This is a proposal to extend work on the function of microtubules in cellular morphogenesis and cell division. We will study the function of microtubules in neurite outgrowth in pheochromacytoma cells and in particular the role of tau protein. In addition we will investigated the complexity of tau protein expression and the heterogeneity of tau protein in brain through cDNA cloning and sequencing. To understand how microtubules are reorganized in the cell we will extend our studies of the dynamics of assembly in vitro using methods which look at individual microtubules, such as EM, immunofluorescence, assembly with biotinylated tubulin, and autoradiography of Gamma-32P GTP. To interpret macroscopic dynamics in terms of microscopic processes we will continue our theoretical collaboration with T. Hill. We will pursue further our studies of microtubule dynamics off purified centrosomes and kinetochore. We will extend our search for the nucleating and structural components of both organelles using monoclonal antibodies. Starting material for the kinetochore will be interphase nuclei from which we have already accomplished a considerable purification. We will study other tubulin interactions with metaphase kinetochores including capture and capping biochemically and by electron microscopy. We will search using appropriate assays for microtubule capping proteins in cellular extracts and attempt to purify them, study in vitro and assay their biological role. Finally we will attempt to integrate microtubule assembly and the cell cycle using Xenopus eggs and occytes. We will continue our studies in this system to purify an oocyte inhibitory factor and an egg activating factor and determine how they are regulated by the endogenous cell cycle timing machinery.
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