Kinetochore Specification and Function

描述（由申请人提供）：在细胞分裂过程中，将复制的基因组精确地分配给子细胞对于所有生物体的发育和繁殖至关重要。基因组分布的错误是癌细胞的一个标志，参与这一过程的机制是癌症化疗的目标。因此，阐明确保准确基因组分布的机制将有助于我们了解癌症的起源，并指导新的治疗策略的发展。在基因组分布中起主要作用的是着丝点，它在有丝分裂过程中建立在染色体的着丝粒区域上，与纺锤体微管产生动态的端偶联界面。这个界面的机制与一个被称为纺锤体检查点的信号通路紧密结合，它阻止细胞周期的进展，直到所有染色体都连接到纺锤体上。微管结合和检查点信号通过保守的Knl1/Mis12复合体/Ndc80复合体（KMN）蛋白网络在着丝点协调。在该蛋白中，Ndc80复合体直接与微管末端相互作用。本文提出的工作有三个目标：1)阐明确保ndc80介导的着丝点-微管附件准确形成的机制；2)定义核心检查点途径组分与着丝点无关的基本功能；3)确定增强内聚疲劳是否是靶向癌细胞分裂的可行策略。这些目标将分为4个具体目标。目的1将重点了解ndc80介导的末端偶联附着物的形成，该附着物通过着丝酶定位的动力蛋白（细胞中主要的负端定向微管马达）对微管的侧向捕获而加速。我们将研究着丝点动力蛋白模块和Ndc80复合体之间的串扰，并确定其对染色体分离保真度的意义。目的2将解决保守的Bub1激酶和Ndc80复合物亚基Nuf2在着丝点-微管附着物形成中的基本功能。基于我们最近的发现，Aim 3将关注纺锤体检查点蛋白Mad1和Mad2的基本功能，这些功能独立于它们在着丝点检查点信号传导中的作用。本研究目的是利用Mad1/Mad2复合体在生物体背景下的基于机制的工程来阐明线虫中与Mad1/Mad2缺失相关的严重发育和生育缺陷的基础，我们已经证明这些缺陷与着丝点定位依赖的检查点信号信号无关。最后，Aim 4建立在我们的观察上，当有丝分裂延长时，附着在纺锤体上的染色体的姐妹染色单体最终以不协调的方式脱胶；这使细胞处于一种异常状态，称为内聚疲劳，它无法恢复。我们将广泛评估癌细胞系中黏合疲劳的倾向是否会增加，正如初步数据所表明的那样，并确定增强黏合疲劳是否可以作为靶向癌细胞分裂的策略。

DESCRIPTION (provided by applicant): Accurate partitioning of the replicated genome to daughter cells during cell division is essential for the development and propagation of all living organisms. Errors in genome distribution are a hallmark of cancer cells and the machinery involved in this process is targeted in cancer chemotherapy. Thus, elucidating the mechanisms ensuring accurate genome distribution will inform our understanding of the genesis of cancer and guide development of new therapeutic strategies. A major player in genome distribution is the kinetochore, the machine built on the centromere regions of chromosomes during mitosis to generate a dynamic end- coupled interface with spindle microtubules. The mechanics at this interface are tightly integrated with a signaling pathway, termed the spindle checkpoint, which prevents cell cycle progression until all chromosomes are connected to the spindle. Microtubule binding and checkpoint signaling are coordinated at the kinetochore by the conserved Knl1/Mis12 complex/Ndc80 complex (KMN) protein network. Within this protein set, the Ndc80 complex interacts directly with microtubule ends. The work proposed here has three goals: 1) to elucidate the mechanisms ensuring accurate formation of Ndc80-mediated kinetochore-microtubule attachments, 2) to define kinetochore-independent essential functions of core checkpoint pathway components, and 3) to determine if enhancing cohesion fatigue is a viable strategy to target cancer cell division. These goals will be addressed in 4 specific aims. Aim 1 will focus on understanding the formation of Ndc80-mediated end-coupled attachments, which are accelerated by lateral capture of microtubules by kinetochore-localized dynein, the major minus end-directed microtubule motor in cells. The proposed work will investigate crosstalk we discovered between the kinetochore dynein module and the Ndc80 complex and determine its significance to chromosome segregation fidelity. Aim 2 will address poorly understood essential functions of the conserved Bub1 kinase and the Ndc80 complex subunit Nuf2 in the formation of kinetochore- microtubule attachments. Aim 3 will focus on the provocative idea, based on our recent findings, that there are essential functions of the spindle checkpoint proteins Mad1 and Mad2 independent of their role in kinetochore- based checkpoint signaling. This aim exploits mechanism-based engineering of the Mad1/Mad2 complex in an organismal context to elucidate the basis for the severe developmental and fertility defects associated with loss of Mad1/Mad2 in C. elegans, which we have shown are independent of kinetochore-localization dependent checkpoint signaling. Finally, Aim 4 builds on our observation that sister chromatids of chromosomes attached to the spindle ultimately become unglued in an uncoordinated manner when mitosis is prolonged; this traps the cell in an aberrant state, termed cohesion fatigue, from which it cannot recover. We will broadly assess if the propensity for cohesion fatigue is increased in cancer cell lines, as suggested by preliminary data, and determine whether enhancing cohesion fatigue could serve as a strategy to target cancer cell division.