Centrosome Maturation In Vitro

中心体是独特的亚细胞细胞器，参与从酵母到人类的细胞结构组织。作为微管组织中心（MTOCs），中心体直接或间接参与了许多基本的细胞过程，包括细胞复制、细胞迁移、定向细胞器运输以及细胞形状和极性的维持。因此，了解中心体的功能将影响我们对癌症、转移、趋化性和早期发展的理解。这项工作的长期目标是了解中心体的组成，微管成核的分子基础和中心体功能的生化调节。利用固体Spisula卵母细胞的独特特性，已经开发出了以下方法：1)在体外诱导细胞周期特异性中心体成熟；2)从卵母细胞周期的不同阶段分离中心体，3)分解和重组中心体组织微管的能力，4)产生特异性识别中心体蛋白质的抗体，5)分离中心体蛋白质编码的基因。资金要求继续工作，以确定所需的蛋白质中心体组装和中心体依赖的微管成核。中心体蛋白磷酸化将被研究，以确定控制中心体的蛋白激酶，并开始揭示蛋白质磷酸化调节中心体功能的机制。该提案将完成鉴定重要中心体蛋白的工作，并开始利用这种独特的体外系统来纯化调节中心体功能的分子。

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.