Effect of phosphorylation of myosin light chains on the interaction of myosin minifilaments with F-actin

The effect of phosphorylation of light chains-2 (LC2) of rabbit skeletal muscle myosin on the interaction of myosin minifilaments with F-actin as well as on the actin-stimulated Mg2+-ATPase of minifilaments was studied. It was shown that in the absence of KCl the degree of F-actin-induced stimulation of myosin minifilament Mg2+-ATPase with phosphorylated LC2 exceeds 2-4-fold that with unphosphorylated LC2. Phosphorylation of LC2 considerably increases the rate of actin-stimulated Mg2+-ATPase reaction of myosin minifilaments but exerts only a very weak influence on the affinity of minifilaments for F-actin. After addition of KCl the differences in the actin-stimulated Mg2+-ATPase activity disappear in a great degree; in the presence of 50 mM KCl they do not exceed 50%. It was assumed that the observed specific influence of LC2 phosphorylation on the kinetic parameters of actin-stimulated Mg2+-ATPase reaction of myosin minifilaments is due to unique properties of the minifilaments (e.g., their ability to ordered self-assembly as a result of interaction between the heads of myosin molecules) which reflect their structural peculiarities.     

The effect of myosin light chain phosphorylation on the actin-stimulated ATPase activity of myosin minifilaments

It has been shown that in the absence of KCl, the actin-stimulated Mg2+-ATPase activity of rabbit skeletal myosin minifilaments with phosphorylated regulatory lights chains (LC2) exceeds 3-4-fold that of myosin minifilaments with dephosphorylated LC2. Addition of KCl leads to a decrease in the difference between the two ATPase activities. LC2 phosphorylation considerably increases the rate of ATPase reaction and only slightly decreases the affinity of myosin minifilaments for F-actin. It is suggested that the unusual effect of LC2 phosphorylation on the kinetic parameters of the actin-stimulated ATPase reaction of myosin minifilaments can be accounted for by its influence on the interaction between myosin heads which results in the ordered self-assembly of minifilaments.     

Effect of the filamentous structure of myosin on the actomyosin ATPase activity

The ATPase activities of acto-heavy meromyosin and of acto-myosin minifilaments have been compared under the same conditions at low ATP (0.1 mM) and at several KC1 concentrations. The activities, which are strongly salt-dependent in both systems, have been found to be similar at high ionic strength (about 0.16 M) but different at lower ionic strength (0.06-0.07 M). Under this last condition, the catalytic constants kcat and Km are lower for acto-myosin minifilaments than for acto-heavy meromyosin ATPase. In addition, at low ionic strength, any decrease in the concentration of any of the ionic species (ATP, citrate, etc.) induces an increase in the interaction strength between myosin and actin filaments, as revealed by the Km changes. The presence of the troponintropomyosin complex and of Ca2+ also enhances the strength of this interaction. On the other hand, the occurrence of particular interactions between F-actin and myosin minifilaments is further substantiated by the phenomenon of superprecipitation which occurs when the ATP concentration decreases. The favourable effect of the organized structure of the myosin minifilaments on the ATPase activity of actomyosin is discussed.     

Catalytic cooperativity induced by SH1 labeling of myosin filaments

Modifications of SH1 groups on isolated myosin subfragment 1 (S-1) and myosin in muscle fibers affect differently the acto-S-1 ATPase and the fiber properties. Consistent with the findings of earlier work on fibers, the modification of SH1 groups in relaxed myofibrils with phenylmaleimide caused a loss of their shortening. This loss paralleled the decrease in the Vmax of extracted myosin but was not linear with the extent of SH1 labeling. Strikingly, the decrease in Vmax of S-1 prepared from the modified myofibrils was directly proportional to the extent of SH1 labeling. The specificity of SH1 labeling in myofibrils was verified by ATPase activities, thiol titrations, radiolabeling experiments, and comparisons to myosin labeled on SH1 in solution. To test for intermolecular interactions in the myosin filaments and their contribution to the differences between S-1 and myosin, the catalytic properties of copolymers of myosin were examined. Copolymers of myosin and rod minifilaments were formed in 5 mM citrate-Tris (pH 8.0) buffer, and their homogeneity was verified by sedimentation velocity analysis. The inhibition of actomyosin ATPase by rod particles was related to the decrease in the Km value. When rod particles were replaced in these minifilaments by SH1-modified myosin, the ATPase of the copolymers was increased over that of the combined ATPases of the individual filaments. The actomyosin ATP turnover rates on the unmodified heads were increased severalfold by the modified heads.(ABSTRACT TRUNCATED AT 250 WORDS)     

The actomyosin ATPase of synthetic myosin minifilaments, filaments, and heavy meromyosin

The actin-activated ATPase activities of myosin minifilaments and heavy meromyosin are similar at high actin concentrations. Under low ionic strength conditions, the minifilaments in Tris citrate buffer yield the same maximal turnover rate (Vmax) and apparent dissociation constant of actin from myosin (Kapp) as heavy meromyosin in standard low salt conditions. The time course of actin-activated ATP hydrolysis of minifilaments is similar to that observed for standard myosin preparations. Depending on the exact protein composition of the assay mixture, either the ATPase activity declines continuously with time, or is accelerated at the onset of superprecipitation. In analogy with myosin filaments, the ATPase of minifilaments shows a biphasic dependence on actin concentration. Super-precipitation of minifilaments follows a well resolved clearing phase during which their structural integrity appears to be fully preserved. These results indicate that minifilaments or similar small assemblies of myosin can fulfill contractile functions.     

Structural changes in synthetic myosin minifilaments and their dissociation by adenosine triphosphate and pyrophosphate

Morphologically similar short myosin and rod filaments (minifilaments) have been prepared in 10 mM Tris--citrate buffer, pH 8.0, in the absence of other myosin or rod forms. Both minifilament systems are dissociated in the same manner in the presence of ATP or pyrophosphate. Identical binding of these ligands to myosin and rod minifilaments suggests that myosin heads play no role in substrate-induced destabilization of the minifilaments. The effects of ATP and pyrophosphate on minifilaments are similar to their dissociating effect on synthetic filaments [Harrington, W. F., & Himmelfarb, S. (1972) Biochemistry 11, 2945--2952], thus justifying their use in conformational studies in lieu of filaments. In view of their small size and homogeneity, the minifilaments constitute an appropriate material for such studies. The binding of pyrophosphate to myosin and rod minifilaments decreases their alpha-helical content, as measured by circular dichroism. No change in the secondary structure of subfragment 1 and light meromyosin is observed upon binding of pyrophosphate, but substantial changes (10%) are detected in subfragment 2. The structural changes in myosin, possibly relevant to contraction, are localized in the subfragment 2 region of the molecule. These results emphasize the importance of charge interactions in the functional behavior of thick filaments.     

Cold-sensitive regulatory mutants of simian virus 40

A preparation of short synthetic myosin filaments (minifilaments) in the absence of other myosin forms is reported. Myosin minifilaments have been prepared by dialysing myosin from vertebrate striated muscle into 10 mm-citrate/Tris buffer (pH 8.0 at 4 °C) containing no other salt. These polymers of myosin are very stable and show little tendency to aggregate or dissociate in the original solvent. Sedimentation velocity, diffusion and viscosity measurements indicate that the minifilaments are composed of 16 to 18 molecules. Examination of electron micrographs reveals that the bare central region of minifilaments extends over 1600 to 1800 Å and the entire particles are about 3000 Å long with a diameter of 80 Å across the smooth region. They have the appearance of short bipolar filaments (Huxley, 1963). In solution the minifilaments are homogeneous in terms of size distribution and exhibit normal MgATPase and CaATPase activities. When examined in the ultracentrifuge, the minifilaments sediment in the form of a hypersharp peak (or bar) with a sedimentation coefficient independent of rotor speed. The minifilaments can be dissociated by ATP, hardly by MgATP; whereas KCl (between 0.04 and 0.2 m) induces further polymerization. It is suggested that the minifilaments are an intermediate in the assembly of myosin filaments.     

Calculation of a Gap restoration in the membrane skeleton of the red blood cell: possible role for myosin II in local repair

Human red blood cells contain all of the elements involved in the formation of nonmuscle actomyosin II complexes (V. M. Fowler. 1986. J. Cell. Biochem. 31:1-9; 1996. Curr. Opin. Cell Biol. 8:86-96). No clear function has yet been attributed to these complexes. Using a mathematical model for the structure of the red blood cell spectrin skeleton (M. J. Saxton. 1992. J. Theor. Biol. 155:517-536), we have explored a possible role for myosin II bipolar minifilaments in the restoration of the membrane skeleton, which may be locally damaged by major mechanical or chemical stress. We propose that the establishment of stable links between distant antiparallel actin protofilaments after a local myosin II activation may initiate the repair of the disrupted area. We show that it is possible to define conditions in which the calculated number of myosin II minifilaments bound to actin protofilaments is consistent with the estimated number of myosin II minifilaments present in the red blood cells. A clear restoration effect can be observed when more than 50% of the spectrin polymers of a defined area are disrupted. It corresponds to a significant increase in the spectrin density in the protein free region of the membrane. This may be involved in a more complex repair process of the red blood cell membrane, which includes the vesiculation of the bilayer and the compaction of the disassembled spectrin network.     

Assembly of smooth muscle myosin minifilaments: effects of phosphorylation and nucleotide binding

Small bipolar filaments, or "minifilaments," are formed when smooth muscle myosin is dialyzed against low ionic strength pyrophosphate or citrate/Tris buffers. Unlike synthetic filaments formed at approximately physiological ionic conditions, minifilaments are homogeneous as indicated by their hypersharp boundary during sedimentation velocity. Electron microscopy and hydrodynamic techniques were used to show that 20-22S smooth muscle myosin minifilaments are 380 nm long and composed of 12-14 molecules. By varying solvents, a continuum of different size polymers in the range of 15-30S could be obtained. Skeletal muscle myosin, in contrast, preferentially forms a stable 32S minifilament (Reisler, E., P. Cheung, and N. Borochov. 1986. Biophys. J. 49:335-342), suggesting underlying differences in the assembly properties of the two myosins. Addition of salt to the smooth muscle myosin minifilaments caused unidirectional growth into a longer "side-polar" type of filament, whereas bipolar filaments were consistently formed by skeletal muscle myosin. As with synthetic filaments, addition of 1 mM MgATP caused dephosphorylated minifilaments to dissociate to a mixture of folded monomers and dimers. Phosphorylation of the regulatory light chain prevented disassembly by nucleotide, even though it had no detectable effect on the structure of the minifilament. These results suggest that differences in filament stability as a result of phosphorylation are due largely to conformational changes occurring in the myosin head, and are not due to differences in filament packing.     

Growth cones contain myosin II bipolar filament arrays

Nonmuscle myosin II is among the most abundant forms of myosin in nerve growth cones. At least two isoforms of myosin II (A and B) that have overlapping but distinct distributions are found in growth cones. It appears that both myosin IIA and IIB may be necessary for normal nerve outgrowth and motility, but the molecular interactions responsible for their activity remain unclear. For instance, it is unknown if these myosin II isoforms produce bipolar "minifilaments" in growth cones similar to those observed in other nonmuscle cells. To determine if minifilaments are present in growth cones, we modified the electron microscopy preparative procedures used to detect minifilaments in other cell types. We found structures that appeared very similar to bipolar minifilaments found in noneuronal cells. They also labeled with antibodies to either myosin IIA or IIB. Thus, the activity of myosin II in growth cones is likely to be similar to that in other nonmuscle cells. Bipolar filaments interacting with oppositely oriented actin filaments will produce localized contractions or exert tension on actin networks. This activity will be responsible for the myosin II dependent motility in growth cones.     

Mechanism of action of myosin X, a membrane-associated molecular motor

We have performed a detailed biochemical kinetic and spectroscopic study on a recombinant myosin X head construct to establish a quantitative model of the enzymatic mechanism of this membrane-bound myosin. Our model shows that during steady-state ATP hydrolysis, myosin X exhibits a duty ratio (i.e. the fraction of the cycle time spent strongly bound to actin) of around 16%, but most of the remaining myosin heads are also actin-attached even at moderate actin concentrations in the so-called "weak" actin-binding states. Contrary to the high duty ratio motors myosin V and VI, the ADP release rate constant from actomyosin X is around five times greater than the maximal steady-state ATPase activity, and the kinetic partitioning between different weak actin-binding states is a major contributor to the rate limitation of the enzymatic cycle. Two different ADP states of myosin X are populated in the absence of actin, one of which shows very similar kinetic properties to actomyosin.ADP. The nucleotide-free complex of myosin X with actin shows unique spectral and biochemical characteristics, indicating a special mode of actomyosin interaction.     

Superprecipitation of actomyosin with p-chloromercuribenzoate-modified myosin reconstituted from rabbit skeletal muscle

Myosin head modified with p-chloromercuribenzoate (CMB) forms rigor-like complex with actin in the presence of ATP. Actomyosins with CMB-modified myosin were reconstituted to study the effect of rigor-like complexes on superprecipitation. As native myosin was increasingly replaced by CMB-modified myosin, superprecipitation of the actomyosin was strongly suppressed. Further, the suppression of superprecipitation occurred in a different fashion depending on how CMB-modified myosin was incorporated in myosin filaments of the reconstituted actomyosin. The present results indicate that superprecipitation requires the dissociation of actin and myosin head to take place (i.e., the presence of molecular rearrangements of actomyosin network), and further suggest that superprecipitation is associated with dynamic rearrangements of actomyosin network along myosin filaments.     

Macromolecular assemblies of myosin.

The self-assembly of myosin into filamentous structures is a highly cooperative and rapid process. Nevertheless, the presence of nonequivalent bonding interactions within the filament permits differential stabilization of several macromolecular assemblies of myosin under well-controlled ionic conditions in citrate/Tris buffer at pH 8.0. We have detected and characterized bipolar myosin minifilaments, myosin octamers, and tetramers by using light scattering, analytical ultracentrifugation, and viscosity techniques. These structures have molecular weights of 8.0 X 10(6), 3.9 X 10(6) g/mol, sedimentation coefficients of 32S, 22S, and 18S, and radii of gyration of 990 A, 890 A and 790, A, respectively. The similar radii of gyration indicate similar bipolar geometry for all these particles. The 32S minifilaments in 10 mM citrate/Tris buffer (pH 8.0) are the most stable species. The smaller 18S and 22S assemblies in 2 mM and 5 mM citrate/Tris, pH 8.0, are readily affected by low concentrations of KCl and fuse into the minifilament particles. The instability of the 18S and 22S forms of myosin assembly is also revealed by their titration with ATP. These structures are dissociated at lower ATP concentrations than the minifilaments and do not show the cooperative dissociation transitions characteristic of filaments and minifilaments. Sedimentation velocity analysis of the 18S and 22S species in the presence of ATP reveals the involvement of 10S myosin dimer in the dissociation of assembled myosin. The different forms of assembled myosin are discussed in the context of formation of myosin minifilaments.     

Kinetics of ADP dissociation from the trail and lead heads of actomyosin V following the power stroke

Myosin V is a cellular motor protein, which transports cargos along actin filaments. It moves processively by 36-nm steps that require at least one of the two heads to be tightly bound to actin throughout the catalytic cycle. To elucidate the kinetic mechanism of processivity, we measured the rate of product release from the double-headed myosin V-HMM using a new ATP analogue, 3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxy-ATP (deac-aminoATP), which undergoes a 20-fold increase in fluorescence emission intensity when bound to the active site of myosin V (Forgacs, E., Cartwright, S., Kovács, M., Sakamoto, T., Sellers, J. R., Corrie, J. E. T., Webb, M. R., and White, H. D. (2006) Biochemistry 45, 13035-13045). The kinetics of ADP and deac-aminoADP dissociation from actomyosin V-HMM, following the power stroke, were determined using double-mixing stopped-flow fluorescence. These used either deac-aminoATP as the substrate with ADP or ATP chase or alternatively ATP as the substrate with either a deac-aminoADP or deac-aminoATP chase. Both sets of experiments show that the observed rate of ADP or deac-aminoADP dissociation from the trail head of actomyosin V-HMM is the same as from actomyosin V-S1. The dissociation of ADP from the lead head is decreased by up to 250-fold.     

Comparison of kinetic properties of the ATPase reaction of arterial smooth muscle myosin with skeletal muscle myosin

Myosin was prepared from arterial smooth muscle, and a hybrid actomyosin was formed from arterial myosin and rabbit skeletal muscle F-actin. We performed kinetics on the ATPase reaction [EC 3.6.1.3] of arterial myosin and the hybrid actomyosin at high ionic strength, and compared the kinetic properties of arterial myosin ATPase with those of skeletal muscle myosin ATPase. No significant difference was found between these two myosins in the size of the initial Pi burst, the amount of bound nucleotides, and the rates of various elementary steps in the ATPase reaction. On the other hand, two important differences were observed between the hybrid actomyosin and skeletal muscle actomyosin: (i) The amounts of ATP necessary for complete dissociation of the hybrid and skeletal muscle actomyosins were 2 and 1 mol/mol of myosin, respectively. (ii) The rate of dissociation of the hybrid actomyosin induced by ATP was much lower than that of skeletal muscle actomyosin and also was lower than that of fluorescence enhancement.     

Crossbridge release and alpha-helix-coil transition in myosin and rod minifilaments

The relationship between crossbridge release and alpha-helix-coil transition in myosin has been investigated by employing synthetic myosin and rod minifilaments prepared in 10 mM-citrate/Tris buffer at pH 7.0 and 8.0. Initial sedimentation velocity and turbidity measurements have established that the minifilament structures obtained at pH 7.0 and 8.0 are relatively similar in size and homogeneity, and can be used in comparative circular dichroism studies. Chemical crosslinkings and proteolytic digestions carried out at pH 7.0 and 8.0 verify that myosin and rod minifilaments undergo the same pH-induced changes as myosin filaments, i.e. a decrease in the rate of subfragment-2 crosslinking to the filament surface, and an increase in proteolytic susceptibility of the light meromyosin-heavy meromyosin hinge at alkaline pH. These results suggest charge-induced release of the S-2 element from the myosin and rod minifilament surface. Circular dichroism measurements reveal a reduced alpha-helical content of myosin (5%) and rod minifilaments (10%) at pH 8.0 compared to the respective pH 7.0 structures. These results establish a direct link between crossbridge release and alpha-helix-coil transition in myosin.     

Human myosin Vc is a low duty ratio, nonprocessive molecular motor

Myosin Vc is the product of one of the three genes of the class V myosin found in vertebrates. It is widely found in secretory and glandular tissues, with a possible involvement in transferrin trafficking. Transient and steady-state kinetic studies of human myosin Vc were performed using a truncated, single-headed construct. Steady-state actin-activated ATPase measurements revealed a V(max) of 1.8 +/- 0.3 s(-1) and a K(ATPase) of 43 +/- 11 microm. Unlike previously studied vertebrate myosin Vs, the rate-limiting step in the actomyosin Vc ATPase pathway is the release of inorganic phosphate (~1.5 s(-1)), rather than the ADP release step (~12.0-16.0 s(-1)). Nevertheless, the ADP affinity of actomyosin Vc (K(d) = 0.25 +/- 0.02 microm) reflects a higher ADP affinity than seen in other myosin V isoforms. Using the measured kinetic rates, the calculated duty ratio of myosin Vc was approximately 10%, indicating that myosin Vc spends the majority of the actomyosin ATPase cycle in weak actin-binding states, unlike the other vertebrate myosin V isoforms. Consistent with this, a fluorescently labeled double-headed heavy meromyosin form showed no processive movements along actin filaments in a single molecule assay, but it did move actin filaments at a velocity of approximately 24 nm/s in ensemble assays. Kinetic simulations reveal that the high ADP affinity of actomyosin Vc may lead to elevations of the duty ratio of myosin Vc to as high as 64% under possible physiological ADP concentrations. This, in turn, may possibly imply a regulatory mechanism that may be sensitive to moderate changes in ADP concentration.     

Identification of functional regions on the tail of Acanthamoeba myosin- II using recombinant fusion proteins. I. High resolution epitope mapping and characterization of monoclonal antibody binding sites

We used a series of COOH-terminally deleted recombinant myosin molecules to map precisely the binding sites of 22 monoclonal antibodies along the tail of Acanthamoeba myosin-II. These antibodies bind to 14 distinguishable epitopes, some separated by less than 10 amino acids. The positions of the binding sites visualized by electron microscopy agree only approximately with the physical positions of these sites on the alpha-helical coiled-coil tail. On the other hand, the epitope map agrees precisely with competitive binding studies: all antibodies that share an epitope compete with each other for binding to myosin. Antibodies with adjacent epitopes can compete with each other at linear distances up to 5 or 6 nm, and many antibodies that bind 3-7- nm apart can enhance the binding of each other to myosin. Most of the antibodies that bind to the distal 37 nm of the tail disrupt assembly of octameric minifilaments and, depending upon the exact location of the binding site, stop assembly at specific steps yielding, for example, monomers, antiparallel dimers, parallel dimers or antiparallel tetramers. The effects of these antibodies on assembly identify sites on the tail that are required for individual steps in minifilament assembly. Experiments on the assembly of truncated myosin-II tails have revealed a complementary group of sites that participate in the assembly reactions (Sinard, J.H., D.L. Rimm, and T.D. Pollard. 1990. J. Cell Biol. 111:2417-2426). Antibodies that bind to the distal tail but do not affect assembly appear to have a low affinity for myosin-II. Antibodies that bind to the proximal 50 nm of the tail do not inhibit the assembly of minifilaments. Many antibodies that bind to the tail of myosin-II, even some that have no obvious effect on minifilament assembly, can inhibit the actomyosin ATPase activity and the contraction of an actin gel formed in crude extracts. An antibody that binds between amino acids 1447 and 1467 inhibits the phosphorylation of serine residues distal to residue 1483.     

Ordered assemblies of myosin minifilaments

Electron microscopic observations of negatively stained myosin minifilaments in a solution containing 10 mM-citrate buffer (pH 8.0), 4 mM-MgCl2 reveal ordered assemblies. They reveal bundles of parallel minifilaments of about 330 nm in length, aggregated into periodic linear and hexagonal structures. These structures are formed by means of interaction between myosin heads, arranged at the ends of minifilament bundles. The addition of 1 mM-ATP to the above solution causes dissociation of minifilament bundles into individual minifilaments without preventing the latter from association into linear "end-to-end" assemblies. Phosphorylation of myosin light chains does not exert any noticeable effect on the structure of the ordered minifilament assemblies but significantly increases their quantity. The interaction between minifilaments observed by us should be taken into consideration in studies on the properties of myosin which make use of minifilament systems.     

Myosin VIIB from Drosophila is a high duty ratio motor

Myosin VII is an unconventional myosin widely expressed in organisms ranging from amoebae to mammals that has been shown to play vital roles in cell adhesion and phagocytosis. Here we present the first study of the mechanism of action of a myosin VII isoform. We have expressed a truncated single-headed Drosophila myosin VIIB construct in the baculovirus-Sf9 system that bound calmodulin light chains. By using steady-state and transient kinetic methods, we showed that myosin VIIB exhibits a fast release of phosphate and a slower, rate-limiting ADP release from actomyosin. As a result, myosin VIIB will be predominantly strongly bound to actin during steady-state ATP hydrolysis (its duty ratio will be at least 80%). This kinetic pattern is in many respects similar to that of the single-molecule vesicle transporters myosin V and VI. The enzymatic properties of myosin VIIB provide a kinetic basis for processivity upon possible dimerization via the C-terminal domains of the heavy chain. Our experiments also revealed conformational heterogeneity of the actomyosin VIIB complex in the absence of nucleotide.