CENTROSOME MATURATION IN VITRO

体外中心体成熟

• 批准号: 2734645
• 负责人: ROBERT E PALAZZO
• 金额: $ 19.12万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734645
• 关键词: Bivalvia; cell biology; cell free system; centrosome; egg /ovum; microtubules; monoclonal antibody; nucleoproteins; phosphorylation; protein kinase; protein structure function; saltwater environment

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.

描述：中心体是独特的亚细胞器， 从酵母到人类的微管细胞结构。作为微管 组织中心（MTOC），中心体参与许多基本的 包括细胞复制、细胞迁移、定向 细胞器运输和维持细胞形状和极性。 因此，在本发明中， 对中心体功能的理解将影响我们对癌症的理解， 转移、趋化性和早期发育。 这一长期目标 工作是了解中心体的组成， 微管成核与中心体的生化调控 功能 利用刺果的独特性质 在卵母细胞中，申请人已经开发了以下方法：1）诱导细胞分化， 体外细胞周期特异性中心体成熟和中心粒复制，2） 从卵母细胞细胞周期的不同阶段分离中心体，以及3） 产生特异性识别中心体的单克隆抗体 proteins. 要求提供资金， SpiCen #14蛋白，并测试SpiCen #14是否为 中心体依赖的微管成核。 此外，实验 提出了确定中心体蛋白结合微管获得 深入了解微管成核的性质，并确定 蛋白质被募集到中心体，因为它们增加了它们的能力， 有核微管 最后，中心体蛋白磷酸化将 研究以确定蛋白激酶是否与分离的 中心体，如果中心体蛋白作为已知细胞的底物， 周期调节激酶，开始解开的机制， 蛋白质磷酸化调节中心体成熟。 这项建议会 完成鉴定对功能重要的中心体蛋白的工作， 开始利用这种独特的体外系统来纯化分子， 调节中心体功能。