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

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.