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 in vitro and to determine whether selected mitotic events are dependent upon the dynamic properties. A variety of experimental approaches will be used including immunofluorescence, microsurgery, fluorescent and hapten tagging of proteins, microinjection and fluorescence photobleaching recovery. The principal aims will be to identify the transformations occurring in the microtubule network at the transition between interphase and mitosis; to determine the extent and sites of exchange of tubulin subunits and microtubule associated proteins with the microtubules: to test for the existence and significance of microtubule treadmilling; and through the use of tubulin-colchicine complex, a specific inhibitor of tubulin exchange, to test the dependence of anaphase motion on microtubule disassembly. Novel methods including hapten-mediated immunoelectorn microscopy and fluorescence photobleaching of fluorescently-tagged proteins incorporated into microtubule polymer will be employed. The results should help elucidate the precise nature and cellular significance of microtubule lability. The results should also be of direct relevance for mechanisms regulating division in normal cells. An understanding of normal cell division may assist in the analysis of cancer cells where the mechanisms restricting division are aberrant.