Investigating the regulation and mechanism of tension-sensors Stu2 & Ndc80c

项目摘要 正确的染色体分离是维持真核生物基因组的关键，但其分子机制 这一根本过程背后的原因仍不清楚。这一过程必须以绝对的保真度进行，因为 当它出错时，就会产生有害的非整倍体。非整倍体是许多不同癌症的统一标志。 类型，在那里它似乎是一种治疗的脆弱性。了解黄曲霉毒素的分子机制 染色体分离将使人们深入了解非整倍体是如何发生的，以及如何防止它。我们的 研究的目的是通过探索染色体分离的规律和机制来了解染色体分离的规律和机制 负责分离染色体的蛋白质因子的功能。为了进行适当的染色体分离 发生时，复制的染色体必须附着在起源于相反细胞极的微管上。 当染色体以正确的方式从相对的两极附着在微管上时，就会产生一种很大的力 是生成的。相比之下，染色体错误地附着在同一细胞极的微管上 感受到了低力。动粒复合体中的蛋白质，形成染色体之间的连接 和微管，感知这些力并对其做出反应。动心点稳定“正确”的高力附件 并通过未知机制破坏“不正确的”低力附着物的稳定性，这一活动对 适当的染色体分离。过去的工作表明，两个保守的真核因子Stu2和ITS 动粒受体Ndc80复合体(Ndc80c)是张力感应活动所必需的，我们开始 了解这些因素的张力传感机制。利用结构和生化手段，我们 鉴定了Stu2和Ndc80c之间的物理相互作用，并表明这些蛋白质必须相互作用 在酵母中进行适当的染色体分离。这些发现是最近发表的一篇文章的主题。在这 提案，我们将确定Stu2和Ndc80c是如何被监管以控制张力传感的，我们将调查 张力传感机理受这些因素的影响。我们将分析磷酸化对Stu2-Ndc80c的影响 酵母中的结合和活性。初步数据显示，Stu2在Ndc80c结合位点附近被磷酸化，并且 这种磷酸化可能影响Ndc80c的结合和张力感觉。对Stu2-的进一步拓扑分析- NDC80C组件指向细胞所需的这些因素之间的额外交互界面 生存能力和可能的张力感应。我们还将研究Stu2-Ndc80c的张力传感机制 通过重建动粒微管界面并用生物物理方法探测它。这些加在一起 方法将揭示重要的机械细节，这些细节即使不是不可能，也是很难通过其他方法获得的 实验手段。我们过去的工作和初步数据使我们能够在这些调查中取得成功。两者都有 Stu2和Ndc80c在人类癌症中发生突变，在它们的结合界面发现了几个突变，以及 了解这些机制可能会提供新的治疗策略。总的来说，这项工作将增加我们的 了解染色体分离以及当这个过程出错时非整倍体是如何导致的。

Project Summary Proper chromosome segregation is vital for maintenance of eukaryotic genomes, yet the molecular mechanisms underlying this fundamental process remain unclear. This process must occur with absolute fidelity as detrimental aneuploidies result when it goes awry. Aneuploidy is a unifying hallmark of many different cancer types, where it appears to be a therapeutic vulnerability. Understanding the molecular mechanisms of chromosome segregation would give insight into how aneuploidy occurs and how it might be prevented. Our research goal is to understand the regulation and mechanisms of chromosome segregation by probing the function of protein factors responsible for segregating chromosomes. For proper chromosome segregation to occur, duplicated chromosomes must become attached to microtubules originating from opposite cell poles. When a chromosome becomes attached to microtubules from opposing poles in a correct manner, a high force is generated. By contrast, a chromosome attached incorrectly to microtubules from the same cell pole experiences low force. Proteins in the kinetochore complex, which forms the attachment between chromosomes and microtubules, sense and respond to these forces. Kinetochores stabilize “correct” high-force attachments and destabilize “incorrect” low-force attachments through unknown mechanisms, and this activity is vital for proper chromosome segregation. Past work had shown that two conserved eukaryotic factors, Stu2 and its kinetochore receptor the Ndc80 complex (Ndc80c), are required for tension-sensing activity, and we set out to understand the tension-sensing mechanisms of these factors. Using structural and biochemical means, we characterized the physical interaction between Stu2 and Ndc80c and showed that these proteins must interact for proper chromosomes segregation in yeast. These findings are the subject of a recent publication. In this proposal, we will determine how Stu2 and Ndc80c are regulated to control tension-sensing and we will investigate the tension-sensing mechanism by these factors. We will analyze the effects of phosphorylation on Stu2-Ndc80c binding and activity in yeast. Preliminary data showed Stu2 is phosphorylated near the Ndc80c binding site, and that this phosphorylation may affect Ndc80c binding and tension-sensing. Further topology analysis of the Stu2- Ndc80c assembly pointed to an additional interaction interface between these factors that is required for cell viability and possibly tension-sensing. We will also investigate the tension-sensing mechanism of Stu2-Ndc80c by reconstituting the kinetochore microtubule interface and probing it with biophysical methods. These combined approaches will reveal important mechanistic details that are difficult, if not impossible, to obtain by other experimental means. Our past work and preliminary data prime us to be successful in these investigations. Both Stu2 and Ndc80c are mutated in human cancers, with several mutations found at their binding interface, and understanding these mechanisms may provide novel treatment strategies. Overall, this work will increase our understanding of chromosome segregation and how aneuploidy results when this process goes wrong.