UCSF

For a cell to divide correctly, the spindle must connect to and align chromosomes and then generate force to move them into two daughter cells. The kinetochore is the macromolecular machine that connects chromosomes to a bundle of dynamic microtubules, the kinetochore-fiber (k-fiber). While we have a nearly complete parts list of kinetochore components and regulators, how they together give rise to the robust mechanics of the kinetochore-microtubule interface remains poorly understood. This is due to the fact that mammalian kinetochores and k-fibers cannot yet be reconstituted in vitro and there are few tools to perturb forces and measure the mechanics at this interface in vivo. In my thesis work I have addressed this gap using direct biophysical assays in mammalian cells to focus on two main questions about the kinetochore-microtubule interface. First, how do kinetochores hold on to microtubules that grow and shrink? Using live imaging to monitor spindle dynamics and laser ablation to challenge kinetochore grip, I show that regulation of the key microtubule binding protein Ndc80/Hec1 at the outer kinetochore by the kinase Aurora B specifically affects kinetochore movement on polymerizing microtubules without disrupting coupling to depolymerizing microtubules that generate force to move chromosomes. Second, at the other side of the interface, how do kinetochore-fibers remodel under force? I directly exert forces on individual mammalian k-fibers and find that even under high force for minutes they do not lose grip. Instead, k-fibers bend and elongate by polymerizing at normal rates at plus-ends and inhibiting depolymerization at minus-ends – thus ensuring robust connection to kinetochores. Altogether I find that robust kinetochore grip emerges from underlying properties of both the kinetochore and k-fiber microtubules – specialized regulation at the kinetochore allows the cell to adjust grip while still allowing force generation and dynamic mechanical feedback of k-fiber microtubules locally dissipates force, protecting spindle connections. These fundamental physical properties of the kinetochore-microtubule interface allow the spindle to faithfully segregate chromosomes and more broadly suggest a model for how force-generating cellular machines can also robustly maintain their structure.