Kinetochore Function in Vertebrate Cells

 描述（由申请人提供）：动粒是组装在着丝粒染色质上的大型蛋白质结构，在有丝分裂期间驱动和调节染色体分离。正常发挥功能的着丝粒对于有丝分裂细胞分裂和维持人类健康是必不可少的，因为有缺陷的染色体分离被认为是出生缺陷形成以及癌症发生和发展的致病因素。动粒被认为是有丝分裂的“管弦乐队”有几个原因。动粒将有丝分裂染色体与纺锤体微管（MT）物理连接，并通过这种连接与国会染色体（congress chromosomes）产生反作用力;它们调节染色体与MT之间的结合强度，从而纠正不适当的附着;最后，它们通过调节纺锤体组装检查点（SAC）确保如果染色体未能附着或不正确地附着到MT上，细胞不会退出有丝分裂，其监测着丝粒-MT附着并防止在这种不利条件下的后期。NDC 80复合物在附件的产生和调节中起着核心作用：它是着丝粒和MT之间的主要联系，并且由必需的极光B激酶（ABK）对其Hec 1亚基的磷酸化调节着丝粒-MT附着强度。本研究的目的是了解如何在有丝分裂进程中精确调节kinetochore-MT附件，并确定如何kinetochore-MT稳定性与SAC信号。这些问题将在三个目标中解决。第一个目标将研究如何NDC 80介导的kinetochore-MT连接的磷酸化和去磷酸化的调节，以及如何SAC检测稳定的NDC 80-MT连接。使用体内，体外和计算机模拟方法的组合，在有丝分裂过程中Hec 1去磷酸化的机制将被确定，ABK和非ABK有丝分裂激酶对Hec 1磷酸化的贡献将被映射，以及Hec 1如何调节上游SAC激活剂的募集和驱逐将被确定。在第二个目标中，ABK介导的调节kinetochore-MT附着稳定性的机制将通过建立令人兴奋的初步数据来解决，这些数据表明，动粒底物，包括Hec 1，被特定于动粒招募的ABK群体磷酸化，而不是被内部着丝粒的ABK群体磷酸化。为了确定这种活性的分子基础，将采用细胞生物学方法和使用纯化蛋白质和核小体阵列的体外重建测定。在第三个目标中，将研究动粒支架蛋白KNL 1的未探索的作用。KNL 1如何招募SAC沉默剂和激活剂将被确定，最近发现的KNL 1相互作用蛋白的功能将被表征。总之，该提案将解决在有丝分裂期间如何调节和监测着丝粒-MT附着，以使用多种方法确保准确的染色体分离，包括基于定量细胞的高分辨率和超分辨率显微镜、生化重建、单分子生物物理测定和数学建模。

 DESCRIPTION (provided by applicant): Kinetochores are large protein structures assembled on centromeric chromatin that power and regulate chromosome segregation during mitosis. Properly functioning kinetochores are essential for mitotic cell division and for maintaining human health, as defective chromosome segregation is implicated as a causative factor in the formation of birth defects and in the initiation and progression of cancer. Kinetochores are considered the "orchestrators" of mitosis for several reasons. Kinetochores physically connect mitotic chromosomes to spindle microtubules (MTs) and transduce forces through this connection to congress chromosomes; they regulate the binding strength between chromosomes and MTs so that improper attachments are corrected; and, finally, they ensure that cells do not exit mitosis if chromosomes fail to attach or are incorrectly attached to MTs by regulating the spindle assembly checkpoint (SAC), which monitors kinetochore-MT attachment and prevents anaphase in such unfavorable conditions. The NDC80 complex plays a central role in the generation and regulation of attachments: it is the primary linkage between kinetochores and MTs, and phosphorylation of its Hec1 subunit by the essential Aurora B kinase (ABK) regulates kinetochore-MT attachment strength. The goals of this study are to understand how kinetochore-MT attachments are precisely regulated during mitotic progression and to determine how kinetochore-MT stability is linked to SAC signaling. These issues will be addressed in three Aims. The first Aim will investigate how NDC80-mediated kinetochore-MT attachment is regulated by phosphorylation and dephosphorylation and how the SAC detects stable NDC80-MT connections. Using a combination of in vivo, in vitro, and in silico approaches, the mechanisms governing Hec1 dephosphorylation during mitosis will be determined, the contributions of ABK and non-ABK mitotic kinases to Hec1 phosphorylation will be mapped, and how Hec1 regulates the recruitment and eviction of upstream SAC activators will be determined. In the second Aim, the mechanism for ABK-mediated regulation of kinetochore-MT attachment stability will be addressed by building on exciting preliminary data suggesting that kinetochore substrates, including Hec1, are phosphorylated by a population of ABK recruited specifically to kinetochores, rather than by a population of ABK at inner centromeres. To determine the molecular basis for this activity, cell biological approaches and in vitro reconstitution assays using purified proteins and nucleosome arrays will be employed. In the third Aim, unexplored roles of the kinetochore scaffold protein KNL1 will be investigated. How KNL1 recruits both SAC silencers and activators will be determined, and the functions of recently identified KNL1-interacting proteins will be characterized. In summary, this proposal will address how kinetochore-MT attachments are regulated and monitored during mitosis to ensure accurate chromosome segregation using multiple approaches, including quantitative cell-based high-resolution and super-resolution microscopy, biochemical reconstitution, single- molecule biophysical assays, and mathematical modeling.