Organization of the Mammalian Mitotic Spindle

哺乳动物有丝分裂纺锤体的组织

• 批准号: 6325590
• 负责人: Duane A. Compton
• 金额: $ 27.83万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6325590
• 关键词: antibody; cell free system; cytoskeletal proteins; genetic library; immunoprecipitation; mass spectrometry; microinjections; microtubules; mitotic spindle apparatus; nucleic acid sequence; polymerase chain reaction; protein structure function

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.

描述（申请人摘要的逐字记录）：长期目标 该项目的目的是鉴定蛋白质并表征分子机制 参与哺乳动物纺锤体极微管负端的组织 细胞。准确的染色体分离对于繁殖至关重要 物种和所有细胞的活力。染色体在以下过程中分离 有丝分裂和减数分裂是通过一种复杂的基于微管的上层结构进行的，称为 主轴。纺锤体内的微管的正端延伸到 朝向细胞皮层或细胞赤道，在那里它们接触 染色体或与其他微管相互作用。微管负端是 集中在两个独特的位置，称为主轴极。主轴极数 是姐妹染色单体在过程中分离的功能位点 后期。最近的证据表明微管负端被释放 和/或从与相关的主要成核位点切断 中心体。从中心体解离后，收集 结构蛋白和运动蛋白共同作用，紧密聚焦微管 终止于主轴杆。我们设计了一种无细胞测定法，忠实地 再现纺锤体组织的中心体独立方面。 将该测定与体内方法相结合以监测纺锤体组织 为我的实验室提供了表征蛋白质的独特机会 以及参与哺乳动物纺锤体组织和功能的机制 细胞。该项目的目标是识别参与聚焦的蛋白质 微管负端位于纺锤体极，可在体外和体内使用 生化实验来剖析参与这一过程的分子机制 的必要过程。本研究的具体目的是：1）确定 NuMA 与纺锤体微管负端关联的动力学 电线杆； 2）描述大型复合体的功能特性 含有负端导向的微管运动蛋白HSET； 3）执行 使用微管紫苑对纺锤体极组件进行分子解剖 组装在无细胞有丝分裂提取物中。