CENTROSOME MATURATION IN VITRO

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DESCRIPTION: Centrosomes are unique subcellular organelles which organize the microtubule cytoarchitecture from yeast to man. As microtubule organizing centers (MTOCs), centrosomes are involved in numerous fundamental cell processes including cell replication, cell migration, directed organelle traffic and maintenance of cell shape and polarity. Thus, understanding centrosome function will impact our understanding of cancer, metastasis, chemotaxis and early development. The long term goals of this work are to understand centrosome composition, the molecular basis of microtubule nucleation and the biochemical regulation of centrosome function. Taking advantage of the unique properties of Spicula solidissima oocytes, the applicant has developed methods to: 1) induce cell cycle-specific centrosome maturation and centriole duplication in vitro, 2) isolate centrosomes from distinct phases of the oocyte cell cycle and 3) generate monoclonal antibodies which specifically recognize centrosome proteins. Funds are requested to complete the characterization of the SpiCen #14 protein and to test if SpiCen #14 is necessary for centrosome-dependent microtubule nucleation. Further, experiments are proposed to identify centrosome proteins which bind microtubules to gain insights into the nature of microtubule nucleation and to determine which proteins are recruited to centrosomes as they increase their ability to nucleate microtubules. Finally centrosome protein phosphorylation will be studied to determine if protein kinase enzymes are associated with isolated centrosomes,and if centrosome proteins serve as substrates for known cell cycle regulatory kinases, to begin to unravel the mechanisms by which protein phosphorylation regulates centrosome maturation. This proposal will complete work to identify centrosome proteins important for function, and begin to exploit this unique in vitro system to purify molecules which regulate centrosome function.