Actin concentration and monomer-polymer ratio in developing chicken skeletal muscle

The actin concentration and monomer-polymer ratio in developing chicken skeletal muscle were determined by means of a DNase I inhibition assay. The concentration of G-actin in embryonic muscle was much higher than the critical concentration for polymerization of purified actin. As muscle development progressed, the amount of total actin remarkably increased, whereas the concentration of G-actin markedly decreased, and finally in adults reached the critical concentration for polymerization of purified actin. When the monomeric actin in the soluble fraction of embryonic muscle was purified, the critical concentration for polymerization of the embryonic actin decreased to the same value as that of adult skeletal muscle actin. On the other hand, there was no difference between the crude and purified actin in the type of actin. They consisted of alpha-, beta-, and gamma-actins; their amounts were in the order, beta greater than gamma greater than alpha. Furthermore, polymerization of the monomeric actin in the soluble fraction of embryonic muscle was induced by the addition of myosin or HMM. The large amount of monomeric actin in the embryonic skeletal muscle may be due to the presence of some factor(s) which inhibits actin polymerization and also to an insufficiency of myosin.     

A cofilin-like protein is involved in the regulation of actin assembly in developing skeletal muscle

An actin-binding protein of 20 kDa (called 20K protein) was purified from the sarcoplasmic fraction of embryonic chicken skeletal muscle. The properties of this protein were very similar to cofilin, which was discovered in porcine brain (Nishida et al. (1984) Biochemistry, 23, 5307-5313): it bound to both G- and F-actin, inhibited actin polymerization in a pH-dependent manner, inhibited binding of tropomyosin to F-actin, and had almost the same molecular size and pI as cofilin. A specific monoclonal antibody to 20K protein (MAB-22) was prepared to examine the expression and location of 20K protein during skeletal muscle development. When the whole protein lysates of embryonic and post-hatched chicken skeletal muscles were examined by means of immunoblotting combined with SDS-PAGE, 20K protein was detected in skeletal muscle through the developmental stages. Location of 20K protein in the cells differed between the embryonic and adult tissues; immunofluorescence staining of the cryosections of embryonic muscle with MAB-22 visualized irregular dot-like structures, but adult muscle sections were stained faintly and uniformly. 20K protein was present as a complex with actin in embryonic muscle, as judged by the ability to bind to a DNase I affinity column, while the same protein was free from actin in the cytoplasm of adult muscle. From these results, it is suggested that 20K protein regulates actin assembly transiently in developing skeletal muscle.     

An 18K protein from ascites hepatoma cell depolymerizes actin filaments rapidly

Several actin binding proteins were isolated from ascites hepatoma cells AH7974 by DNase I affinity chromatography. Among them, a protein having a molecular weight of 18,000 was further purified by DEAE cellulose and hydroxyapatite column chromatographies and gel filtration on a Sephadex G-75 column. The 18K protein not only inhibits actin polymerization but also depolymerizes actin filaments. This conclusion was supported by viscosity and fluorescence intensity measurements and the DNase I inhibition assay. A chemical cross-linking experiment suggested that the 18K protein binds to monomeric actin and forms and 18K-actin 1:1 complex. The net depolymerization rate by the 18K protein measured by the DNase I inhibition assay was slower than the rapid reduction of the fluorescence intensity of pyrene-labeled F-actin upon addition of the 18K protein. This result suggests that the 18K protein not only binds to monomeric actin but also binds to actin filaments directly. The sedimentation assay showed that a part of the 18K protein was cosedimented with actin filaments. Electron microscopic observations demonstrated that the 18K protein decreased the amount of actin filaments and the remaining filaments appeared to be decorated and distorted by the 18K protein. The 18K protein had no Ca2+ ion sensitivity and exhibited the same effect on both this tumor actin and muscle actin.     

Interaction of bacterial flagella with myosin

The interaction of isolated flagellar filaments of Bacillus brevis var. G.-B. P+ with skeletal muscle myosin has been investigated. Bacterial flagellar filaments co-precipitate with myosin at low ionic strength (at the conditions of myosin aggregation). Addition of bacterial flagellar filaments to myosin led to inhibition of its K+-EDTA- and Ca2+-ATPase activity, but had no influence on Mg2+-ATPase. Monomeric protein of bacterial flagella filaments (flagellin) did not co-precipitate with myosin and had no influence on its ATPase activity. The flagella filaments did not co-precipitate with myosin in the presence of F-actin if it was mixed with myosin before the filaments. If the flagella filaments were added to myosin solution before the addition of F-actin the amount of filaments and actin in myosin precipitate were comparable. In this case the presence of flagella filaments decreased activation of myosin Mg2+-ATPase by actin to 25-30%. Thus the bacterial flagellar filaments are able to interact with myosin and modify its ATPase activity. Probably, these properties of filaments are caused by resemblance of flagellin and actin. For instance, the unique origin of these proteins may be the reason of such resemblance.     

Inhibition of the relative movement of actin and myosin by caldesmon and calponin.

Contractile activity of myosin II in smooth muscle and non-muscle cells requires phosphorylation of myosin by myosin light chain kinase. In addition, these cells have the potential for regulation at the thin filament level by caldesmon and calponin, both of which bind calmodulin. We have investigated this regulation using in vitro motility assays. Caldesmon completely inhibited the movement of actin filaments by either phosphorylated smooth muscle myosin or rabbit skeletal muscle heavy meromyosin. The amount of caldesmon required for inhibition was decreased when tropomyosin is present. Similarly, calponin binding to actin resulted in inhibition of actin filament movement by both smooth muscle myosin and skeletal muscle heavy meromyosin. Tropomyosin had no effect on the amount of calponin needed for inhibition. High concentrations of calmodulin (10 microM) in the presence of calcium completely reversed the inhibition. The nature of the inhibition by the two proteins was markedly different. Increasing caldesmon concentrations resulted in graded inhibition of the movement of actin filaments until complete inhibition of movement was obtained. Calponin inhibited actin sliding in a more "all or none" fashion. As the calponin concentration was increased the number of actin filaments moving was markedly decreased, but the velocity of movement remained near control values.     

Some motile properties of fast characean myosin

We improved a motility assay system by using an affinity-purified antibody against the C-terminal globular domain of characean myosin. This improvement allowed us to study the sensitivity to ionic strength or the processivity of characean myosin. The sliding velocity of actin filaments on a characean myosin-coated surface was unaffected by ionic strength. This property is unlike that of skeletal or smooth muscle myosin and suggests that the binding manner of characean myosin to actin is different from that in other muscle myosins. The sliding velocity decreased when the MgADP concentration was raised. The extent of inhibition by MgADP on the motile activity of characean myosin was almost the same as in skeletal muscle or cardiac myosin. The number of sliding filaments on the characean myosin-coated surface decreased drastically with a decrease in the motor density. The motor density required to produce a successful movement of actin filament was about 200 molecules/microm(2). These results suggest that the characean myosin is not a processive motor protein.     

Gelsolin sensitivity of microfilaments as a marker for muscle differentiation

The ability of porcine smooth muscle gelsolin to sever actin filaments was used to study alterations in the organization of F-actin containing structures during skeletal myogenesis. In permeabilized fibroblasts and unfused myoblasts, gelsolin induced complete degradation of the actin cytoskeleton. After fusion of myoblasts to multinucleated myotubes, gelsolin removed a substantial amount of actin, revealing fibers with a sarcomere-like arrangement of gelsolin-insensitive actin. These fibrils were much thinner and had shorter sarcomeres than fully differentiated myofibrils. The proportion of gelsolin-resistant fibrils increased during differentiation, resulting in almost complete inertness of mature myofibrils. Fibrils isolated from adult muscle were also found nearly resistant to gelsolin. Extraction of tropomyosin and myosin in buffer of high ionic strength prior to gelsolin treatment reestablished the susceptibility to the severing protein, both in myotubes and isolated myofibrils. Only small remnants of phalloidin-stainable material were retained. We therefore conclude that during myotube differentiation either an increased interaction of actin with actin-binding proteins (e.g., myosin and tropomyosin), or the assembly of muscle-specific isoforms of these proteins protect the filaments against degradation by actin severing proteins.     

Scaffolds and chaperones in myofibril assembly: putting the striations in striated muscle

Sarcomere assembly in striated muscles has long been described as a series of steps leading to assembly of individual proteins into thick filaments, thin filaments and Z-lines. Decades of previous work focused on the order in which various structural proteins adopted the striated organization typical of mature myofibrils. These studies led to the view that actin and α-actinin assemble into premyofibril structures separately from myosin filaments, and that these structures are then assembled into myofibrils with centered myosin filaments and actin filaments anchored at the Z-lines. More recent studies have shown that particular scaffolding proteins and chaperone proteins are required for individual steps in assembly. Here, we review the evidence that N-RAP, a LIM domain and nebulin repeat protein, scaffolds assembly of actin and α-actinin into I-Z-I structures in the first steps of assembly; that the heat shock chaperone proteins Hsp90 & Hsc70 cooperate with UNC-45 to direct the folding of muscle myosin and its assembly into thick filaments; and that the kelch repeat protein Krp1 promotes lateral fusion of premyofibril structures to form mature striated myofibrils. The evidence shows that myofibril assembly is a complex process that requires the action of particular catalysts and scaffolds at individual steps. The scaffolds and chaperones required for assembly are potential regulators of myofibrillogenesis, and abnormal function of these proteins caused by mutation or pathological processes could in principle contribute to diseases of cardiac and skeletal muscles.     

Protein phosphatase activity is necessary for myofibrillogenesis

During myofibrillogenesis, myosin light-chain kinase (MLCK) phosphorylates the regulatory light chain (RLC) of myosin II, enabling patterned assembly of myosin thick filaments. A protein phosphatase (PP) has been shown to mediate RLC dephosphorylation in adult smooth and striated muscle. A role for PP activity in regulating myofibrillogenesis during embryonic development, however, has not been investigated. Tautomycin (TM) was used to inhibit both PP1 and PP2A activities, whereas okadaic acid (OA) and fostriecin (FOS) were used to inhibit PP2A. TM affected both actin and myosin assembly at 5nM; the IC50 value was 20 and 8.5nM, respectively. In contrast, OA applied at 10 times above its reported Ki for PP2A caused no significant disruption. There was also no disruption when FOS was applied at a concentration 30 times above its reported Ki for PP2A. Thus, our results suggest a primary role for PP1 isoforms during myofibrillogenesis. Although rho kinase (RK) regulates PP activity in embryonic smooth and cardiac muscle, application of the RK inhibitor Y27632 did not affect actin or myosin assembly in skeletal myocytes. Collectively, our pharmacological results suggest that PP1 is involved in dynamic regulation of RLC phosphorylation. To specifically test involvement of the myosin-targeted isoform (PP1M), we used a morpholino antisense approach to knock down the myosin targeting (M) subunit of PP1. Embryos injected with morpholino targeted to the 110-kDa M targeting subunit had fewer somites, and myosin organization was significantly perturbed. The combined pharmacological and molecular results suggest a dynamic equilibrium between MLCK and PP1M activities is required for proper myofibrillogenesis.     

Myosin and actin from Escherichia coli K12 C600.

Myosin-like protein and actin-like protein from E. coli formed filaments very similar in structure to those of myosin and actin from skeletal muscle. At 0.2 M KCl, a large number of "thick filaments" of uniform size (about 0.6-0.7 micron long and about 20 nm wide) was present. These thick filaments aggregated as the KCl concentration decreased to less than 0.2 M. Filaments of actin-like protein were decorated with muscle heavy meromyosin, showing "arrowheads". The arrowhead structure disappeared in the presence of ATP. A mixture of E. coli myosin-like protein and rabbit skeletal actin exhibited a gelation phenomenon on the additon of ATP. The phenomenon was reversible and showed ATP specificity. However, the gelation phenomenon was not observed with the mixture of E. coli actin-like protein and E. coli myosin-like protein. These results provide compelling evidence that the E. coli myosin-like protein and actin-like protein we isolated are essentially identical to myosin and actin, respectively.     

Interaction of bacterial flagellum filaments with skeletal muscle myosin

Interaction of isolated bacterial flagellum filaments (BFF) and intact flagella from E. coli MS 1350 and B. brevis G.-B.p+ with rabbit skeletal myosin was studied. BFF were shown to coprecipitate with myosin (but not with isolated myosin rod) at low ionic strength, that is, under conditions of myosin aggregation. The data of electron microscopy indicate that filaments of intact bacterial flagella interact with isolated myosin heads (myosin subfragment 1, S1), and this interaction is fully prevented by addition of Mg2+ -ATP. Addition of BFF inhibited both K+ -EDTA- and Ca2+ -ATPase activity of skeletal muscle myosin, but had no effect on its Mg2+ -ATPase activity. Monomeric flagellin did not coprecipitate with myosin and had no effect on its ATPase activities. BFF were shown to compete with F-actin in myosin binding. It is concluded that BFF interact with myosin heads and affect their ATPase activity. Thus, BFF composed of a single protein flagellin are in many respects similar to actin filaments. Common origin of actin and flagellin may be a reason for this similarity.     

A kinetic mechanism for the fast movement of Chara myosin

Endoplasmic streaming of characean cells of Nitella or Chara is known to be in the range 30-100 microm/second. The Chara myosin extracted from the cells and fixed onto a glass surface was found to move muscle actin filaments at a velocity of 60 microm/second. This is ten times faster than that of skeletal muscle myosin (myosin II). In this study, the displacement caused by single Chara myosin molecules was measured using optical trapping nanometry. The step size of Chara myosin was approximately 19nm. This step size is longer than that of skeletal muscle myosin but shorter than that of myosin V. The dwell time of the steps was relatively long, and this most likely resulted from two rate-limiting steps, the dissociation of ADP and the binding of ATP. The rate of ADP release from Chara myosin after the completion of the force-generation step was similar to that of myosin V, but was considerably slower than that of skeletal muscle myosin. The 19nm step size and the dwell time obtained could not explain the fast movement. The fast movement could be explained by the load-dependent release of ADP. As the load imposed on the myosin decreased, the rate of ADP release increased. We propose that the interaction of Chara myosin with an actin filament resulted in a negative load being imposed on other myosin molecules interacting with the same actin filament. This resulted in an accelerated release of ADP and the fast sliding movement.     

A new fiber-forming protein from Tetrahymena pyriformis

A new fiber-forming protein was isolated from the acetone powder of Tetrahymena pyriformis by co-precipitating with skeletal muscle myosin while trials were made to find actin or actin-like protein in Tetrahymena. It has a molecular weight of 38000 D and forms a tetramer (140000 D, 9 S) in physiological conditions. Its isoelectric point (pH 6.7), amino acid composition and antigenic determinant(s) differ significantly from those of non-muscle actin and skeletal muscle actin. It does not undergo G-F conversion while actin does, and does not activate Mg²⁺-ATPase of skeletal muscle myosin. The protein localizes in the oral apparatus and division furrow as revealed by fluorescent antibody method. The protein can be assembled into 14-nm filaments in a reassembly buffer. The in vitro filaments appear to correspond to some filaments included in the oral apparatus and the contractile ring. The fiber-forming protein from Tetrahymena may play important roles in cell motility including cell division.     

Actin and myosin-linked calcium regulation in the nematode Caenorhabditis elegans. Biochemical and structural properties of native filaments and purified proteins.

Calcium regulation of actomyosin activity in the nematode, Caenorhabditis elegans, has been studied with purified proteins and crude thin filaments. Actin and tropomyosin have been purified from C. elegans and shown to be similar in most respects to actin and tropomyosin from rabbit skeletal muscle. The actin comigrates with rabbit actin on polyacrylamide-sodium dodecyl sulfate gel electrophoresis, forms similar filaments and paracrystals, and activates the Mg2+-ATPase of rabbit myosin heads as efficiently as rabbit actin. Nematode tropomyosin has a greater apparent molecular weight (estimated by mobility on polyacrylamide-sodium dodecyl sulfate gels) than the rabbit protein, yet it forms Mg2+-paracrystals with a slightly shorter periodicity. Native thin filaments extracted from nematodes activate rabbit myosin subfragment 1 Mg2+-ATPase in a calcium sensitive manner; the extent of activation is threefold greater in 0.2 mM CaCl2 than in the absence of calcium. This observation suggests that the thin filaments contain components which are functionally equivalent to vertebrate troponins. Calcium is also required for maximal activation of the Mg2+-ATPase of purified nematode myosin by pure rabbit F-actin. C. elegans therefore has both myosin and thin filament-linked calcium regulatory systems. The origin of the actin, tropomyosin, and myosin from different tissues and the use of genetic analysis to answer questions about assembly and function in vivo are discussed.     

Chick brain actin and myosin. Isolation and characterization

Brain actin extracted from an acetone powder of chick brains was purified by a cycle of polymerization-depolymerization followed by molecular sieve chromatography. The brain actin had a subunit molecular weight of 42,000 daltons as determined by co-electrophoresis with muscle actin. It underwent salt-dependent g to f transformation to form double helical actin filaments which could be "decorated" by muscle myosin subfragment 1. A critical concentration for polymerization of 1.3 microM was determined by measuring either the change in viscosity or absorbance at 232 nm. Brain actin was also capable of stimulating the ATPase activity of muscle myosin. Brain myosin was isolated from whole chick brain by a procedure involving high salt extraction, ammonium sulfate fractionation and molecular sieve chromatography. The purified myosin was composed of a 200,000-dalton heavy chain and three lower molecular weight light chains. In 0.6 M KCl the brain myosin had ATPase activity which was inhibited by Mg++, stimulated by Ca++, and maximally activated by EDTA. When dialyzed against 0.1 M KCl, the brain myosin self-assembled into short bipolar filaments. The bipolar filaments associated with each other to form long concatamers, and this association was enhanced by high concentrations of Mg++ ion. The brain myosin did not interact with chicken skeletal muscle myosin to form hybrid filaments. Furthermore, antibody recognition studies demonstrated that myosins from chicken brain, skeletal muscle, and smooth muscle were unique.     

Characterization of caldesmon binding to myosin

Caldesmon inhibits the binding of skeletal muscle subfragment-1 (S-1).ATP to actin but enhances the binding of smooth muscle heavy meromyosin (HMM).ATP to actin. This effect results from the direct binding of caldesmon to myosin in the order of affinity: smooth muscle HMM greater than skeletal muscle HMM greater than smooth muscle S-1 greater than skeletal muscle S-1 (Hemric, M. E., and Chalovich, J. M. (1988) J. Biol. Chem. 263, 1878-1885). We now show that the difference between skeletal muscle HMM and S-1 is due to the presence of the S-2 region in HMM and is unrelated to light chain composition or to two-headed versus single-headed binding. Differences between the binding of smooth and skeletal muscle myosin subfragments to actin do not result from the lack of light chain 2 in skeletal muscle S-1. In the presence of ATP, caldesmon binds to smooth muscle myosin filaments with a stoichiometry of 1:1 (K = 1 x 10(6) M-1). Similar results were obtained for the binding of caldesmon to smooth muscle rod as well as the binding of the purified myosin-binding fragment of caldesmon to smooth muscle myosin. The binding of caldesmon to intact myosin is ATP sensitive. The interaction of caldesmon with myosin is apparently specific and sensitive to the structure of both proteins.     

Actin-facilitated assembly of smooth muscle myosin induces formation of actomyosin fibrils

To identify regulatory mechanisms potentially involved in formation of actomyosin structures in smooth muscle cells, the influence of F-actin on smooth muscle myosin assembly was examined. In physiologically relevant buffers, AMPPNP binding to myosin caused transition to the soluble 10S myosin conformation due to trapping of nucleotide at the active sites. The resulting 10S myosin-AMPPNP complex was highly stable and thick filament assembly was suppressed. However, upon addition to F-actin, myosin readily assembled to form thick filaments. Furthermore, myosin assembly caused rearrangement of actin filament networks into actomyosin fibers composed of coaligned F-actin and myosin thick filaments. Severin-induced fragmentation of actin in actomyosin fibers resulted in immediate disassembly of myosin thick filaments, demonstrating that actin filaments were indispensable for mediating myosin assembly in the presence of AMPPNP. Actomyosin fibers also formed after addition of F-actin to nonphosphorylated 10S myosin monomers containing the products of ATP hydrolysis trapped at the active site. The resulting fibers were rapidly disassembled after addition of millimolar MgATP and consequent transition of myosin to the soluble 10S state. However, reassembly of myosin filaments in the presence of MgATP and F-actin could be induced by phosphorylation of myosin P-light chains, causing regeneration of actomyosin fiber bundles. The results indicate that actomyosin fibers can be spontaneously formed by F-actin-mediated assembly of smooth muscle myosin. Moreover, induction of actomyosin fibers by myosin light chain phosphorylation in the presence of actin filament networks provides a plausible hypothesis for contractile fiber assembly in situ.     

Alternative exon-encoded regions of Drosophila myosin heavy chain modulate ATPase rates and actin sliding velocity

To investigate the molecular functions of the regions encoded by alternative exons from the single Drosophila myosin heavy chain gene, we made the first kinetic measurements of two muscle myosin isoforms that differ in all alternative regions. Myosin was purified from the indirect flight muscles of wild-type and transgenic flies expressing a major embryonic isoform. The in vitro actin sliding velocity on the flight muscle isoform (6.4 microm x s(-1) at 22 degrees C) is among the fastest reported for a type II myosin and was 9-fold faster than with the embryonic isoform. With smooth muscle tropomyosin bound to actin, the actin sliding velocity on the embryonic isoform increased 6-fold, whereas that on the flight muscle myosin slightly decreased. No difference in the step sizes of Drosophila and rabbit skeletal myosins were found using optical tweezers, suggesting that the slower in vitro velocity with the embryonic isoform is due to altered kinetics. Basal ATPase rates for flight muscle myosin are higher than those of embryonic and rabbit myosin. These differences explain why the embryonic myosin cannot functionally substitute in vivo for the native flight muscle isoform, and demonstrate that one or more of the five myosin heavy chain alternative exons must influence Drosophila myosin kinetics.     

Four and a half LIM protein 1 binds myosin-binding protein C and regulates myosin filament formation and sarcomere assembly

Four and a half LIM protein 1 (FHL1/SLIM1) is highly expressed in skeletal and cardiac muscle; however, the function of FHL1 remains unknown. Yeast two-hybrid screening identified slow type skeletal myosin-binding protein C as an FHL1 binding partner. Myosin-binding protein C is the major myosin-associated protein in striated muscle that enhances the lateral association and stabilization of myosin thick filaments and regulates actomyosin interactions. The interaction between FHL1 and myosin-binding protein C was confirmed using co-immunoprecipitation of recombinant and endogenous proteins. Recombinant FHL2 and FHL3 also bound myosin-binding protein C. FHL1 impaired co-sedimentation of myosin-binding protein C with reconstituted myosin filaments, suggesting FHL1 may compete with myosin for binding to myosin-binding protein C. In intact skeletal muscle and isolated myofibrils, FHL1 localized to the I-band, M-line, and sarcolemma, co-localizing with myosin-binding protein C at the sarcolemma in intact skeletal muscle. Furthermore, in isolated myofibrils FHL1 staining at the M-line appeared to extend partially into the C-zone of the A-band, where it co-localized with myosin-binding protein C. Overexpression of FHL1 in differentiating C2C12 cells induced "sac-like" myotube formation (myosac), associated with impaired Z-line and myosin thick filament assembly. This phenotype was rescued by co-expression of myosin-binding protein C. FHL1 knockdown using RNAi resulted in impaired myosin thick filament formation associated with reduced incorporation of myosin-binding protein C into the sarcomere. This study identified FHL1 as a novel regulator of myosin-binding protein C activity and indicates a role for FHL1 in sarcomere assembly.     

Metastasis-associated protein Mts1 (S100A4) inhibits CK2-mediated phosphorylation and self-assembly of the heavy chain of nonmuscle myosin

A role for EF-hand calcium-binding protein Mts1 (S100A4) in the phosphorylation and the assembly of myosin filaments was studied. The nonmuscle myosin molecules form bipolar filaments, which interact with actin filaments to produce a contractile force. Phosphorylation of the myosin plays a regulatory role in the myosin assembly. In the presence of calcium, Mts1 binds at the C-terminal end of the myosin heavy chain close to the site of phosphorylation by protein kinase CK2 (Ser1944). In the present study, we have shown that interaction of Mts1 with the human platelet myosin or C-terminal fragment of the myosin heavy chain inhibits phosphorylation of the myosin heavy chain by protein kinase CK2 in vitro. Mts1 might also bind directly the beta subunit of protein kinase CK2, thereby modifying the enzyme activity. Our results indicate that myosin oligomers were disassembled in the presence of Mts1. The short C-terminal fragment of the myosin heavy chain was totally soluble in the presence of an equimolar amount of Mts1 at low ionic conditions (50 mM NaCl). Depolymerization was found to be calcium-dependent and could be blocked by EGTA. Our data suggest that Mts1 can increase myosin solubility and therefore suppress its assembly.