The goal of this proposal is to identify and characterize proteins that reguate the interaction of the membrane-bound motor molecule, myosin-I, with phospholipid surfaces such as cell membranes. Myosin-I is the only motor molecule known to use a lipid surface as a substrate for motility, and because cellular movement is accompanied by motile events at the leading edge of the cell where myosin-I is concentrated, myosin-I is a likely candidate for driving cell movement. To search for proteins that regulate myosin-I, extracts from Acanthamoeba castellanii will be screened with three in vitro assays that measure different activities of myosin-I: (1), a direct binding assay, to determine the effect of regulatory proteins on myosin-I's association with membranes; (2), ATPase activity to evaluate the effect of regulatory proteins on myosin-I's enzyme activity; and (3), a motility assay to observe the effect of regulatory proteins on myosin-I's ability to move actin filaments. After regulatory proteins have been identified in these assays, specific proteins will be purified using standard biochemical procedures and polyclonalantibodies to each of these proteins will be produced. These antibodies will be used in the above assays to test their effect on regulatory protein activities and in cells to localize the regulatory proteins by immunostaining. These experiments will provide important information on how myosin-I is organized at the cell membrane and how myosin-I's activity is regulated. %%% Cellular motility is fundamental to the life processes of unicellular organisms and essential to many physiological activities of higher organisms, such as development, neuronal growth, angiogenesis, defense mechanisms, wound healing, and organismal locomotion. Cellular movement requires the coordination of membrane and cytoskeletal systems. As a result of its lipid- binding properties, the cytoskeletal motor protein myosin-I has been implicated in many membrane-mediated motile processes. However, unlike other motor proteins (e.g., myosin-II, kinesin, or dynein) it has not been possible thus far to show that mysoin-I is required for some generalized cellular process. The problem of what myosin-I does may not be easily solved solely by studying mysoin-I, since it appears likely that multiple genes encode a family of myosin-I molecules with overlapping functions. The results of this study will contribute to the understanding of myosin-I's role in cell motility.

Agency
National Science Foundation (NSF)
Institute
Division of Molecular and Cellular Biosciences (MCB)
Application #
9205344
Program Officer
Eve Ida Barak
Project Start
Project End
Budget Start
1992-08-15
Budget End
1996-07-31
Support Year
Fiscal Year
1992
Total Cost
$290,190
Indirect Cost
Name
University of Texas Southwestern Medical Center at Dallas
Department
Type
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390