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

Abstract

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

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

“…The reaction was stopped by adding DTT to a final concentration of 10 mM, and unreacted NEM was removed by exhaustive dialysis. ATPase activities of the treated proteins were measured as described previously (24). As with NEM-HMM, the calcium ATPase activity of NEM-S, was elevated above that of S,, and the actin-activated and K*/EDTAstimulated ATPase activity of NEM-S, was < 10% that of the untreated proteins.…”

“…We have previously reported that NEM-HMM forms rigorlike bonds even in the presence of ATP and blocks actomyosin interactions in several model systems by preventing unmodified myosin from interacting with the actin microfilament (24). NEM-S, probably inhibits cleavage by a similar mechanism, that is, it may prevent native myosin from interacting with filaments in the contractile ring.…”

“…NEM-S, was prepared by the same method used previously to prepare NEM-HMM (22,24). S, at 5-10 mg/ml was incubated in 10 mM imidazole-Cl, 0.2 mM dithiothreitol (DTT) with 1 mM freshly dissolved NEM, pH 7.0, at room temperature for 75 min.…”

“…As has been described previously for NEM-HMM (22,24), NEWS, inhibits super precipitation of actomyosin solutions, the contraction of glycerinated myofibrils in the presence of MgATP, and the contraction of strands of Chaos cytoplasm . NEWS, at a concentration of 1-2 mg/ml completely blocks reinitiation of cleavage in permeabilized cells after a 90-s incubation in the protein (Figs.…”

Myosin-V is a mechanical ratchet

“…26. Beads, sparsely covered with motor protein, were positioned over tetramethylrhodaminephalloidin-labeled and aligned actin filaments attached to a coverslip by myosin-II treated with N-ethylmaleimide (26,47). Before each experiment the trap stiffness was calibrated for the trapped bead from the amplitude of the thermal diffusion (48).…”