of supernate with spectrin-actin seeds) was estimated to be approximately 1-2 microM. Thus, there appeared to be enough high affinity capper to cap all the barbed ends in vivo. Nevertheless, immediately after lysis with detergent, neutrophils contained sites that nucleate barbed-end elongation of pyrenyl-G-actin. These barbed ends subsequently become capped with a time course and concentration dependence similar to that of spectrin-F-actin seeds in high speed supernates. These observations suggest that, despite the excess of high affinity capper, some ends either are not capped in vivo or are transiently uncapped upon lysis and dilution.

Actin Cytoskeleton↗
PubMed1996

Mutational analysis of capping protein function in Saccharomyces cerevisiae.

To investigate physiologic functions and structural correlates for actin capping protein (CP), we analyzed site-directed mutations in CAP1 and CAP2, which encode the alpha and beta subunits of CP in Saccharomyces cerevisiae. Mutations in four different regions caused a loss of CP function in vivo despite the presence of mutant protein in the cells. Mutations in three regions caused a complete loss of all aspects of function, including the actin distribution, viability with sac6, and localization of CP to actin cortical patches. Mutation of the fourth region led to partial loss of only one function-formation of actin cables. Some mutations retained function and exhibited the complete wild-type phenotype, and some mutations led to a complete loss of protein and therefore loss of function. The simplest hypothesis that can explain these results is that a single biochemical property is necessary for all in vivo functions. This biochemical property is most likely binding to actin filaments, because the nonfunctional mutant CPs no longer co-localize with actin filaments in vivo and because direct binding of CP to actin filaments has been well established by studies with purified proteins in vitro. More complex hypotheses, involving the existence of additional biochemical properties important for function, cannot be excluded by this analysis.

Actin Capping Proteins↗
PubMed2003

A balance of capping protein and profilin functions is required to regulate actin polymerization in Drosophila bristle.

Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of the cpb loss-of-function phenotype. The interaction between cpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell.

Actin Depolymerizing Factors↗
PubMed2004

Cyclase-associated protein 1 (CAP1) promotes cofilin-induced actin dynamics in mammalian nonmuscle cells.

Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, the mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. Here, we show that CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B16F1 cells, CAP1 is a highly abundant protein that colocalizes with cofilin-1 to dynamic regions of the cortical actin cytoskeleton. Analysis of CAP1 knockdown cells demonstrated that this protein promotes rapid actin filament depolymerization and is important for cell morphology, migration, and endocytosis. Interestingly, depletion of CAP1 leads to an accumulation of cofilin-1 into abnormal cytoplasmic aggregates and to similar cytoskeletal defects to those seen in cofilin-1 knockdown cells, demonstrating that CAP1 is required for proper subcellular localization and function of ADF/cofilin. Together, these data provide the first direct in vivo evidence that CAP promotes rapid actin dynamics in conjunction with ADF/cofilin and is required for several central cellular processes in mammals.

Actin Cytoskeleton↗
PubMed2004

Role for actin filament turnover and a myosin II motor in cytoskeleton-driven disassembly of the epithelial apical junctional complex.

Disassembly of the epithelial apical junctional complex (AJC), composed of the tight junction (TJ) and adherens junction (AJ), is important for normal tissue remodeling and pathogen-induced disruption of epithelial barriers. Using a calcium depletion model in T84 epithelial cells, we previously found that disassembly of the AJC results in endocytosis of AJ/TJ proteins. In the present study, we investigated the role of the actin cytoskeleton in disassembly and internalization of the AJC. Calcium depletion induced reorganization of apical F-actin into contractile rings. Internalized AJ/TJ proteins colocalized with these rings. Both depolymerization and stabilization of F-actin inhibited ring formation and disassembly of the AJC, suggesting a role for actin filament turnover. Actin reorganization was accompanied by activation (dephosphorylation) of cofilin-1 and its translocation to the F-actin rings. In addition, Arp3 and cortactin colocalized with these rings. F-actin reorganization and disassembly of the AJC were blocked by blebbistatin, an inhibitor of nonmuscle myosin II. Myosin IIA was expressed in T84 cells and colocalized with F-actin rings. We conclude that disassembly of the AJC in calcium-depleted cells is driven by reorganization of apical F-actin. Mechanisms of such reorganization involve cofilin-1-dependent depolymerization and Arp2/3-assisted repolymerization of actin filaments as well as myosin IIA-mediated contraction.

Actin Cytoskeleton↗
PubMed2004

Tropomyosin and troponin are required for ovarian contraction in the Caenorhabditis elegans reproductive system.

Ovulation in the nematode Caenorhabditis elegans is coordinated by interactions between the somatic gonad and germ cells. Myoepithelial sheath cells of the proximal ovary are smooth muscle-like cells, but the regulatory mechanism of their contraction is unknown. We show that contraction of the ovarian muscle requires tropomyosin and troponin, which are generally major actin-linked regulators of contraction of striated muscle. RNA interference of tropomyosin or troponin C caused sterility by inhibiting ovarian contraction that is required for expelling mature oocytes into the spermatheca where fertilization takes place, thus causing accumulation of endomitotic oocytes in the ovary. Tropomyosin and troponin C were associated with actin filaments in the myoepithelial sheath, and the association of troponin C with actin was dependent on tropomyosin. A mutation in the actin depolymerizing factor/cofilin gene suppressed the ovulation defects by RNA interference of tropomyosin or troponin C. These results strongly suggest that tropomyosin and troponin are the actin-linked regulators for contraction of ovarian muscle in the C. elegans reproductive system.

Actin Cytoskeleton↗
PubMed2004

Actin-depolymerizing factor and cofilin-1 play overlapping roles in promoting rapid F-actin depolymerization in mammalian nonmuscle cells.

Actin-depolymerizing factor (ADF)/cofilins are small actin-binding proteins found in all eukaryotes. In vitro, ADF/cofilins promote actin dynamics by depolymerizing and severing actin filaments. However, whether ADF/cofilins contribute to actin dynamics in cells by disassembling "old" actin filaments or by promoting actin filament assembly through their severing activity is a matter of controversy. Analysis of mammalian ADF/cofilins is further complicated by the presence of multiple isoforms, which may contribute to actin dynamics by different mechanisms. We show that two isoforms, ADF and cofilin-1, are expressed in mouse NIH 3T3, B16F1, and Neuro 2A cells. Depleting cofilin-1 and/or ADF by siRNA leads to an accumulation of F-actin and to an increase in cell size. Cofilin-1 and ADF seem to play overlapping roles in cells, because the knockdown phenotype of either protein could be rescued by overexpression of the other one. Cofilin-1 and ADF knockdown cells also had defects in cell motility and cytokinesis, and these defects were most pronounced when both ADF and cofilin-1 were depleted. Fluorescence recovery after photobleaching analysis and studies with an actin monomer-sequestering drug, latrunculin-A, demonstrated that these phenotypes arose from diminished actin filament depolymerization rates. These data suggest that mammalian ADF and cofilin-1 promote cytoskeletal dynamics by depolymerizing actin filaments and that this activity is critical for several processes such as cytokinesis and cell motility.

Actins↗
PubMed2005

Profilin-mediated competition between capping protein and formin Cdc12p during cytokinesis in fission yeast.

Fission yeast capping protein SpCP is a heterodimer of two subunits (Acp1p and Acp2p) that binds actin filament barbed ends. Neither acp1 nor acp2 is required for viability, but cells lacking either or both subunits have cytokinesis defects under stressful conditions, including elevated temperature, osmotic stress, or in combination with numerous mild mutations in genes important for cytokinesis. Defects arise as the contractile ring constricts and disassembles, resulting in delays in cell separation. Genetic and biochemical interactions show that the cytokinesis formin Cdc12p competes with capping protein for actin filament barbed ends in cells. Deletion of acp2 partly suppresses cytokinesis defects in temperature-sensitive cdc12-112 cells and mild overexpression of capping protein kills cdc12-112 cells. Biochemically, profilin has opposite effects on filaments capped with Cdc12p and capping protein. Profilin depolymerizes actin filaments capped by capping protein but allows filaments capped by Cdc12p to grow at their barbed ends. Once associated with a barbed end, either Cdc12p or capping protein prevents the other from influencing polymerization at that end. Given that capping protein arrives at the division site 20 min later than Cdc12p, capping protein may slowly replace Cdc12p on filament barbed ends in preparation for filament disassembly during ring constriction.

Actin Depolymerizing Factors↗
PubMed1987

Beta-actinin is not distinguishable from an actin barbed-end capping protein in chicken breast muscle.

beta-Actinin is an actin-pointed end capping protein in skeletal muscle. Casella et al. have reported that a protein isolated from muscle acetone powder by procedures similar to those used for beta-actinin purification caps the barbed end of an actin filament (J. Biol. Chem. 261, 10915-10921 (1986)). We have confirmed the above results. However, it turned out that the two proteins were identical as to subunit sizes, peptide maps, and cross-reactivities with anti-beta-actinin IgG. The binding of the two proteins to opposite ends of an actin filament remains unexplained.

Actin Depolymerizing Factors↗
PubMed1988

A novel actin filament-capping protein from sea urchin eggs: a 20,000-molecular-weight protein-actin complex.

A novel protein factor which reduced the low-shear viscosity of rabbit skeletal muscle actin was purified from a 0.6 M KCl-extract of an insoluble fraction of sea urchin eggs by ammonium sulfate fractionation, gel filtration column chromatography, DNase I column chromatography, and hydroxylapatite column chromatography. This protein factor was shown to be a one-to-one complex of a 20,000-molecular-weight protein and egg actin. This protein complex accelerated the initial rate of actin polymerization, but reduced the steady-state viscosity of F-actin. It inhibited at substoichiometric amounts the elongation of actin filaments on sonicated F-actin fragments and depolymerization of F-actin induced by dilution. In addition, it increased the critical concentration of actin for polymerization. All these effects of this protein complex on actin could be explained by the "capping the barbed end" of the actin filament by the complex. The 20,000-molecular-weight protein which was separated from actin also possessed the barbed end-capping activities, but differed from the complex in that it did not accelerate the polymerization of actin.

Actin Depolymerizing Factors↗
PubMed1989

An actin-depolymerizing protein in embryonic chicken skeletal muscle: purification and characterization.

In embryonic skeletal muscle, a large amount of non-polymerized actin exists in the cytoplasm (Shimizu and Obinata [1986] J. Biochem. 99, 751-759). A 19-kDa protein (called 19K protein) which binds to G-actin was purified by sequential chromatography on DNase I-agarose, hydroxylapatite, SP-Sephadex, and Sephadex G-75, from the sarcoplasmic fraction of embryonic chicken skeletal muscle. This protein decreased the extent of actin polymerization at a steady state and increased the monomeric actin in a concentration-dependent fashion; it also caused quick depolymerization of F-actin, as determined by spectrophotometry at 237 nm, viscometry, DNase I inhibition assay, and electron microscopy. The molar ratio of 19K protein and actin interacting with each other was estimated to be 1:1. From these results, 19K protein was regarded as being actin depolymerizing protein. The amount of 19K protein in muscle decreased during development. The inhibitory action of 19K protein was removed by myosin or heavy meromyosin, and actin filaments were formed on the surface of myosin filaments when myosin filaments were added to a mixture of actin and 19K protein in a physiological salt solution. We propose that actin assembly is dually controlled in the developing muscle by the inhibitor(s) and an accelerator (myosin); this mechanism may enable the ordered assembly of actin and myosin in the early phase of myofibrillogenesis.

Actin Cytoskeleton↗
PubMed1993

Increased expression of cofilin in dystrophic chicken and mouse skeletal muscles.

A monoclonal antibody (McAb) specific for actin depolymerizing factor (ADF) was prepared. With this and previously prepared anti-cofilin McAb (MAB-22) and other antibodies, the expression of cofilin and ADF in the muscles of dystrophic (NH-413) chicken and dystrophic (C57BL/6J dy/dy) mice was compared with that in normal control animals by immunoblotting and immunocytochemical methods. Since cofilin expression is down-regulated during normal postnatal development of skeletal muscles [Abe et al. (1989) J. Biochem. 106, 696-702], cofilin was detected in the breast (pectoralis) muscle of normal adult chicken and the leg (femoris and tibialis anterior) muscles of normal mice only at a low level. ADF was not detectable in adult skeletal muscles. However, a significant increase of cofilin amount, but not of ADF amount, was observed in these muscles of the dystrophic animals, when the symptom of muscular dystrophy became evident. In order to localize cofilin in individual muscle fibers, serial cryosections of the dystrophic chicken muscle were examined with anti-cofilin antibody (MAB-22). The antibody stained cells of different size in the dystrophic muscle, indicating that cofilin expression was induced in the regenerating muscle cells as well as in the pre-existing myofibers. We suggest that cofilin is involved in disassembly or reorganization of actin in the dystrophic muscle.

Actin Depolymerizing Factors↗
PubMed1982

Effects of muscle proteins on the interaction between actin and an actin-depolymerizing protein from starfish oocytes.

The effects of myosin, heavy meromyosin (HMM), alpha-actinin, tropomyosin, and calmodulin on the interaction between an actin-depolymerizing protein (depactin) from starfish oocytes and rabbit skeletal actin were investigated. 1. Alpha-actinin or tropomyosin did not affect the inhibitory effect of depactin on salt-induced polymerization of actin and did not induce actin polymerization when added to a mixture of actin and depactin in a buffer solution which was designed to keep actin in F-form. Myosin or HMM allowed polymerization of actin under similar conditions except that ATP was not included. Addition of ATP, inorganic pyrophosphate, adenyl-5'-yl imidodiphosphate, or ADP abolished the effect of myosin. 2. None of these proteins could keep actin in a polymerized form when depactin was added to the mixture of actin and these proteins in the presence of ATP. In the absence of ATP, however, myosin protected actin against depactin. 3. The association constant between actin and depactin was estimated from competitive binding experiments using HMM to be around 4.5 X 10(6) M-1 assuming the association constant between actin and HMM to be 3 X 10(9) M-1 (Greene, L.E. & Eisenberg, E. (1980) J. Biol. Chem. 255, 549-555). 4. Actin did not activate Mg-ATPase activity of HMM in the presence of depactin. From the above results, the mode of interaction of myosin with actin in the presence of depactin and the possible role of myosin in actin assembly in the cell are discussed.

Actin Depolymerizing Factors↗
PubMed1984

Action of a 19K protein from porcine brain on actin polymerization: a new functional class of actin-binding proteins.

A 19K protein isolated from porcine brain not only inhibits actin polymerization but depolymerizes actin filaments quickly. The protein reacts stoichiometrically with actin in a 1:1 molar ratio. When actin is induced to polymerize with salts in the presence of the brain 19K protein, the lag phase is prolonged, and the extent of polymerization is decreased, but the half-polymerization time is not increased. This can be explained by assuming that the 19K protein severs growing actin filaments and thus causes an increase in the number of filament ends during the polymerization process, thereby accelerating the overall polymerization. Moreover, the low-shear viscosity of actin filaments is reduced much more than the high-shear viscosity by the 19K protein, suggesting that actin filaments become shorter in the presence of the 19K protein than in its absence. Actin filament depolymerization by the 19K protein is much faster than that by brain profilin or than spontaneous depolymerization. This indicates that the 19K protein depolymerizes actin filaments not only by sequestering actin monomers but also by directly attacking the filaments. The number of actin filaments, measured by assaying the nucleating ability, is increased by substoichiometric concentrations of the 19K protein, irrespective of whether the protein is added to actin monomers before polymerization or added to performed actin filaments. These results suggest that the brain 19K protein not only stabilizes actin monomers but also cuts actin filaments, thereby decreasing the extent of actin polymerization and also changing the filament length. The action on actin of the actin-depolymerizing protein from starfish oocytes resembles that of the brain 19K protein, although the molecular weight of the starfish protein is slightly smaller. The brain 19K protein and starfish protein should be classified into a new functional group of actin-binding proteins.

Actin Depolymerizing Factors↗