Muscle contraction and other forms of cell motility are believed to be driven by myosin molecules pulling themselves along actin filaments. Although the understanding of ATP-dependent movement of myosin along actin filaments is paramount to the understanding of motility, the molecular basis of this movement remains obscure. Recently, Sheetz and Spudich (1983. Nature 303:31-35) directly visualized the movement of myosin in vitro and devised a quantitative assay to measure this movement. The development of this assay provides the basis for this proposal. The overall goal is to define in molecular terms the parameters of the myosin molecular necessary for its ability to move. First we will attempt to develop a totally defined assay for myosin movement using an organized polar array of purified actin filaments. We will then characterize the biochemical and biophysical parameters of the myosin movement and search for activities in crude extracts of both Dictyostelium amoebae and skeletal muscle that modify the rate of the myosin movement. Such regulatory activities will be purified and characterized. Of primary importance will be studies to determine the regions of the myosin necessary for or involved in movement. For this part of the overall program we will examine the effects of various monoclonal antibodies against Dictyostelium myosin, the movement capabilities of myosin fragments prepared by proteolysis, and the movement capabilities of myosins altered by site-specific mutagenesis using a molecular genetics approach.