MECHANISM AND CONTROL OF MICROTUBULE ASSEMBLY

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Microtubules are dynamic structures that serve to organize the cytoplasm in interphase cells and to provide the structural framework and possibly the motive force for the spindle in mitotic cells. The broad objectives of this proposal are to understand microtubule dynamics in vivo and vitro and to determine whether selected mitotic events are dependent upon the dynamic properties. A variety of experimental approaches will be used including fluorescent and hapten tagging of proteins, microinjection, hapten- mediated immunocytochemistry, fluorescence photobleaching, and digital imaging microscopy. These methods allow determination of the sites of tubulin subunit addition into an loss from pre- existing microtubules. They also permit a determination of the kinetics of turnover of the microtubule network. Posttranslationally modified subunits will be identified by indirect immunofluorescence with peptide specific antibodies. The principal specific aims will be to complete our analysis of anaphase dynamics; to determine the mechanism of turnover in metaphase; to identify sites of subunit addition and loss during congression in prometaphase; to investigate further microtubule dynamics in the leading lamellum of fibroblasts and their relationship to cell motility; to obtain direct evidence for the mechanism of microtubule turnover near the cell center; to test the role of two posttranslational modification, detyrosination and acetylation, on microtubule turnover; to evaluate the stability of microtubules in a highly differentiated cell type, the PC-12 neuronal cell line; and to directly test the structural hypothesis of slow axonal transport. The results should help elucidate the precise nature and significance of microtubule lability. The results should also be of relevance for mechanisms regulating motility and division in normal cells. An understanding of normal cell motility and division may assist in the analysis of cancer cells where the mechanisms restricting division are aberrant.