9514248 Zot The Zot laboratory has discovered the first non-actin protein that binds a nonfilamentous myosin. This protein, Acan125, from Acanthamoeba, binds to myosin-I through a src homology domain, SH3. Another protein, Acan47, copurifies with Acan125 and may also be a myosin-binding protein. The goals of this project are to determine the functions of Acan125 and Acan47 in order to determine cellular roles for myosin-I. The specific aims are to: 1, determine the structure of Acan125 and its SH3 binding site by cloning and sequencing the cDNA; 2, determine the structure and myosin-I binding characteristics of Acan47; and 3, establish a role for Acan125 in Acanthamoeba using monospecific high-titer antibodies to localize Acan125 by immunofluorescence and to inhibit its function in living cells by microinjection. Antibody-injected cells will be monitored for myosin-associated functions such as shape changes and cell motility, intracellular vesicular transport, and vacuolar contraction. In addition, Acan125 and myosin-I will be localized simultaneously by double-label immunofluorescence, including isoform-specific antibodies to myosin-I to detect changes in patterns of associations between specific isoforms of myosin-I and Acan125. This project will explore not only issues of myosin-I function, but also the broader issue of SH3 function. SH3-mediated associations form transiently between proteins of diverse functions on the surfaces of cellular membranes. Cellular movement requires the coordination of membrane and cytoskeletal systems. As a result of its lipid binding properties, the actin-based motor protein myosin-I has been implicated in many membrane-mediated motile processes. The characterization of SH3 binding partners will likely reveal novel roles for myosin-I and the microfilament system. the overall goal is to understand the processes involved in actin-based cell motility that are common among all cells. ***
