Probing the mechanical integrity of the mammalian k-fiber in live cells by laser ablation and speckle microscopy

When cells divide, a microtubule-based machine called the mitotic spindle delivers chromosomes to two new daughter cells. Microtubule bundles called kinetochore-fibers (k-fibers) attach chromosomes to the spindle, and forces transmitted through k-fibers ultimately segregate chromosomes. Thus, the mechanical integrity of the k-fiber is critical to accurate chromosome segregation, which in turn is essential for cell and organismal health. Yet, the k-fiber itself as a mechanical object is not well understood. We do not fully understand how or where k-fiber microtubules attach to each other along their lengths, nor what molecules mediate these connections. To address these questions, we are probing the mechanics of the k-fiber by severing it via laser ablation, and by using single-molecule speckle microscopy to analyze intra-k-fiber microtubule movements. Speckle microscopy suggests that k-fiber microtubules move poleward together as a single mechanical unit. However, splaying of the k-fiber in response to laser ablation demonstrates that these intra-k-fiber connections can be disrupted. Furthermore, initial evidence suggests more splaying after perturbation of the molecular motor kinesin Kif15, supporting its role as an inter-k-fiber crosslinker.