Organization of the Mammalian Mitotic Spindle

描述(逐字摘自申请者的摘要)：长期目标 这个项目的主要目的是鉴定蛋白质和确定分子机制。 参与组织哺乳动物纺锤体极部的微管负端 细胞。准确的染色体分离对黄瓜的繁殖至关重要 物种和所有细胞的活力。染色体在发育过程中分离 以微管为基础的复杂超结构的有丝分裂和减数分裂 纺锤形。纺锤体内的微管的正端延伸到 朝向细胞皮质或细胞赤道，在那里它们与 染色体或与其他微管相互作用。微管减去末端是 集中在两个独特的位置，称为纺锤杆。主轴两极 是姐妹染色单体将分离到的功能部位 后期。最近的证据表明，微管减去末端被释放 和/或从与以下物质相关联的主要成核点切断 中心体。在从中心体解离之后，一系列 结构蛋白和马达蛋白共同作用，紧密聚焦微管负核 在主轴极点结束。我们已经设计出了一种无细胞检测方法 复制了纺锤极组织的中心体独立的方面。 结合体内检测方法监测纺锤体组织 为我的实验室提供了一个独特的机会来表征这些蛋白质 和参与哺乳动物纺锤体极组织和功能的机制 细胞。这个项目的目标是识别与聚焦有关的蛋白质 微管末端位于纺锤体两极，在体外和体内均可使用 生化实验以剖析与此有关的分子机制 必不可少的过程。这项研究的具体目的是：1)确定 NUMA与纺锤体微管减端联系的动力学研究 极点；2)描述一个大的复合体的功能性质 包含负末端导向的微管马达蛋白HSET；和3)执行 用微管紫杉酯对纺锤极组件的分子解剖 组装在无细胞的有丝分裂提取物中。

DESCRIPTION (Verbatim from the applicant's abstract): The long range objective of this project is to identify proteins and characterize molecular mechanisms involved in organizing microtubule minus ends at spindle poles in mammalian cells. Accurate chromosome segregation is essential for the propagation of species and the viability of all cells. Chromosomes are segregated during mitosis and meiosis by a complex microtubule-based superstructure called the spindle. Microtubules within the spindle have their plus ends extending either toward the cell cortex or to the cell equator where they contact the chromosomes or interact with other microtubules. Microtubule minus ends are focused at two unique positions referred to as spindle poles. The spindle poles are the functional sites to which the sister chromatids will segregate during anaphase. Recent evidence has shown that microtubule minus ends are released and/or severed from the primary site of nucleation associated with the centrosomes. Following dissociation from the centrosome, a collection of structural and motor proteins work together to tightly focus microtubule minus ends at the spindle pole. We have devised a cell free assay that faithfully reproduces the centrosome-independent aspects of spindle pole organization. Combining this assay with in vivo approaches to monitor spindle organization provide my laboratory with a unique opportunity to characterize the proteins and mechanisms involved in spindle pole organization and function in mammalian cells. It is the goal of this project to identify proteins involved in focusing microtubule minus ends at spindle poles and to use both in vitro and in vivo biochemical experiments to dissect the molecular mechanisms involved in this essential process. The specific aims of this research are to: 1) determine the dynamics of the association of NuMA with microtubule minus ends at spindle poles; 2) characterize the functional properties of a large complex that contains the minus end-directed microtubule motor protein HSET; and 3) perform a molecular dissection of spindle pole components using microtubule asters assembled in a cell free mitotic extract.