Kinetochore Assembly and Regulation

项目摘要 在染色体分离过程中，每个子细胞都会收到完整的基因组，而这 对每一次细胞分裂都重复进行。因此，染色体分离必须非常准确和 强健，以确保成年人的健康；否则，可能会发生癌症等灾难。癌细胞利用 并重新连接他们的染色体分离机制，以满足他们不受控制的细胞分裂的永无止境的需求。 成功的化疗药物通过破坏这种固有需求来杀死癌细胞。因此，理解 染色体分离的机制对人类健康有着深远的影响。 着丝粒通过将染色体着丝粒连接到纺锤体来执行染色体分离 微管。这种连接必须是灵活的，以适应DNA聚合酶的短暂通过 在S阶段复制着丝粒；它也必须很强，才能承受纺锤的拉力 有丝分裂过程中的微管。细胞在时间上协调这些相互对立的动粒属性，并调节 它们之间的过渡。因为动蛋白和它们相关的调节器在 真核生物的生命王国，我们将使用酵母酿酒酵母作为主要研究生物 来研究动感神经是如何组装的。中心假设是动粒组装是一种高度的 由细胞控制的涉及多个蛋白质-蛋白质和蛋白质-DNA接触的合作过程 循环信号。为了了解运动中枢是如何组装的，特定目标1将应用定量的 确定动粒组装步骤的蛋白质组学平台；利用稳定同位素为基础的质量 光谱学(MS)分析原生动点以及由 浓缩的细胞提取物。管理着动粒组装的两个关键接口：第一个是在 着丝粒和内着丝点，第二个在内着丝点和外着丝点之间。特定的 目标2将探索着丝粒-内部动粒界面，并重点研究特定内部的磷酸化如何 动粒成分可能会对其进行调节。具体目标3将剖析内外动粒界面和 研究它的细胞周期控制，最终目标是重建保持其生理功能的动粒 属性。总而言之，这些研究的目的是了解动觉中枢是如何组装的。 理解动粒组装具有广泛的相关性，因为研究的规则和方法适用于所有人 集成信号以控制大分子组件的系统。我们的协作团队，配备了 拥有跨学科的专业知识和对动毛虫生物学的共同兴趣，是唯一有资格进行 建议的项目，并在这一领域取得有相当大生物医学意义的有影响力的进展。

Project Summary During chromosome segregation, each daughter cell receives a complete complement of the genome, and this is repeated for every cell division. Therefore, chromosome segregation must be extraordinarily accurate and robust to ensure the health of an adult human; otherwise, disasters like cancers can occur. Cancer cells exploit and rewire their chromosome segregation machinery to meet their insatiable need of uncontrolled cell division. Successful chemotherapeutic drugs kill cancer cells through disrupting this obligate need. Thus, understanding mechanisms of chromosome segregation has far-reaching implications to human health. Kinetochores execute chromosome segregation by connecting chromosomal centromeres to spindle microtubules. This connection must be flexible to accommodate the fleeting passage of the DNA polymerases that replicate centromeres during the S phase; it must also be strong to withstand the pulling force of spindle microtubules during mitosis. Cells coordinate these opposing attributes of kinetochores temporally and regulate the transition between them. Because kinetochores and their associated regulators are highly conserved among the eukaryotic kingdoms of life, we will use the yeast Saccharomyces cerevisiae as a primary research organism to study how kinetochores are assembled. The central hypothesis is that kinetochore assembly is a highly cooperative process that involves multiple protein-protein and protein-DNA contacts, which are controlled by cell cycle signals. To understand how kinetochores are assembled, Specific Aim 1 will apply a quantitative proteomics platform to define the steps of kinetochore assembly; making use of stable isotope based mass spectrometry (MS) to analyze native kinetochores as well as reconstituted kinetochores assembled from concentrated cell extracts. Two key interfaces govern kinetochore assembly: the first one is between centromeres and inner kinetochores, while the second one is between inner and outer kinetochores. Specific Aim 2 will probe the centromere-inner kinetochore interface and focus on how phosphorylation of specific inner kinetochore components may regulate it. Specific Aim 3 will dissect the inner-outer kinetochore interface and study its cell cycle control with an ultimate goal of reconstituting the kinetochore that retains its physiological properties. All together, these studies are aimed at understanding how kinetochores are assembled. Understanding kinetochore assembly has broad relevance, because the rules and methods of study apply to all systems in which signals are integrated to control macromolecular assemblies. Our collaborative team, equipped with interdisciplinary expertise and shared interest in kinetochore biology, is uniquely qualified to carry out the proposed projects and to make impactful advance in this area of considerable biomedical significance.