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.

项目概要 在染色体分离过程中，每个子细胞都会接收到完整的基因组，并且这 每次细胞分裂都会重复。因此，染色体分离必须非常准确并且 坚固以确保成年人的健康；否则，就会发生癌症等灾难。癌细胞利用 并重新连接它们的染色体分离机制，以满足不受控制的细胞分裂的永不满足的需求。 成功的化疗药物通过破坏这种必然需求来杀死癌细胞。因此，了解 染色体分离机制对人类健康具有深远影响。 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。这种连接必须灵活，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，能够承受主轴的拉力 有丝分裂期间的微管。细胞暂时协调动粒的这些相反属性并调节 它们之间的过渡。因为着丝粒及其相关调节因子在生物体中高度保守 在真核生命王国中，我们将使用酿酒酵母作为主要研究生物体 研究动粒如何组装。中心假设是动粒组装是高度 涉及多个蛋白质-蛋白质和蛋白质-DNA 接触的合作过程，这些接触由细胞控制 循环信号。为了了解动粒是如何组装的，具体目标 1 将应用定量方法 蛋白质组学平台定义着丝粒组装的步骤；利用基于稳定同位素的质量 光谱法 (MS) 用于分析天然动粒以及由重组体组装的动粒 浓缩细胞提取物。两个关键接口控制着丝粒组装：第一个接口位于 着丝粒和内着丝粒，而第二个位于内着丝粒和外着丝粒之间。具体的 目标 2 将探测着丝粒-内着丝粒界面，并重点关注特定内着丝粒的磷酸化如何 着丝粒成分可能对其进行调节。具体目标 3 将剖析内外着丝粒界面并 研究其细胞周期控制，最终目标是重建保留其生理功能的动粒 特性。总而言之，这些研究旨在了解动粒是如何组装的。 了解动粒组装具有广泛的相关性，因为研究规则和方法适用于所有 集成信号以控制大分子组装的系统。我们的协作团队，装备精良 拥有跨学科的专业知识和对着丝粒生物学的共同兴趣，具有独特的资格来开展 拟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。