Myosin behavior in non-muscle cells does not mimic that of purified myosin in vitro. In the cytoplasm, myosin thick filaments are reversibly assembled, disassembled and directed to sites of contractility in the constant presence of MgATP and networks of actin filaments; conditions that normally result in continual contraction of stationary actomyosin complexes in vitro. The broad goal of this research is therefore to establish the biochemical mechanisms regulating Dictyostelium myosin thick filament assembly, disassembly, and actin association under conditions relevant to the cytoskeletal environment of non-muscle cells. First, by combining fluorescence energy transfer, light scattering, and electron microscopy, we will determine basic mechanisms of myosin assembly, disassembly, and exchange for purified Dictyostelium myosin under physiologic buffer conditions. Second, we will investigate the effects of diverse actin cytoskeletal structures created by a 40kD actin disassembly protein (severin), 95kD (alpha-actinin) and 120 kD actin cross-linking proteins, and 34kD actin bundling protein on myosin assembly and binding to actin. Third, cytoplasmic myosin-binding proteins from Dictyostelium that promote or inhibit thick filament assembly, disassembly, exchange and F-actin interactions will be identified and isolated. Fourth, since phosphorylation of Dictyostelium myosin heavy chains is known to chain phosphorylation to cause reversible disassembly and reassembly of myosin during phosphorylation in the presence of actin filaments will be tested. Finally, an in vitro simulation of myosin movement and contraction in the cortical cytoplasm of Dictyostelium will be conducted using a model actin filament cytogel system. Myosin movement through actin filament networks, regulated formation of actomyosin contractile complexes, and the effects of myosin HC phosphorylation and actin cytoskeleton structure on myosin movements will be visualized and quantitated. It is our ultimate goal to establish mechanisms that control myosin movement and contraction in dividing and locomotory cells.
