CENTROSOME MATURATION IN VITRO

描述：中心体是一种独特的亚细胞细胞器， 从酵母到人的微管细胞构筑。作为微管 组织中心(MTOC)，中心体参与了许多基本的 细胞过程，包括细胞复制、细胞迁移、定向 细胞器的运输和维持细胞的形状和极性。因此， 了解中心体功能将影响我们对癌症的理解， 转移、趋化和早期发育。这样做的长期目标是 工作是了解中心体的组成，即中心体的分子基础 微管成核与中心体的生化调控 功能。利用三棱草的独特性质 卵母细胞，申请人已经开发出方法：1)诱导细胞 体外周期特异性中心体成熟和中心粒复制，2) 从卵母细胞周期的不同阶段分离中心体 产生特异性识别中心体的单抗 蛋白质。要求提供资金，以完成对 SpiCen#14蛋白，并测试SpiCen#14是否是 中心体依赖的微管成核。此外，实验是 建议鉴定与微管结合的中心体蛋白以获得 对微管成核的本质的洞察和确定 蛋白质被招募到中心体，因为它们增加了自己的能力 有核的微管。最后中心体蛋白的磷酸化将是 研究以确定蛋白激酶酶是否与分离的 中心体，以及中心体蛋白是否作为已知细胞的底物 循环调节激酶，开始解开 蛋白质磷酸化调控中心体成熟。这项提议将 完成确定对功能重要的中心体蛋白的工作，以及 开始开发这种独特的体外系统来纯化分子 调节中心体功能。

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.