Cell motility is essential for many biological processes. During development, cells move in a organized manner to give rise to specific organs, and disruption of this process can lead to serious abnormalities. Directed cell movements are also associated with angiogenesis, neuronal re-arrangements, wound healing, and the movement of macrophages to sites of infection. One of the most lethal manifestations of uncontrolled cell motility is metastasis. In muscle, myosis is the protein which converts the chemical energy of ATP into the mechanical energy of movement. Myosin is also present in nonmuscle cells and is believed to be responsible for most types of cell locomotion. In order to understand how cell motility is regulated, therefore, it is essential to study regulation of the myosin molecule. Dictyostelium is an excellent system since cells can be grown easily, are amenable to genetic analysis, and exhibit chemotaxis. A major goal is to understand how myosin assembly and ATPase activity are controlled by the cell. Since filament structure can directly regulate ATPase activity, it is also important to study the relationship between these two processes. The main techniques for studying assembly will be sedimentation in an airfuge and turbidity changes in a stopped-flow spectrophotometer. With the later instrument it should be possible to identify subtle effects which influence the rate of assembly or disassembly. In addition to the effect of various solution conditions on assembly, a search will be made for possible myosin- associated proteins which regulate the assembly or stability of myosin thick filaments. A known regulator of assembly is heavy chain phosphorylation, and detailed studies of phosphorylation effects will be carried out using partially purified kinases. Additionally, an attempt will be made to purify the kinase which phosphorylates a specific in vivo site. To identify domains essential for assembly, a variety of well-defined proteolytic fragments of the tail will be studied for their effect on assembly and ATPase activity. Since filament stability and shape are very sensitive to solution conditions and modifications of the myosin molecule, attempts will be made to stabilize the filaments in a known conformation by use of chemical cross-linkers. This should provide important new information on the relationship between filament structure and ATPase activity.
