Kinetochore Function in Vertebrate Cells

 描述(申请人提供)：动粒是在着丝粒染色质上组装的大型蛋白质结构，在有丝分裂过程中为染色体分离提供动力并调节染色体分离。正常功能的动粒对于有丝分裂细胞分裂和维持人类健康是必不可少的，因为染色体分离缺陷是导致出生缺陷形成和癌症发生和发展的原因之一。动点被认为是有丝分裂的“管弦乐团”，原因有几个。动粒在物理上将有丝分裂染色体连接到纺锤体微管(MTS)，并通过这种连接传递力量到国会染色体；它们调节染色体和MTS之间的结合强度，以便纠正不适当的附着；最后，它们通过调节纺锤体组装检查点(SAC)，确保如果染色体无法附着或错误附着到MTS，细胞不会退出有丝分裂，该检查点监控着动粒-微管附着，并防止在这种不利条件下的后期。NDC80复合体在附着的产生和调节中起着核心作用：它是动粒和MT之间的主要联系，其Hec1亚单位被必需的Aurora B激酶(ABK)磷酸化调节着动粒-MT的附着强度。这项研究的目的是了解动粒-MT连接在有丝分裂过程中是如何被精确调控的，并确定动粒-MT的稳定性如何与SAC信号联系在一起。这些问题将通过三个目标加以解决。第一个目的将研究NDC80介导的动粒-MT连接如何受到磷酸化和去磷酸化的调节，以及SAC如何检测稳定的NDC80-MT连接。采用体内、体外和电子方法相结合的方法，将确定在有丝分裂过程中控制Hec1去磷酸化的机制，ABK和非ABK有丝分裂酶对Hec1磷酸化的贡献，以及Hec1如何调节上游SAC激活剂的招募和驱逐。在第二个目标中，ABK介导的动粒-MT连接稳定性的调节机制将通过建立令人兴奋的初步数据来解决，该数据表明，包括HEC1在内的动粒底物是由专门招募到动粒的ABK群体磷酸化的，而不是由内部着丝粒的ABK群体磷酸化的。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和核小体阵列的体外重建分析。在第三个目标中，将研究动粒支架蛋白KNL1的未知角色。将确定KNL1如何招募SAC消音器和激活剂，以及最近发现的KNL1相互作用蛋白的功能。综上所述，这项建议将解决如何在有丝分裂过程中调节和监测动粒-MT连接，以确保使用多种方法准确地分离染色体，包括基于细胞的定量高分辨率和超分辨率显微镜、生化重建、单分子生物物理分析和数学建模。

 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.