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 在染色体隔离期间，每个子细胞都会获得基因组的完整补体，这 为每个细胞分裂重复。因此，染色体隔离必须非常准确，并且 坚固的人，以确保成年人的健康；否则，可能会发生像癌症这样的灾难。癌细胞利用 并将其染色体隔离机制重新连接，以满足他们对不受控制的细胞分裂的无限需求。 成功的化学治疗药物通过破坏这种义务需求而杀死癌细胞。因此，理解 染色体分离的机制对人类健康具有深远的影响。 Kinetochores通过将染色体centromeres连接到主轴来执行染色体隔离 微管。这种连接必须灵活以适应DNA聚合酶的短暂通过 在S阶段复制中心粒；承受主轴的拉力也必须很强大 有丝分裂过程中的微管。细胞在时间上协调这些相反的属性并进行调节 他们之间的过渡。因为动力学及其相关调节器在 真核王国的生活，我们将使用酵母糖酿酒酵母作为主要研究生物 研究如何组装动力学。中心假设是动力学组装是一个高度 涉及多种蛋白质 - 蛋白质和蛋白-DNA接触的合作过程，由细胞控制 周期信号。为了了解动力学如何组装，特定的目标1将应用定量 蛋白质组学平台来定义动力学组装的步骤；利用稳定同位素的质量 光谱法（MS）分析天然动物学和重组的动力学 浓缩细胞提取物。两个关键接口控制动物学组装：第一个是在之间 Centromeres和内部动物学体，而第二个则在内部和外部动物学之间。具体的 AIM 2将探测Centromere-Inner Kinetochore界面，并专注于特定内部的磷酸化 动力学组件可能会调节它。特定的AIM 3将剖析内部的动力学界面和 研究其细胞周期控制，其最终目标是重构保留其生理的动力学 特性。这些研究旨在了解动力学的组装方式。 理解动力学大会具有广泛的相关性，因为研究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。我们的合作团队配备了 凭借跨学科的专业知识和对Kinetochore生物学的共同兴趣，具有独特的资格来执行 拟议的项目，并在这一具有相当大的生物医学意义的领域取得了影响力。