Probing the mechanical organization of k-fiber microtubule bundles within the mammalian mitotic spindle via targeted laser ablation and speckle microscopy

In dividing cells, a self-assembling microtubule-based machine called the mitotic spindle segregates chromosomes, ensuring that each new daughter cell has exactly one copy of its genetic information. Spindle microtubule organization imparts shape and structural robustness. For example, bundles of microtubules called kinetochore-fibers (k-fibers) attach chromosomes to the spindle and exert force to align and ultimately segregate them. Thus, the mechanical integrity of the k-fiber is critical to spindle function, yet its organization is not well-understood. For example, we do not fully understand how molecules that crosslink microtubules along k-fiber lengths contribute to overall k-fiber mechanics, nor whether k-fiber microtubules remain rigidly associated or fluidly slide past each other in response to spindle dynamics. To address these questions, we are probing the mechanics of inter-microtubule associations within the k-fiber by single-molecule speckle microscopy and by severing k-fibers via laser ablation. Our initial results suggest that k-fiber microtubules associate along their lengths and primarily act in the spindle as single rigid objects, but that connections between their microtubules are weak enough to be disrupted by our mechanical perturbations.