Muscle Myosin Filaments Research Articles

Filamentous smooth muscle myosin is regulated by phosphorylation.

The enzymatic activity of filamentous dephosphorylated smooth muscle myosin has been difficult to determine because the polymer disassembles to the folded conformation in the presence of MgATP. Monoclonal antirod antibodies were used here to "fix" dephosphorylated myosin in the filamentous state. The steady-state actin-activated ATPase of phosphorylated filaments was 30-100-fold higher than that of antibody-stabilized dephosphorylated filaments, suggesting that phosphorylation can activate ATPase activity independent of changes in assembly. The degree of regulation may exceed 100-fold, because steady-state measurements slightly overestimate the rate of product release from dephosphorylated filaments. Single-turnover experiments in the absence of actin showed that although dephosphorylated folded myosin released products at the low rate of 0.0005 s-1 (Cross, R. A., K. E. Cross, A. Sobieszek. 1986. EMBO [Eur. Mol. Biol. Organ.] J. 5:2637-2641) the rate of product release from dephosphorylated filaments was only 3-12-fold higher, depending on the ionic strength. The addition of actin did not increase this rate to any appreciable extent. Dephosphorylated filaments and dephosphorylated heavy meromyosin (Sellers, J. R. 1985. J. Biol. Chem. 260:15815-15819) thus have similar low rates of phosphate release both in the presence and absence of actin. These results show that light chain phosphorylation alone, without invoking other mechanisms, is an effective switch for regulating the activity of smooth muscle myosin filaments.

Influence of Reconstituted Dark and Light Chicken Muscle Myosin Filaments on the Morphology and Strength of Heat‐Induced Gels

ABSTRACTThe morphological characteristics and heat‐induced gel properties of filaments reconstituted from dark and light chicken muscle myosin by dialysis (slow filamentogenesis) and dilution (fast filamentogenesis) methods were studied. The solubility/turbidity properties of myosin isolated from dark and light muscles were quite similar at different pH values and temperatures, but the morphological characteristics of reconstituted filaments of dark and light myosin varied markedly, depending upon whether they were formed by dialysis or dilution.

Substructure and accessory proteins in scallop myosin filaments.

Native myosin filaments from scallop striated muscle fray into subfilaments of approximately 100-A diameter when exposed to solutions of low ionic strength. The number of subfilaments appears to be five to seven (close to the sevenfold rotational symmetry of the native filament), and the subfilaments probably coil around one another. Synthetic filaments assembled from purified scallop myosin at roughly physiological ionic strength have diameters similar to those of native filaments, but are much longer. They too can be frayed into subfilaments at low ionic strength. Synthetic filaments share what may be an important regulatory property with native filaments: an order-disorder transition in the helical arrangement of myosin cross-bridges that is induced on activation by calcium, removal of nucleotide, or modification of a myosin head sulfhydryl. Some native filaments from scallop striated muscle carry short "end filaments" protruding from their tips, comparable to the structures associated with vertebrate striated muscle myosin filaments. Gell electrophoresis of scallop muscle homogenates reveals the presence of high molecular weight proteins that may include the invertebrate counterpart of titin, a component of the vertebrate end filament. Although the myosin molecule itself may contain much of the information required to direct its assembly, other factors acting in vivo, including interactions with accessory proteins, probably contribute to the assembly of a precisely defined thick filament during myofibrillogenesis.

Myosin filaments isolated from skinned amphibian smooth muscle cells are side-polar

The structure of myosin filaments isolated from skinned toad stomach smooth muscle cells has been examined by electron microscopy as a step toward identifying the in vivo structure. When negatively stained following exposure to relaxing conditions, the filaments exhibited a continuous 14-nm axial repeat of crossbridge projections with no central bare zone. The filaments thus differed from the bipolar filaments found in striated muscle and displayed instead features resembling side-polar and mixed-polarity filament models. By rotation of isolated filaments around their longitudinal axes it was found that cross bridges occurred only along two sides of the filament, an arrangement consistent with the side-polar but not the mixed-polarity model. The polarity is thus similar to that proposed for ribbons (Small & Squire, J. molec. Biol. 67, (1972) 17-149) and for synthetic smooth muscle myosin filaments (Craig and Megerman, J. Cell Biol. 75, (1977) 990-996); their appearance in cross-section, however, shows that these structures are filaments (i.e. with two axes of similar dimensions) and not broad ribbons. As the filaments were derived directly from skinned cells which contracted and relaxed in response to physiological levels of MgATP and Ca2+ at rates comparable to those of native, isolated cells, this unusual arrangement of cross bridges appears to be an effective, functional form of myosin in the contractile apparatus. Side-polar filaments therefore merit consideration as plausible candidates for the native organization of myosin in vertebrate smooth muscle cells.

Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections

For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In principle, good preservation of native structure may be achieved with fast freezing, followed by low-dose electron imaging of unstained vitrified cryosections. In practice, however, cryosections undergo large-scale distortions, including irreversible compression; furthermore, phase contrast imaging results in a nonlinear relationship between the projected density of the specimen and the optical density of the micrograph. To overcome these limitations, we have devised methods of image restoration and generalized correlation averaging, and applied them to cryosections of rabbit psoas fibers in both the relaxed and rigor states. Thus visualized, myosin filaments appear thicker than actin filaments by a much smaller margin than in conventional thin sections, and particularly so for rigor muscle. This may result from a significant fraction of the myosin S1-cross-bridges averaging out in projection and thus contributing only to the baseline of projected density. Entering rigor incurs a loss of density from an annulus around the myosin filament, with a compensating accumulation of density around the actin filament. This redistribution of mass represents attachment of the fraction of cross-bridges that are visible above background. Myosin filaments in the "nonoverlap" zone appear to broaden on entering rigor, suggesting that on deprivation of ATP, cross-bridges in situ move outwards even without actin in their immediate proximity.

What is 10S myosin for?

Myosin filaments in smooth muscle and in non-muscle cells exchange molecules with a pool of free monomers. Regulatory light chain phosphorylation, which regulates the force-generating interaction of these filaments with actin, also regulates the transition of the free monomers into a functionally inert, folded (10S) conformation. It seems likely that the regulated formation of 10S in these systems is a means of coupling force generation to myosin filament assembly.

Subunit exchange between smooth muscle myosin filaments.

Filaments formed from phosphorylated smooth muscle myosin are stable in the presence of MgATP, whereas dephosphorylated filaments are disassembled to a mixture of folded monomers and dimers. The stability of copolymers of phosphorylated and dephosphorylated myosin was, however, unknown. Gel filtration, sedimentation velocity, and pelleting assays were used to show that MgATP could dissociate dephosphorylated myosin from copolymers containing either rod and myosin or dephosphorylated and phosphorylated myosin. Copolymers were typically formed by dialyzing monomeric mixtures into filament-forming buffer but, unexpectedly, could also be formed within minutes of mixing preformed rod and myosin minifilaments. This result suggested that molecules can rapidly and extensively exchange between filaments, presumably via the monomeric pool of myosin in equilibrium with polymer. An exchange of molecules between filaments was demonstrated directly by electron microscopy using gold-labeled streptavidin or antibody to detect the exchanged species. By this approach it was shown that smooth muscle myosin filaments, like other macromolecular assemblies, are dynamic structures that can readily alter their composition in response to changing solvent conditions. Moreover, because folded monomeric myosin is unable to polymerize, these experiments suggest a mechanism for the disassembly of the filament by MgATP.

Polymerization of vertebrate non-muscle and smooth muscle myosins

We investigated how light chain phosphorylation controls the stability of filaments of vertebrate non-muscle myosins (from bovine thymocytes and chicken intestine epithelial brush border cells) and smooth muscle myosin (from chicken gizzard) in vitro. Using a sedimentation assay, the solubilities of the myosins were determined by measuring the amounts of myosin monomers ( C m) and filaments ( C p) present under a given set of conditions as a function of the total myosin concentration ( C t). Below 200 m m-NaCl, each myosin displayed distinct “critical monomer concentrations” ( C c) for polymerization, which were dependent on the salt concentration, the state of light chain phosphorylation and the presence of MgATP. At 150 m m-NaCl, MgATP increased the C c of non-phosphorylated brush border myosin approximately five to tenfold, thymus myosin approximately 10 to 15-fold, and gizzard myosin approximately 25 to 50-fold. When these myosins were phosphorylated, MgATP had little effect on their solubilities, and their C c values remained low. Analytical ultracentrifugation and electron microscopy demonstrated that the myosins were present in three different conformational states under the conditions used in the sedimentation assays, i.e. filaments, extended monomer (6 S) and folded monomer (10 S). Since at equilibrium only filaments and monomers were observed, we suggest that the polymerization pathway for these myosins can be analysed in terms of a dynamic monomer-polymer equilibrium (polymer ⇔ 6 S monomer ⇔ 10 S monomer). At roughly physiological ionic strength, light chain dephosphorylation (in the presence of MgATP) promotes the folded state (10 S), whereas phosphorylation promotes the extended state (6 S), and thereby favours filament assembly. The relevance of the monomer-polymer equilibrium to the state of organization of the myosin in vivo is discussed.

Structural changes accompanying phosphorylation of tarantula muscle myosin filaments.

Electron microscopy has been used to study the structural changes that occur in the myosin filaments of tarantula striated muscle when they are phosphorylated. Myosin filaments in muscle homogenates maintained in relaxing conditions (ATP, EGTA) are found to have nonphosphorylated regulatory light chains as shown by urea/glycerol gel electrophoresis and [32P]phosphate autoradiography. Negative staining reveals an ordered, helical arrangement of crossbridges in these filaments, in which the heads from axially neighboring myosin molecules appear to interact with each other. When the free Ca2+ concentration in a homogenate is raised to 10(-4) M, or when a Ca2+-insensitive myosin light chain kinase is added at low Ca2+ (10(-8) M), the regulatory light chains of myosin become rapidly phosphorylated. Phosphorylation is accompanied by potentiation of the actin activation of the myosin Mg-ATPase activity and by loss of order of the helical crossbridge arrangement characteristic of the relaxed filament. We suggest that in the relaxed state, when the regulatory light chains are not phosphorylated, the myosin heads are held down on the filament backbone by head-head interactions or by interactions of the heads with the filament backbone. Phosphorylation of the light chains may alter these interactions so that the crossbridges become more loosely associated with the filament backbone giving rise to the observed changes and facilitating crossbridge interaction with actin.

Subfilament organization in myosin filaments of the fast abdominal muscles of the lobster, Homarus americanus

The myosin filaments of the fast abdominal muscle of the lobster are about 2.7 μm long with a diameter of about 20 run. They have a low density core in transverse sections except for a short portion in the middle of the filaments about 140 nm in length which is solid. In the solid region the diameter of the filaments is 25 nm. The wall of the filaments is made up of 12 subfilaments arranged in six pairs in a single layer around the wall. The spacing between the subfilaments of a pair is 3.4 nm and the spacing between successive pairs is 8.4 nm. An extra density is present on the inner surface of the wall of the filament along the entire length of the tubular portion of the filament. This density is always adherent to the wall and in serial transverse sections of the same filament its position changes from section to section without any apparent pattern to the change. No structural organization could be detected in this extra density.

Amino acid sequence and structural repeats in schistosome paramyosin match those of myosin.

The cDNA encoding about half of an antigenic non-surface schistosome parasite protein of Mr 97 K has recently been cloned and sequenced (Lanar, Pearce, James and Sher (1986) Science 234:593-596). Analysis of this sequence, together with the properties of the native protein, reveals that this protein is paramyosin, the hitherto unsequenced core protein of myosin filaments in invertebrate muscle. In this report we analyze in more detail the partial amino acid sequence of schistosome paramyosin and describe electron microscope studies of the native protein and its aggregates. We show a close correspondence between the structures of paramyosin and the myosin rod that is required for these proteins to assemble together in muscle thick filaments.

Identification of paramyosin as schistosome antigen recognized by intradermally vaccinated mice.

Mice immunized intradermally with extracts of Schistosoma mansoni in combination with the adjuvant BCG are significantly protected against subsequent infection with living larval forms of the parasite. Remarkably, these vaccinated animals produce antibodies predominantly against a single parasite protein of molecular weight 97 kilodaltons (Sm-97). A complementary DNA that encodes about half of the Sm-97 molecule has now been cloned and sequenced. Analysis of the deduced amino acid sequence reveals a protein containing periodic repeats of hydrophobic amino acids characteristic of an alpha-helical coiled-coil structure. The deduced amino acid composition of the cloned gene and several properties of the native protein are similar to that of paramyosin, an alpha-helical protein that forms the core for myosin filaments in invertebrate muscle. Paramyosin was isolated from Schistosoma mansoni adult worms and antibodies to Sm-97 were shown to react with this molecule as well as with a known paramyosin from molluscan muscle.

ATP-linked monomer-polymer equilibrium of smooth muscle myosin: the free folded monomer traps ADP.Pi.

In vitro and at physiological ionic strength, unphosphorylated smooth muscle myosin filaments dissolve on addition of ATP, forming folded (10S) myosin monomers. By following the fate of ATP and the time course of filament disassembly we have established details of the mechanism of this process. Myosin filaments first bind and hydrolyse 2.0 mol/mol of ATP before significant filament dissolution occurs. Following dissolution, the hydrolysis products ADP.Pi are retained on the heads of the folded myosin monomers, and are released so slowly (half time approximately 100 min at 100 mM KCl) as to be effectively trapped. The straight (6S) conformation of myosin, stable at greater than 225 mM KCl, did not exhibit this product trapping, and neither did myosin filaments held under conditions which disfavour ATP-induced disassembly. The implications of these results for filament stability in vivo are discussed in terms of a simple, testable model for smooth muscle myosin self-assembly.

Purealin, a novel stabilizer of smooth muscle myosin filaments that modulates ATPase activity of dephosphorylated myosin.

Effects of purealin isolated from a sea sponge, Psammaplysilla purea, on the enzymatic and physiochemical properties of chicken gizzard myosin were studied. At 0.15 M KCl, 40 microM purealin increased the Ca2+- and Mg2+-ATPase activity of dephosphorylated gizzard myosin to 2.5- and 3-fold, respectively, but decreased the K+-EDTA-ATPase activity of the myosin to 0.25-fold. In contrast, purealin had little effect on the ATPase activities of phosphorylated gizzard myosin. The ATP-induced decrease in light scattering of dephosphorylated gizzard myosin at 0.15 M KCl was lessened by 40 microM purealin. Electron microscopic observations indicated that thick filaments of dephosphorylated myosin were disassembled immediately by addition of 1 mM ATP at 0.15 M KCl, although they were preserved by purealin for a long time even after addition of ATP. Upon ultracentrifugation, dephosphorylated myosin sedimented as two components, the 10 S species and myosin filaments, in the solution containing 0.18 M KCl and 1 mM Mg X ATP in the presence of 60 microM purealin. These results suggest that purealin modulates the ATPase activities of dephosphorylated gizzard myosin by enhancing the stability of myosin filaments against the disassembling action of ATP.

The reconstruction of myosin filaments in rabbit psoas muscle from solubilized myosin.

Using rabbit psoas muscle strips, A-bands with their myosin-containing thick filaments have been substantially reconstructed in situ (as judged by electron and light microscopy and by low-angle X-ray diffraction analysis) after prior solubilization of the myosin filaments in high ionic strength potassium phosphate solution. The maintenance of a very high local concentration of soluble myosin, by means of a closely apposed artificial semi-permeable membrane is necessary for reconstruction of full-length filaments. This reconstruction effect can be totally abolished by pre-glycerolation of the muscle, or (reversibly) by pre-depletion of Ca2+. Reconstruction at longer sarcomere lengths (greater than 2.6 micron) is anomalous, part-length 'stub filaments' being formed, with their stub tails projecting out from the I-Z-I lattice. A model is proposed to explain this reconstruction effect.

Muscle contraction and free energy transduction in biological systems.

Muscle contraction occurs when the actin and myosin filaments in muscle are driven past each other by a cyclic interaction of adenosine triphosphate (ATP) and actin with cross-bridges that extend from myosin. Current biochemical studies suggest that, during each adenosine triphosphatase cycle, the myosin cross-bridge alternates between two main conformations, which differ markedly in their strength of binding to actin and in their overall structure. Binding of ATP to the cross-bridge induces the weak-binding conformation, whereas inorganic phosphate release returns the cross-bridge to the strong-binding conformation. This cross-bridge cycle is similar to the kinetic cycle that drives active transport and illustrates the general principles of free energy transduction by adenosine triphosphatase systems.

Conformational states of smooth muscle myosin. Effects of light chain phosphorylation and ionic strength.

Stoichiometric amounts of MgATP disassemble dephosphorylated smooth muscle and nonmuscle myosin filaments to a 10 S monomer. Phosphorylation of the regulatory light chain reassembles the myosin into filaments (Suzuki, H., Onishi, H., Takahashi, K., and Watanabe, S. (1978) J. Biochem. (Tokyo) 84, 1529-1542). The conformation of the dephosphorylated 10 S monomer is highly unusual in that the 1500 A long myosin tail is folded into approximately equal thirds (Onishi, H., and Wakabayashi, T. (1982) J. Biochem. (Tokyo) 92, 871-879; Trybus, K. M., Huiatt, T. W., and Lowey, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6151-6155). It was recently reported that phosphorylation of the regulatory light chain causes the bent monomer to unfold to the extended conformation characteristic of 6 S myosin in high salt (Craig, R., Smith, R., and Kendrick-Jones, J. (1983) Nature (Lond.) 302, 436-439). Here we show that phosphorylated myosin can exist in a stable 10 S conformation provided that the salt concentration is kept sufficiently low. Only in a narrow range of salt concentration does the monomer conformation depend on the state of phosphorylation. Above 0.3 M KCl, all myosins revert to the extended form; below 0.1 M KCl, all monomeric myosin is folded. As the salt concentration is decreased to 0.05 M KCl, the 10 S monomers form antiparallel folded dimers. Because phosphorylation increases filament formation even when 10 S monomer remains in equilibrium with polymer, assembly could proceed via the association of 10 S monomers or by a transient 6 S intermediate.

Light-chain phosphorylation controls the conformation of vertebrate non-muscle and smooth muscle myosin molecules.

Phosphorylation of the 20,000-molecular weight (Mr) light chains of vertebrate non-muscle (thymus) and smooth muscle (gizzard) myosins regulates the assembly of these myosins into filaments in vitro. At physiological ionic strength and pH, nonphosphorylated smooth muscle and non-muscle myosin filaments are disassembled by stoichiometric levels of MgATP, forming species having sedimentation coefficients of approximately 11S (range 10-12S; myosin monomers in high salt sediment at 6S). When the 20,000 (20K)-Mr light chains on these 11S myosin species are phosphorylated by the light-chain kinase/calmodulin-Ca2+ complex, the inhibitory effect of the light chains on filament formation is removed and the myosins reassemble into filaments which are stable in MgATP. It was originally suggested that the 11S myosin species was a dimer, previously suggested as a building block for smooth muscle and non-muscle myosin filaments. It has since been shown, however, that 11S smooth muscle myosin is monomeric and has a folded conformation rather than the extended shape characteristic of monomeric myosin in high salt. Here we show that 11S non-muscle myosin is also folded and that phosphorylation of the 20K-Mr light chains of both vertebrate non-muscle (thymus) and vertebrate smooth muscle (gizzard) myosins causes these folded 11S molecules to unfold into the conventional extended monomeric form, which is able to assemble into filaments.

A bent monomeric conformation of myosin from smooth muscle.

Smooth muscle myosin filaments formed in 0.15 M KCl are depolymerized by MgATP to a 10S component, rather than to the 6S component typical of myosin monomer in high salt concentrations. This 10S species is also monomeric as determined by sedimentation equilibrium and calculated from the diffusion and sedimentation coefficients. The conformation of 10S myosin is, however, very different from that of 6S myosin, which has a flexible but extended rod. The Stokes radius and the viscosity of 10S myosin are less than those of 6S myosin, consistent with a structure in which the rod is bent. Electron microscopy of rotary-shadowed preparations confirmed that the light meromyosin region of the rod is bent back on subfragment 2, that region of the rod adjacent to the two globular heads. MgATP and dephosphorylation of the 20,000 molecular weight light chain increase the amount of 10S myosin present in 0.15 M KCl; addition of salt converts 10S myosin back to the typical 6S conformation. We conclude that smooth muscle myosin preferentially forms a bent or folded conformation instead of the extended shape usually associated with skeletal muscle myosin, provided that the salt concentration is kept sufficiently low.

Organization of native and in vitro-reassembled myosin filaments from lobster tonic muscle

Tonic muscle of the crusher claw of the American lobster ( Homarus amencanus) was investigated with respect to sarcomeric organization and the capacity for self-assembly of extracted myosin for comparison with the same properties of rabbit muscle. Native myosin filaments in the lobster muscle are much longer than in rabbit skeletal fibers, and differ further in sarcomeric organization in showing an actinto-myosin relationship in which two actin filaments are shared between adjacent myosins in a 12-membered orbital. The self-assembly of lobster myosin into filaments comparable in length and fine structure to the natural filament was achieved in the presence of excess Mg 2+, a condition not required for rabbit myosin self-assembly. Results of in situ and self-assembly studies indicate a difference in molecular organization between lobster and rabbit myosin filaments and of the inferred presence of regulatory factors in the formation of these ultrastructural elements. These studies represent the groundwork for an investigation of in vitro polymerization of actin in association with the synthetic lobster myosin filament.