Assembly and Function of Mammalian Centrosomes

哺乳动物中心体的组装和功能

• 批准号: 6829771
• 负责人: RYOKO KURIYAMA
• 金额: $ 21.88万
• 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 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6829771
• 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（由申请人提供）：中心体是主要的微管组织中心，负责动物细胞中微管组装的启动。微管对于有丝分裂/减数分裂、细胞运动和形态发生等过程很重要，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。该应用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。该研究重点关注三种中心体蛋白：Aurora-A、CNN 和 Cep135。 Aurora-A 是一种有丝分裂特异性激酶，参与细胞分裂过程中中心体的成熟。它与 CNN（中心体蛋白）结合，具有独特的特性，可通过与 γ-微管蛋白复合物相互作用诱导微管成核位点。 CNN 还与高度卷曲的蛋白质 Cep135 结合，对于维持中心体的结构完整性非常重要。第一个具体目标是确定 Aurora-A 如何控制 CNN 形成微管成核位点的活性。将检查 CNN 与 Aurora-A 相互作用的变化及其被 Aurora-A 激酶磷酸化的影响。将使用组合的两种杂交筛选和功能性 RNAi 测定来搜索其他 CNN 和 Aurora-A 相互作用分子。第二个目标是剖析参与微管成核位点组织的分子。 CNN 过表达诱导的细胞质组件上微管的形成将在体内和体外进行表征。微管成核所需的蛋白质将通过在分离的 CNN 组件上进行微管聚合的体外测试来鉴定，其中不同的分子被剥离并重新添加回来。将尝试将微管聚合到涂有 CNN 和其他潜在分子的微珠上。第三个目标是通过用外源 Cep135 和 CNN 补充 Cep135 耗尽的细胞来检查哺乳动物中心体的组装。功能中心体的分解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，其过度表达会导致癌症形成。由于中心体异常与染色体不稳定和癌症发病机制的起源有关，因此该研究将有助于制定人类癌症的预防、诊断和治疗策略。