A comparison of the effect of vanadate on the binding of myosin-subfragment-1.ADP to actin and on actomyosin subfragment 1 ATPase activity

Equilibrium binding studies were used to determine the binding constant of vanadate ion (Vi), to the complex of actomyosin subfragment 1 (S1) with ADP and Vi and of actin to the myosin S1.ADP.Vi complex. The proteins used were obtained from rabbit skeletal muscle. Pre-steady-state measurements were also performed to determine the rates of Vi association and dissociation from the actomyosin S1.ADP.Vi complex. Using these measured values in a simple model, the steady-state actomyosin S1 ATPase activity was predicted over a range of Vi concentrations. This model predicted that Vi would have little effect on the actomyosin S1 ATPase activity. In agreement with this prediction, the measured ATPase activity of actomyosin S1 was not greatly inhibited by Vi, except at high concentrations at which polymeric species of Vi may occur (greater than 900 microM).     

Intermediate states of actomyosin adenosine triphosphatase.

The early kinetic steps of actomyosin subfragment 1 (acto-S1) adenosine triphosphatase have been investigated by simultaneous monitoring of fluorescence and light scattering and also by observation of the time course of the production of phosphate. The results show that fluorescence enhancement occurs after the dissociation of actomyosin and that the rate of enhancement is similar to the maximum rate of enhancement for S1 alone, under similar conditions of pH and temperature. The maximum rate of the phosphate burst for acto S1 is also approximately the same as that for S1 alone. The maximum rates for fluorescence enhancement or phosphate formation are reached at much lower adenosine triphosphate concentrations for acto-S1 than for S1. An extension of the actomyosin scheme is presented which accounts for these results.     

Modeling the establishment of PAR protein polarity in the one-cell C. elegans embryo

At the one-cell stage, the C. elegans embryo becomes polarized along the anterior-posterior axis. The PAR proteins form complementary anterior and posterior domains in a dynamic process driven by cytoskeletal rearrangement. Initially, the PAR proteins are uniformly distributed throughout the embryo. After a cue from fertilization, cortical actomyosin contracts toward the anterior pole. PAR-3/PAR-6/PKC-3 (the anterior PAR proteins) become restricted to the anterior cortex. PAR-1 and PAR-2 (the posterior PAR proteins) become enriched in the posterior cortical region. We present a mathematical model of this polarity establishment process, in which we take a novel approach to combine reaction-diffusion dynamics of the PAR proteins coupled to a simple model of actomyosin contraction. We show that known interactions between the PAR proteins are sufficient to explain many aspects of the observed cortical PAR dynamics in both wild-type and mutant embryos. However, cytoplasmic PAR protein polarity, which is vital for generating daughter cells with distinct molecular components, cannot be properly explained within such a framework. We therefore consider additional mechanisms that can reproduce the proper cytoplasmic polarity. In particular we predict that cytoskeletal asymmetry in the cytoplasm, in addition to the cortical actomyosin asymmetry, is a critical determinant of PAR protein localization.     

Preparation and properties of vertebrate smooth-muscle myofibrils and actomyosin.

A new technique for obtaining a myofibril-like preparation from vertebrate smooth muscle has been developed. An actomyosin can be readily extracted from these myofibrils at low ionic strength and in yields 20 times as high as previously reported. The protein composition of all preparations has been monitored using dodecylsulfate-gel electrophoresis. By this method smooth muscle actomyosin showed primarily only the major proteins, myosin, actin and tropomyosin, while the myofibrils contained, additionally, three new proteins not previously described with polypeptide chain weights of 60000, 110000 and 130000. The ATPase activities of both the myofibrils and actomyosin preparations are considerably higher than previously described for vertebrate smooth muscle. They are sensitive to micromolar Ca2+ ion concentrations to the same degree as comparable skeletal and cardiac muscle preparations, even though troponin-like proteins could not be identified in these smooth muscle preparations. From the latter observation and the presence of Ca2+-sensitivity in tropomyosin-free actomyosin it is suggested that this calcium sensitivity is, as in some invertebrate muscles, a property of the myosin molecule.     

Cross-bridge model of muscle contraction. Quantitative analysis

We recently presented, in a qualitative manner, a cross-bridge model of muscle contraction which was based on a biochemical kinetic cycle for the actomyosin ATPase activity. This cross-bridge model consisted of two cross-bridge states detached from actin and two cross-bridge states attached to actin. In the present paper, we attempt to fit this model quantitatively to both biochemical and physiological data. We find that the resulting complete cross-bridge model is able to account reasonably well for both the isometric transient data observed when a muscle is subjected to a sudden change in length and for the relationship between the velocity of muscle contraction in vivo and the actomyosin ATPase activity in vitro. This model also illustrates the interrelationship between biochemical and physiological data necessary for the development of a complete cross-bridge model of muscle contraction.     

Modeling myosin-dependent rearrangement and force generation in an actomyosin network

Actomyosin contractility is a major force-generating mechanism that drives rearrangement of actomyosin networks; it is fundamental to cellular functions such as cellular reshaping and movement. Thus, to clarify the mechanochemical foundation of the emergence of cellular functions, understanding the relationship between actomyosin contractility and rearrangement of actomyosin networks is crucial. For this purpose, in this study, we present a new particulate-based model for simulating the motions of actin, non-muscle myosin II, and α‐actinin. To confirm the model's validity, we successfully simulated sliding and bending motions of actomyosin filaments, which are observed as fundamental behaviors in dynamic rearrangement of actomyosin networks in migrating keratocytes. Next, we simulated the dynamic rearrangement of actomyosin networks. Our simulation results indicate that an increase in the density fraction of myosin induces a higher-order structural transition of actomyosin filaments from networks to bundles, in addition to increasing the force generated by actomyosin filaments in the network. We compare our simulation results with experimental results and confirm that actomyosin bundles bridging focal adhesions and the characteristics of myosin-dependent rearrangement of actomyosin networks agree qualitatively with those observed experimentally.     

Actomyosin interactions with insulin-storage granules in vitro.

Interactions between actomyosin and insulin storage granules isolated from rat islets of Langerhans have been examined in a simple system in vitro, which allows comparison of the sedimentation of the granules in the presence of absence of actomyosin in various conditions. Actomyosin altered granule-sedimentation rates in a manner consistent with the binding of the granules of actomyosin filaments. This interaction was enhanced by addition of ATP (1.5 mM) but unaltered by addition of CaCl2, by calmodulin or by calmodulin in the presence of 10 microM-CaCl2. Addition of EGTA (0.1 mM), cyclic AMP (10 microM) of cytochalasin B (10 microgram/ml) were also without effects in these conditions. Pre-incubation of granules with phospholipase c did not affect granule-actomyosin interaction. Ultrastructural studies showed close contacts between the membranes of the granules and actomyosin filaments. The results indicate the possibility that actomyosin might provide the motile force for granule translocation during the insulin secretory process.     

Dinitrophenylation of rabbit skeletal actomyosin.

Dinitrophenylated reconstituted or natural actomyosin effected changes in the Ca2+ sensitivity which were dependent upon the ionic strength of the reaction medium. Dinitrophenylation of reconstituted actomyosin in 0.6 M KCl led to the incorporation of 2-6 mol of the reagent per 5-10(5) g of protein and it possessed considerable Ca2+ sensitivity. Dinitrophenylated natural actomyosin under the same conditions lost most of its Ca2+ sensitivity when 1.3-5.4 mol of the dinitrophenyl group were bound. The myosin from these modified actomyosins did not lose Ca2+ sensitivity and the myosin was labeled only with 0.4-1.7 mol of the dinitrophenyl group. Dinitrophenylation of both kinds of actomyosin in 0.06 M KCl abolished the Ca2+ sensitivity; the myosin from the modified actomyosins also lost Ca2+ sensitivity. Myosin alone was more susceptible to a loss of Ca2+ sensitivity than myosin in actomyosin. Actin protected the ability of myosin to sense Ca2+ regulated actin in modified actomyosin at 0.6 M KCl but not at 0.06 M KCl. Actomyosin dinitrophenylated in the presence of ATP lost Ca2+ sensitivity. However, the myosin from this actomyosin possessed Ca2+ sensitivity. Thiolysis of the dinitrophenylated actomyosin by 2-mercaptoethanol at low ionic strength did not restore the Ca2+ sensitivity of this actomyosin or its myosin although there was a significant loss of the dinitrophenyl group.     

Nuclear magnetic resonance investigation of the state of water in protein solution.

The line width of the NMR signal of water protons in solutions of native actomyosin and actomyosin denatured by heat, acetone or urea was measured over the temperature range from -10 degrees to below the freezing point. The line widths of the water band which increased exponentially with decreasing temperature were compared with each other and also with those of the corresponding control solution without actomyosin. The line broadening observed for native actomyosin solution on lowering the temperature was significantly smaller than that for heat-denatured actomyosin solution. This difference implies that this signal is sensitive to conformational perturbations of the protein. In addition, the temperature dependence of the line width for heat-, acetone-, or urea-denatured actomyosin solution was similar to that for the corresponding control solution. These phenomena can be interpreted in terms of the state of water associated with the hydrophobic and hydrophilic residues. Similar NMR studies of actomyosin solution containing dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) showed that DMSO and DMF prevent the formation of ice crystals until about -70 degrees, suggesting that the cryoprotective effects of DMSO and DMF are due to the change in the state of water described above. These differences in temperature dependence between the sample and control solutions are well-correlated with the viscosity of the solution. This correlation is useful for elucidation of the mechanism of the protein denaturation.     

The synergetic enzyme theory of muscular contraction: II Relation between Hill's equations and functions of two-headed myosin.

Based on the assumption of nonidentical two heads of myosin it is pointed out that a strong motive force is generated in actomyosin pair only when ATP-decomposition occurs co-operatively at the both heads and that the tension-independent part of shortening heat is liberated when an ATP molecule is decomposed only at the burst head. These two actions of actomyosin pair are related to the two states of force-generator in Huxley-Simmons' model. Elementary cycles at different positions in a sarcomere are interacted each other through feedback loop via sliding motion of muscular filaments. Due to this synergetic interaction the rate constant for the rate-determining step of elementary cycle has a dependence on velocity v of shortening such as k = k ° + κv. From these functions and properties of actomyosin system in vivo, the following properties of muscle are explained consistently in a quantitative manner: (1) Hill's equation on the relationship between tension and velocity of shortening, (2) damped oscillations in tension and in muscular length around steady state, (3) Hill's energy equation improved in 1964, (4) the chemical equivalence of shortening heat, (5) the influence of tension on the incorporation of radio-active phosphate into ATP and (6) the asymmetric activation by actomyosin system only for the forward reaction, the decomposition of ATP.     

Direct inhibition of the actomyosin motility by local anesthetics in vitro.

Using a recently developed in vitro motility assay, we have demonstrated that local anesthetics directly inhibit myosin-based movement of single actin filaments in a reversible dose-dependent manner. This is the first reported account of the actions of local anesthetics on purified proteins at the molecular level. In this study, two tertiary amine local anesthetics, lidocaine and tetracaine, were used. The inhibitory action of the local anesthetics on actomyosin sliding movement was pH dependent; the anesthetics were more potent at higher pH values, and this reaction was accompanied by an increased proportion of the uncharged form of the anesthetics. QX-314, a permanently charged derivative of lidocaine, had no effect on actomyosin sliding movement. These results indicate that the uncharged form of local anesthetics is predominantly responsible for the inhibition of actomyosin sliding movement. The local anesthetics inhibited sliding movement but hardly interfered with the binding of actin filaments to myosin on the surface or with actomyosin ATPase activity at low ionic strength. To characterize the actomyosin interaction in the presence of anesthetics, we measured the binding and breaking force of the actomyosin complex. The binding of actin filaments to myosin on the surface was not affected by lidocaine at low ionic strength. The breaking force, measured using optical tweezers, was approximately 1.5 pN per micron of an actin filament, which was much smaller than in rigor and isometric force. The binding and breaking force greatly decreased with increasing ionic strength, indicating that the remaining interaction is ionic in nature. The result suggests that the binding and ATPase of actomyosin are governed predominantly by ionic interaction, which is hardly affected by anesthetics; whereas the force generation requires hydrophobic interaction, which plays a major part of the strong binding and is blocked by anesthetics, in addition to the ionic interaction.     

Actomyosin contraction during cellularization is regulated in part by Src64 control of Actin 5C protein levels

Src64 is required for actomyosin contraction during cellularization of the Drosophila embryonic blastoderm. The mechanism of actomyosin ring constriction is poorly understood even though a number of cytoskeletal regulators have been implicated in the assembly, organization, and contraction of these microfilament rings. How these cytoskeletal processes are regulated during development is even less well understood. To investigate the role of Src64 as an upstream regulator of actomyosin contraction, we conducted a proteomics screen to identify proteins whose expression levels are controlled by src64. Global levels of actin are reduced in src64 mutant embryos. Furthermore, we show that reduction of the actin isoform Actin 5C causes defects in actomyosin contraction during cellularization similar to those caused by src64 mutation, indicating that a relatively high level of Actin 5C is required for normal actomyosin contraction and furrow canal structure. However, reduction of Actin 5C levels only slows down actomyosin ring constriction rather than preventing it, suggesting that src64 acts not only to modulate actin levels, but also to regulate the actomyosin cytoskeleton by other means.     

Myosin-linked calcium regulation in vertebrate smooth muscle.

By the use of a new procedure, actomyosin may be extracted in high yield and purity from fowl gizzard which exhibits a calcium-dependent actin-activated ATPase activity comparable to that of the parent myofibril-like preparation. Studies of this vertebrate smooth muscle actomyosin show that the regulation of the actin-myosin interaction is effected, as in molluscan muscles, by the myosin molecule itself and not by an actin-linked regulatory system, as found in vertebrate skeletal muscle.Thus, calcium-sensitive smooth muscle actomyosin is composed of only myosin, actin and tropomyosin, any troponin-like components being absent. Myosin is the only component that binds significant amounts of calcium and shows a calcium-dependent actin-activated ATPase activity in the presence of F-actin from either gizzard or rabbit skeletal muscle.The cross-reaction of gizzard thin filaments with skeletal muscle myosin produces an actomyosin whose actin-activated ATPase is calcium-insensitive, showing that smooth muscle thin filaments do not serve a regulatory function.The effect of Mg²⁺ and pH, and evidence for the involvement of one of the myosin light chains in calcium regulation are described and discussed.     

An Equatorial Contractile Mechanism Drives Cell Elongation but not Cell Division

Cell shape changes and proliferation are two fundamental strategies for morphogenesis in animal development. During embryogenesis of the simple chordate Ciona intestinalis, elongation of individual notochord cells constitutes a crucial stage of notochord growth, which contributes to the establishment of the larval body plan. The mechanism of cell elongation is elusive. Here we show that although notochord cells do not divide, they use a cytokinesis-like actomyosin mechanism to drive cell elongation. The actomyosin network forming at the equator of each notochord cell includes phosphorylated myosin regulatory light chain, α-actinin, cofilin, tropomyosin, and talin. We demonstrate that cofilin and α-actinin are two crucial components for cell elongation. Cortical flow contributes to the assembly of the actomyosin ring. Similar to cytokinetic cells, membrane blebs that cause local contractions form at the basal cortex next to the equator and participate in force generation. We present a model in which the cooperation of equatorial actomyosin ring-based constriction and bleb-associated contractions at the basal cortex promotes cell elongation. Our results demonstrate that a cytokinesis-like contractile mechanism is co-opted in a completely different developmental scenario to achieve cell shape change instead of cell division. We discuss the occurrences of actomyosin rings aside from cell division, suggesting that circumferential contraction is an evolutionally conserved mechanism to drive cell or tissue elongation.     

The phosphorylation-dephosphorylation process as a myosin-linked regulation of superprecipitation of fast skeletal muscle actomyosin

The dependence of the onset and course of turbidity changes ( superprecipitation) induced by ATP were studied in a natural actomyosin suspension with the dephosphorylated and phosphorylated forms of light chains (LC2) of myosin. It was found that the onset and time course of the changes in turbidity of the natural actomyosin suspension are strongly dependent on the (phosphorylated and dephosphorylated) form of these chains of myosin. The ATPase activity of actomyosin with phosphorylated LC2 was lower and the half-time for achieving maximal turbidity of actomyosin suspension after addition of ATP was higher than that of actomyosin with dephosphorylated LC2. Natural actomyosin preparations contain endogenous light-chain kinase and phosphatase. The changes of turbidity induced by ATP in the natural actomyosin suspension are greatly diminished in the presence of phosphate. Thiophosphorylation of LC2 of myosin leads to a decrease of the extent of superprecipitation of natural actomyosin. The release of [32P]phosphate from actomyosin containing [32P]ATP-phosphorylated LC2 of myosin increases with increased turbidity of actomyosin suspension. The change of the form LC2 as a kind of additional myosin-linked regulation of superprecipitation is discussed.     

Single molecule analysis of the actomyosin motor

Progress in imaging techniques and nano-manipulation of single molecules has been remarkable. These techniques have allowed the accurate determination of myosin-head-induced displacements and of how the mechanical cycles of the actomyosin motor are coupled to ATP hydrolysis. This has been achieved by measuring mechanical and chemical events of actomyosin directly at the single molecule level. Recent studies have made detailed measurements of myosin step size and mechanochemical coupling. The results of these studies suggest a new model for the mechanism of motion underlying actomyosin motors, which differs from the currently accepted lever-arm swinging model.     

Reconstruction of Active Regular Motion in Amoeba Extract: Dynamic Cooperation between Sol and Gel States

Amoeboid locomotion is one of the typical modes of biological cell migration. Cytoplasmic sol–gel conversion of an actomyosin system is thought to play an important role in locomotion. However, the mechanisms underlying sol–gel conversion, including trigger, signal, and regulating factors, remain unclear. We developed a novel model system in which an actomyosin fraction moves like an amoeba in a cytoplasmic extract. Rheological study of this model system revealed that the actomyosin fraction exhibits shear banding: the sol–gel state of actomyosin can be regulated by shear rate or mechanical force. Furthermore, study of the living cell indicated that the shear-banding property also causes sol–gel conversion with the same order of magnitude as that of shear rate. Our results suggest that the inherent sol–gel transition property plays an essential role in the self-regulation of autonomous translational motion in amoeba.     

Actin-based centripetal flow: phosphatase inhibition by calyculin-A alters flow pattern, actin organization, and actomyosin distribution

Previous studies have suggested that the actin-based centripetal flow process in sea urchin coelomocytes is the result of a two-part mechanism, actin polymerization at the cell edge coupled with actomyosin contraction at the cell center. In the present study, we have extended the testing of this two-part model by attempting to stimulate actomyosin contraction via treatment of coelomocytes with the phosphatase inhibitor Calyculin A (CalyA). The effects of this drug were studied using digitally-enhanced video microscopy of living cells combined with immunofluorescent localization and scanning electron microscopy. Under the influence of CalyA, the coelomocyte actin cytoskeleton undergoes a radical reorganization from a dense network to one displaying an array of tangential arcs and radial rivulets in which actin and the Arp2/3 complex concentrate. In addition, the structure and dynamics of the cell center are transformed due to the accumulation of actin and membrane in this region and the constriction of the central actomyosin ring. Physiological evidence of an increase in actomyosin-based contractility following CalyA treatment was demonstrated in experiments in which cells generated tears in their cell centers in response to the drug. Western blotting and immunofluorescent localization with antibodies against the phosphorylated form of the myosin regulatory light chain (MRLC) suggested that the demonstrated constriction of actomyosin distribution was the result of CalyA-induced phosphorylation of MRLC. Overall, the results suggest that there is significant cross talk between the two underlying mechanisms of actin polymerization and actomyosin contraction, and indicate that changes in actomyosin tension may be translated into alterations in the structural organization of the actin cytoskeleton.     

On the active streaming experiment of actomyosin solutions in glass microcapillaries.

The active-streaming experiments of Oplatka et al. (Oplatka, A. and Tirosh, R. (1973) Biochim. Biophys. Acta 305, 684-688 and Oplatka, A. Gadasi, H., Tirosh, R. Lamed, Y., Muhlrad, A. and Liron, N. (1974) J. Mechanochem. Cell Motil. 2, 295-306) with actomyosin solution in a glass microcapillary is reexamined under various conditions with several kinds of reference material. It is found that vigorous streaming took place in the actomyosin solution as reported by Oplatka et al. However, streaming which is indistinguishable from that observed in the actomyosin solution in the presence of actomyosin ATPase activity also occurred, even when the ATPase activity was blocked. The streaming also cannot be confirmed as being active when using acto-heavy meromyosin solution. There is a possibility that the streaming experiment provides interesting information on the microscopic state of solutions which is not directly related to the chemo-mechanical conversion.