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PubMed1996

The cortical actin cytoskeleton of unactivated zebrafish eggs: spatial organization and distribution of filamentous actin, nonfilamentous actin, and myosin-II.

Actin and nonmuscle myosin heavy chain (myosin-II) have been identified and localized in the cortex of unfertilized zebrafish eggs using techniques of SDS-polyacrylamide gel electrophoresis, immunoblotting, and fluorescence microscopy. Whole egg mounts, egg fragments, cryosections, and cortical membrane patches probed with rhodamine phalloidin, fluorescent DNase-I, or anti-actin antibody showed the cortical cytoskeleton to contain two domains of actin: filamentous and nonfilamentous. Filamentous actin was restricted to microplicae and the cytoplasmic face of the plasma membrane where it was organized as an extensive meshwork of interconnecting filaments. The cortical cytoplasm deep to the plasma membrane contained cortical granules and sequestered actin in nonfilamentous form. The cytoplasmic surface (membrane?) of cortical granules displayed an enrichment of nonfilamentous actin. An antibody against human platelet myosin was used to detect myosin-II in whole mounts and egg fragments. Myosin-II colocalized with both filamentous and nonfilamentous actin domains of the cortical cytoskeleton. It was not determined if egg myosin was organized into filaments. Similar to nonfilamentous actin, myosin-II appeared to be concentrated over the surface of cortical granules where staining was in the form of patches and punctate foci. The identification of organized and interconnected domains of filamentous actin, nonfilamentous actin, and myosin-II provides insight into possible functions of these proteins before and after fertilization.

Actins↗
PubMed1993

Use of bimanyl actin derivative (TMB-actin) for studying complexation of beta-thymosins. Inhibition of actin polymerization by thymosin beta 9.

By reacting trimethylammoniobromobimane bromide (TMB bromide) with rabbit muscle actin, a fluorescent reporter group was linked to cysteine at position 374. Fluorescence of TMB-actin decreased significantly on addition of thymosin beta 4 (T beta 4), a peptide of 43 amino acid residues reported to bind to monomeric actin and to prevent filament formation. Based on this effect, we determined the KD value of the thymosin beta 4 complex as 0.8 microM, a value that is in agreement with previous determinations. In addition to the main compound thymosin beta 4, bovine tissue contains a related peptide, thymosin beta 9 (T beta 9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that T beta 9, similar to T beta 4, forms a 1:1 complex with monomeric actin, and hereby inhibits actin polymerization. With a KD value of 1.1 microM the affinity of T beta 9 is in the same range as that of T beta 4, suggesting that T beta 9, like T beta 4, contributes to maintaining the pool of monomeric actin in bovine non-muscle cells. Further proof of the interaction of T beta 9 with actin was provided by native PAGE, where the complex showed the reported higher mobility, as well as by crosslinking experiments. Using different crosslinking reagents, like water-soluble carbodiimide (EDC), m-maleimidobenzoyl-N-hydroxysuccinimidate (MBS), and disuccinimidylsuberate (DSS), we were able to produce conjugates of 47 kDa. In one of these (from MBS) both actin and T beta 9 could be identified by immunoblotting. When, in the MBS crosslinking experiments, native actin was replaced with (374-NEM)-actin, the 47 kDa band was not seen, indicating that Cys-374 takes part in the thiol-specific crosslinking reaction. This suggests that part of the binding site of T beta 9 must be located close to the carboxy-terminus.

Actins↗
PubMed1976

The polymerization of actin. III. Aggregates of nonfilamentous actin and its associated proteins: a storage form of actin.

When echinoderm sperm are treated with the detergent Triton X-100 at pH 6.4 in 10 mM phosphate buffer, the membranes are solubilized, but the actin which is located in the periacrosomal region remains as a phase-dense cup. These cups can be isolated free from the flagella and chromatin and can be solubilized by increasing the pH to 8.0 and by changing the ionic strength and type of buffer used. Since the actin does not exist in the "F" state in unreacted sperm, and since the actin remains as a unit that does not diffuse away, it must be present in the mature sperm in a bound or storage state. The actin is, in fact, associated with a pair of proteins whose mol wt are 250,000 and 230,000. When the isolated cups are digested with trypsin, these high molecular weight proteins are digested, thereby liberating the actin. The actin will polymerize if heavy meromyosin or subfragment 1 is added to a preparation of isolated cups. Evidence is presented that this pair of high molecular weight proteins is similar in molecular weight and properties to erythrocyte spectrin. Attempts at transforming the storage form of actin in the cup into filaments were only moderately successful. The best conditions for filament formation involve incubating the cup in ATP and divalent salts. Careful examination of these cups reveals that the actin polymerized preferentially on either end of oriented filaments that already exist in the cup, indicating that self-nucleation is inefficacious. I conclude that the actin can exist in the storage form by its association with spectrin-like molecules and that the actin in this state polymerizes preferentially onto existing filaments.

Actins↗
PubMed2001

The interaction of plectin with actin: evidence for cross-linking of actin filaments by dimerization of the actin-binding domain of plectin.

Plectin is a major component of the cytoskeleton and is expressed in a wide variety of cell types. It plays an important role in the integrity of the cytoskeleton by cross-linking the three filamentous networks and stabilizing cell-matrix and cell-cell contacts. Sequence analysis showed that plectin contains a highly conserved actin-binding domain, consisting of a pair of calponin-like subdomains. Using yeast two-hybrid assays in combination with in vitro binding experiments, we demonstrate that the actin-binding domain of plectin is fully functional and preferentially binds to polymeric actin. The sequences required for actin binding were identified at the C-terminal end of the first calponin homology domain within the actin-binding domain of plectin. We found that the actin-binding domain of plectin is able to bundle actin filaments and we present evidence that this is mediated by the dimerization of this domain. In addition we also show that plectin and another member of the plakin family, dystonin, can heterodimerize by their actin-binding domains. We propose a new mechanism by which plectin and possibly also other actin-binding proteins can regulate the organization of the F-actin network in the cell.

Actins↗
PubMed1975

Interactions between actin, myosin, and an actin-binding protein from rabbit alveolar macrophages. Alveolar macrophage myosin Mg-2+-adenosine triphosphatase requires a cofactor for activation by actin.

The interactions were analyzed between actin, myosin, and a recently discovered high molecular weight actin-binding protein (Hartwig, J. H., and Stossel, T. P. (1975) J. Biol Chem.250,5696-5705) of rabbit alveolar macrophages. Purified rabbit alveolar macrophage or rabbit skeletal muscle F-actins did not activate the Mg2+ATPase activity of purified rabbit alveolar macrophage myosin unless an additional cofactor, partially purified from macrophage extracts, was added. The Mg2+ATPase activity of cofactor-activated macrophage actomyosin was as high as 0.6 mumol of Pi/mg of myosin protein/min at 37 degrees. The macrophage cofactor increased the Mg2+ATPase activity of rabbit skeletal muscle actomyosin, and calcium regulated the Mg2+ATPase activity of cofactor-activited muscle actomyosin in the presence of muscle troponins and tropomyosin. However, the Mg2+ATPase activity of macrophage actomyosin in the presence of the cofactor was inhibited by muscle control proteins, both in the presence and absence of calcium. The Mg2+ATPase activity of the macrophage actomyosin plus cofactor, whether assembled from purified components or studied in a complex collected from crude macrophage extracts, was not influenced by the presence of absence of calcium ions. Therefore, as described for Acanthamoeba castellanii myosin (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem. 248, 4691-4697), rabbit alveolar macrophage myosin requires a cofactor for activation of its Mg2+ATPase activity by F-actin; and no evidence was found for participation of calcium ions in the regulation of this activity. In macrophage extracts containing 0.34 M sucrose, 0.5 mM ATP, and 0.05 M KCl at pH 7.0,the actin-binding protein bound F-actin into bundles with interconnecting bridges. Purified macrophage actin-binding protein in 0.1 M KCl at pH 7.0 also bound purified macrophage F-actin into filament bundles. Macrophage myosin bound to F-actin in the absence but not the presence of Mg2+ATP, but the actin-binding protein did not bind to macrophage myosin in either the presence or absence of Mg2+ATP.

Actins↗
PubMed2001

Actin depolymerizing factor stabilizes an existing state of F-actin and can change the tilt of F-actin subunits.

Proteins in the actin depolymerizing factor (ADF)/cofilin family are essential for rapid F-actin turnover, and most depolymerize actin in a pH-dependent manner. Complexes of human and plant ADF with F-actin at different pH were examined using electron microscopy and a novel method of image analysis for helical filaments. Although ADF changes the mean twist of actin, we show that it does this by stabilizing a preexisting F-actin angular conformation. In addition, ADF induces a large ( approximately 12 degrees ) tilt of actin subunits at high pH where filaments are readily disrupted. A second ADF molecule binds to a site on the opposite side of F-actin from that of the previously described ADF binding site, and this second site is only largely occupied at high pH. All of these states display a high degree of cooperativity that appears to be an integral part of F-actin.

Actin Depolymerizing Factors↗
PubMed1989

The cortical actin-membrane cytoskeleton of unfertilized sea urchin eggs: analysis of the spatial organization and relationship of filamentous actin, nonfilamentous actin, and egg spectrin.

Whole mounts, cryosections, and isolated cortices of unfertilized sea urchin eggs were probed with fluorescent phalloidin, anti-actin and anti-egg spectrin antibodies to investigate the organizational state of the cortically associated actin-membrane cytoskeleton. Filamentous actin and egg spectrin were localized to the plasma membrane, within microvillar and nonmicrovillar domains. The nonmicrovillar filamentous actin was located immediately subjacent to the microvilli forming an extensive interconnecting network along the inner surface of the plasma membrane. The organization of this filamentous actin network precisely correlated with the positioning of the underlying cortical granules. The cortical cytoplasm did not contain any detectable filamentous actin, but instead contained a sequestered domain of nonfilamentous actin. Spectrin was localized to the cytoplasmic surface of the plasma membrane with concentrated foci co-localized with the filamentous actin present in microvilli. Spectrin was also observed to coat the surfaces of cortical granules as well as other populations of intracellular vesicles. On the basis of light microscopic morphology, intracellular distribution, and co-isolation with the egg cortex, some of these spectrin-coated organelles represent acidic vesicles. Identification of an elaborate organization of inter-related domains of actin (filamentous and nonfilamentous) and spectrin forming the cortical membrane cytoskeleton provides insight into the fundamental mechanisms for early membrane restructuring during embryogenesis. Additionally, the localization of spectrin to the surface of intracellular vesicles is indicative of its newly identified functional roles in membrane trafficking, membrane biogenesis and cellular differentiation.

Actins↗
PubMed1984

Chordate muscle actins differ distinctly from invertebrate muscle actins. The evolution of the different vertebrate muscle actins.

A total of 30 actins from various chordate and invertebrate muscle sources were either characterized by full amino acid sequence data or typed by those partial sequences in the NH2-terminal tryptic peptide which are known to be specific markers for different actin isoforms. The results show that most, if not all, invertebrate muscle actins are homologous to each other and to the isoforms recognized as vertebrate cytoplasmic actins. In contrast the actin forms typically found in muscle cells of warm-blooded vertebrates are noticeably different from invertebrate muscle actins and seem to have appeared in evolution already with the origin of chordates. During subsequent vertebrate evolution there has been a high degree of sequence conservation similar or stronger than that seen in histone H4. Urochordates, Cephalochordates and probably also Agnathes express only one type of muscle actin. Two types, a striated muscle-specific form and a smooth muscle form, are already observed in Chondrichthyes and Osteichthyes. Later in evolution, with the origin of reptiles, both muscle actins seem to have duplicated again; the striated muscle type branched into a skeletal- and cardiac-specific form, while the smooth muscle form duplicated into a vascular- and stomach-specific type. These findings support the hypothesis that each of the four muscle actins of warm-blooded vertebrates are coded for by a small number and possibly only one functional gene.

Actins↗
PubMed1987

Polyphosphoinositide micelles and polyphosphoinositide-containing vesicles dissociate endogenous gelsolin-actin complexes and promote actin assembly from the fast-growing end of actin filaments blocked by gelsolin.

The Ca2+-activated actin-binding protein gelsolin regulates actin filament length by severing preformed filaments and by binding actin monomers, stabilizing nuclei for their assembly into filaments. Gelsolin binds to phosphatidylinositol 4,5-bisphosphate (PIP2), with consequent inhibition of its filament severing activity and dissociation of EGTA-resistant complexes made with rabbit macrophage or human plasma gelsolin and rabbit muscle actin. This study provides evidence for an interaction of gelsolin with phosphatidylinositol monophosphate (PIP) as well as PIP2 and further describes their effects on gelsolin's function. Both phosphoinositides completely dissociate EGTA-insensitive rabbit macrophage cytoplasmic gelsolin-actin complexes and inhibit gelsolin's severing activity. The magnitude of inhibition depends strongly on the physical state of the phosphoinositides, being maximal in preparations that contain small micelles of either purified PIP or PIP2. Aggregation of PIP or PIP2 micelles by divalent cations or insufficient sonication or their incorporation into vesicles containing other phospholipids decreases but does not eliminate the inhibitory properties of the polyphosphoinositides. The presence of gelsolin partly inhibits the divalent cation-induced aggregation of PIP2 micelles. PIP2 in combination with EGTA inactivates gelsolin molecules that block the fast-growing end of actin filaments, thereby accelerating actin polymerization. Regulation of gelsolin by the intracellular messengers Ca2+ and polyphosphoinositides allows for the formation of several different gelsolin-actin intermediates with distinct functional properties that may be involved in changes in the state of cytoplasmic actin following cell stimulation.

Actins↗
PubMed1987

Conversion of ATP-actin to ADP-actin reverses the affinity of monomeric actin for Ca2+ vs Mg2+.

Monomeric ATP-actin binds Ca2+ 3-4-times more strongly than Mg2+ at pH 8. On conversion of G-ATP-actin to G-ADP-actin, the relative affinity of actin for the divalent cations is reversed, so that Mg2+ is bound 6-times more strongly than Ca2+. The dissociation rate constant of Ca2+ from Ca-ADP-actin is 50-fold higher than that for Ca2+ from Ca-ATP-actin, suggesting that this reversal of divalent cation affinities is due primarily to a higher equilibrium dissociation constant for Ca-ADP-actin. These results demonstrate an interaction between the actin-bound nucleotide and divalent cation or their binding sites.

Actins↗
PubMed1999

Tropomodulin increases the critical concentration of barbed end-capped actin filaments by converting ADP.P(i)-actin to ADP-actin at all pointed filament ends.

The pointed end capping protein, tropomodulin, increases the critical concentration of barbed end capped actin, i.e. it lowers the apparent affinity of pointed ends for actin monomers. We show here that this is due to the conversion of pointed end ADP. P(i)-actin (low critical concentration) to ADP-actin (high critical concentration) when 70-98% of the ends are capped by tropomodulin. We propose that this is due to the low affinity of tropomodulin for pointed ends (K(d) approximately 0.3 microM), which allows tropomodulin to rapidly exchange binding sites and transiently block access of actin monomers to all pointed ends. This leaves time for ATP hydrolysis and phosphate release to go to completion between successive monomer additions to the pointed end. When the affinity of tropomodulin for pointed ends was increased about 1000-fold by the presence of tropomyosin (K(d) < 0.05 nM), capping of 95% of the ends by tropomodulin did not alter the critical concentration. However, the critical concentration did increase when the tropomodulin concentration was raised to the high values effective in the absence of tropomyosin. This may reflect transient tropomodulin binding to tropomyosin-free actin molecules at the pointed ends of the tropomyosin-actin filaments without a high affinity tropomodulin cap, i.e. the ends that determine the value of the actin critical concentration.

Actins↗
PubMed2003

FHL3 is an actin-binding protein that regulates alpha-actinin-mediated actin bundling: FHL3 localizes to actin stress fibers and enhances cell spreading and stress fiber disassembly.

Four and a half LIM domain (FHL) proteins are members of the LIM protein superfamily. Several FHL proteins function as co-activators of CREM/CREB transcription factors and the androgen receptor. FHL3 is highly expressed in skeletal muscle, but its function is unknown. FHL3 localized to the nucleus in C2C12 myoblasts and, following integrin engagement, exited the nucleus and localized to actin stress fibers and focal adhesions. In mature skeletal muscle FHL3 was found at the Z-line. Actin was identified as a potential FHL3 binding partner in yeast two-hybrid screening of a skeletal muscle library. FHL3 complexed with actin both in vitro and in vivo as shown by glutathione S-transferase pull-down assays and co-immunoprecipitation of recombinant and endogenous proteins. FHL3 promoted cell spreading and when overexpressed in spread C2C12 cells disrupted actin stress fibers. Increased FHL3 expression was detected in highly motile cells migrating into an artificial wound, compared with non-motile cells. The molecular mechanism by which FHL3 induced actin stress fiber disassembly was demonstrated by low speed actin co-sedimentation assays and electron microscopy. FHL3 inhibited alpha-actinin-mediated actin bundling. These studies reveal FHL3 as a significant regulator of actin cytoskeletal dynamics in skeletal myoblasts.

Actinin↗
PubMed2004

Identification of functional residues on Caenorhabditis elegans actin-interacting protein 1 (UNC-78) for disassembly of actin depolymerizing factor/cofilin-bound actin filaments.

Actin-interacting protein 1 (AIP1) is a WD40 repeat protein that enhances actin filament disassembly in the presence of actin-depolymerizing factor (ADF)/cofilin. AIP1 also caps the barbed end of ADF/cofilin-bound actin filament. However, the mechanism by which AIP1 interacts with ADF/cofilin and actin is not clearly understood. We determined the crystal structure of Caenorhabditis elegans AIP1 (UNC-78), which revealed 14 WD40 modules arranged in two seven-bladed beta-propeller domains. The structure allowed for the mapping of conserved surface residues, and mutagenesis studies identified five residues that affected the ADF/cofilin-dependent actin filament disassembly activity. Mutations of these residues, which reside in blades 3 and 4 in the N-terminal propeller domain, had significant effects on the disassembly activity but did not alter the barbed end capping activity. These data support a model in which this conserved surface of AIP1 plays a direct role in enhancing fragmentation/depolymerization of ADF/cofilin-bound actin filaments but not in barbed end capping.

Actin Depolymerizing Factors↗
PubMed1994

Interaction of Cap Z with actin. The NH2-terminal domains of the alpha 1 and beta subunits are not required for actin capping, and alpha 1 beta and alpha 2 beta heterodimers bind differentially to actin.

Cap Z is a widely distributed, highly conserved, heterodimeric protein that binds to the barbed ends of actin filaments, but does not sever filaments. In chicken, two variant cDNAs (alpha 1 and alpha 2) encoding proteins homologous to the alpha subunit of Cap Z have been described versus one for the beta subunit. To establish the effect of each subunit and of the two potential heterodimers (alpha 1 beta and alpha 2 beta) on actin and to explore the functional domains of the proteins, RNA transcripts derived from these cDNAs were studied using in vitro translation. Sequential deletion mutants at the carboxyl and amino termini of the alpha subunit and at the amino terminus of the beta subunit were constructed, and the ability of each mutant to recombine with its heterologous subunit was assessed by gel filtration. The interaction of the individual subunits, heterodimers, and mutants with actin was studied using cosedimentation and quantitative binding assays. This study demonstrates that 1) both alpha 1 beta and alpha 2 beta heterodimers assemble in vitro after translation and bind selectively to the barbed ends of actin filaments; 2) the affinity of alpha 1 beta heterodimers for actin (KD = 1.6 x 10(-10) M) is approximately 4-fold higher than that of alpha 2 beta heterodimers (KD = 6.3 x 10(-10) M); 3) the amino-terminal 40% of the alpha 1 subunit (amino acids 1-115) and the amino-terminal 31% of the beta subunit (amino acids 1-86) are not required for high affinity binding of Cap Z to the barbed ends of actin filaments; and 4) the carboxyl-terminal 55 amino acids of the alpha subunit appear to be required for binding to actin, as are the carboxyl-terminal 15 amino acids of the beta subunit.

Actins↗
PubMed1983

The genes coding for the cardiac muscle actin, the skeletal muscle actin and the cytoplasmic beta-actin are located on three different mouse chromosomes.

The actins are a group of highly conserved proteins encoded by a multigene family. We have previously reported that the skeletal muscle actin gene is located on mouse chromosome 3, together with several other unidentified actin DNA sequences. We show here that the gene coding for the cardiac muscle actin, which is closely related to the skeletal muscle actin (1.1% amino acid replacements), is located on mouse chromosome 17. The gene coding for the cytoplasmic beta-actin is located on mouse chromosome 5. Thus, these three actin genes are located on three different chromosomes.

Actins↗
PubMed1994

Characterisation of the F-actin binding domains of villin: classification of F-actin binding proteins into two groups according to their binding sites on actin.

The F-actin binding properties of chicken villin, its headpiece and domains 2-3 (V2-3) have been analysed to identify sites involved in bundle formation. Headpiece and V2-3 bind actin with Kd values of approximately 7 microM and approximately 0.3 microM, respectively, at low ionic strength. V2-3 binding, like that of villin, is weakened with increasing salt concentration; headpiece binding is not. Competition experiments show that headpiece and V2-3 bind to different sites on actin, forming the two cross-linking sites of villin. Headpiece does not compete with the F-actin binding domains of gelsolin or alpha-actinin, but it dissociates actin depolymerizing factor. We suggest that the F-actin binding domains of actin severing, crosslinking and capping proteins can be organized into two classes.

Actins↗
PubMed1984

Microinjection of actin-binding proteins and actin antibodies demonstrates involvement of nuclear actin in transcription of lampbrush chromosomes.

Nuclei of amphibian oocytes contain large amounts of actin, mostly in unpolymerized or short-polymer form. When antibodies to actin or actin-binding proteins (fragmin and the actin modulator from mammalian smooth muscle) are injected into nuclei of living oocytes of Pleurodeles waltlii, transcription of the lampbrush chromosomes, but not of the rRNA genes, is inhibited. When transcription is repressed by drugs or RNA is digested by microinjection of RNAase into oocyte nuclei, an extensive meshwork of actin filament bundles is seen in association with the isolated lampbrush chromosomes. These observations indicate a close relationship between the state of nuclear actin and transcriptional activity and suggest that nuclear actin may be involved in transcriptional events concerning protein-coding genes.

Actins↗
PubMed1993

Cooperativity in F-actin: chemical modifications of actin monomers affect the functional interactions of myosin with unmodified monomers in the same actin filament.

We have chemically modified a fraction of the monomers in actin filaments, and then measured the effects on the functional interaction of myosin with unmodified monomers within the same filament. Two modifications were used: (a) covalent attachment of various amounts of myosin subfragment-1 (S1) with the bifunctional reagent disuccinimidyl suberate and (b) copolymerization of unmodified actin monomers with monomers cross-linked internally with 1-ethyl-3-(dimethylaminopropyl)-carbodiimide. Each of these modifications abolished the interaction of the modified monomers with myosin, so the remaining interactions were exclusively with unmodified monomers. The two modifications had similar effects on the interaction of actin with myosin in solution: decreased affinity of myosin heads for unmodified actin monomers, without a change in the Vmax of actin-activated myosin ATPase activity. However, modification (b) produced much greater inhibition of actin sliding on a myosin-coated surface, as measured by an in vitro motility assay. These results provide insight into the functional consequences of cooperative interactions within the actin filament.

Actins↗