ADP-ribosylated actin caps the barbed ends of actin filaments

The mode of action on actin polymerization of skeletal muscle actin ADP-ribosylated on arginine 177 by perfringens iota toxin was investigated. ADP-ribosylated actin decreased the rate of nucleated actin polymerization at substoichiometric ratios of ADP-ribosylated actin to monomeric actin. ADP-ribosylated actin did not tend to copolymerize with actin. Actin filaments were depolymerized by the addition of ADP-ribosylated actin. The maximal monomer concentration reached by addition of ADP-ribosylated actin was similar to the critical concentration of the pointed ends of actin filaments. ADP-ribosylated actin had no effect on the rate of polymerization of gelsolin-capped actin filaments which polymerize at the pointed ends. The results suggest that ADP-ribosylated actin acts as a capping protein which binds to the barbed ends of actin filaments to inhibit polymerization. Based on an analysis of the depolymerizing effect of ADP-ribosylated actin, the equilibrium constant for binding of ADP-ribosylated actin to the barbed ends of actin filaments was determined to be about 10(8) M-1. As actin is ADP-ribosylated by perfringens iota toxin and by botulinum C2 toxin, it appears that conversion of actin into a capping protein by ADP-ribosylation is a pathophysiological reaction catalyzed by bacterial toxins which ultimately leads to inhibition of actin assembly.     

Kinetic evidence for insertion of actin monomers between the barbed ends of actin filaments and barbed end-bound insertin, a protein purified from smooth muscle

An actin polymerization-retarding protein was isolated from chicken gizzard smooth muscle. This protein copurified with vinculin on DEAE-cellulose and gel filtration columns. The polymerization-retarding protein could be separated from vinculin by hydroxylapatite chromatography. The isolated polymerization-retarding protein lost its activity within a few days, but was stable for weeks when it was not separated from vinculin. We termed the polymerization-retarding protein "insertin". Because of the instability of the isolated insertin, we investigated the effect of insertin-vinculin on actin polymerization. Insertin-vinculin retarded nucleated actin polymerization maximally fivefold. Polymerization at the pointed ends of gelsolin-capped actin filaments was not affected by insertin-vinculin, suggesting that insertin-vinculin binds to the barbed ends, but not to the pointed ends, of actin filaments. Retarded polymerization was observed even if the actin monomer concentration was between the critical concentrations of the ends of treadmilling actin filaments. As at this low monomer concentration the pointed ends depolymerize, monomers appeared to be inserted at the barbed ends between the terminal subunit and barbed end-bound insertin molecules. Insertin-vinculin was found not to increase the actin monomer concentration to the value of the pointed ends. These observations support the conclusion that insertin is not a barbed end-capping protein but an actin monomer-inserting protein. According to a quantitative analysis of the kinetic data, all observations could be explained by a model in which two insertin molecules were assumed to bind co-operatively to the barbed ends of actin filaments. Actin monomers were found to be inserted between the barbed ends and barbed end-bound insertin molecules at a rate of about 1 x 10(6) M-1 s-1. Insertin may be an essential part of the machinery of molecules that permit treadmilling of actin filaments in living cells by insertion of actin molecules between membranes and actin filaments.     

Tropomodulin caps the pointed ends of actin filaments

Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining to a site at or near the pointed ends of skeletal muscle thin filaments (Fowler, V. M., M. A., Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120: 411-420). Our experiments demonstrate that tropomodulin in conjunction with tropomyosin is a pointed end capping protein: it completely blocks both elongation and depolymerization at the pointed ends of tropomyosin-containing actin filaments in concentrations stoichiometric to the concentration of filament ends (Kd < or = 1 nM). In the absence of tropomyosin, tropomodulin acts as a "leaky" cap, partially inhibiting elongation and depolymerization at the pointed filament ends (Kd for inhibition of elongation = 0.1-0.4 microM). Thus, tropomodulin can bind directly to actin at the pointed filament end. Tropomodulin also doubles the critical concentration at the pointed ends of pure actin filaments without affecting either the rate of extent of polymerization at the barbed filament ends, indicating that tropomodulin does not sequester actin monomers. Our experiments provide direct biochemical evidence that tropomodulin binds to both the terminal tropomyosin and actin molecules at the pointed filament end, and is the long sought-after pointed end capping protein. We propose that tropomodulin plays a role in maintaining the narrow length distributions of the stable, tropomyosin-containing actin filaments in striated muscle and in red blood cells.     

Arabidopsis capping protein (AtCP) is a heterodimer that regulates assembly at the barbed ends of actin filaments

The precise regulation of actin filament polymerization and depolymerization is essential for many cellular processes and is choreographed by a multitude of actin-binding proteins (ABPs). In higher plants the number of well characterized ABPs is quite limited, and some evidence points to significant differences in the biochemical properties of apparently conserved proteins. Here we provide the first evidence for the existence and biochemical properties of a heterodimeric capping protein from Arabidopsis thaliana (AtCP). The purified recombinant protein binds to actin filament barbed ends with Kd values of 12-24 nM, as assayed both kinetically and at steady state. AtCP prevents the addition of profilin actin to barbed ends during a seeded elongation reaction and suppresses dilution-mediated depolymerization. It does not, however, sever actin filaments and does not have a preference for the source of actin. During assembly from Mg-ATP-actin monomers, AtCP eliminates the initial lag period for actin polymerization and increases the maximum rate of polymerization. Indeed, the efficiency of actin nucleation of 0.042 pointed ends created per AtCP polypeptide compares favorably with mouse CapZ, which has a maximal nucleation of 0.17 pointed ends per CapZ polypeptide. AtCP activity is not affected by calcium but is sensitive to phosphatidylinositol 4,5-bisphosphate. We propose that AtCP is a major regulator of actin dynamics in plant cells that, together with abundant profilin, is responsible for maintaining a large pool of actin subunits and a surprisingly small population of F-actin.     

Identification of a novel tropomodulin isoform, skeletal tropomodulin, that caps actin filament pointed ends in fast skeletal muscle.

Tropomodulin (E-Tmod) is an actin filament pointed end capping protein that maintains the length of the sarcomeric actin filaments in striated muscle. Here, we describe the identification and characterization of a novel tropomodulin isoform, skeletal tropomodulin (Sk-Tmod) from chickens. Sk-Tmod is 62% identical in amino acid sequence to the previously described chicken E-Tmod and is the product of a different gene. Sk-Tmod isoform sequences are highly conserved across vertebrates and constitute an independent group in the tropomodulin family. In vitro, chicken Sk-Tmod caps actin and tropomyosin-actin filament pointed ends to the same extent as does chicken E-Tmod. However, E- and Sk-Tmods differ in their tissue distribution; Sk-Tmod predominates in fast skeletal muscle fibers, lens, and erythrocytes, while E-Tmod is found in heart and slow skeletal muscle fibers. Additionally, their expression is developmentally regulated during chicken breast muscle differentiation with Sk-Tmod replacing E-Tmod after hatching. Finally, in skeletal muscle fibers that coexpress both Sk- and E-Tmod, they are recruited to different actin filament-containing cytoskeletal structures within the cell: myofibrils and costameres, respectively. All together, these observations support the hypothesis that vertebrates have acquired different tropomodulin isoforms that play distinct roles in vivo.     

Eps8 controls actin-based motility by capping the barbed ends of actin filaments

Actin filament barbed-end capping proteins are essential for cell motility, as they regulate the growth of actin filaments to generate propulsive force. One family of capping proteins, whose prototype is gelsolin, shares modular architecture, mechanism of action, and regulation through signalling-dependent mechanisms, such as Ca(2+) or phosphatidylinositol-4,5-phosphate binding. Here we show that proteins of another family, the Eps8 family, also show barbed-end capping activity, which resides in their conserved carboxy-terminal effector domain. The isolated effector domain of Eps8 caps barbed ends with an affinity in the nanomolar range. Conversely, full-length Eps8 is auto-inhibited in vitro, and interaction with the Abi1 protein relieves this inhibition. In vivo, Eps8 is recruited to actin dynamic sites, and its removal impairs actin-based propulsion. Eps8-family proteins do not show any similarity to gelsolin-like proteins. Thus, our results identify a new family of actin cappers, and unveil novel modalities of regulation of capping through protein-protein interactions. One established function of the Eps8-Abi1 complex is to participate in the activation of the small GTPase Rac, suggesting a multifaceted role for this complex in actin dynamics, possibly through the participation in alternative larger complexes.     

Rate constant for capping of the barbed ends of actin filaments by the gelsolin-actin complex

The rate of capping of actin filaments by the gelsolin-actin complex was measured by inhibition of elongation of the barbed ends of actin filaments. Polymeric actin (0.1-1.0 microM) was added to 0.5 microM monomeric actin and various concentrations of the gelsolin-actin complex (0.08-2.4 nM) to induce nucleated polymerization. As under the experimental conditions (2 mM MgCl2, 100 mM KCl, 37 degrees C, actin monomer concentration less than or equal to 0.5 microM) actin filaments treadmilled, filaments elongated only at the barbed ends and the gelsolin-actin complex did not nucleate actin filaments to polymerize towards the pointed ends. The rate of nucleated actin polymerization in the presence of the gelsolin-actin complex was quantitatively analyzed. The rate constant for capping of the barbed ends of actin filaments by the gelsolin-actin complex was found to be about 10(7) M-1 s-1.     

Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments

Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.     

Cap100, a novel phosphatidylinositol 4,5-bisphosphate-regulated protein that caps actin filaments but does not nucleate actin assembly.

The fast and transient polymerization of actin in nonmuscle cells after stimulation with chemoattractants requires strong nucleation activities but also components that inhibit this process in resting cells. In this paper, we describe the purification and characterization of a new actin-binding protein from Dictyostelium discoideum that exhibited strong F-actin capping activity but did not nucleate actin assembly independently of the Ca2+ concentration. These properties led at physiological salt conditions to an inhibition of actin polymerization at a molar ratio of capping protein to actin below 1:1,000. The protein is a monomer, with a molecular mass of approximately 100 kDa, and is present in growing and in developing amoebae. Based on its F-actin capping function and its apparent molecular weight, we designated this monomeric protein cap100. As shown by dilution-induced depolymerization and by elongation assays, cap100 capped the barbed ends of actin filaments and did not sever F-actin. In agreement with its capping activity, cap100 increased the critical concentration for actin polymerization. In excitation or emission scans of pyrene-labeled G-actin, the fluorescence was increased in the presence of cap100. This suggests a G-actin binding activity for cap100. The capping activity could be completely inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), and bound cap100 could be removed by PIP2. The inhibition by phosphatidylinositol and the Ca(2+)-independent down-regulation of spontaneous actin polymerization indicate that cap100 plays a role in balancing the G- and F-actin pools of a resting cell. In the cytoplasm, the equilibrium would be shifted towards G-actin, but, below the membrane where F-actin is required, this activity would be inhibited by PIP2.     

The capactins, a class of proteins that cap the ends of actin filaments

A number of proteins that bind specifically to the barbed ends of actin filaments in a cytochalasin-like manner have been purified to various degrees from a variety of muscle and non-muscle cells and tissues. Preliminary evidence also indicates that proteins that interact with the pointed ends of filaments are present in skeletal muscle. Because of their ability to cap one or the other end of an actin filament, we have designated this class of proteins as the 'capactins'. On the basis of their effect on actin filament assembly and interaction in vitro, we propose that the capactins play important roles in cellular regulation of actin-based cytoskeletal and contractile functions. Our finding that the disappearance of actin filament bundles in virally transformed fibroblasts can be correlated with an increase in capactin activity in the extracts of these cells is consistent with this hypothesis.     

Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein

Exposure of cryptic actin filament fast growing ends (barbed ends) initiates actin polymerization in stimulated human and mouse platelets. Gelsolin amplifies platelet actin assembly by severing F-actin and increasing the number of barbed ends. Actin filaments in stimulated platelets from transgenic gelsolin-null mice elongate their actin without severing. F-actin barbed end capping activity persists in human platelet extracts, depleted of gelsolin, and the heterodimeric capping protein (CP) accounts for this residual activity. 35% of the approximately 5 microM CP is associated with the insoluble actin cytoskeleton of the resting platelet. Since resting platelets have an F- actin barbed end concentration of approximately 0.5 microM, sufficient CP is bound to cap these ends. CP is released from OG-permeabilized platelets by treatment with phosphatidylinositol 4,5-bisphosphate or through activation of the thrombin receptor. However, the fraction of CP bound to the actin cytoskeleton of thrombin-stimulated mouse and human platelets increases rapidly to approximately 60% within 30 s. In resting platelets from transgenic mice lacking gelsolin, which have 33% more F-actin than gelsolin-positive cells, there is a corresponding increase in the amount of CP associated with the resting cytoskeleton but no change with stimulation. These findings demonstrate an interaction between the two major F-actin barbed end capping proteins of the platelet: gelsolin-dependent severing produces barbed ends that are capped by CP. Phosphatidylinositol 4,5-bisphosphate release of gelsolin and CP from platelet cytoskeleton provides a mechanism for mediating barbed end exposure. After actin assembly, CP reassociates with the new actin cytoskeleton.     

Equilibrium constant for binding of an actin filament capping protein to the barbed end of actin filaments

Depolymerization of treadmilling actin filaments by a capping protein isolated from bovine brain was used for determination of the equilibrium constant for binding of the capping protein to the barbed ends of actin filaments. When the capping protein blocks monomer consumption at the lengthening barbed ends, monomers continue to be produced at the shortening pointed ends until a new steady state is reached in which monomer production at the pointed ends is balanced by monomer consumption at the uncapped barbed ends. In this way the ratio of capped to uncapped filaments could be determined as a function of the capping protein concentration. Under the experimental conditions (100 mM KCl and 2 mM MgCl2, pH 7.5, 37 degrees C) the binding constant was found to be about 2 X 10(9) M-1. Capping proteins effect the actin monomer concentration only at capping protein concentrations far above the reciprocal of their binding constant. Half-maximal increase of the monomer concentration requires capping of about 99% of the actin filaments. A low proportion of uncapped filaments has a great weight in determining the monomer concentration because association and dissociation reactions occur at the dynamic barbed ends with higher frequencies than at the pointed ends.     

Variable patterns of actin filaments in mammalian skeletal muscle

Summary The ultrastructural organization of myofilaments in skeletal muscle was studied in four mammalian species (mouse, rat, hamster, goat). In all these species, myofibrils showing irregularly distributed arrays of a variable number of actin filaments (from 6 to 11) were observed. The proportion of such myofibrils and the predominant patterns of actin filaments varied from one species to another. These results are in agreement with those previously reported for human skeletal muscle.     

Purification and characterization of the parvalbumin from monkey skeletal muscle

1. A high affinity Ca2+ binding and low mol. wt protein, parvalbumin, was purified from monkey skeletal muscle. 2. As compared with other animals, only one component and a lower content of monkey parvalbumin were found. 3. This may suggest that both the component and the content of parvalbumin decreases with biological evolution. 4. The parvalbumin was found to have a mol. wt of 11,400, a pI of 5.1, a high aspartic acid and lysine content, maximum absorption at around 260 nm, a blocked amino-terminal, an immunological distinction, 2 mol Ca2+ binding/mol, and a conformational change by Ca2+ binding. 5. Parvalbumin was shown to have alpha type properties.     

Purification and characterization of a new mammalian serum protein with the ability to inhibit actin polymerization and promote depolymerization of actin filaments

A protein with capacity to bind G-actin and the ability to inhibit polymerization and promote depolymerization of actin filaments has been isolated from the serum of rabbit. The protein, SAIP (for serum actin inhibitory protein), has been purified by affinity chromatography of serum over actin-Sepharose followed by protein fractionation with ammonium sulfate and chromatography over DEAE-cellulose. Five milligrams of purified SAIP is obtained from 100 mL of serum. Rabbit SAIP is resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two closely related polypeptides of 60000 and 56000 daltons, respectively (ratio 5.1:1). Each of these polypeptides consists of two isoelectric variants. SAIP binds to monomeric actin with a stoichiometry of 1:1 and a Kd of 0.12 microM. The SAIP-actin complex binds to DNase I. Actin polymerization is completely inhibited by incubation of actin with an equal concentration of SAIP. At equimolar concentrations to F-actin, SAIP induces complete depolymerization of the actin filaments. SAIP is also present in calf serum.     

Extra actin filaments at the periphery of skeletal muscle myofibrils.

Myofibrils isolated from a variety of vertebrate muscle fibers have a set of peripheral filaments associated with the periphery of the Z line free to move away from the surface of the myofibril. Decoration with myosin subfragment 1 shows that these are actin filaments.     

Dynamics of capping protein and actin assembly in vitro: uncapping barbed ends by polyphosphoinositides

Bursts of actin polymerization in vivo involve the transient appearance of free barbed ends. To determine how rapidly barbed ends might appear and how long they might remain free in vivo, we studied the kinetics of capping protein, the major barbed end capper, binding to barbed ends in vitro. First, the off-rate constant for capping protein leaving a barbed end is slow, predicting a half-life for a capped barbed end of approximately 30 min. This half-life implies that cells cannot wait for capping protein to spontaneously dissociate from capped barbed ends in order to create free barbed ends. However, we find that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4- mono-phosphate (PIP) cause rapid and efficient dissociation of capping protein from capped filaments. PIP2 is a strong candidate for a second messenger regulating actin polymerization; therefore, the ability of PIP2 to remove capping protein from barbed ends is a potential mechanism for stimulating actin polymerization in vivo. Second, the on- rate constant for capping protein binding to free barbed ends predicts that actin filaments could grow to the length of filaments observed in vivo during one lifetime. Third, capping protein beta-subunit isoforms did not differ in their actin binding properties, even in tests with different actin isoforms. A major hypothesis for why capping protein beta-subunit isoforms exist is thereby excluded. Fourth, the proposed capping protein regulators, Hsc70 and S100, had no effect on capping protein binding to actin in vitro.     

Purification of a latent protein phosphatase from rabbit skeletal muscle

A high molecular weight protein phosphatase (Mr = 260K) has been isolated from rabbit skeletal muscle. The enzyme has a very low activity towards phosphorylase a isolated from the same tissue, but its activity towards this substrate is stimulated several fold after dissociation by 2-mercaptoethanol treatment. The purified phosphatase shows one major protein staining band on non denaturing polyacrylamide gel electrophoresis, and contains four subunits with molecular weights of 95K, 75K, 65K and 38K. The catalytic activity resides in the Mr = 38K subunit and is not sensitive to inhibition by the heat stable protein phosphatase inhibitor-1 or modulator protein. Polyamines stimulate the holoenzyme in a dose dependent, biphasic manner, but inhibit the activity of the dissociated Mr = 38K catalytic subunit.     

A calcium- and pH-regulated protein from Dictyostelium discoideum that cross-links actin filaments

We have purified an actin binding protein from amebas of Dictyostelium discoideum which we call 95,000-dalton protein (95K). This protein is rod shaped, approximately 40 nm long in the electron microscope, contains two subunits measuring 95,000 daltons each, and cross-links actin filaments. Cross-linking activity was demonstrated by using falling-ball viscometry, Ostwald viscometry, and electron microscopy. Cross-linking activity is optimal at 0.1 microM Ca++ and pH 6.8, but is progressively inhibited at higher Ca++ and pH levels over a physiological range. Half-maximal inhibition occurs at 1.6 microM free Ca++ and pH 7.3, respectively. Sedimentation experiments demonstrate that elevated Ca++ and pH inhibit the binding of 95K to F-actin which explains the loss of cross-linking activity. Electron microscopy demonstrates that under optimal conditions for cross-linking, 95K protein bundles actin filaments and that this bundling is inhibited by microM Ca++. Severing of actin filaments by 95K was not observed in any of the various assays under any of the solution conditions used. Hence, 95K protein is a rod-shaped, dimeric, Ca++- and pH-regulated actin binding protein that cross-links but does not sever actin filaments.     

Purification and characterization of a Cl- -activated aminopeptidase from bovine skeletal muscle

To elucidate the mechanisms involved in the increase in free amino acids during postmortem storage of meat, a novel aminopeptidase was purified from bovine skeletal muscle by ammonium sulfate fractionation and successive chromatographies such as DEAE-cellulose, Sephacryl S-200, Hydroxyapatite, Phenyl-Sepharose, and Hi-Trap affinity column chromatography. The molecular mass of the enzyme was found to be 58 kDa on SDS-PAGE. This enzyme had optimum pH at around 7.5, and preferably hydrolyzed Ala-beta-naphthylamide (-NA) in amino acid-NAs. The activity was strongly inhibited by phenylmethansulfonyl fluoride (PMSF) and bestatin, suggesting that it is to be classified as a serine protease. Moreover, the activity was enhanced by chloride and nitrate ions, which is the most remarkable property of this enzyme. The enzyme appeared to be involved in the increase in free amino acids during postmortem storage of meat.