UCSF

When a cell divides, it builds the mitotic spindle, a micron-scale bipolar structure made of dynamic microtubules that must attach to chromosomes and segregate them evenly into two daughter cells. Segregation relies on microtubule-generated force to capture and move chromosomes, yet how this force contributes to a cell’s decision-making during division has been challenging to discern. In my thesis work, I have addressed how biophysical cues at the kinetochore contribute to two key decisions during mitosis: first, whether to hold on to chromosome attachments or let go and second, when to stop building the spindle and divide. The kinetochore is the macromolecular interface that connects chromosomes to spindle microtubules. For accurate chromosome segregation, sister kinetochores need to become bioriented or attach to opposite spindle poles. Biorientation occurs by a constant feedback loop, destabilizing incorrect attachments while reinforcing correct ones. How the kinetochore distinguishes between correct and incorrect attachments is not clear. In this work, I address the long-standing hypothesis that tension at the kinetochore dictates which attachments to maintain. Using live imaging to monitor outcomes, I directly perturb force on kinetochores globally by chromokinesin overexpression and locally by individual chromosome arm ablation. Together with experiments enriching for attachment errors, this work demonstrates that elevated force at the kinetochore promotes attachment stabilization, and this effect is not chromosome-agnostic but leads to impaired error correction on long chromosomes. In parallel with local error correction decisions, the cell must globally prevent anaphase until all chromosomes are attached correctly. The spindle assembly checkpoint (SAC) generates a diffusible signal at unattached kinetochores to prevent premature segregation. The precise cues that trigger SAC satisfaction at an individual kinetochore and how the cell measures and integrates these signals is not well understood. Here, I use laser ablation to generate unpaired, sisterless kinetochores and monitor anaphase entry timing. I find a progressive, titratable delay in mitosis with increasing number of unpaired kinetochores, suggesting that without a sister kinetochore or a discrete opposing force, attachments may not be sufficiently stable to satisfy the SAC with normal dynamics. Still, these unpaired kinetochores are insufficient to prevent anaphase entirely, suggesting either that the SAC does satisfy eventually or that the cell cannot detect this low level of SAC signal. Altogether, I find that altering the physical landscape of the kinetochore by perturbing opposing force on it, either by manipulating chromokinesins or removing its sister, alters two critical pathways: error correction and the SAC. I find that force directly impacts the fidelity of cell division by signaling the error correction machinery and may alter dynamics of SAC satisfaction either directly or indirectly.