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At least 685 records · Page 38Linked to original sources
PubMed2001

Actin filament cross-linking by MARCKS: characterization of two actin-binding sites within the phosphorylation site domain.

We recently identified conformational changes that occur upon phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) that preclude efficient cross-linking of actin filaments (Bubb, M. R., Lenox, R. H., and Edison, A. S. (1999) J. Biol. Chem. 274, 36472-36478). These results implied that the phosphorylation site domain of MARCKS has two actin-binding sites. We now present evidence for the existence of two actin-binding sites that not only mutually compete but also specifically compete with the actin-binding proteins thymosin beta(4) and actobindin to bind to actin. The effects of substitution of alanine for phenylalanine within a repeated hexapeptide segment suggest that the noncharged region of the domain contributes to binding affinity, but the binding affinity of peptides corresponding to each binding site has a steep dependence on salt concentration, consistent with presumed electrostatic interactions between these polycationic peptides and the polyanionic N terminus of actin. Phosphorylation decreases the site-specific affinity by no more than 0.7 kcal/mol, which is less than the effect of alanine substitution. However, phosphorylation has a much greater effect than alanine substitution on the loss of actin filament cross-linking activity. These results are consistent with the hypothesis that the compact structure resulting from conformational changes due to phosphorylation, in addition to modest decreases in site-specific affinity, explains the loss of cross-linking activity in phosphorylated MARCKS.

Actins↗
PubMed1989

Actin-binding proteins from Drosophila embryos: a complex network of interacting proteins detected by F-actin affinity chromatography.

By using F-actin affinity chromatography columns to select proteins solely by their ability to bind to actin filaments, we have identified and partially purified greater than 40 proteins from early Drosophila embryos. These proteins represent approximately 0.5% of the total protein present in soluble cell extracts, and 2 mg are obtained by chromatography of an extract from 10 g of embryos. As judged by immunofluorescence of fixed embryos, 90% of the proteins that we have detected in F-actin column eluates are actin-associated in vivo (12 of 13 proteins tested). The distributions of antigens observed suggest that groups of these proteins cooperate in generating unique actin structures at different places in the cell. These structures change as cells progress through the cell cycle and as they undergo the specializations that accompany development. The variety of different spatial localizations that we have observed in a small subset of the total actin-binding proteins suggests that the actin cytoskeleton is a very complex network of interacting proteins.

Actins↗
PubMed1992

How Listeria exploits host cell actin to form its own cytoskeleton. II. Nucleation, actin filament polarity, filament assembly, and evidence for a pointed end capper.

After Listeria, a bacterium, is phagocytosed by a macrophage, it dissolves the phagosomal membrane and enters the cytoplasm. The Listeria than nucleates actin filaments from its surface. These newly assembled actin filaments show unidirectional polarity with their barbed ends associated with the surface of the Listeria. Using actin concentrations below the pointed end critical concentration we find that filament elongation must be occurring by monomers adding to the barbed ends, the ends associated with the Listerial surface. If Listeria with tails are incubated in G actin under polymerizing conditions, the Listeria is translocated away from its preformed tail by the elongation of filaments attached to the Listeria. This experiment and others tell us that in vivo filament assembly must be tightly coupled to filament capping and cross-bridging so that if one process outstrips another, chaos ensues. We also show that the actin filaments in the tail are capped on their pointed ends which inhibits further elongation and/or disassembly in vitro. From these results we suggest a simple picture of how Listeria competes effectively for host cell actin. When Listeria secretes a nucleator, the host's actin subunits polymerize into a filament. Host cell machinery terminate the assembly leaving a short filament. Listeria overcomes the host control by nucleating new filaments and thus many short filaments assemble. The newest filaments push existing ones into a growing tail. Thus the competition is between nucleation of filaments caused by Listeria and the filament terminators produced by the host.

Actins↗
PubMed1995

A posttranslational modification of beta-actin contributes to the slow dissociation of the spectrin-protein 4.1-actin complex of irreversibly sickled cells.

Irreversibly sickled cells (ISCs) remain sickled even under conditions where they are well oxygenated and hemoglobin is depolymerized. In our studies we demonstrate that triton extracted ISC core skeletons containing only spectrin, protein 4.1, and actin also retain their sickled shape; while reversibly sickled cell (RSC) skeletons remodel to a round or biconcave shape. We also demonstrate that these triton extracted ISC core skeletons dissociate more slowly upon incubation at 37 degrees C than do RSC or control (AA) core skeletons. This observation may supply the basis for the inability of the ISC core skeleton to remodel its shape. Using an in vitro ternary complex dissociation assay, we demonstrate that a modification in beta-actin is the major determinant of the slow dissociation of the spectrin-protein 4.1-actin complex isolated from the ISC core skeleton. We demonstrate that the difference between ISC and control beta-actin is the inaccessibility of two cysteine residues in ISC beta-actin to labeling by thiol reactive reagents; due to the formation of a disulfide bridge between cysteine284 and cysteine373 in ISC beta-actin, or alternatively another modification of cysteine284 and cysteine373 which is reversible with DTT and adds less than 100 D to the molecular weight of beta-actin.

Actins↗
PubMed1995

Actin-based movement of Listeria monocytogenes: actin assembly results from the local maintenance of uncapped filament barbed ends at the bacterium surface.

The thermodynamic basis for actin-based motility of Listeria monocytogenes has been investigated using cytoplasmic extracts of Xenopus eggs, initially developed by Theriot et al. (Theriot, J. A., J. Rosenblatt, D. A. Portnoy, P. J. Goldschmidt-Clermont, and T. J. Mitchison. 1994. Cell. 76:505-517) as an in vitro cell-free system. A large proportion (75%) of actin was found unpolymerized in the extracts. The amount of unassembled actin (12 microM) is accounted for by the sequestering functions of T beta 4Xen (20 microM) and profilin (5 microM), the barbed ends being capped. Movement of Listeria was not abolished by depletion of over 99% of the endogenous profilin. The proline-rich sequences of ActA are unlikely to be the target of profilin. All data support the view that actin assembly at the rear of Listeria results from a local shift in steady state due to a factor, keeping filaments uncapped, bound to the surface of the bacterium, while barbed ends are capped in the bulk cytoplasm. Movement is controlled by the energetic difference (i.e., the difference in critical concentration) between the two ends of the filaments, hence a constant ATP supply and the presence of barbed end capped F-actin in the medium are required to buffer free G-actin at a high concentration. The role of membrane components is demonstrated by the facts that: (a) Listeria movement can be reconstituted in the resuspended pellets of high speed-centrifuged extracts that are enriched in membranes; (b) Actin-based motility of endogenous vesicles, exhibiting the same rocketing movement as Listeria, can be observed in the extracts.

Actins↗
PubMed1979

Polymerization of actin. VI. The polarity of the actin filaments in the acrosomal process and how it might be determined.

The polarity of the actin filaments which assemble from the nucleating body or actomere of Thyone and Pisaster sperm was determined using myosin subfragment 1 decoration. The polarity was found to be unidirectional with the arrowheads pointing towards the cell center. When polymerization is induced at low temperature with concentrations of actin near the critical concentration for polymerization, elongation of filaments occurs preferentially off the apical end. If the sperm are induced to undergo the acrosomal reaction with an ionophore, the polarity of the actin filaments attached to the actomere is the same as that already described, but the filaments which polymerize parallel to, but peripheral to, those extending from the actomere are randomly polarized. These randomly polarized filaments appear to result from spontaneous nucleation. When sperm are induced to undergo the acrosomal reaction with eggs, the polarity of the actin filaments is also unidirectional with the arrowheads pointing towards the cell center. From these results we conclude: (a) that the actomere, by nucleating the polymerization of actin filaments, controls the polarity of the actin filaments in the acrosomal process, (b) that the actomere recognizes a surface of the actin monomer that is different from that surface recognized by the dense material attached to membranes, and (c) that egg myosin could not act to pull the sperm into the egg. Included is a discussion of how the observation that monomers add largely to one end of a decorated filament in vitro relates to these in vivo observations.

Acrosome↗
PubMed1981

Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks.

The ordered structure of the leading edge (lamellipodium) of cultured fibroblasts is readily revealed in cells extracted briefly in Triton X-100-glutaraldehyde mixtures, fixed further in glutaraldehyde, and then negatively stained for electron microscopy. By this procedure, the leading edge regions show a highly organised, three-dimensional network of actin filaments together with variable numbers of radiating actin filament bundles or microspikes. The use of Phalloidin after glutaraldehyde fixation resulted in a marginal improvement in filament order. Processing of the cytoskeletons though the additional steps generally employed for conventional electron microscopy resulted in a marked deterioration or complete disruption of the order of the actin filament networks. In contrast, the actin filaments of the stress fiber bundles were essentially unaffected. Thus, postfixation in osmium tetroxide (1% for 7 min at room temperature) transformed the networks to a reticulum of kinked fibers, resembling those produced by the exposure of muscle F-actin to OsO4 in vitro (P. Maupin-Szamier and T. D. Pollard. 1978. J. Cell Biol. 77:837--852). While limited exposure to OsO4 (0.2+ for 20 min at 0 degrees C) obviated this destruction, dehydration in acetone or ethanol, with or without post-osmication, caused a further and unavoidable disordering and aggregation of the meshwork filaments. The meshwork regions of the leading edge then showed a striking resemblance to the networks hitherto described in critical point-dried preparations of cultured cells. I conclude that much of the "microtrabecular lattice" described by Wolosewick and Porter (1979. J. Cell Biol. 82:114--139) in the latter preparations constitutes actin meshworks and actin filament arrays, with their associated components, that have been distorted and aggregated by the preparative procedures employed.

Actins↗
PubMed2002

Utrophin binds laterally along actin filaments and can couple costameric actin with sarcolemma when overexpressed in dystrophin-deficient muscle.

Dystrophin is widely thought to mechanically link the cortical cytoskeleton with the muscle sarcolemma. Although the dystrophin homolog utrophin can functionally compensate for dystrophin in mice, recent studies question whether utrophin can bind laterally along actin filaments and anchor filaments to the sarcolemma. Herein, we have expressed full-length recombinant utrophin and show that the purified protein is fully soluble with a native molecular weight and molecular dimensions indicative of monomers. We demonstrate that like dystrophin, utrophin can form an extensive lateral association with actin filaments and protect actin filaments from depolymerization in vitro. However, utrophin binds laterally along actin filaments through contribution of acidic spectrin-like repeats rather than the cluster of basic repeats used by dystrophin. We also show that the defective linkage between costameric actin filaments and the sarcolemma in dystrophin-deficient mdx muscle is rescued by overexpression of utrophin. Our results demonstrate that utrophin and dystrophin are functionally interchangeable actin binding proteins, but that the molecular epitopes important for filament binding differ between the two proteins. More generally, our results raise the possibility that spectrin-like repeats may enable some members of the plakin family of cytolinkers to laterally bind and stabilize actin filaments.

Actins↗
PubMed1999

GCS1, an Arf guanosine triphosphatase-activating protein in Saccharomyces cerevisiae, is required for normal actin cytoskeletal organization in vivo and stimulates actin polymerization in vitro.

Recent cloning of a rat brain phosphatidylinositol 3,4, 5-trisphosphate binding protein, centaurin alpha, identified a novel gene family based on homology to an amino-terminal zinc-binding domain. In Saccharomyces cerevisiae, the protein with the highest homology to centaurin alpha is Gcs1p, the product of the GCS1 gene. GCS1 was originally identified as a gene conditionally required for the reentry of cells into the cell cycle after stationary phase growth. Gcs1p was previously characterized as a guanosine triphosphatase-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects. Here, we have shown that similar to centaurin alpha, recombinant Gcs1p bound phosphoinositide-based affinity resins with high affinity and specificity. A novel GCS1 disruption strain (gcs1Delta) exhibited morphological defects, as well as mislocalization of cortical actin patches. gcs1Delta was hypersensitive to the actin monomer-sequestering drug, latrunculin-B. Synthetic lethality was observed between null alleles of GCS1 and SLA2, the gene encoding a protein involved in stabilization of the actin cytoskeleton. In addition, synthetic growth defects were observed between null alleles of GCS1 and SAC6, the gene encoding the yeast fimbrin homologue. Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vitro. These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S. cerevisiae.

ADP-Ribosylation Factor 1↗
PubMed2005

A Plasmodium actin-depolymerizing factor that binds exclusively to actin monomers.

ADF/cofilins (AC) are essential F- and G-actin binding proteins that modulate microfilament turnover. The genome of Plasmodium falciparum, the parasite causing malaria, contains two members of the AC family. Interestingly, P. falciparum ADF1 lacks the F-actin binding residues of the AC consensus. Reverse genetics in the rodent malaria model system suggest that ADF1 performs vital functions during the pathogenic red blood cell stages, whereas ADF2 is not present in these stages. We show that recombinant PfADF1 interacts with monomeric actin but does not bind to actin polymers. Although other AC proteins inhibit nucleotide exchange on monomeric actin, the Plasmodium ortholog stimulates nucleotide exchange. Thus, PfADF1 differs in its biochemical properties from previously known AC proteins and seems to promote turnover exclusively by interaction with actin monomers. These findings provide important insights into the low cytosolic abundance and unique turnover characteristics of actin polymers in parasites of the phylum Apicomplexa.

Actins↗
PubMed1987

Purification of an 80 kDa Ca2+-dependent actin-modulating protein, which severs actin filaments, from bovine adrenal medulla.

A protein with a molecular weight of 80 kDa, which binds Ca2+-dependently to actin, was purified chromatographically from bovine adrenal medulla by using Sephacryl S-300, DEAE-Sepharose, actin-DNase I Sepharose, and Sephacryl S-200. This protein was retained on an actin-DNase I affinity column only in the presence of Ca2+, and could be eluted from this column by EGTA. The 80 kDa protein is a monomer and binds to G-actin in a Ca2+-dependent manner at an equimolar ratio. It caused fragmentation of actin filaments at more than 4 X 10(-7) M free Ca2+ concentration, as determined by low-shear viscometry and electron microscopy. Saturating amounts of tropomyosin showed a slight protective effect on the fragmentation of actin filaments by the 80 kDa protein. Considering the mode of action on actin filaments, the 80 kDa protein reported here seems to be a gelsolin-like protein. Gel electrophoresis of this protein revealed changes in mobility depending upon the concentration of Ca2+. This result also indicates that the 80 kDa protein itself is a Ca2+-binding protein.

Actins↗
PubMed1986

Stoichiometry of actin and phalloidin binding: one molecule of the toxin dominates two actin subunits.

Sonication of F-actin in the absence of added ATP in the solvent induces denaturation of the actin. When phalloidin is added to actin at the molar ratio of 1:2, the denaturation is completely inhibited. More directly, pelleting experiments have indicated that the binding of phalloidin to actin subunits is saturated at the same molar ratio. The protection of F-actin from heat denaturation or depolymerization with 0.6 M KI is also complete with one molecule of the toxin over two actin subunits. Therefore, it is concluded that the binding ratio of phalloidin and the actin subunit is not 1:1 but 1:2.

Actins↗
PubMed1989

A fragmin-like protein from plasmodium of Physarum polycephalum that severs F-actin and caps the barbed end of F-actin in a Ca2+-sensitive way.

Many protein factors regulating actin polymerization can be extracted from plasmodia of Physarum polycephalum in the presence of a high EGTA concentration (30 mM). A protein factor with the molecular weight of 60,000 (60 kDa protein) was especially interesting because of its fragmin-like properties. We purified and characterized this 60 kDa protein in the present study. The purified 60 kDa protein enhanced the initial rate of G-actin polymerization, severed F-actin, and capped the barbed end of F-actin in a Ca2+-dependent way. The threshold concentration for Ca2+ was around 10(-6) M. The flow birefringence measurement showed that the length of F-actin decreased from 2.8 to 1.0 microns depending on the concentration of 60 kDa protein added to F-actin. These properties were identical to those of fragmin (Mr 42,000) isolated from plasmodia (Hasegawa et al. (1980) Biochemistry 19, 2677-2683). However, the molecular weight, the tryptic peptide map, and the cross-reactivities with polyclonal anti-fragmin antibodies were different from those of fragmin. We concluded from these results that 60 kDa protein is a new Ca2+-sensitive F-actin-severing protein. Considering its similarity to fragmin, we termed the 60 kDa protein fragmin 60.

Actins↗
PubMed1979

Interaction of myosin subfragment-1 with actin. I. Effect of actin binding on the susceptibility of subfragment-1 to trypsin.

The heavy chain of myosin subfragment-1 prepared by chymotrypsin treatment had a molecular weight of about 96 K. It was split into 26 K, 50K, and 21 K fragments on trypsin treatment. The effect of actin binding on the susceptibilities of the junctions between 26 K and 50 K and between 50 K and 21 K, and on that of alkali light chain 1 to trypsin was studied. The addition of actin increased the viscosity of the solution, and the apparent activity of trypsin decreased. We estimated this decrease as 35% by measuring the degradation of gamma-globin heavy chain, which is known not to interact with actin and subfragment-1 but is known to be susceptible to trypsin, in actin-subfragment-1 solution. Taking this value into consideration, we concluded that the 26 K-50 K junction became 5 times more and the 50 K-21 K junction became 3 times less susceptible to tryptic attack upon the binding of actin. We also observed that alkali light chain 1 became resistant to trypsin upon the binding of actin to subfragment-1. The relation between this conformational change in subfragment-1 and the cyclic interaction of subfragment-1 with actin and ATP is discussed.

Actins↗
PubMed1982

Shear stress modulates the action of the 88 K protein-actin complex on actin polymerization.

An actin-regulatory protein, 88 K protein-actin complex (88 K-actin complex) isolated from porcine brain, increases the rate of actin polymerization as measured by viscometry which gives rise to shearing stresses. In contrast, when measured by the optical absorption method without solvent flow, the complex does not promote actin polymerization but inhibits both the initial rate and final extent of polymerization. These results suggest that shearing stresses are capable of modulating association of actin with the 88 K-actin complex.

Actins↗
PubMed1984

Interaction between myosin and F-actin. Correlation with actin-binding sites on subfragment-1.

F-Actin bindings to subfragment-1 (S-1) and S-1 after limited proteolysis by trypsin (S-1t) were studied in the absence and presence of ATP by means of ultracentrifugation. No significant difference in the affinities for F-actin was observed between S-1 and S-1t in the absence of ATP. In contrast, the affinity for F-actin in the presence of ATP was decreased about 50 times by the limited proteolysis of the S-1 heavy chain. The S-1 whose SH1 and SH2 groups were cross-linked by N,N'-p-phenylenedimaleimide bound F-actin weakly. The affinity for F-actin was similar to that of unmodified S-1 in the presence of ATP and was also decreased markedly by limited proteolysis of the cross-linked S-1. Reciprocals of the dissociation constant of acto-S-1 complex decreased markedly with increase of ionic strength in the presence of ATP, but decreased only slightly at the rigor state. All these results are consistent with our proposal that S-1 has two different actin binding sites, as reported previously (Katoh, T., Imae, S., & Morita, F. (1984) J. Biochem. 95, 447-454). The mechanism of activation of S-1 ATPase by F-actin is discussed.

Actins↗
PubMed1999

Actin surface structure revealed by antibody imprints: evaluation of phage-display analysis of anti-actin antibodies.

Phage-display peptide library analysis of an anti-F actin polyclonal antibody identified 12 amino acid residues of actin that appear, in its X-ray crystal structure, to be grouped together in a surface accessible conformational epitope. Phage epitope mapping was carried out by isolating immune complexes containing members of the J404 nonapeptide phage-display library formed in diluted antiserum and isolated on a protein A affinity matrix. Immunoreactive clones were grown as plaques, replica plated onto nitrocellulose, and labeled with anti-actin immune serum. One hundred and forty-four positively staining clones identified in this way were sequenced. Of these, 54 displayed peptides with sequence similarities. When the most abundantly selected sequence, KQTWQQLWD, was produced as a synthetic peptide and derivatized to ovalbumin, the complex was strongly recognized by the antiserum on Western blots and inhibited the binding of the antibody to immobilized F-actin by 60%. A scrambled version of this sequence WQDK WLQTQ, when coupled to ovalbumin, was not recognized by the antiserum and minimally inhibited binding of antiserum to immobilized F-actin by 10%. KQTWQQLWD contained four residues that corresponded, in frame, to a highly conserved six residue region of the chicken beta-actin sequence 351TFQQMW356 (identical residues are shown in bold). Examination of the rabbit skeletal muscle X-ray crystal structure suggested that within a 15 A radius of W356, nine additional residues were arranged on the actin surface in such a way that they could be mimicked by several of the selected phage sequences with root-mean-square deviation fits of 2.1-2.5 A. We conclude that phage-display analysis can provide information about the relative location of amino acids on the surfaces of proteins using antibody imprints of the protein surface structure.

Actins↗
PubMed1984

Actin pools and actin microfilament organization in cultured human endothelial cells after exposure to thrombin.

Human umbilical vein endothelial cells (HUVEC) in primary confluent cultures lost their normal polygonal shape and assumed a 'contracted' appearance as judged by phase contrast microscopy when exposed to highly purified bovine thrombin (2 N.I.H. u/ml). Total actin in thrombin-exposed cells did not differ from that of control cells, as measured by the deoxyribonuclease I inhibition assay. However, the monomeric actin pool (unpolymerized actin) in thrombin-treated HUVEC was c. 15% smaller (P less than 0.01) than in control HUVEC (in which it represented approximately 50% of total actin). Transmission electron microscopy showed that thrombin-stimulated HUVEC contained more and thicker bundles of filamentous actin than control cells. Polymerization of actin and reorganization of actin microfilaments may contribute to the shape changes of HUVEC induced by thrombin.

Actins↗