Structural and mechanistic understanding of kinetochore-mediated control of microtubule dynamics (360G-Wellcome-308895_Z_23_Z)

Microtubules are conserved cytoskeletal polymers that play crucial roles in processes ranging from cell division to cell migration. Microtubule ends are constantly adding or losing tubulin, a process that is tightly regulated by end-binding proteins. Vitally important for cell division, microtubule end- generated forces are balanced by kinetochores, protein super-complexes assembled at the centromeric chromatin. Here, force is necessary to silence the spindle assembly checkpoint through a poorly understood mechanism. I have previously established that the minimal human outer kinetochore model featuring oligomerised Ndc80 complex can stabilise microtubules in vitro while simultaneously resisting microtubule-generated force. However, flexibility of microtubule ends and Ndc80 oligomers, as well as the transience of interactions between them present a challenge in studying this unique force- sensitive connection using conventional structural methods, and therefore significantly limit our understanding of kinetochore function. Here I propose to directly investigate the kinetochore-microtubule interface reconstituted in vitro under force using electron cryo-tomography. A multi-scale experimental approach that I have established previously, featuring a combination of light and electron microscopy, in vitro reconstitution, single-molecule force- sensing, and cutting-edge image processing, will enable me to understand the mechanisms underlying force-sensitivity of kinetochore-microtubule interface, its regulation by post-translational modifications, and its evolutionary conservation.