Kinetochore Specification and Function

描述(由申请人提供)：在细胞分裂过程中，准确地将复制的基因组分割成子细胞，对于所有活着的生物体的发育和繁殖是至关重要的。基因组分布错误是癌细胞的一个标志，而参与这一过程的机制是癌症化疗的目标。因此，阐明确保基因组准确分布的机制将有助于我们理解癌症的发生，并指导新的治疗策略的发展。基因组分布的主要参与者是着丝粒，这是一种在有丝分裂期间建立在染色体着丝粒区域上的机器，以产生与纺锤体微管的动态末端耦合界面。这一界面上的机制与称为纺锤体检查点的信号通路紧密结合，该通路阻止细胞周期进展，直到所有染色体都连接到纺锤体。Knl1/Mis12复合体/Ndc80复合体(KMN)蛋白网络在着丝粒上协调微管结合和检查点信号传递。在这组蛋白质中，Ndc80复合体直接与微管末端相互作用。这项工作有三个目标：1)阐明确保Ndc80介导的动粒-微管连接的准确形成的机制，2)确定核心检查点通路组件的动粒非依赖的基本功能，以及3)确定增强凝聚力疲劳是否是靶向癌细胞分裂的可行策略。这些目标将在4个具体目标中得到解决。目的1将重点了解Ndc80介导的末端偶联连接的形成，它是通过动粒定位的动力蛋白侧向捕获微管而加速的，动力蛋白是细胞中主要的负端导向微管马达。这项拟议的工作将调查我们发现的动粒动力蛋白模块和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将阐述鲜为人知的保守的Bub1激酶和Ndc80复合亚单位Nuf2在动粒-微管连接形成过程中的基本功能。根据我们最近的发现，目的3将重点放在挑衅性的想法上，即纺锤体检查点蛋白MAD1和MAD2的基本功能独立于它们在基于动粒的检查点信号中的作用。本研究的目的是利用在生物体环境中对Mad1/MAD2复合体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不依赖于动粒定位依赖的检查点信号。最后，目标4建立在我们的观察基础上，当有丝分裂延长时，附着在纺锤体上的染色体姐妹染色单体最终会以不协调的方式脱胶；这会使细胞处于一种异常状态，称为凝聚力疲劳，无法从中恢复。我们将广泛评估癌细胞系的凝聚力疲劳倾向是否如初步数据所表明的那样增加，并确定增强凝聚力疲劳是否可以作为针对癌细胞分裂的一种策略。

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.