Kinetochore Specification and Function

着丝粒规格及功能

• 批准号: 6910352
• 负责人: Arshad Desai
• 金额: $ 27.49万
• 依托单位: LUDWIG INSTITUTE FOR CANCER RES LTD
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6910352
• 关键词: Caenorhabditis elegans; cell component structure /function; cell cycle; centromere; chromosomes; fertilization; functional /structural genomics; histones; immunologic assay /test; intermolecular interaction; laboratory rabbit; microtubules; mitotic spindle apparatus; molecular assembly /self assembly; protein localization

DESCRIPTION (provided by applicant): Eukaryotic chromosome segregation requires kinetochores, organelles that assemble on condensing chromosomes to form dynamic attachment sites for spindle microtubules. Errors in kinetochore function contribute to chromosomal instability during tumorigenesis and potentially also to birth defects. Because of their specific roles in cell division, kinetochore components are attractive targets for anti-mitotic chemotherapy. Molecular analysis of kinetochores in metazoans has been limited by their essential nature, rarity of their constituents, and difficulties in translating mechanochemical functions into biochemical assays. Two central unanswered questions are: (1) how is the localized region of the chromosome where the kinetochore assembles specified? and (2) how is the initiation of kinetochore assembly translated into the formation of an interface that interacts with spindle microtubules to direct chromosome segregation? The goal of the proposed work is to address these questions using functional analysis in the C. elegans embryo, and extend some of the studies to human cells. The work will capitalize on the unique access provided by the one-cell stage C. elegans embryo for analyzing the function of essential gene products which, in combination with genomics and biochemistry, is rapidly providing a comprehensive component list for kinetochores. In addition, current assays provide a powerful classification scheme to place newly identified as well as known proteins into specific functional groups and to define their site of action within the substructure of the kinetochore. Kinetochore specification is intimately associated with CENP-A, a centromere-specific histone H3 variant. The first specific aim is directed towards defining the mechanism of CENP-A deposition following fertilization in C. elegans, particularly to distinguish between inheritance versus de novo deposition. The second aim will continue this theme to analyze a protein identified by functional genomics as a likely central player in CENP-A targeting and organization. The third aim will focus on a conserved multi-subunit complex identified by a combination of genomics and biochemistry as playing a key role in propagating initiation of kinetochore assembly to form the interface with spindle microtubules. The final aim will focus on other conserved outer kinetochore proteins to define their specific roles at the interface with spindle microtubules in vivo and to investigate their interactions with microtubules in vitro.

描述（由申请人提供）：真核生物染色体分离需要着丝点，细胞器聚集在凝聚的染色体上，形成纺锤体微管的动态附着位点。着丝点功能的错误有助于肿瘤发生期间染色体的不稳定，也可能导致出生缺陷。由于它们在细胞分裂中的特殊作用，着丝点成分是抗有丝分裂化疗的有吸引力的靶点。后生动物中着丝点的分子分析受到其本质、成分罕见以及将机械化学功能转化为生化分析的困难的限制。两个悬而未决的核心问题是：(1)染色体中着丝点组装的局部区域是如何指定的？(2)着丝点组装的起始如何转化为与纺锤体微管相互作用以指导染色体分离的界面的形成？这项工作的目标是利用秀丽隐杆线虫胚胎的功能分析来解决这些问题，并将一些研究扩展到人类细胞。这项工作将利用单细胞阶段秀丽隐杆线虫胚胎提供的独特途径来分析基本基因产物的功能，结合基因组学和生物化学，迅速提供一个完整的着丝点成分清单。此外，目前的分析提供了一个强大的分类方案，将新鉴定的和已知的蛋白质放入特定的功能基团中，并确定它们在着丝点亚结构中的作用位点。着丝粒规格与着丝粒特异性组蛋白H3变体CENP-A密切相关。第一个具体目标是确定秀丽隐杆线虫受精后CENP-A沉积的机制，特别是区分遗传与新生沉积。第二个目标将继续这一主题，分析一种由功能基因组学鉴定的蛋白质，该蛋白质可能是CENP-A靶向和组织的核心参与者。第三个目标将集中在一个保守的多亚基复合体上，该复合体由基因组学和生物化学结合鉴定，在着丝粒组装的起始繁殖中起关键作用，从而形成与纺锤体微管的界面。最后的目标将集中在其他保守的外着丝点蛋白上，以确定它们在体内与纺锤体微管界面上的特定作用，并研究它们在体外与微管的相互作用。