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.

摘要/摘要 生成需要精确的细胞分裂遗传物质的精确分离 和所有生物的生存。这种复制的遗传物质或染色体的隔离取决于 正确的时机和附件位于保守的Megadalton大小的蛋白质网络，称为 动力学。一旦连接到动力学，将重复的染色体拉开以均匀分布 在细胞分裂之后导致的子细胞。在此过程中的错误可能导致错误的迅速积累 隔离的染色体，导致细胞状态称为非整倍性，这是癌细胞的标志。到 确保生产性动力学附件可产生适当的染色体隔离，启动和 动力学组装的维护在细胞中受到严格调节。尽管动力学守恒很高 真核生物之间的蛋白质支架，该过程的启动和调节的基本机制是 不太了解。该建议旨在使用整合酵母遗传学的跨学科方法， 分子生物学，蛋白质生物化学和单分子成像，以解决几种关键的未偿还 问题：确定内部动元组装的调节和动力学，并阐明密钥 调节动力学开始的磷酸化位点。使用最近开发的实时技术 通过共定位光谱法监测酿酒酵母中的动脉组合体，该项目 将首先映射动力学组装启动的精确动力学和调节。这将完成 通过监测动力学形成的第一步，组蛋白变体蛋白CSE4的沉积 centromeric DNA实时。在同时，该项目将依靠新颖的本地人组装技术 丝粒DNA上的动力学，以确定磷酸化和相关调节的作用 CSE4沉积和动力学组装启动中的机制。这些研究在一起将严格 确定如何通过分子细节启动动力学组装。重要的是，这些细节将提供 框架更好地了解癌症开始和进展的潜在机制，这对于 对治疗这种毁灭性疾病的疗法的未来发展。通过指导与协作 在这项奖学金的促进的情况下，我将在Kinetochore生物学领域获得宝贵的专业知识以及 了解如何解决该领域的关键问题。这项培训，加上我的经验 在我对重组蛋白，遗传密码扩张和单分子显微镜研究的研究生研究中， 将提供一个研究基金会，以便我准备进行专注于的独立研究 阐明调节有丝分裂纺锤体功能以驱动染色体分离的机制 分配。此外，弗雷德·哈钦森癌症研究中心是拟议的理想环境 由于获得领先的技术和资源以及高度互动的科学环境而导致的研究 周围的生物化学和生物物理学专家。