Elucidating the mechanisms of kinetochore assembly initiation

阐明着丝粒组装起始机制

• 批准号: 9909687
• 负责人: Andrew R Popchock
• 金额: $ 6.49万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9909687
• 关键词: Address; Aneuploidy; Animal Model; Area; Biochemistry; Biological Assay; Biology; Biophysics; Cancerous; Cell Cycle; Cell Cycle Progression; Cell division; Cells; Centromere; Chromosome Segregation; Chromosomes; Collaborations; Complex; Coupled; Cytoskeletal Filaments; DNA; DNA-Binding Proteins; Deposition; Development; Disease; Ensure; Environment; Eukaryota; Event; Fellowship; Fluorescence Microscopy; Foundations; Fred Hutchinson Cancer Research Center; Future; Generations; Genetic Code; Genetic Materials; Genome Stability; Histones; In Vitro; Kinetochores; Life; Location; Macromolecular Complexes; Maintenance; Malignant Neoplasms; Maps; Mechanics; Mentorship; Methods; Microscopy; Microtubules; Mitotic; Mitotic spindle; Molecular; Molecular Biology; Molecular Chaperones; Monitor; Organism; Phosphorylation; Phosphorylation Site; Phosphotransferases; Planet Earth; Process; Property; Protein Biochemistry; Proteins; Recombinant Proteins; Regulation; Research; Resources; Role; Saccharomyces cerevisiae; Saccharomycetales; Scaffolding Protein; Spectrum Analysis; Techniques; Technology; Testing; Time; Training; Variant; Yeasts; base; cancer cell; cancer initiation; daughter cell; experience; experimental study; imaging platform; in vivo; insight; interdisciplinary approach; macromolecular assembly; molecular imaging; novel; novel therapeutics; protein complex; real time monitoring; segregation; single molecule; spatiotemporal; therapy development; tumor progression; yeast genetics

Summary/Abstract Precise separation of replicated genetic material during cell division is required for the generation, development and survival of all organisms. Segregation of this replicated genetic material, or chromosomes, relies on the correct timing and location of attachment to a conserved megadalton-sized protein network called the kinetochore. Once attached to the kinetochore, duplicated chromosomes are pulled apart to be distributed evenly to resulting daughter cells after cell division. Errors in this process can result in the rapid accumulation of mis- segregated chromosomes resulting in a cellular condition called aneuploidy, a hallmark of cancerous cells. To ensure productive kinetochore attachments that yield proper segregation of chromosomes, the initiation and maintenance of kinetochore assembly is tightly regulated in cells. Despite high conservation of the kinetochore protein scaffold among eukaryotes, the fundamental mechanics of the initiation and regulation of this process are not well understood. This proposal aims to use an interdisciplinary approach that integrates yeast genetics, molecular biology, protein biochemistry, and single-molecule imaging to address several key outstanding questions: to determine the regulation and dynamics of inner kinetochore assembly, and to elucidate key phosphorylation sites that regulate kinetochore initiation. Using a recently developed technique of real-time monitoring of kinetochore assembly in Saccharomyces cerevisiae via colocalization spectroscopy, this project will first map the precise dynamics, and regulation of kinetochore assembly initiation. This will be accomplished by monitoring the first steps of kinetochore formation, deposition of the histone variant protein Cse4 onto centromeric DNA in real-time. In tandem, this project will rely on a novel technique of de novo assembly of native kinetochores on centromeric DNA to determine the role of phosphorylation and associated regulatory mechanisms in Cse4 deposition and kinetochore assembly initiation. Together, these studies will rigorously determine how kinetochore assembly is initiated in molecular detail. Importantly, these details will provide a framework to better understand potential mechanisms of cancer initiation and progression that are critical for future development of therapies to treat this devastating disease. Through the mentorship and collaboration facilitated by this fellowship, I will gain valuable expertise in the field of kinetochore biology as well as an understanding of how to address key outstanding questions in the field. This training, coupled to my experience during my graduate study with recombinant proteins, genetic code expansion, and single molecule microscopy, will provide a research foundation such that I will be prepared to perform independent research focused on elucidating the mechanisms that regulate mitotic spindle function to drive chromosome separation during cell division. Additionally, the Fred Hutchinson Cancer Research Center is an ideal environment for the proposed studies due to access to leading technologies and resources as well as a highly interactive scientific environment with surrounding experts in biochemistry and biophysics.

总结/摘要 在细胞分裂过程中，复制的遗传物质的精确分离是产生、发育 所有生物的生存。这种复制的遗传物质或染色体的分离依赖于 正确的时间和位置连接到一个保守的兆道尔顿大小的蛋白质网络，称为 动粒一旦附着在动粒上，复制的染色体被拉开， 到细胞分裂后产生的子细胞。这一过程中的错误可能会导致错误的快速积累。 分离的染色体导致称为非整倍体的细胞状况，这是癌细胞的标志。到 确保生产性的动粒附件，产生染色体的适当分离，起始和 着丝粒装配的维持在细胞中受到严格调节。尽管动粒高度保守 蛋白质支架在真核生物中，启动和调节这一过程的基本机制是 没有很好地理解。该提案旨在使用跨学科的方法，整合酵母遗传学， 分子生物学、蛋白质生物化学和单分子成像，以解决几个关键的突出问题， 问题：确定内部动粒组装的调节和动力学，并阐明关键 磷酸化位点调节动粒起始。使用最近开发的实时技术 通过共定位光谱学监测酿酒酵母中的动粒组装，该项目 将首先绘制动粒组装启动的精确动力学和调节。这将是完成 通过监测动粒形成的第一步，组蛋白变体蛋白Cse 4沉积到 实时检测着丝粒DNA同时，该项目将依赖于一种新的技术， 着丝粒DNA上的着丝粒，以确定磷酸化和相关的调控作用 Cse 4沉积和动粒组装启动的机制。总之，这些研究将严格 确定动粒组装是如何在分子细节上启动的。重要的是，这些细节将提供一个 框架，以更好地了解癌症发生和发展的潜在机制，这对 治疗这种毁灭性疾病的疗法的未来发展。通过指导和协作， 通过这项奖学金的促进，我将获得动粒生物学领域的宝贵专业知识， 了解如何解决该领域的关键未决问题。这种训练加上我的经验 在我的研究生学习重组蛋白质，遗传密码扩展，和单分子显微镜， 我将提供一个研究基础，这样我将准备进行独立的研究，重点是 阐明了调节有丝分裂纺锤体功能以驱动细胞分裂期间染色体分离的机制， 师.此外，弗雷德哈钦森癌症研究中心是一个理想的环境， 由于获得领先的技术和资源以及高度互动的科学环境， 与周围的生物化学和生物物理学专家一起工作。