Elucidating the mechanisms of kinetochore assembly initiation

阐明着丝粒组装起始机制

• 批准号: 10645456
• 负责人: Andrew R Popchock
• 金额: $ 0.52万
• 依托单位: FRED HUTCHINSON CANCER 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/10645456
• 关键词: Elucidating; mechanisms; kinetochore; assembly; initiation

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 细胞分裂过程中复制的遗传物质的精确分离是产生、发育所必需的 以及所有生物体的生存。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保守的兆道尔顿大小的蛋白质网络，称为 动粒。一旦附着到着丝粒上，复制的染色体就会被拉开并均匀分布 细胞分裂后产生的子细胞。此过程中的错误可能会导致错误的快速积累 分离的染色体导致一种称为非整倍性的细胞状况，这是癌细胞的标志。到 确保有效的动粒附着，从而产生染色体的正确分离、起始和 着丝粒组装的维持在细胞中受到严格调节。尽管着丝粒高度保守 真核生物中的蛋白质支架，这一过程的启动和调节的基本机制是 不太理解。该提案旨在采用跨学科方法，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，解决几个关键的突出问题 问题：确定内着丝粒组装的调节和动力学，并阐明关键 调节着丝粒起始的磷酸化位点。使用最近开发的实时技术 通过共定位光谱监测酿酒酵母中着丝粒的组装，该项目 首先将绘制出动粒组装起始的精确动力学和调节图。这将实现 通过监测动粒形成的第一步，组蛋白变异蛋白 Cse4 沉积在 实时着丝粒 DNA。与此同时，该项目将依赖于一种新颖的技术，即原生的从头组装 着丝粒 DNA 上的着丝粒以确定磷酸化和相关调节的作用 Cse4 沉积和着丝粒组装启动的机制。这些研究将共同​​严格 确定着丝粒组装如何在分子细节上启动。重要的是，这些细节将提供 框架，以更好地了解癌症发生和进展的潜在机制，这对于癌症治疗至关重要 未来开发治疗这种毁灭性疾病的疗法。通过指导和合作 在这项奖学金的帮助下，我将获得动粒生物学领域的宝贵专业知识以及 了解如何解决该领域的关键悬而未决的问题。这次培训，结合我的经验 在我对重组蛋白、遗传密码扩展和单分子显微镜进行研究生学习期间， 将提供一个研究基础，以便我准备进行独立研究，重点关注 阐明调节有丝分裂纺锤体功能以驱动细胞过程中染色体分离的机制 分配。此外，弗雷德哈钦森癌症研究中心是拟议的理想环境 研究得益于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。