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. KEY WORDS actomyosin N-ethylmaleimide -cell motility -heavy meromyosinMicrofilament systems are ubiquitous components of eukaryotic cells, serving as cytoskeletal elements and functioning in cellular processes such as cell movements, cytoplasmic streaming, and morphogenetic shape changes (4, 16, 25). The major molecular components of these systems are actin, which is usually organized in 5-to 7-nm filaments; myosin, an ATPase which can generate shear forces by interacting with actin; and control proteins capable of regulating the functioning of actin and myosin . The interactions of these components are best understood in the highly ordered structures found in muscle (11) . Actomyosin systems of nonmuscle cells contain similar protein components, but the organization is less ordered, less stable, and varies greatly among cell types (4, 25). Moreover, it is unclear how the arrangement of these proteins contributes to cell motility .Nonmuscle cells also contain other elements potentially capable of serving cytoskeletal and contractile functions. Microtubules have been J. CELT . BIOLOGY

After lysis in a Brij 58-polyethylene glycol medium, PtK1 cells are permeable to small molecules, such as erythrosin B, and to proteins, such as rhodamine-labeled FAB, myosin subfragment-1, and tubulin. Holes are present in the plasma membrane, and the mitochondria are swollen and distorted, but other membrane-bounded organelles of the lysed cell model are not noticeably altered . After lysis, the mitotic apparatus is functional ; chromosomes move poleward and the spindle elongates . Cells lysed while in cytokinesis will continue to divide for several minutes. Addition of crude tubulin extracts, MAP-free tubulin, or taxol to the lysis medium retards anaphase chromosome movements but does not affect cleavage . On the other hand, N-ethylmaleimide-modified myosin subfragment-1, phalloidin, and cytochalasin B inhibit cleavage but have no effect on anaphase chromosome movements under identical lysis conditions . These results suggest that actomyosin plays no functional role in anaphase chromosome movement in mammalian tissue culture cells and that microtubule depolymerization is a rate-limiting step for chromosome-to-pole movements.

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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.Cytokinesis in animal cells is accomplished by an equatorial constriction of the cortex that progressively pinches the cell in two, much like the action of a purse string . The discovery that a band of actin microfflaments, the contractile ring, forms in association with the developing cleavage furrow gave rise to a model of cytokinesis based on the role of actomyosin in the muscle sarcomere (1, 3, 26, 27, 29-32, 35, 38, 40).Evidence supporting an actomyosin-based model for cytokinesis has come largely from studies at the light and electron microscope levels that demonstrate that both actin and myosin are present in the cleavage furrow (11,17,28). Microinjection of an antibody against myosin has been shown to prevent cytokinesis in starfish (18) and sea urchin blastomeres (15), and studies using cytochalasin B, although limited by questions regardingthe drug's specificity, likewise suggest an actomyosinbased mechanism for cleavage (33) .We have previously demonstrated that rabbit skeletal muscle heavy meromyosin (HMM) treated with the sulfhydryl reagent N-ethylmaleimide (NEM) serves as a specific inhibitor of muscle-type force-generating systems (21) . N-Ethylmaleimidemodified heavy meromyosin (NEM-HMM) binds tightly to actin and does not release in the presence of MgATP. After decoration with NEM-HMM, the actin becomes unavailable to native myosin. Inhibition of in vitro actomyosin interactions, the contraction of demembranated muscle myofibrils, and contractility of Chaos cytoplasm also result (21) . It is not known whether NEM-HMM interferes with actin polymerization and the organization of actin gels . However, NEM-HMM inhibits neither the beat of demembranated cilia nor in vitro microtubule polymerization (21) .We report here that microinjection of NEM-HMM into blastomeres of fertilized Rana pipiens eggs inhibits cleavage in a zone surrounding the injection point and can prevent wound closure. We also report that changes in microvilli distribution are inhibited by NEM-HMM.Our findings confirm the involvement of an actomyosin force-generating mechanism in cleavage and are consistent with the...

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