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）：真核细胞染色体分离需要着丝粒，即在浓缩染色体上组装形成纺锤体微管动态附着位点的细胞器。动粒功能的错误导致肿瘤发生过程中染色体的不稳定性，也可能导致出生缺陷。由于它们在细胞分裂中的特定作用，动粒组分是抗有丝分裂化疗的有吸引力的靶点。后生动物中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。有两个中心问题没有得到解答：（1）染色体上着丝粒组装的局部区域是如何确定的？以及（2）动粒组装的启动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？建议的工作的目标是解决这些问题，使用功能分析在C。elegans胚胎，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C提供的独特通道。elegans胚胎的功能分析的必要基因产物，结合基因组学和生物化学，是迅速提供一个全面的成分清单为动粒。此外，目前的测定提供了一个强大的分类方案，将新鉴定的以及已知的蛋白质放入特定的官能团，并定义其在动粒的亚结构内的作用位点。动粒特化与CENP-A密切相关，CENP-A是一种着丝粒特异性组蛋白H3变体。第一个具体目标是定义C受精后CENP-A沉积的机制。elegans，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，作为CENP-A靶向和组织的可能核心参与者。第三个目标将集中在一个保守的多亚基复合物确定的基因组学和生物化学的结合，在传播启动动粒组装，形成与纺锤体微管的接口发挥关键作用。最终的目标将集中在其他保守的外动粒蛋白，以确定其在体内与纺锤体微管的接口的具体作用，并在体外研究它们与微管的相互作用。