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

项目概要/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个 基因完全相同的子细胞动粒是连接新复制的 染色体到有丝分裂纺锤体，纺锤体在细胞体积上收缩，以完成定向和 每个子细胞的染色体分离相等。在人类细胞中，两个大的蛋白质复合物 称为SKA和HEC 1，形成了着丝粒结合的动粒和着丝粒之间连接的基础。 使形成有丝分裂纺锤体的微管解聚。细胞分裂过程中染色体的正确分离 分裂是所有生物体保持基因组完整性的基础，而这一过程的突变 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（冷冻EM）和冷冻电子断层扫描（冷冻ET） 方法和分子生物学工具来定义人类动粒的结构和分子基础 用于附着在有丝分裂纺锤体上。在K99/R 00期间，我将， 1)确定SKA-微管复合物的高分辨率分子视图，以深入了解SKA 寡聚化并结合微管以确保着丝粒附着在有丝分裂纺锤体上 2)可视化HEC 1复合物在与SKA协调以介导“末端”动粒附着中的作用 微管末端完成定向染色体分离 3)为分离的着丝粒-染色体复合体的完整结构提供机制性见解 直接进入正在进行有丝分裂的人体细胞 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），冷冻电子显微镜（cryo-electron microscopy） 断层扫描（cryo-ET）和低温聚焦离子束（cryo-FIB）技术，以提供对 大分子复合物直接在细胞内。在诺加莱斯实验室的博士后培训期间，我开始 通过专门的培训来提高我在cryo-EM和cryo-ET方面的技能，以生化制备和分析微管 组件.在K99/R 00阶段，我将接受冷冻EM和冷冻ET的进一步培训，以及 细胞和分子生物学工具来研究有丝分裂过程中的关键过程。我相信我的低温训练- EM加上伊娃诺加莱斯、苏·比金斯以及我的顾问团队的其他成员的出色指导， 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其成为我的K99/R 00的理想环境 训练在我的R 00阶段，我将深入了解染色体的分子机制， 有丝分裂纺锤体的附着、分离和解体。我设想开发一个跨学科的 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 问题，并了解这些机制的缺陷如何导致染色体疾病和癌症。

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.