Kinetochore Specification and Function

着丝粒规格及功能

• 批准号: 8000170
• 负责人: Arshad Desai
• 金额: $ 7.98万
• 依托单位: LUDWIG INSTITUTE FOR CANCER RES LTD
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-28 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8000170
• 关键词: Address; Binding; Biochemical; Biochemistry; Biological Assay; Caenorhabditis elegans; Cell division; Cells; Centromere; Characteristics; Chromatin; Chromosomal Instability; Chromosome Condensation; Chromosome Segregation; Chromosomes; Classification Scheme; Complex; Congenital Abnormality; Dependence; Deposition; Electron Microscopy; Embryo; Epigenetic Process; Essential Genes; Fertilization; Genome; Genomics; Germ Cells; Goals; Histone H3; Histones; Human; In Vitro; Inheritance Patterns; Kinetochores; Location; Microtubules; Mitotic; Molecular Analysis; Nature; Nucleosomes; Oocytes; Organelles; Outcome; Phenotype; Physical condensation; Play; Proteins; RNA Interference; Research Personnel; Role; Screening procedure; Site; Specific qualifier value; Staging; Structure; Testing; Time; Translating; Variant; Work; base; centromere protein A; chemotherapy; condensin; functional genomics; functional group; in vivo; insight; novel; programs; protein complex; protein function; reconstitution; research study; segregation; tumorigenesis

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）：真核染色体分离需要着丝粒，即在浓缩染色体上组装的细胞器，以形成纺锤体微管的动态附着位点。着丝粒功能的错误会导致肿瘤发生过程中的染色体不稳定，并可能导致出生缺陷。由于其在细胞分裂中的特殊作用，动粒成分是抗有丝分裂化疗的有吸引力的靶标。后生动物动粒的分子分析受到其基本性质、其成分的稀有性以及将机械化学功能转化为生化测定的困难的限制。两个尚未解答的核心问题是：（1）着丝粒组装的染色体局部区域是如何指定的？ （2）着丝粒组装的启动如何转化为与纺锤体微管相互作用的界面的形成以指导染色体分离？拟议工作的目标是利用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必需基因产物的功能，结合基因组学和生物化学，快速提供动粒的全面成分列表。此外，当前的测定提供了强大的分类方案，将新鉴定的以及已知的蛋白质放入特定的功能组中，并定义它们在着丝粒子结构内的作用位点。着丝粒规范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，特别是区分遗传与从头沉积。第二个目标将继续这一主题，分析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。第三个目标将集中于基因组学和生物化学相结合鉴定的保守多亚基复合物，该复合物在传播着丝粒组装起始以形成与纺锤体微管的界面方面发挥着关键作用。最终目标将集中于其他保守的外着丝粒蛋白，以确定它们在体内与纺锤体微管界面的特定作用，并研究它们在体外与微管的相互作用。