Viscosity Of F-actin Research Articles

Regulation of Actin Dynamics by Tropomyosin

It is widely accepted that tropomyosin stabilizes actin filaments mechanically and also by protecting against the action of filament destabilizing proteins. The mechanisms underlying these effects are still unclear. To investigate the influence of tropomyosin on the actin filament severing activity of gelsolin we measured both the F-actin viscosity and the relative number concentrations of filaments after fragmentation by either gelsolin alone or by gelsolin/tropomyosin complexes. Our results show that the association of muscle tropomyosin with F-actin did not significantly protect the filaments from being severed by gelsolin. On the other hand, preceding interaction of gelsolin with tropomyosin reduced the severing activity of gelsolin by up to 80%. These results suggest that tropomyosin is involved in the modulation of actin dynamics by binding gelsolin in solution to prevent it from severing. We also show that in proteolytically modified F-actin where the turnover of subunits is strongly enhanced, tropomyosin restored the stability of this actin but did not stabilize it against the disruptive effects of centrifugal forces and shear stress. Instead of this, tropomyosin inhibited the steady-state ATP hydrolysis of proteolytically modified actin in a cooperative manner, with half-maximal and maximal effects observed at TM:actin molar ratios of about 1:50 and 1:8, respectively. Thus, stabilization of actin filaments by tropomyosin involves conformational changes which seem to modify the monomer-monomer contacts along the filament. We believe that both interaction with gelsolin and stabilization of intermonomer contacts within F-actin may contribute to the regulation by tropomyosin of cytoskeleton dynamics.

Isolation and characterization of 190K protein from aorta smooth muscle.

Molecular assemblies of actin and myosin for the contractility of smooth muscle are quite different from those of striated muscle. Another striking difference is that vascular smooth muscle has a potential to transform to migratory synthetic cell type. At this point of view, smooth muscle cell has properties similar to those of non-muscle. In fact, myosin Ic, a single headed unconventional myosin, was identified in aorta smooth muscle. During the studies on myosin Ic, we have found another calmodulin related 190kDa protein. This protein binds to calmodulin irrespective on calcium ion and to F-actin in an ATP independent manner. Furthermore, the F-actin binding stoichiometry diminished to half upon the addition of exogenous calmodulin. Partial amino acid sequence indicated a high homology to those of GRD (GTPase Related Domain) of human brain IQGAP1. Western blot analysis using anti-human IQGAP1 antibody also indicated a strong cross-reactivity with the protein. We have tested the protein with respect to the characteristic F-actin gelation by IQGAP1. In the presence of cdc42 and GTPgammaS, 190kDa protein could cause a high viscosity of F-actin. These data indicate a close similarity to human brain IQGAP1. The presence of IQGAP1 in aorta smooth muscle suggests contributions for cellular processes such as actin reorganization during contraction-relaxation cycle, association of cytoskeletal structure to cell membrane, organelle movement.

Interaction of Talin with Actin: Sensitive Modulation of Filament Crosslinking Activity

Talin is an adhesion plaque protein believed important in linking actin filaments to the plasma membrane. The nature of a direct talin–actin interaction, however, is complex and has remained unclear. We have systematically characterized the effects of pH, ionic strength, temperature, and protein molar ratio on the interaction between highly purified talin and actin. The ability of talin to increase viscosity of F-actin at 25°C and low ionic strength increased with decreasing pH from 7.3 to 6.4 and increasing molar ratio of talin to actin. At pH 6.4 and low ionic strength, talin could extensively crosslink actin filaments into ordered bundles as shown by negative staining and could cosediment with F-actin at molar ratios as high as one talin to two actin monomers. Talin crosslinked prepolymerized actin filaments to a similar extent as actin filaments polymerized in its presence. The 190-kDa calpain-generated proteolytic fragment of talin bound poorly to actin under conditions favorable for intact talin, but was able to crosslink actin filaments at a lower pH. Increasing the ionic strength within a relatively narrow range significantly decreased ability of talin to bind to actin, regardless of pH. The effects of pH and ionic strength on the talin-actin interaction were rapid and reversible. Low-shear-viscosity studies revealed a strong temperature dependence in the talin–actin interaction with significant crosslinking activity at physiological-like ionic conditions and temperature (37°C). Our results consistently demonstrated that talin crosslinks actin filaments and that this direct interaction is highly sensitive to, and dependent upon, ionic conditions and temperature.

Actin depolymerizing action by marine toxin, pectenotoxin-2

Pectenotoxin-2 (PCTX-2), which is one of Diarrhetic Shellfish Poisoning (DSP), is a family of cyclic polyether macrolide toxin isolated from scallop Patinopecten yessoensis. Although PCTX-2 has a potent cytotoxic activities against several cancer cell lines, the biochemical activity of PCTX-2 has not been determined yet. To clarify the biochemical activity of PCTX-2 is the aime in this study. PCTX-2 inhibited the contractions elicited by 72.7 mM KCl or 1 microM phenyrephrine in a concentration dependent manner in the isolated rat aorta. In A10 cells, actin stressfiber in center but not in periphery of the cell was disrupted by PCTX-2 without any visible shape change. By monitoring fluorescent intensity of pyrenyl-actin, PCTX-2 was found to inhibit the velocity and the degree of actin polymerization in a concentration dependent manner. In addition, PCTX-2 decreased viscosity of F-actin measured with falling ball viscometry. Stoichiometric analysis indicated that PCTX-2 forms 1:4 complex with G-actin. These results suggest that PCTX-2 is a potent natural actin depolymerizing compound with unique mode of action.

Correlation Between Conformational and Binding Properties of Nebulin Repeats

Nebulin, a large protein (600 to 800 kDa) located in the thin filament of striated vertebrate muscle, is assumed to bind and stabilise F-actin. Complete sequence determination of human nebulin has only recently been accomplished showing a uniform modular structure along the whole length of the molecule. Up to 97% of the sequence is assembled from repeats of a sequence motif 35 amino acid residues long. This architecture suggests that a structural and functional understanding of such a large molecule may be possible by characterising single repeats and reconstructing from them the behaviour of the whole molecule. In the present study, we extend and generalise to the whole molecule previous work carried out on single repeats from a limited region of nebulin. Knowledge of the complete sequence allowed extensive analysis of the single repeats revealing a progressive N to C-terminal divergence that is mirrored by an increase of the α-helix propensity. A number of synthetic peptides spanning the sequences of selected repeats were obtained and their conformational and binding properties studied in detail. All the peptides showed a tendency to fold as transient helices in aqueous solution with helix content as observed by CD and NMR studies in excellent agreement with predictions. A higher helical tendency of repeats near the C terminus was observed. Analysis of the influence of charged media as well as trifluoroethanol on the folding of single repeats strongly suggested that the mechanism by which the nebulin α-helix is stabilised is mostly electrostatic. Peptides with higher helical content also showed a higher binding affinity to F-actin. Considerably varying effects were observed for the peptides on F-actin viscosity and polymerisation. We discuss the divergence in sequence and helical tendency and its correlation to the functional data with regard to their significance for the assembly of the thin filament during myogenesis.

Microtubule associated protein MAP1A is an actin-binding and crosslinking protein.

High molecular weight microtubule-associated proteins MAP1A and MAP2 form thin projections from microtubule surfaces and have been implicated in crosslinking microtubules and other cytoskeletal components. We have purified native MAP1A from bovine brain and have studied its interaction with G- and F-actin. Using a solid-phase immunoassay we show that MAP1A binds in a dose-dependent manner to both G-actin and F-actin. Addition of MAP1A to F-actin causes gelation of F-actin and SDS-PAGE analysis shows that MAP1A co-sediments with the gelled network, under conditions where F-actin alone does not pellet. The low apparent viscosity of F-actin is markedly increased in the presence of MAP1A, suggesting that MAP1A can crosslink F-actin. Co-incubation experiments indicate that MAP1A and MAP2 may bind to common or overlapping sites on the actin molecule. The widespread distribution of MAP1A and its interaction with microtubules, actin, and intermediate filaments suggests that it may constitute an important determinant of neuronal and non-neuronal cellular morphology.

Urea-Denaturation of Myosin B Prepared from Smooth Dogfish Muscle.

To study the effects of urea on the denaturation of myosin B prepared from smooth dogfish, myosin B was kept at 20°C for 10 min with various concentrations (0-3.0M) of urea, and the reduced viscosity was measured as a parameter for structural changes in myosin B. The decrease in the reduced viscosity of myosin B containing urea above 1.0M was similar to that of myosin in the presence of urea, However, the reduced viscosity of F-actin was slightly affected by urea even above 1.0M. The reduced viscosity of myosin B did not recovered to its initial level through the removal of urea at concentrations above 1.0M urea, but that of myosin recovered perfectly through the re-moval of urea at concentrations up to 3.0M urea. In the presence of urea above 1.0M, myosin B irreversibly dissociated into myosin and actin. Therefore, it is concluded that the urea denaturation of myosin B would be attributed to its dissociation into myosin and actin as well as the conforma-tional changes of myosin molecules in the presence of urea above 1.0M. Accordingly, shark myosin B is fairly stable under physiological concentrations of urea below 1.0M.

Viscoelasticity of F-actin and F-actin/gelsolin complexes.

Actin is the major protein of eukaryote peripheral cytoplasm where its mechanical effects could determine cell shape and motility. The mechanical properties of purified F-actin, whether it is a viscoelastic fluid or an elastic solid, have been a subject of controversy. Mainstream polymer theory predicts that filaments as long as those found in purified F-actin are so interpenetrated as to appear immobile in measurements over a reasonable time with available instrumentation and that the fluidity of F-actin could only be manifest if the filaments were shortened. We show that the static and dynamic elastic moduli below a critical degree of shear strain are much higher than previously reported, consistent with extreme interpenetration, but that higher strain or treatment with very low concentrations of the F-actin severing protein gelsolin greatly diminish the moduli and cause F-actin to exhibit rheologic behavior expected for independent semidilute rods, and defined by the dimensions of the filaments, including shear rate independent viscosity below a critical shear rate. The findings show that shortening of actin filaments sufficiently to permit reasonable measurements brings out their viscoelastic fluid properties. Since gelsolin shortens F-actin, it is likely that the effect of high strain is also to fragment a population of long actin filaments. We confirmed recent findings that the viscosity of F-actin is inversely proportional to the shear rate, consistent with an indeterminate fluid, but found that gelsolin abolishes this unusual shear rate dependence, indicating that it results from filament disruption during the viscosity measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Salt dependent dimerisation of caldesmon

Using analytical gel filtration (FPLC) we show here that avian gizzard caldesmon (chain molecular mass 150 kDa) self-associates to form end-to-end dimers. Increasing salt concentration promotes dimerisation: at 150 mM KCl, about 40% of the caldesmon was dimeric. Freshly gel filtered caldesmon had an actin gelating activity which decreased with increasing ionic strength. At 150 mM KCl, caldesmon at a 1:90 molar ratio to actin doubled the low shear viscosity of F-actin. Sixfold less filamin was required to produce the same effect.

F-actin and microtubule suspensions as indeterminate fluids.

The viscosity of F-actin and microtubule suspensions has been measured as a function of shear rate with a Weissenberg rheogoniometer. At shear rates of less than 1.0 per second the viscosity of suspensions of these two structural proteins is inversely proportional to shear rate. These results are consistent with previous in vivo measurements of the viscosity of cytoplasm. This power law implies that shear stress is independent of shear rate; that is, shear stress is a constant at all shear rates less than 1.0 per second. Thus the flow profile of these fluids is indeterminate, or nearly so. This flow property may explain several aspects of intracellular motility in living cells. Possible explanations for this flow property are based on a recent model for semidilute suspensions of rigid rods or a classical friction model for liquid crystals.

Isolation of a 240-kilodalton actin-binding protein from Dictyostelium discoideum.

A high molecular weight actin-binding protein with subunit mass of 240 kilodaltons has been purified from vegetative amoebae of Dictyostelium discoideum. Briefly, a cell extract was prepared by homogenizing vegetative amoebae in 5 mM EGTA, 5 mM 1,4-piperazineethanesulfonic acid, 1 mM dithiothreitol, 0.02% NaN3, pH 7.0, followed by ultracentrifugation at 114,000 X g for 1 h. The 240-kDa protein in this extract was separated from actin by chromatography on ATP-saturated DEAE-cellulose and further purified by chromatography on hydroxylapatite and Sephacryl S-300. The 240-kDa protein increases the low shear viscosity of F-actin. Covalent cross-linking with dimethyl suberimidate demonstrates that the 240-kDa protein can form dimers in high salt (500 mM NaCl). Hydrodynamic studies in high salt demonstrate the presence of an asymmetric dimer (Stokes' radius = 8.6 nm, sedimentation coefficient = 12 S, native molecular weight = 434,000, and frictional ratio = 1.7). Rotary shadowing demonstrates that the monomer is a flexible rod of approximately 70 nm in length that can associate end to end to form a dimer of approximately 140 nm in length. The 240-kDa protein cross-reacts with antibodies to chicken gizzard filamin. The properties of the 240-kDa protein suggest that it is a member of the filamin class of actin-associated proteins.

Calspectin (fodrin or nonerythroid spectrin)-actin interaction: A possible involvement of 4.1-related protein

The calspectin/actin complex extracted from the bovine brain membrane crosslinks F-actin, resulting in the increasing viscosity of F-actin determined by low-shear viscometry. We demonstrated the presence of a protein factor in this complex, which regulated the calspectin-F-actin interaction in a Ca 2+- and calmodulin-dependent manner. Erythrocyte protein 4.1, but not synapsin I, mimics the function of this brain factor using a reconstitution system including purified calspectin, calmodulin and F-actin. In the brain complex, the Mr 120,000 and the Mr 80,000 70,000 polypeptides were detected to crossreact with anti-protein 4.1 antibody.

Binding of pig plasma gelsolin to F-actin and partial fractionation into calcium-dependent and calcium-independent forms.

The interaction of pig plasma gelsolin with F-actin has been studied by a sedimentation assay using 125I-gelsolin in a Beckman Airfuge. Over 90% of the gelsolin bound to F-actin in 0.1 mM CaCl2 in experiments using 24 microM actin and 2-10 nM 125I-gelsolin, but only 40-50% bound in 1 mM EGTA. Addition of more F-actin to the EGTA supernatant does not sediment this gelsolin. Demonstration of this partial calcium sensitivity depends critically on the use of F-actin that has been prepared in the absence of calcium ions. F-actin prepared from G-actin in calcium or pretreated with calcium, binds 125I-gelsolin more completely in EGTA. This suggests that gelsolin activity is influenced by transient exposure of actin to calcium. Further evidence for partial calcium sensitivity in the interactions between gelsolin and F-actin has been obtained by other methods, including viscometry and electron microscopy. The gelsolin present in the EGTA supernatant is complexed to G-actin, predominantly as binary complexes. Very low concentrations of these complexes reduce the viscosity of F-actin in calcium but not in EGTA. Whether this effect is due to severing activity, or capping with consequent depolymerization to establish the new critical concentration, is uncertain. The results suggest the presence of two types of gelsolin, one that requires micromolar concentrations of calcium for binding to F-actin and one that does not. Both bind to G-actin. Partial separation has been achieved using actin-Sepharose. Pig plasma gelsolin is heterogeneous on isoelectric focussing gels in urea, but the two types of gelsolin separated on actin-Sepharose do not correspond to specific isoelectric species.

A novel 40 000 Da Ca 2+-dependent actin modulator from bovine brain

A monomeric protein of M r 40000 that modulates the polymer state of actin has been isolated from bovine brain. When added either to preformed actin filaments or to monomeric actin, prior to polymerization, the modulator reduces the low-shear viscosity of F-actin provided that Ca 2+ is present. The 40 kDa protein also inhibits the rate of actin polymerization. The inhibition is fully suppressed by removal of Ca 2+ and restored by subsequent readdition of Ca 2+, suggesting that the Ca 2+-controlled interaction of actin with the 40 kDa modulator is freely reversible.

Isolation and characterization of a conserved actin-binding domain from rat hepatic actinogelin, rat skeletal muscle, and chicken gizzard alpha-actinins.

A common protease-resistant fragment (Mr = 27,000) was generated from purified rat hepatic actinogelin, and rat skeletal muscle and chicken gizzard alpha-actinins by limited proteolysis using thermolysin. A monoclonal antibody (A-1) which was raised against the rat hepatic actinogelin and has a cross-reactivity with rat skeletal muscle and chicken gizzard alpha-actinins was found to bind to all of the 27-kDa fragments selectively. Furthermore, one-dimensional peptide maps of the 27-kDa fragments showed a close similarity indicating the presence of some conservation in primary structure of the fragments. The 27-kDa fragments were purified to homogeneity by the same procedure using ammonium sulfate fractionation and hydrophobic chromatography. As the fragments were easily separated from other peptides during purification, they might be present as an independent structural domain. Purified 27-kDa fragments were found to bind to F-actin in a Ca2+-insensitive manner. The fragments failed to affect the low-shear viscosity of F-actin up to a molar ratio to actin monomer of 1:3.2, indicating that gelation activity of the parental molecules was lost and the fragments have only a single binding site on F-actin. Binding of the fragments to F-actin was almost completely inhibited by respective parental molecules, while binding of the whole molecules was blocked partly by their 27-kDa fragments. Thus, the interaction of the fragments with F-actin seemed to be specific, although apparent affinity was lower than the parental molecules. Considering these results, we concluded that the 27-kDa fragments are a conserved, monovalent, and Ca2+-insensitive actin-binding domain of the actinogelin and muscle alpha-actinins.

魚類のα‐アクチニンとＦ‐アクチンの相互作用におよぼす温度の影響

α-Actinins which were purified from five different species of fish and rabbit skeletal muscles had the same subunit weight, approximately 100, 000, as determined by SDS-polyacrylamide gel electrophoresis. The effect of various α-actinins on F-actin viscosity and turbidity was compared by alternating incubation temperature. It was found that an increase in rabbit F-actin viscosity with fish α-actinins occured at lower temperatures in warm to cold water adapted species. This order of the temperature dependence for fish α-actinins was the same as obtained by measuring temperature to give the maximum increase in turbidity of F-actin. Similar results were obtained when rabbit F-actin was replaced by atka mackerel F-actin. The effect of temperature was completely re-versible. Similar temperature dependence resulted when the effect of different fish α-actinins was mea-sured in terms of actomyosin Mg2+-ATPase activity enhancement. These findings suggest that the temperature dependence of fish α-actinin-F-actin interaction may be correlated to the environmental temperature at which the fish lives.

The 45K molecular weight actin-modulating protein from sea urchin eggs forms a complex with actin in the presence of calcium ions.

The 45K protein from sea urchin eggs forms a complex with actin in the presence of Ca2+. The fraction was not readily dissociated on depletion of Ca2+ by gel filtration, but was dissociated on dialysis against an EGTA-containing solution. The free 45K protein and the 45K protein-actin complex affect F-actin in different manners in the presence of Ca2+. The former severs F-actin in the presence of Ca2+ but not in its absence, while the latter caps the barbed end of F-actin in a Ca2+-independent manner thereby lowering the viscosity of F-actin slowly.

Bovine serum brevin. Purification by hydrophobic chromatography and properties.

Brevin, an actin-severing protein present in serum from numerous mammals, has been purified to homogeneity from bovine serum, using hydrophobic chromatography as the last purification step. The physicochemical parameters of brevin have been established and some of them studied in the absence and presence of Ca2+. Brevin exhibits an apparent Stokes radius, Rs, of 3.4 nm, an intrinsic sedimentation coefficient S degrees 20, W, of 4.8 S and 4.4 S in the absence and presence of Ca2+ respectively, indicative of calcium-induced conformational change. The native molecular mass of brevin was found to be 68 kDa and the hydrodynamic data suggest that the protein is an asymmetric molecule. Sedimentation equilibrium studies demonstrated that Ca2+ affects the shape (asymmetry) of brevin without altering its molecular mass. Limited tryptic and chymotryptic digestion of brevin distinguishes the Ca2+-induced conformation from the EGTA one. No change in the electrophoretic migration of brevin was seen upon Ca2+ addition. Several isoforms were detected by two-dimensional gel electrophoresis. Brevin increases the rate of nucleation of actin but decreases the rate of elongation of the filaments and the steady-state viscosity of F-actin in substoichiometric amounts, as measured by viscometric assays under high shear conditions. Electron microscopic examination documents these effects. Brevin produces shorter actin filaments and binds to the 'barbed' end of filaments to which monomers add preferentially during elongation, as demonstrated by indirect immunogold staining of antibodies against brevin. Filament elongation occurs only at the slowly growing end. An enzyme-linked immunosorbent assay was developed and used to detect and quantify brevin and related proteins in extracts of different bovine cells and tissues. Liver and smooth muscles were found to contain the highest amounts of the severing protein.

Isolation and some properties of macrophage alpha-actinin: evidence that it is not an actin gelling protein.

We have isolated an actin-binding protein from rabbit alveolar macrophages which by virtue of its physical properties we classify as a nonmuscle alpha-actinin. The protein consists of two subunits of Mr 103 000 and has a Stokes' radius of 7.26 nm and a sedimentation coefficient of 6.83 X 10(-13) s-1. Under the electron microscope, rotary-shadowed molecules appeared as short rods with an average length of 39.9 nm. We have examined the nature of the interaction of macrophage alpha-actinin with F-actin. The binding of radioiodinated macrophage alpha-actinin to F-actin is calcium sensitive. At a low concentration of free calcium (less than 10(-9) M), the binding affinity is 4.2 X 10(6) M-1 and is relatively unaffected by changes in temperature, while in the presence of 0.1 mM Ca2+, binding is reduced more than 5-fold. The stoichiometry of binding suggests that alpha-actinin binds all along the length of the actin filaments. The affinity of 45Ca2+ for macrophage alpha-actinin is 4 X 10(6) M-1 with a capacity of four calcium ions per molecule. Although macrophage alpha-actinin has calcium-inhibitable actin gelation activity at 7 degrees C, its effect on the apparent viscosity of F-actin decreases with increasing temperature, and at 37 degrees C, no gel point is observed. Therefore, at the temperature at which macrophages function in vivo, alpha-actinin probably does not promote the isotropic gelation of actin.