Effect of heavy chain phosphorylation on the polymerization and structure of Dictyostelium myosin filaments.

Abstract

Abstract. In Dictyostelium amebas, myosin appears to be organized into filaments that relocalize during cell division and in response to stimulation by cAME To better understand the regulation of myosin assembly, we have studied the polymerization properties of purified Dictyostelium myosin. In 150 mM KC1, the myosin remained in the supernate following centrifugation at 100,000 g. Rotary shadowing showed that this soluble myosin was monomeric and that ,,o80% of the molecules had a single bend 98 nm from the head-tail junction. In very low concentrations of KC1 (<10 mM) the Dictyostelium myosin was also soluble at 100,000 g.But rather than being monomeric, most of the molecules were associated into dimers or tetramers. At pH 7.5 in 50 mM KCI, dephosphorylated myosin polymerized into filaments whereas myosin phosphorylated to a level of 0.85 tool Pi/mol heavy chain failed to form filaments. The phosphorylated myosin could be induced to form filaments by lowering the pH or by increasing the magnesium concentration to 10 mM. The resulting filaments were bipolar, had blunt ends, and had a uniform length of `o0.43 gm. In contrast, filaments formed from fully dephosphorylated myosin were longer, had tapered ends, and aggregated to form very long, threadlike structures. The Dictyostelium myosin had a very low critical concentration for assembly of ,o5 gg/ml, and this value did not appear to be affected by the level of heavy chain phosphorylation. The concentration of polymer at equilibrium, however, was significantly reduced, indicating that heavy chain phosphorylation inhibited the affinity of subunits for each other. Detailed assembly curves revealed that small changes in the concentration of KCI, magnesium, ATE or H + strongly influenced the degree of assembly. Thus, changes in both the intracellular milieu and the level of heavy chain phosphorylation may control the location and state of assembly of myosin in response to physiological stimuli. IN both striated and smooth muscle, myosin molecules are assembled into stable filaments that slide relative to the actin-containing thin filaments to produce force. It has been suggested that nonmuscle motility involves a sliding filament mechanism, and the fact that isolated nonmuscle myosins retain the ability to self-assemble (39) strongly suggests that filaments are required for some aspects of cell motility. Indeed, Langanger et al. (25) showed that myosin was assembled into bipolar thick filaments which were distributed along the length of stress fibers, and studies by Yumura and Fukui (51) demonstrated that in Dictyostelium amebas myosin formed discrete structures having the dimensions of thick filaments.Recent studies have revealed that the state of myosin assembly can directly regulate ATPase activity. For example, dephosphorylated smooth muscle (47, 48) and thymus (46) myosins do not form filaments and have low actin-activated ATPase activity. Phosphorylation of the light chain, however, induces myosin assembly concomitant with an increase in ATPase activity. The a...

Role of the C‐terminal extremities of the smooth muscle myosin heavychains" implication for assembly properties

“…It is well known that the smooth muscle LMM has a high solubility at low ionic strength [17], and behavior comparable to that of LMM1 at pH 7.0 has been observed for Dictyostelium non-muscle myosin [18,19]. Thus the solubility features of LMM1 and LMM2 are similar except at pH 7.0 in the absence of monovalent salt (NaC1 or LiC1 or KCI as experimentally verified).…”

Myosin Heavy Chain Phosphorylation Sites Regulate Myosin Localization during Cytokinesis in Live Cells

“…They disassemble into soluble myosin molecules at 0 and 200 mM NaCl (Kuczmarski and Spudich, 1980;Kuczmarski et al, 1987). GFP-myosin formed thick filaments with the same dependence on ionic conditions as untagged Dictyostelium myosin (our unpublished observations).…”

WD Repeat Domains Target Dictyostelium Myosin Heavy Chain Kinases by Binding Directly to Myosin Filaments

“…The resulting supernatants were transferred to clean centrifuge tubes and then mixed with the other binding assay components to give 40-l reaction mixes containing 0.3 M kinase fusion protein, 1.0 M Dictyostelium myosin II, 0.1% BSA, 2.5 mM TES, pH 7.0, 40 mM KCl, 2 mM MgCl 2 , and 5% sucrose. Under these conditions, greater than 80% of the myosin II is in filaments (25). After incubation for 10 min at 4°C, the binding reactions were centrifuged for 10 min at 95,000 ϫ g, and equal volumes of pellet and supernatant fractions were resolved by SDS-PAGE.…”

“…This WD domain-mediated binding facilitates the phosphorylation of MHC. For MHCK A, the functional consequence of this phosphorylation is to promote myosin II filament disassembly and ultimately to inhibit myosin II-mediated contraction in cells (9,19,25). Further studies are needed to characterize the physiological roles of MHCK B and to determine whether MHCK B phosphorylation of MHC is restricted to the same or different sites phosphorylated by MHCK A.…”