FUNCTION OF THE CYTOSKELETAL PROTEINS IN VIVO

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The goal of the proposed research is to understand the molecular basis for different cell shape, and why that shape is disrupted upon malignant transformation. The work focuses on one central component of the cytoskeleton common to all eukaryotic cells, the microtubules. These structures are virtually identical in all cell types and their major components, the tubulins, are among the most conserved of proteins. Nevertheless, microtubules are arranged in organelles of dramatically diverse properties, structures, and functions. Where the information resides that specifies this diversity while still maintaining the highly conserved aspects of microtubule structure is a major question in understanding normal and pathological organization of the cytoplasm. The proposed research is directed at two possible explanations for these events: first, that the diversity that does exist among the tubulins is sufficient to explain the diversity of microtubules; and second, that minor microtubule components, interacting with microtubules are the origin of specialized organization. These experiments proposed take advantage of the powerful genetics of yeast, S. cerevisiae, to analyze functional consequences of structural changes in tubulins, and to use those consequences to identify gene products that interact with tubulins in vivo. A scheme for generating mutations with a broad range of phenotypes will be used to study alpha tubulins, and to identify second-site revertants. The cytoplasmic differentiation of two functionally-interchangeable tubulin gene products will be studied in detail. Domain structure of another tubulin will be studied, to identify the functional role of a stretch of sequence of particular interest. Introduction of foreign tubulin genes into both yeast and animal cells will allow permit identification of proteins that interact by rescuing function. Overall, the proposed research should provide significant information about the detailed molecular interaction that underlie structure and function in cytoplasmic and mitotic microtubules. Eventually, this understanding should lead to a detailed mechanism for loss of function in pathological states, such as cancer.