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，其中一个细胞复制其遗传物质并产生两种 属于属的子细胞。动力学是连接新复制的大蛋白质组件 染色体到有丝分裂纺锤体，该纺锤体在整个细胞体积上收缩以完成方向性和 染色体与每个子细胞的等效分离。在人类细胞中，两个大蛋白质复合物 称为SKA和HEC1，构成了Centromere结合的动物学和 形成有丝分裂主轴的去聚合微管。细胞期间染色体的适当分离 分裂对于所有生物体维持基因组完整性都是基础，并且对此过程的突变具有 对疾病和癌症至关重要。 我的建议结合了创新的低温电子显微镜（Cryo-EM）和冷冻电子层析成像（Cryo-ET） 方法和分子生物学工具来定义人类动物学和分子基础的结构 用于附着有丝分裂主轴。在K99/R00期间，我将 1）确定SKA-Microtube复合物的高分辨率分子视图，以洞悉SKA 寡聚并结合微管，以确保在有丝分裂主轴上附着的动力学附着 2）可视化Hec1复合物在与SKA协调中的作用，以介导“终端”动力学附件 到达到方向性染色体分离的微管末端 3）提供机械洞察，以隔离隔离动型染色体复合体的完整体系结构 直接发生有丝分裂的人类细胞内 在MIT的博士后期间，我获得了低温电子显微镜（Cryo-EM），Cryo-Electron的培训 断层扫描（冷冻-ET）和以低温为中心的离子束（冷冻FIB）技术，以提供结构性见解 大分子复合物直接在细胞内部。在Nogales实验室的博士后培训期间，我已经开始 为了完善我在Cryo-EM和Cryo-ET方面的技能，并通过特定的培训以生化准备和分析微管 集会。在我的K99/R00阶段，我将在Cryo-EM和Cryo-Et以及 细胞和分子生物学工具，用于研究有丝分裂过程中的关键过程。我相信我在冷冻方面的培训 Em加上Eva Nogales，Sue Biggins和我的其他咨询团队的出色精神 帮助我过渡到独立的研究职业。我相信我可以进入顶级科学基础设施 加州大学伯克利分校的真正合作科学界使其成为我的K99/R00的理想环境 训练。在我的R00阶段，我将洞悉有关染色体的分子机制 有丝分裂主轴的附着，分离和拆卸。我设想开发跨学科 利用电子显微镜，生物化学和计算工具来解决这些困难的研究小组 问题，并了解这些机制的缺陷如何导致染色体疾病和癌症。