Structural and mechanistic understanding of kinetochore-mediated control of microtubule dynamics

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.