Assembly and Function of Mammalian Centrosomes

哺乳动物中心体的组装和功能

• 批准号: 6929714
• 负责人: RYOKO KURIYAMA
• 金额: $ 22.28万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6929714
• 关键词: RNA interference; cell component structure /function; cell cycle proteins; centrosome; confocal scanning microscopy; electron microscopy; fluorescence microscopy; laboratory mouse; laboratory rabbit; laboratory rat; microtubules; mitotic spindle apparatus; molecular assembly /self assembly; nuclear proteins; phosphorylation; protein localization; protein protein interaction; protein structure function; time resolved data; yeast two hybrid system

DESCRIPTION (provided by applicant): The centrosome is a major microtubule-organizing center responsible for initiation of microtubule assembly in animal cells. Microtubules are important for such processes as mitosis/meiosis, cell motility, and morphogenesis, thus centrosome dysfunction causes serious human diseases, including cancers, metastasis, and birth defects. The overall aim of this application is to understand how assembly and function of the centrosome are controlled at the molecular level. The research focuses on three centrosome proteins: Aurora-A, CNN, and Cep135. A mitosis-specific kinase, Aurora-A is involved in centrosome maturation during cell division. It binds to CNN (centrosomin), which has unique properties to induce microtubule-nucleating sites through interacting with the gamma-tubulin complex. CNN also binds to a highly coiled-coiled protein, Cep135 important for maintaining the structural integrity of the centrosome. The first specific aim is to determine how the CNN activity to form microtubule-nucleating sites is controlled by Aurora-A. Effects of the change in CNN interaction with Aurora-A and its phosphorylation by the Aurora-A kinase will be examined. Additional CNN- and Aurora-A-interacting molecules will be searched using a combined two-hybrid screens and functional RNAi assays. The second objective is to dissect molecules involved in organization of microtubule-nucleating sites. Microtubule formation onto cytoplasmic assemblies induced by CNN overexpression will be characterized in vivo and in vitro. Proteins required for microtubule nucleation will be identified by in vitro tests of microtubule polymerization onto isolated CNN assemblies from which different molecules are stripped off and added back. Attempts will be made to polymerize microtubules onto microbeads coated with CNN and other potential molecules. The third objective is to examine assembly of mammalian centrosomes by supplementing Cep135-depleted cells with exogenous Cep135 and CNN. The process of disintegration and reformation of the functional centrosome will be monitored by fluorescence and electron microscopy. The Aurora-A gene is a tumor-susceptibility gene and its overexpression causes cancer formation. As centrosome abnormalities are implicated in the origin of chromosome instability and cancer pathogenesis, the study will be useful in establishing strategies for prevention, diagnosis, and treatment of human cancers.

描述（申请人提供）：中心体是动物细胞中负责启动微管组装的主要微管组织中心。微管对于有丝分裂/减数分裂、细胞运动和形态发生等过程非常重要，因此中心体功能障碍会导致严重的人类疾病，包括癌症、转移和出生缺陷。本申请的总体目标是了解如何在分子水平上控制中心体的组装和功能。研究重点是三种中心体蛋白：Aurora-A，CNN和Cep 135。Aurora-A是一种有丝分裂特异性激酶，参与细胞分裂过程中的中心体成熟。它与CNN（centrosomin）结合，CNN具有通过与γ-微管蛋白复合物相互作用诱导微管成核位点的独特性质。CNN还与一种高度卷曲的蛋白质Cep 135结合，Cep 135对维持中心体的结构完整性很重要。第一个具体目标是确定形成微管成核位点的CNN活性如何由Aurora-A控制。将检查CNN与Aurora-A相互作用的变化及其通过Aurora-A激酶的磷酸化的影响。其他CNN和Aurora-A相互作用分子将使用组合的双杂交筛选和功能性RNAi测定进行搜索。第二个目标是剖析参与微管成核位点组织的分子。将在体内和体外表征由CNN过表达诱导的细胞质组装体上的微管形成。微管成核所需的蛋白质将通过微管聚合到分离的CNN组装体上的体外测试来鉴定，从所述分离的CNN组装体中剥离并添加回不同的分子。将尝试将微管包被到用CNN和其他潜在分子包被的微珠上。第三个目标是通过补充Cep 135和CNN来检查哺乳动物中心体的组装。功能中心体的解体和重组过程将通过荧光和电子显微镜监测。Aurora-A基因是一种肿瘤易感基因，其过表达导致癌症形成。由于中心体异常与染色体不稳定性和癌症发病机制的起源有关，因此该研究将有助于建立预防，诊断和治疗人类癌症的策略。