Elucidating the mechanisms of kinetochore assembly initiation

摘要/摘要 在细胞分裂期间复制的遗传物质的精确分离是发生、发育所必需的 以及所有有机体的生存。这种复制的遗传物质或染色体的分离取决于 正确的时间和位置连接到一个保守的百万吨大小的蛋白质网络，称为 动毛虫。一旦附着在着丝粒上，复制的染色体就被拉开，均匀分布。 在细胞分裂后转移到产生的子代细胞。这一过程中的错误可能会导致错误的快速积累- 分离的染色体导致一种称为非整倍体的细胞状况，这是癌细胞的标志。至 确保高效的动粒连接产生适当的染色体分离、起始和 动粒组装的维持在细胞中受到严格的调控。尽管动毛虫的高度保护 真核生物中的蛋白质支架，启动和调节这一过程的基本机制是 不是很清楚。这项提议旨在使用一种融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 调节动粒起始的磷酸化位点。使用最新开发的实时技术 本项目利用共定位光谱技术监测酿酒酵母的动粒组装 将首先绘制出动粒组装启动的精确动力学和调控图。这将会实现的 通过监测动粒形成的第一步，组蛋白变异蛋白Cse4沉积到 着丝粒DNA的实时检测。同时，这个项目将依赖于一种新的技术，即从新组装本机 着丝粒DNA上的运动中枢，以确定磷酸化及其相关调节的作用 Ce4沉积和动粒组装启动的机制。总之，这些研究将严格地 在分子细节上确定动粒组装是如何启动的。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，这些机制对 未来治疗这种毁灭性疾病的疗法的发展。通过指导和合作 在这个奖学金的帮助下，我将在动粒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悬而未决的问题。这次培训，再加上我的经历 在我对重组蛋白质、遗传密码扩展和单分子显微镜的研究生学习中， 将提供一个研究基础，以便我将准备进行专注于以下方面的独立研究 阐明细胞中调节有丝分裂纺锤体功能以驱动染色体分离的机制 组织。此外，弗雷德·哈钦森癌症研究中心是拟议中的 由于获得领先的技术和资源以及高度互动的科学环境而进行的研究 与周围的生物化学和生物物理学专家一起。

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.