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

研究张力传感器 Stu2 的调节和机制

• 批准号: 10605085
• 负责人: MICHAEL STEWART
• 金额: $ 4.14万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605085
• 关键词: Adult; Affect; Affinity; Anaphase; Aneuploidy; Binding; Binding Sites; Biochemical; Biological Assay; Biophysics; Cell Survival; Cell division; Cells; Chromosome Segregation; Chromosomes; Complex; DNA; Data; Defect; Development; Disease; Ensure; Face; Genetic; Genome; Goals; Human; In Vitro; Investigation; Kinetochores; Location; Maintenance; Malignant Neoplasms; Microtubules; Mitosis; Mitotic; Molecular; Molecular Conformation; Mutate; Mutation; Phenotype; Phosphorylation; Phosphotransferases; Positioning Attribute; Post-Translational Protein Processing; Process; Protein Region; Proteins; Publications; Publishing; Recombinant Proteins; Regulation; Research; Saccharomyces cerevisiae; Sirolimus; Sister Chromatid; System; Testing; Therapeutic; Work; Yeasts; biophysical analysis; biophysical techniques; cancer type; daughter cell; experience; experimental study; genetic information; in vivo; insight; intermolecular interaction; mechanical force; model organism; mutant; novel; optic trap; optical traps; prevent; protein complex; protein function; receptor; reconstitution; response; segregation; sensor; tool; treatment strategy

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.

项目摘要 正确的染色体分离对真核生物基因组的维持至关重要，但其分子机制 这一基本进程的基础仍不清楚。这一过程必须以绝对的保真度进行， 当它出错时会导致有害的非整倍性。非整倍体是许多不同癌症的统一标志 类型，它似乎是一个治疗的弱点。了解的分子机制 染色体分离将使我们深入了解非整倍性是如何发生的，以及如何预防。我们 研究目的是通过探索染色体分离的调控机制， 负责分离染色体的蛋白质因子的功能。为了正确的染色体分离， 当染色体发生复制时，复制的染色体必须附着在来自相反细胞两极的微管上。 当一个染色体以正确的方式从相反的两极附着在微管上时， 生成。相比之下，一条染色体错误地附着在同一细胞极的微管上， 力量小。着丝粒复合体中的蛋白质，形成染色体之间的附着 和微管，感知并响应这些力量Kinetochores稳定“正确的”高力附件 并通过未知的机制破坏“不正确的”低力附件，这种活动对于 染色体分离。过去的研究表明，两个保守的真核生物因子，Stu 2和其 动粒受体Ndc 80复合物（Ndc 80 c）是张力感应活性所必需的，我们开始研究 了解这些因素的张力感知机制。利用结构和生物化学手段，我们 表征了Stu 2和Ndc 80 c之间的物理相互作用，并表明这些蛋白质必须相互作用， 在酵母中正确的染色体分离。这些发现是最近出版物的主题。在这 建议，我们将确定如何调节Stu 2和Ndc 80 c来控制张力传感，我们将调查 张力感应机制受到这些因素的影响。我们将分析磷酸化对Stu 2-Ndc 80 c的影响， 结合和活性。初步数据显示Stu 2在Ndc 80 c结合位点附近被磷酸化， 这种磷酸化可能会影响Ndc 80 c的结合和张力感应。进一步的拓扑分析Stu 2- Ndc 80 c组装体指出了这些因子之间的额外相互作用界面，这是细胞生长所必需的。 可行性和可能的张力感应。我们还将探讨Stu 2-Ndc 80 c的张力传感机理 通过重组动粒微管界面并用生物物理方法探测它。这些组合 方法将揭示重要的机械细节，这些细节很难，如果不是不可能的话，通过其他方法获得。 实验手段。我们过去的工作和初步数据使我们能够成功地进行这些调查。两 Stu 2和Ndc 80 c在人类癌症中突变，在其结合界面处发现了几个突变， 了解这些机制可以提供新的治疗策略。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如何导致非整倍体。