Uncovering the structural mechanisms of chromosome attachment to the mitotic spindle by SKA/HEC1

通过 SKA/HEC1 揭示染色体附着在有丝分裂纺锤体上的结构机制

• 批准号: 10507380
• 负责人: Anthony P Schuller
• 金额: $ 0.8万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10507380
• 关键词: Advisory Committees; Aneuploidy; Architecture; Binding; Biochemical; Cell Division Process; Cell division; Cells; Centromere; Chromosome Segregation; Chromosome abnormality; Chromosomes; Communities; Complex; Congenital chromosomal disease; Coupled; Couples; Cryo-electron tomography; Cryoelectron Microscopy; Defect; Disease; Dissection; Electron Microscopy; Ensure; Environment; Excision; Foundations; Future; Genetic Materials; Human; Imaging Techniques; Infrastructure; Investigation; Ions; Kinetochores; Lateral; Lead; Light; Macromolecular Complexes; Malignant Neoplasms; Mediating; Mentorship; Metaphase Plate; Methods; Microtubule Depolymerization; Microtubule Polymerization; Microtubules; Mitosis; Mitotic; Mitotic spindle; Modeling; Molecular; Molecular Biology; Molecular and Cellular Biology; Mutation; Organism; Phase; Process; Proteins; Recombinants; Research; Resolution; Rest; Role; Technology; To specify; Training; Tubulin; Weight-Bearing state; Work; biochemical tools; career; chromosome movement; computerized tools; daughter cell; experimental study; genome integrity; innovation; insight; nanometer resolution; notch protein; post-doctoral training; protein complex; reconstitution; segregation; skills; three dimensional structure; tool

PROJECT SUMMARY/ABSTRACT Mitosis is the process of cell division in which one cell replicates its genetic material and gives rise to two genetically identical daughter cells. Kinetochores are large protein assemblies that connect the newly replicated chromosomes to the mitotic spindle that constricts across the cellular volume to accomplish directional and equivalent segregation of chromosomes to each daughter cell. In human cells, two large protein complexes called SKA and HEC1, form the basis of connection between the centromere-bound kinetochore and the depolymerizing microtubules that form the mitotic spindle. The proper segregation of chromosomes during cell division is fundamental for all living organisms to maintain genome integrity, and mutations to this process have shown critically important to disease and cancer. My proposal combines innovative cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) methods and molecular biology tools to define the architecture of the human kinetochore and molecular basis for attachment to the mitotic spindle. During the K99/R00 period I will, 1) Determine a high-resolution molecular view of the SKA-microtubule complex to provide insight into how SKA oligomerizes and binds microtubules to ensure kinetochore attachment at the mitotic spindle 2) Visualize the role of the HEC1 complex in coordinating with SKA to mediate “end-on” kinetochore attachments to the microtubule ends that accomplish directional chromosome segregation 3) Provide mechanistic insight to the complete architecture of segregating kinetochore-chromosome complexes directly inside human cells undergoing mitosis During my postdoctoral period at MIT, I obtained training in cryo-electron microscopy (cryo-EM), cryo-electron tomography (cryo-ET), and cryo-focused ion beam (cryo-FIB) technologies to provide structural insights into macromolecular complexes directly inside cells. During my postdoctoral training in the Nogales lab, I have begun to refine my skills in cryo-EM and cryo-ET, with specific training to biochemically prepare and analyze microtubule assemblies. During my K99/R00 phase, I will undertake further training in cryo-EM and cryo-ET, as well as cellular and molecular biology tools to study critical processes during mitosis. I am confident my training in cryo- EM coupled with the excellent mentorship of Eva Nogales, Sue Biggins, and the rest of my advisory team, will help me transition to an independent research career. I believe my access to top notch scientific infrastructure and a truly collaborative scientific community at UC Berkeley makes it the ideal environment for my K99/R00 training. During my R00 phase, I will provide insight into the molecular mechanisms governing chromosome attachment, segregation, and disassembly at the mitotic spindle. I envision developing a cross-disciplinary research group utilizing electron microscopy, biochemistry, and computational tools to tackle these difficult problems, and to understand how defects in these mechanisms lead to chromosomal disorders and cancer.

项目摘要/摘要 有丝分裂是细胞分裂的过程，在这个过程中一个细胞复制它的遗传物质并产生两个 基因上完全相同的子细胞。动点是连接新复制的 染色体到有丝分裂纺锤体，它收缩整个细胞体积，以完成定向和 对每个子细胞进行等量的染色体分离。在人类细胞中，两个大型蛋白质复合体 被称为SKA和HEC1，形成着丝粒结合着丝粒和着丝粒之间联系的基础 解聚形成有丝分裂纺锤体的微管。细胞内染色体的适当分离 分裂是所有生物维持基因组完整性的基础，而这一过程的突变 被证明对疾病和癌症至关重要。 我的建议结合了创新的冷冻电子显微镜(Cryo-EM)和冷冻电子断层扫描(Cryo-ET) 确定人类动粒结构和分子基础的方法和分子生物学工具 用于连接有丝分裂纺锤体。在K99/R00期间，我将， 1)确定SKA-微管复合体的高分辨率分子视图，以深入了解SKA是如何 寡聚并结合微管以确保动粒附着在有丝分裂纺锤体上 2)形象化HEC1复合体在协调SKA调节“端接”动粒连接中的作用 到完成定向染色体分离的微管末端 3)提供对分离着丝粒-染色体复合体的完整结构的机械见解 直接进入正在进行有丝分裂的人类细胞内 在麻省理工学院的博士后期间，我接受了冷冻电子显微镜(Cryo-EM)、冷冻电子 层析成像(CRYO-ET)和低温聚焦离子束(CRYO-FIB)技术，以提供对 直接在细胞内的大分子复合体。在我诺加莱斯实验室的博士后培训期间，我开始 通过生物化学制备和分析微管的专门培训，提高我在冷冻-EM和冷冻-ET方面的技能 装配。在我的K99/R00阶段，我将接受冷冻-EM和冷冻-ET的进一步培训，以及 研究有丝分裂过程中关键过程的细胞和分子生物学工具。我对我的低温训练很有信心- EM再加上Eva Nogales、Sue Biggins和我的顾问团队其他人的出色指导，将 帮助我过渡到独立的研究生涯。我相信我能接触到一流的科学基础设施 加州大学伯克利分校真正的协作科学社区使它成为我的K99/R00的理想环境 训练。在我的R00阶段，我将提供关于控制染色体的分子机制的见解 有丝分裂纺锤体的附着、分离和解体。我设想发展一种跨学科的 研究小组利用电子显微镜、生物化学和计算工具来解决这些困难 了解这些机制的缺陷是如何导致染色体紊乱和癌症的。