Kinetochore Assembly and Regulation

着丝粒组装和调控

• 批准号: 10717202
• 负责人: Arshad Desai
• 金额: $ 44.35万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10717202
• 关键词: Accounting; Acute; Address; Adult; Aneuploidy; Area; Auxins; Benchmarking; Biochemical; Biochemistry; Biological Assay; Biology; Cell Cycle; Cell Cycle Regulation; Cell Extracts; Cell division; Cells; Cellular biology; Centromere; Characteristics; Chromosome Segregation; Complement; Complex; Congenital Abnormality; DNA; DNA-Directed DNA Polymerase; Diagnosis; Disasters; Ensure; Event; Genetic; Genome; Goals; Health; Human; Isotopes; Kinetochores; Life; Malignant Neoplasms; Mass Spectrum Analysis; Mental disorders; Methods; Microtubules; Mitosis; Molecular; Nucleosomes; Organism; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Physiological; Process; Property; Protein Degradation Induction; Proteins; Proteomics; Qualifying; Reaction; Regulation; Research; Role; S phase; Saccharomyces cerevisiae; Signal Transduction; Site; Specific qualifier value; System; Techniques; Testing; Therapeutic; Therapeutic Intervention; Yeasts; cancer cell; daughter cell; experimental study; flexibility; innovation; interest; macromolecular assembly; reconstitution; stable isotope; stoichiometry; technology platform

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.

项目摘要 在染色体分离过程中，每个子细胞都得到了完整的基因组， 每一次细胞分裂都是如此因此，染色体分离必须非常精确， 健康，以确保成年人的健康;否则，可能发生癌症等灾难。癌细胞利用 并重新连接它们的染色体分离机制，以满足它们对不受控制的细胞分裂的贪得无厌的需求。 成功的化疗药物通过破坏这种专性需求来杀死癌细胞。因此，理解 染色体分离机制对人类健康有着深远的影响。 动粒通过将染色体着丝粒连接到纺锤体上来完成染色体分离 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期复制着丝粒;它还必须坚固以承受纺锤体的拉力 有丝分裂时的微管。细胞在时间上协调这些相反的着丝粒属性， 他们之间的过渡。由于着丝粒及其相关的调节因子在 真核生物界的生命，我们将使用酵母酿酒酵母作为主要的研究生物体 来研究动粒是如何组装的中心假设是动粒组装是一个高度的 由细胞控制的蛋白质-蛋白质和蛋白质-DNA多重接触的协同过程 循环信号为了理解着丝粒是如何组装的，具体目标1将应用定量分析。 蛋白质组学平台来定义动粒组装的步骤;利用基于稳定同位素的质量 用质谱法（MS）分析天然动粒以及由 浓缩的细胞提取物。两个关键的接口控制动粒组装：第一个是在 第二种介于内着丝粒和外着丝粒之间。具体 Aim 2将探索着丝粒-内动粒界面，并关注特定的内动粒的磷酸化是如何发生的。 具体目标3将解剖内外动粒界面， 研究其细胞周期控制，最终目标是重建动粒， 特性.总之，这些研究旨在了解动粒是如何组装的。 理解动粒装配具有广泛的相关性，因为研究的规则和方法适用于所有 集成信号以控制大分子集合的系统。我们的协作团队，配备 具有跨学科的专业知识和共同的兴趣，在动粒生物学，是唯一有资格进行 建议的项目，并在这一具有重大生物医学意义的领域取得有影响力的进展。