Organization of the Mammalian Mitotic Spindle

哺乳动物有丝分裂纺锤体的组织

• 批准号: 6652913
• 负责人: Duane A. Compton
• 金额: $ 4.08万
• 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/6652913
• 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（来自申请人摘要的逐字记录）：长期目标 这个项目的目的是鉴定蛋白质和表征分子机制 参与哺乳动物纺锤体极微管负末端的组织 细胞准确的染色体分离对烟草的繁殖至关重要 物种和所有细胞的活力。染色体分离的过程中， 有丝分裂和减数分裂由一个复杂的微管为基础的超结构称为 纺锤体。纺锤体内的微管有其正端延伸， 向细胞皮层或细胞赤道移动， 染色体或与其他微管相互作用。微管负末端是 集中在两个独特的位置，称为主轴极。主轴杆 是姐妹染色单体分离的功能位点， 后期最近的证据表明，微管负末端释放， 和/或从与所述聚合物相关联的成核的主要部位切断。 中心体从中心体解离后， 结构蛋白和运动蛋白共同作用， 在主轴杆处结束。我们设计了一种无细胞测定法， 再现了纺锤体极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一个独特的机会来描述 哺乳动物纺锤极组织和功能的机制 细胞本项目的目标是确定参与聚焦的蛋白质 微管负末端在纺锤体极点和使用在体外和体内 生物化学实验，以剖析参与这一过程的分子机制， 必要的过程。本研究的具体目的是：1）确定 NuMA与纺锤体微管负末端结合的动力学 极点; 2）表征一个大的复杂的功能特性， 含有负末端定向微管马达蛋白HSET;和3）进行 用微管星状体对纺锤极成分的分子解剖 在无细胞有丝分裂提取物中组装。