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

Abstract

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...

Evidence that myosin does not contribute to force production in chromosome movement.

“…It is corroborated by the failure of Nethylmaleimide-modified myosin subfragment 1, cytochalasin and antimyosin, agents reported to block actomyosin function, to inhibit chromosome movements in permeabillzed cell models (6,39,51). Our data are consistent with the observation that ATP is not required for chromosomal fiber shortening in permeabilized cell models (4).…”

The Rho GTP exchange factor Lfc promotes spindle assembly in early mitosis

“…The function of ROCK during spindle assembly seems to be highly dependent on cell type because the ROCK inhibitor Y-27632 was not reported to affect mitotic events in HeLa or Rat-1a cells (25,26,30). Furthermore, the activity of ROCK's primary target in spindle assembly, myosin II, does not seem to be required in all cell types and organisms for spindle formation (31)(32)(33)(34)(35). Coinjection of the RhoA mutant G14V͞ F39A, which couples only to mDia-related formins but not to other known Rho effectors, was capable of rescuing spindle defects resulting from anti-Lfc antibody injection (Fig.…”

Analysis of the role of microtubules and actin in erythrophore intracellular motility.

“…The pigment granules continue to move normally after NEM-HMM injection, presumably providing ample opportunity for the injected actomyosin inhibitor to locate its binding site on actin. These probes for actin and actomyosin have been shown to inhibit a diverse set of motility events including cytokinesis (7,22), some forms of axoplasmic transport (11,14), and cytoplasmic streaming (25,39,43). It seems improbable to us that none of them would be successful in affecting pigment transport if it were actin-or actomyosin-based.…”

“…As a result of this specific association with actin, NEM-HMM serves as a high fidelity probe for actomyosin-based motility events. It has been shown to inhibit teleost retinal cone shortening (28) as well as cytokinesis in vivo (22).…”

“…DNase I (essentially RNase-free, specific activity >3,000 U/rag) was from Worthington Biochemical Corp. (Freehold, N J). N-ethyl maleimide-modified heavy meromyosin (NEM-HMM) was a generous gift ofZac Cande (University of California, Berkeley) and was handled as described previously (22). All other materials were purchased from Sigma Chemical Co. (St. Louis, MO).…”