Spatial and Temporal Control of Nonmuscle My0sinLocalization: Identification of a Domain That Is Necessary for Myosin Filament Disassembly In Vivo

Myosin null mutants of Dictyostelium are defective for cytoldnesis, multicellular development, and capping of surface proteins. We have used these cells as transformation recipients for an altered myosin heavy chain gene that encodes a protein bearing a carboxy-terminal 34-kD truncation. This truncation eliminates threonine phosphorylation sites previously shown to control filament assembly in vitro. Despite restoration of growth in suspension, development, and ability to cap cell surface proteins, these AC34-truncated myosin transformants display severe cytoskeletal abnormalities, including excessive localization of the truncated myosin to the cortical cytoskeleton, impaired cell shape dynamics, and a temporal defect in myosin dissociation from beneath capped surface proteins. These data demonstrate that the carboxyterminal domain of myosin plays a critical role in regulating the disassembly of the protein from contractile structures in vivo. T HE production of myosin-deficient mutants in Dictyosteliura by generation of antisense RNA (Knecht and Loomis, 1987) and by homologous recombination (De Lozarme and Spudich, 1987; Manstein et al., 1989) has provided direct genetic evidence that myosin is essential for cytoldnesis and for completion ofDictyostelium's multicellular developmental program. Studies with these mutants have also demonstrated that myosin is involved in movements of cortically linked membrane proteins, as evidenced by the inability of myosin-deficient cells to cap con A cross-linked surface proteins (Pasternak and Spudich, 1989; Fukui et al., 1990). Although a reasonably clear picture of the roles of nonmuscle myosin is beginning to emerge, the mechanisms controlling assembly and localization of myosin structures in vivo are not well understood. Relocalization of myosin in response to cellular signals occurs during cytokinesis, chemotaxis, and capping of surface receptors (Kitanishi-Yumura and Fukui, 1989; Yumura et al., 1984; Carboni and Condeelis, 1985; Schreiner et ai., 1977). In all of these examples, myosin-containing contractile structures must be correctly localized, assembled, activated for contraction, and eventually disassembled. The feasibility of introducing altered myosin genes into myosin null cells now makes it possible to identify domains of the protein that are critical for these in vivo activities. Several lines of evidence suggest that the myosin domains that drive assembly and regulate assembly lie within the ~-helical coiled-coil tail. Muscle myosin can be proteolytically fragmented into heavy meromyosin (HMM) ~ and light meromyosin (LMM), where the HMM fragment contains the globular head and the amino-terminal third of the tail, and the LMM fragment contains the carboxy-terminal two thirds of the tail. Numerous studies have demonstrated that the domains that drive filament assembly lie within the LMM portion of the molecule (Harrington and Rodgers, 1984). These observations have been confirmed and extended with Dictyostelium myosin HMM and LMM fragments. When Dictyostelium HMM is produced in vivo in cells devoid of normal myosin, a diffuse distribution is observed by immunomicroscopy, indicating that the HMM molecule cannot assemble into thick filaments, and is not capable of subcellular localization (Fukui et al., 1990). Conversely, an assembly domain has been identified within the LMM region by an mAb study (Pagh et al., 1984) and by recombinant genetics (O'HaUoran et al., 1990). A genetically engineered subfragment located from 34 to 68 kD from the carboxy terminus of the tail (N34; see Fig. 1) can assemble into LMM-type paracrystals. The salt dependence of this assembly is similar to that of intact myosin. Other LMM subfragments are not capable of assembling, suggesting that this domain may be the primary region driving assembly of the intact protein. The last 34 kD of the tail (C34; see Fig. 1) may regulate assembly. Many cytoplasmic myosins are phosphorylated by heavy chain kinases, with consequent changes in their as1. Abbreviations used in this paper: HMM, heavy meromyosin; LMM, light meromyosin. © The Rockefeller University Press, 0021-9525/91/02/677/12 $2.00 The Journal of Cell Biology, Volume 112, Number 4, February 1991 677-688 677 on Jne 4, 2017 D ow nladed fom Published February 15, 1991