Assembly and Function of Mammalian Centrosomes

描述（由申请人提供）：中心体是动物细胞中主要的微管组织中心，负责启动微管组装。微管在有丝分裂/减数分裂、细胞运动和形态发生等过程中起着重要作用，因此中心体功能障碍导致严重的人类疾病，包括癌症、转移和出生缺陷。本应用程序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Cep135。作为一种有丝分裂特异性激酶，Aurora-A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，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特性。CNN还与一种高度卷曲的蛋白Cep135结合，Cep135对于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活性是如何由Aurora-A控制的。我们将研究CNN与Aurora-A相互作用的变化及其被Aurora-A激酶磷酸化的影响。其他与CNN和aurora - a相互作用的分子将使用组合的双杂交筛选和功能性RNAi分析进行搜索。第二个目标是剖析参与微管成核位点组织的分子。由CNN过表达诱导的细胞质上的微管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微管聚合到分离的CNN组件上的体外测试来鉴定，其中不同的分子被剥离并添加回来。将尝试将微管聚合到涂有CNN和其他潜在分子的微珠上。第三个目的是通过外源性Cep135和CNN补充Cep135缺失的细胞来检测哺乳动物中心体的组装。荧光和电子显微镜将监测功能中心体的解体和重组过程。Aurora-A基因是一种肿瘤易感基因，它的过度表达会导致癌症的形成。由于中心体异常与染色体不稳定的起源和癌症的发病机制有关，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。

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.