Microfilaments are cytoskeletal elements involved in the determination of cell shape as well as in many aspects of cell motility. We have chosen two projects to contribute to an understanding of microfilament organization and function. The first is a functional analysis of the components of the isolated intestinal microvillus cytoskeleton, and an investigation of how these findings relate to microfilament arrangements in other cells. We propose studies on two microvillus proteins. One is the 110K-calmodulin complex that links the microvillus core bundle to the plasma membrane. The complex can be specifically solubilized from the microvillus by a nucleoside triphosphate. The complex has been purified and shown to have a nucleoside triphosphatase activity. We propose to characterize the complex and explore the relationship between these properties, and its interaction with F-actin. We also propose to identify and characterize membrane components with which the complex associates. The other microvillus cytoskeletal protein we will study is ezrin, whose function is not known. In cultured cells, ezrin is a major substrate for the inducible tyrosine kinase activity of the epidermal growth factor receptor and certain kinases of tumor viruses. We propose to determine the function of ezrin in the micrivillus and explore the effects of in vitro tyrosine phosphorylation on its properties. The second project involves characterization of caldesmon from smooth and non-muscle cells. Current evidence indicates that it is a key component of a thin-filament Ca2+-regulatory system in smooth muscle. It is a potent F-actin bundling protein whose F-actin binding activity can be inhibited by the presence of Ca2+-calmodulin. Caldesmon will be characterized in detail and the molecular mechanism that gives it these properties explored. Proteins immunologically and functionally related to smooth muscle caldesmon are present in other cells and show considerable molecular weight heterogeneity. We propose to purify and characterize these proteins and compare them to smooth muscle caldesmon. Since cloned cultured cell lines contain more than one caldesmon species, we propose to explore the functional differences between classes of caldesmon species. The work set out in this study focusses on three proteins that play key roles in microfilament organizations. The results should provide some important basic information about the regulation and molecular organization of microfilaments.
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