Probing Forces on Newly Generated Spindle Microtubule Minus-Ends

Abstract

The mitotic spindle is a dynamic self-organizing machine that coordinates cell division and preserves genomic stability. The ability to focus microtubule minus-ends into poles is crucial to spindle structure and function. However, our understanding of pole-focusing forces has been limited by the challenges of labeling and imaging microtubule minus-ends in established spindles. Here, we used laser ablation to sever kinetochore-fiber microtubules in mammalian cells and probe how the cell detects and organizes newly generated microtubule minus-ends. Within a few seconds of ablation, the cell recognizes new minus-ends and begins pulling them poleward. These pole-focusing forces exist throughout metaphase and anaphase and can move chromosomes rapidly, dominating other spindle forces. Opposing forces on chromosomes from the other half-spindle are able to slow, though not stop, the pole-focusing response, as indicated by faster pole-focusing speeds in monopolar spindles and during anaphase than in metaphase bipolar spindles. Together, our data indicate that microtubule minus-end focusing forces operate broadly and rapidly and are of similar magnitude to other spindle forces. These pole-focusing forces are thus well-suited to robustly maintain spindle structural integrity despite rapid turnover of spindle components and mechanical challenges.