Centrosome Maturation In Vitro

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Centrosomes are unique subcellular organelles involved in the organization of cytoarchitecture from yeast to man. Ass microtubule organizing centers (MTOCs), centrosomes are directly or indirectly 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 Spisula solidissima oocytes, methods have been developed to: 1) induce cell cycle-specific centrosome maturation in vitro; 2) isolate centrosomes from distinct phases of the oocyte cell cycle, 3) disassemble and reassemble a centrosome's ability to organize microtubules, 4) generate antibodies which specifically recognize centrosome proteins, and 5) isolate genes that code for centrosome proteins. Funds are requested to continue work to identify proteins required for centrosome assembly and centrosome- dependent microtubule nucleation. Centrosome protein phosphorylation will be studied to identify protein kinase enzymes that control centrosomes, to begin to unravel the mechanisms by which protein phosphorylation regulates centrosome function. This proposal will complete work to identify important centrosome proteins, and begin to exploit this unique in vitro system to purify molecules that regulate centrosome function.