Kinetochore Specification and Function

描述(申请人提供)：真核细胞的染色体分离需要动点，即细胞器聚集在浓缩的染色体上，形成纺锤体微管的动态附着点。动粒功能的错误会导致肿瘤发生过程中染色体的不稳定，也可能导致出生缺陷。动粒成分因其在细胞分裂中的特殊作用，成为抗有丝分裂化疗的靶点。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力化学功能转化为生化分析的困难等因素的限制。两个中心悬而未决的问题是：(1)染色体上组装着动粒的局部区域是如何指定的？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的界面的形成，从而指导染色体分离？这项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期线虫胚胎为分析基本基因产物的功能提供的独特途径，这些基因产物与基因组学和生物化学相结合，迅速为动粒提供全面的成分清单。此外，目前的分析方法提供了一个强大的分类方案，可以将新鉴定的和已知的蛋白质归入特定的功能基团，并确定它们在动粒亚结构中的作用位置。着丝粒规范与CENP-A密切相关，CENP-A是着丝粒特异的组蛋白H3变体。第一个具体目标是确定线虫受精后CENP-A沉积的机制，特别是区分遗传和从头沉积。第二个目标将继续这一主题，以分析一种被功能基因组学确定为可能在CENP-A靶向和组织中发挥核心作用的蛋白质。第三个目标将集中在一个保守的多亚基复合体上，基因组学和生物化学相结合的方法确定它在传播启动动粒组装以形成与纺锤体微管的界面方面发挥关键作用。最终的目标将集中在其他保守的外部动粒蛋白上，以确定它们在体内与纺锤体微管界面上的特定作用，并在体外研究它们与微管的相互作用。

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.