CENTROSOME MATURATION IN VITRO

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Centrosome are involved in the organization of cytoarchitecture in cells from yeast to man. As organizers of microtubules (microtubule organizing centers, MTOCs), centrosomes are involved in numerous fundamental cell processes including cell migration and cell division. Thus, and understanding of the composition and biochemical regulation of centrosomes will have an impact on understanding cancer, metastasis, chemotaxis, and aspects of early development. Methods have been developed to take advantage of the unique characteristics of clam oocytes which allow the purification of sufficient quantities of centrosomes for preparative and analytical biochemistry. The experiments outlined in this proposal will define the protein composition of centrosomes and determine if compositional changes are a function of the meiotic cell cycle. Functional in vitro assays coupled with video- and electron-microscopy will be used to quantitate centriole content and the ability of purified centrosomes to direct microtubule polymerization. Monoclonal antibodies will be generated for the identification and purification of molecules (MTOCs) which direct microtubule organization. Finally, key experiments will be carried out to determine if centrosomes can be induced to mature in vitro, as indicated by changes in their ability to form and replicate centrioles or their ability to organize microtubules. These studies will open the way for classic biochemical approaches designed to purify molecules which regulate centrosomes during the cell cycle.