Velocity-dependent actomyosin ATPase cycle revealed by in vitro motility assay with kinetic analysis.

The actomyosin interaction plays a key role in a number of cellular functions. Single-molecule measurement techniques have been developed to study the mechanism of the actomyosin contractile system. However, the behavior of isolated single molecules does not always reflect that of molecules in a complex system such as a muscle fiber. Here, we developed a simple method for studying the kinetic parameters of the actomyosin interaction using small numbers of molecules. This approach does not require the specialized equipment needed for single-molecule measurements, and permits us to observe behavior that is more similar to that of a complex system. Using an in vitro motility assay, we examined the duration of continuous sliding of actin filaments on a sparsely distributed heavy meromyosin-coated surface. To estimate the association rate constant of the actomyosin motile system, we compared the distribution of experimentally obtained duration times with a computationally simulated distribution. We found that the association rate constant depends on the sliding velocity of the actin filaments. This technique may be used to reveal new aspects of the kinetics of various motor proteins in complex systems.

Effects of myosin and heavy meromyosin on actin-related gelation of HeLa cell extracts.

The gelation induced by warming (to 25 degrees C) the 100,000 g supernatant fraction (extract) of HeLa cells lysed in a buffer containing sucrose, ATP, DTE, EGTA, imidazole, and Triton X-100 was studied in the presence of myosin and heavy meromyosin (HMM). Myosin mixed with extract induces shrinkage of the gel, but jelled extract or myosin alone does not shrink. In the concentration range, 0.14-1.04 mg/ml of myosin, the degree of shrinkage is roughly proportional to the concentration of myosin. Supplementa MgCl2 also promotes shrinkage. HMM (0.4-0.8 mg/ml) can inhibit gel formation by extract in tubes or floated on a sucrose cushion. Gel electrophoresis of gels shrunken by added myosin or electrophoresis of the proteins which can be sedimented from extract after incubation in the presence of HMM indicate that both myosin and HMM interfere with the changes in sedimentability of the high molecular weight protein (HMWP) thought to participate (together with actin) in gel formation in HeLa cell extracts (R. R. Weihing, 1976. J. Cell Biol. 71:303-307). These results, together with previous results showing that actin is present and that HMWP is enriched in the plasma membrane fraction of HeLa cells (R. R. Weihing, 1976. Cold Spring Harbor Conf. Cell Proliferation. 3:671-684), point to the possibility of dynamic changes in the interactions of HMWP or myosin with actin in processes of movement occurring at the cell surface.

A permeabilized cell model for studying cytokinesis using mammalian tissue culture cells.

PtK1 cells lysed late in cell division in a medium containing the nonionic detergent Brij 58 and polyethylene glycol with continue to undergo cleavage after lysis. Maintenance of cleavage after lysis is dependent on the composition of the lysis medium; the pH must be around neutrality, MgATP must be present, and the free Ca++ concentration should be 1 microM for optimal constriction to occur. Cleavage can be stopped and reinitiated by raising and lowering the Ca++ levels in the lysis medium. Cleavage in the permeabilized cell is blocked by addition of phalloidin, cytochalasin B, and N-ethylmaleimide-modified myosin subfragment-1 to the lysis medium. This represents the first cell model system for studying cleavage since the pioneering studies of Hoffman-Berling in 1954.

Actin in erythrocyte ghosts and its association with spectrin. Evidence for a nonfilamentous form of these two molecules in situ.

Actin was isolated from erythrocyte ghosts. It is identical to muscle actin in its molecular weight, net charge, ability to polymerize into filaments with the double helical morphology, and its decoration with heavy meromyosin (HMM). when erythrocyte ghosts are incubated in 0.1 mM EDTA, actin and spectrin are solubilized. Spectrin has a larger molecular weight than muscle myosin. When salt is added to the EDTA extract, a branching filamentous polymer is formed. However, when muscle actin and the EDTA extract are mixed together in the presence of salt, the viscosity achieved is less than the viscosity of the solution if spectrin is omitted. Thus, spectrin seems to inhibit the polymerization of actin. If the actin is already polymerized, the addition of spectrin increases the viscosity of the solution, presumably by cross-linking the actin filaments. The addition of HMM of trypsin to erythrocyte ghosts results in filament formation in situ. These agents apparently act by detaching erythrocyte actin from spectrin, thereby allowing the polmerization of one or both proteins to occur. Since filaments are not present in untreated erythrocyte ghosts, we conclude that erythrocyte actin and spectrin associate to form an anastomosing network beneath the erythrocyte membrane. This network presumably functions in restricting the lateral movement of membrane-penetrating particles.

Electron microscope and experimental investigations of the neurofilamentous network in Deiters' neurons. Relationship with the cell surface and nuclear pores.

The assembly of filamentous elements and their relations to the plasma membrane and to the nuclear pores have been studied in Deiters' neurons of rabbit brain. Electron microscopy of thin sections and of ectoplasm spread preparations have been integrated with physicochemical experiments and differential interference microscopy of freshly isolated cells. A neurofilamentous network extends as a continuous, three-dimensional, semilattice structure throughout the ectoplasm, the "plasma roads," and the perinuclear zone of the perikaryon. This space network consists of approximately 90-A wide neurofilaments arranged in fascicles which are interconnected by an exchange of neurofilaments. The neurofilaments consist of intercoiled approximately 20-A wide unit-filaments and are associated through cross-associating filaments with other neurofilaments of the fascicle and with microfilaments. The approximately 20-50-A wide microfilaments display intimate associations with the plasma membrane and with the nuclear pores. Electron microscopy of thin sections from glycerinated and heavy meromyosin-treated Deiters' neurons shows that actin-like filaments are present in the pre- and postsynaptic regions of synapses terminating on these neurons. It is proposed that the neurofilamentous space network serves a transducing function by linking plasma membrane activities with the genetic machinery of the neuron.

Polarized bundles of actin filaments within microvilli of fertilized sea urchin eggs.

We report on the internal ultrastructure of long, finger-like microvilli which cover the surface of the fertilized sea urchin egg. Eggs were attached to polylysine-coated surfaces; their upper portions were sheared away with a stream of buffer which left behind only their plasma membranes and adjacent cytoplasmic structures. Scanning electron microscopy (EM) of such fragments revealed intact thin protoplasmic projections radiating away from the body of the cortex. By transmission EM of cortices similarly prepared on grids, small bundles of microfilaments appear as cores within the thin cytoplasmic projections. These microfilaments are shown to be composed of actin by their ability to interact with muscle heavy meromyosin (HMM). HMM-decorated microfilaments possess repeating arrowheads which uniformly point toward the cell interior. Actin bundles in the microvilli of sea urchin eggs may mediate microvillus support and elongation.

Effects of exogenous proteins on cytoplasmic streaming in perfused Chara cells.

Cytoplasmic streaming in characean algae is thought to be generated by interaction between subcortical actin bundles and endoplasmic myosin. Most of the existing evidence supporting this hypothesis is of a structural rather than functional nature. To obtain evidence bearing on the possible function of actin and myosin in streaming, we used perfusion techniques to introduce a number of contractile and related proteins into the cytoplasm of streaming Chara cells. Exogenous actin added at concentrations as low as 0.1 mg/ml is a potent inhibitor of streaming. Deoxyribonuclease I (DNase I), an inhibitor of amoeboid movement and fast axonal transport, does not inhibit streaming in Chara. Fluorescein-DNase I stains stress cables and microfilaments in mammalian cells but does not bind to Chara actin bundles, thus suggesting that the lack of effect on streaming is due to a surprising lack of DNase I affinity for Chara actin bundles. Heavy meromyosin (HMM) does not inhibit streaming, but fluorescein-HMM (FL-HMM), having a partially disabled EDTA ATPase, does. Quantitative fluorescence micrography provides evidence that inhibition of streaming by FL-HMM may be due to a tendency for FL-HMM to remain bound to Chara actin bundles even in the presence of MgATP. Perfusion with various control proteins, including tubulin, ovalbumin, bovine serum albumin, and irrelevant antibodies, does not inhibit streaming. These results support the hypothesis that actin and myosin function to generate cytoplasmic streaming in Chara.

N-ethylmaleimide-modified heavy meromyosin. A probe for actomyosin interactions.

Treatment of rabbit skeletal muscle heavy meromyosin (HMM) with the sulfhydryl reagent N-ethylmaleimide (NEM) produces a species of HMM which remains tightly bound to actin in the presence of MgATP. NEM-HMM forms characteristic "arrowhead" complexes with actin which persist despite rinses with MgATP. NEM-HMM inhibits the actin activation of native HMM-ATPase activity, the superprecipitation of actomyosin, the contraction of glycerinated muscle myofibrils, and the contraction of cytoplasmic strands of the soil amoeba Chaos carolinensis. However, NEM-HMM does not interfere with in vitro microtubule polymerization or beating of demembranated cilia.

Effect of microinjected N-ethylmaleimide-modified heavy meromyosin on cell division in amphibian eggs.

N-Ethylmaleimide-modified heavy meromyosin (NEM-HMM) microinjected into amphibian eggs inhibits cytokinesis and the cortical contractions associated with wound closure. Injection of NEM-HMM into two-cell Rana pipiens embryos produces a zone of cleavage inhibition around the point of injection. Early furrows followed by time-lapse microcinematography are seen to slow and stop as they enter the NEM-HMM-injected zone. Arrested furrows slowly regress, leaving a large region of cytoplasm uncleaved. Few nuclei are found in these regions of cleavage inhibition. Wound closure is often inhibited by NEM-HMM, especially when this inhibitor is injected just beneath the egg cortex. We observe that the surface of an unfertilized Rana egg is covered with microvilli that disappear during the course of development. The surfaces of NEM-HMM-inhibited zones remain covered with microvilli and resemble the unfertilized egg surface.

The polymerization of actin. III. Aggregates of nonfilamentous actin and its associated proteins: a storage form of actin.

When echinoderm sperm are treated with the detergent Triton X-100 at pH 6.4 in 10 mM phosphate buffer, the membranes are solubilized, but the actin which is located in the periacrosomal region remains as a phase-dense cup. These cups can be isolated free from the flagella and chromatin and can be solubilized by increasing the pH to 8.0 and by changing the ionic strength and type of buffer used. Since the actin does not exist in the "F" state in unreacted sperm, and since the actin remains as a unit that does not diffuse away, it must be present in the mature sperm in a bound or storage state. The actin is, in fact, associated with a pair of proteins whose mol wt are 250,000 and 230,000. When the isolated cups are digested with trypsin, these high molecular weight proteins are digested, thereby liberating the actin. The actin will polymerize if heavy meromyosin or subfragment 1 is added to a preparation of isolated cups. Evidence is presented that this pair of high molecular weight proteins is similar in molecular weight and properties to erythrocyte spectrin. Attempts at transforming the storage form of actin in the cup into filaments were only moderately successful. The best conditions for filament formation involve incubating the cup in ATP and divalent salts. Careful examination of these cups reveals that the actin polymerized preferentially on either end of oriented filaments that already exist in the cup, indicating that self-nucleation is inefficacious. I conclude that the actin can exist in the storage form by its association with spectrin-like molecules and that the actin in this state polymerizes preferentially onto existing filaments.

Myosin drives retrograde F-actin flow in neuronal growth cones.

Actin filaments assembled at the leading edge of neuronal growth cones are centripetally transported via retrograde F-actin flow, a process fundamental to growth cone guidance and other forms of directed cell motility. Here we investigated the role of myosins in retrograde flow, using two distinct modes of myosin inhibition: microinjection of NEM inactivated myosin S1 fragments, or treatment with 2,3-butanedione-2-monoxime, and inhibitor of myosin ATPase. Both treatments resulted in dose-dependent attenuation of retrograde F-actin flow and growth of filopodia. Growth was cytochalasin sensitive and directly proportional to the degree of myosin inhibition, suggesting that retrograde flow results from superimposition of two independent processes: actin assembly and myosin-based filament retraction. These results provide the first direct evidence for myosin involvement in neuronal growth cone function.

Activation dependence of stretch activation in mouse skinned myocardium: implications for ventricular function.

Recent evidence suggests that ventricular ejection is partly powered by a delayed development of force, i.e., stretch activation, in regions of the ventricular wall due to stretch resulting from torsional twist of the ventricle around the apex-to-base axis. Given the potential importance of stretch activation in cardiac function, we characterized the stretch activation response and its Ca2+ dependence in murine skinned myocardium at 22 degrees C in solutions of varying Ca2+ concentrations. Stretch activation was induced by suddenly imposing a stretch of 0.5-2.5% of initial length to the isometrically contracting muscle and then holding the muscle at the new length. The force response to stretch was multiphasic: force initially increased in proportion to the amount of stretch, reached a peak, and then declined to a minimum before redeveloping to a new steady level. This last phase of the response is the delayed force characteristic of myocardial stretch activation and is presumably due to increased attachment of cross-bridges as a consequence of stretch. The amplitude and rate of stretch activation varied with Ca2+ concentration and more specifically with the level of isometric force prior to the stretch. Since myocardial force is regulated both by Ca2+ binding to troponin-C and cross-bridge binding to thin filaments, we explored the role of cross-bridge binding in the stretch activation response using NEM-S1, a strong-binding, non-force-generating derivative of myosin subfragment 1. NEM-S1 treatment at submaximal Ca2+-activated isometric forces significantly accelerated the rate of the stretch activation response and reduced its amplitude. These data show that the rate and amplitude of myocardial stretch activation vary with the level of activation and that stretch activation involves cooperative binding of cross-bridges to the thin filament. Such a mechanism would contribute to increased systolic ejection in response to increased delivery of activator Ca2+ during excitation-contraction coupling.

Strongly binding myosin crossbridges regulate loaded shortening and power output in cardiac myocytes.

This study investigated the possible roles of strongly binding myosin crossbridges in determining loaded shortening and power output in cardiac myocytes. Single skinned cardiac myocytes were attached between a force transducer and position motor, and shortening velocities were measured over a range of loads during varying levels of Ca(2+) activation. Lowering the [Ca(2+)] slowed shortening velocities, decreased relative power output, and increased the curvature of length traces. We tested the hypothesis that Ca(2+) activation dependence of loaded shortening is determined primarily by strongly binding crossbridges or by [Ca(2+)] per se, which was done by measuring loaded shortening before and after addition of N-ethylmaleimide-conjugated myosin subfragment-1 (NEM-S1), a strongly binding myosin analogue that cooperatively enhances thin filament activation. At fixed [Ca(2+)], NEM-S1 reduced the curvature of length traces and sped loaded shortening velocities. Even when [Ca(2+)] was adjusted so that force was equal with and without NEM-S1, myocyte shortening was faster and exhibited less curvature with NEM-S1. In the presence of NEM-S1, peak relative power output was also significantly greater during activations either at the same [Ca(2+)] or when [Ca(2+)] was adjusted to achieve the same force. Consequently, NEM-S1 eliminated any Ca(2+) dependence of relative power output that is normally observed in cardiac myocytes. These results indicate that strongly binding crossbridges play a significant role in determining loaded shortening and power output and suggest that previously observed Ca(2+) dependence of power output is mediated by alterations in numbers of crossbridges bound to the thin filament.

Conformational changes of F-actin in myosin-free ghost single fibre induced by either phosphorylated or dephosphorylated heavy meromyosin.

The changes in F-actin conformation of myosin-free single ghost fibre induced by binding of phosphorylated or dephosphorylated heavy meromyosin have been studied by measuring polarized fluorescence of F-actin intrinsic tryptophan and of phalloidin-rhodamine bound to F-actin. The changes of polarization of both fluorescences were found to be dependent on low or high Ca2+ concentration and on the phosphorylated or dephosphorylated form of heavy meromyosin. Computer analysis of polarized fluorescence has shown that binding of phosphorylated heavy meromyosin with divalent ion binding sites saturated with Mg2 (in the presence of 1 mM MgCl2 and 1 mM EGTA) and dephosphorylated heavy meromyosin with divalent ion binding sites saturated with Ca2+ (in the presence of 1 mM MgCl2 and 0.1 mM Ca2+) decreases the angles of emission and absorption dipoles and the angle between the F-actin axis and the fibre axis, thus suggesting that F-actin in ghost fibre becomes more flexible. On the other hand, the above-mentioned angles increase when phosphorylated heavy meromyosin at high and dephosphorylated heavy meromyosin at low Ca2+ concentration were bound to thin filaments, thus showing the decrease of F-actin flexibility under these conditions.

The myosin molecule--charge response to nucleotide binding.

A decrease in the net fixed electric charge in the A-bands of cross-striated muscle was observed by Bartels and Elliott [2,10] when the muscle went from the rigor to the relaxed condition. The current work localises the source of the charge decrease by following the net charge on myosin (in the form of concentrated gels) and also myosin rod and light meromyosin gels when the gels are exposed to different concentrations of ATP. The work includes a study of muscle A-bands when the muscle is exposed to the same variations in ATP concentrations as the protein gels. The work shows that (i) Only 100-200 microns ATP is needed to initiate the charge decrease between the rigor and relaxed conditions; (ii) the effect of ATP is seen in the muscle A-band and the myosin and myosin rod gels, but not in LMM gels; (iii) pyrophosphate (PPi) shows a similar charge effect to ATP. ADP does not affect the charge on myosin gels, on the other hand. The results suggest that the charge decrease caused by ATP or PPi is due to ligand interaction with one or more sites on the myosin molecule. This interaction causes a disseminated effect in the protein, and a consequent loss in net negative charge either by a decrease in the absorption, of anions to Saroff sites on the protein, or, less probably, by an increase in the absorption of cations at those sites.

The effect of Ca2+ on the conformation of tropomyosin and actin in regulated actin filaments with or without bound myosin subfragment 1.

The effects of Ca2+ and myosin subfragment 1 on the conformation of tropomyosin and actin in regulated actin filaments in ghost fibers were investigated by means of the polarized fluorescence technique. Regulated thin filaments were reconstituted in skeletal muscle ghost fibers by incorporation into the fibers of either skeletal muscle troponin-tropomyosin or smooth-muscle caldesmon-calmodulin-tropomyosin complexes. Tropomyosin and actin were specifically labeled with fluorescent probes, 1,5-IAEDANS and phalloidin-rhodamine, respectively. Analysis of the fluorescence parameters indicated that the binding of Ca2+ to regulated actin filaments induces conformational changes in tropomyosin and actin that lead to the strengthening of the interaction between these two proteins and weakening of the binding of actin monomers in the filament. These changes become larger when regulated actin forms rigor links with myosin subfragment 1. No notable alterations in the position of tropomyosin relative to actin in the frontal plane of the fiber were detected either upon binding of Ca2+ or upon the additional binding of myosin subfragment 1 to regulated actin.

Cooperative mechanisms in the activation dependence of the rate of force development in rabbit skinned skeletal muscle fibers.

Regulation of contraction in skeletal muscle is a highly cooperative process involving Ca(2+) binding to troponin C (TnC) and strong binding of myosin cross-bridges to actin. To further investigate the role(s) of cooperation in activating the kinetics of cross-bridge cycling, we measured the Ca(2+) dependence of the rate constant of force redevelopment (k(tr)) in skinned single fibers in which cross-bridge and Ca(2+) binding were also perturbed. Ca(2+) sensitivity of tension, the steepness of the force-pCa relationship, and Ca(2+) dependence of k(tr) were measured in skinned fibers that were (1) treated with NEM-S1, a strong-binding, non-force-generating derivative of myosin subfragment 1, to promote cooperative strong binding of endogenous cross-bridges to actin; (2) subjected to partial extraction of TnC to disrupt the spread of activation along the thin filament; or (3) both, partial extraction of TnC and treatment with NEM-S1. The steepness of the force-pCa relationship was consistently reduced by treatment with NEM-S1, by partial extraction of TnC, or by a combination of TnC extraction and NEM-S1, indicating a decrease in the apparent cooperativity of activation. Partial extraction of TnC or NEM-S1 treatment accelerated the rate of force redevelopment at each submaximal force, but had no effect on kinetics of force development in maximally activated preparations. At low levels of Ca(2+), 3 microM NEM-S1 increased k(tr) to maximal values, and higher concentrations of NEM-S1 (6 or 10 microM) increased k(tr) to greater than maximal values. NEM-S1 also accelerated k(tr) at intermediate levels of activation, but to values that were submaximal. However, the combination of partial TnC extraction and 6 microM NEM-S1 increased k(tr) to virtually identical supramaximal values at all levels of activation, thus, completely eliminating the activation dependence of k(tr). These results show that k(tr) is not maximal in control fibers, even at saturating [Ca(2+)], and suggest that activation dependence of k(tr) is due to the combined activating effects of Ca(2+) binding to TnC and cross-bridge binding to actin.

An actin-modulating protein from Physarum polycephalum. II. Ca++-dependence and other properties.

Actin modulating (AM-) protein from Physarum binds as monomer and as a stable heterodimer with one actin molecule to actin and induces the formation of oligomeric actin complexes and short-chained filaments with no definite stoichiometry. While inhibiting the extent of actin polymerization by reducing the size of the aggregates formed, AM-protein increases the velocity of polymerization. The AM-protein monomer depolymerizes F-actin very rapidly by breaking the filaments into small pieces and oligomeric complexes. When a heterodimer of actin an AM-protein is reconstituted the polymerization inhibitor activity is identical to that of the monomer, but depolymerization of actin is almost completely abolished. The action of the monomeric AM-protein on actin is highly Ca++-dependent as it requires micromolar amounts of Ca++ for full activation. The inhibitory activity of both the natural and the reconstituted heterodimer has only little Ca++-sensitivity: A prolonged exposure to Ca++-chelating agents is necessary to obtain a partial inactivation of the heterodimers. The depolymerizing effect on actin of the AM-protein monomer is inhibited by tropomyosin and also by heavy meromyosin. Addition of phalloidin to the actin reduces only the velocity of depolymerization by AM-protein. In the presence of AM-protein the actomyosin ATPase or Acto-HMM ATPase is strongly inhibited. The supposed function of the AM-protein in Physarum is that of a powerful regulator of the polymer state of actin. Such a regulation system is necessary for the dynamic actin transformation processes during ectoplasm-endoplasm transitions and the assembly-disassembly of contractile and cytoskeletal structures. The Ca++-sensitivity of the AM-protein indicates that these processes are controlled by calcium.

Both isoforms of skeletal muscle subfragment 1 (S1A1 and S1A2) can induce actin polymerization with equal speed in the absence of ATP.

The ability of myosin subfragment 1 to induce actin polymerization was reinvestigated using the DNase I inhibition assay, by electron microscopy after negative staining, cosedimentation, measurement of viscosity and the fluorescence increase of pyrenyl-labeled actin. Using these techniques we demonstrate that rabbit skeletal muscle myosin subfragment 1 containing either the alkali light chain 1 (S1A1) or the alkali light chain 2 (S1A2) is able to promote actin polymerization even in the absence of divalent cations or salt. In the presence of ATP the rate of induction of actin polymerization by S1A2 is slower than by A1A1. In contrast, in the absence of free ATP, both subfragment 1 variants exhibit equal ability to induce actin polymerization. Evidence is given that the slower rate of induction of actin polymerization by S1A2 in the presence of free ATP is due to a slower rate of ATP-hydrolysis by S1A2 and thus to a slower rate of ATP depletion. We therefore assume that the formation of rigor type complexes involving the subfragment 1 heavy chain is necessary for the induction of actin polymerization. The ability of subfragment 1 to induce actin polymerization is retarded by a synthetic heavy chain mimetic peptide which inhibits its actin binding or after proteolytic cleavage of the subfragment 1 heavy chain by trypsin.

Motility of the oocyte of Helisoma (Mollusca).

Mature oocytes isolated from the ovotestis of Helisoma showed amoeboid movement when treated in vitro with a homogenate of the cerebral ganglia (containing dorsal bodies). This movement ceased when either cytochalasin B or colchicine was added to the preparation. Actin filaments were only visualized in in vitro and in vivo preparation of mature oocytes. We suggest tht in order to ovulate, the oocyte uses its own contractile system to become amoeboid and is thereby separated from the envelope of the follicle cells. We also suggest that the ovulation factor(s) is present in the dorsal bodies and that this factor could be responsible for the induction of microfilaments.