The mitotic spindle, a self-constructed microtubule-based machine, segregates chromosomes during cell division. In mammalian cells, microtubule bundles called kinetochore-fibers (k-fibers) connect chromosomes to the spindle poles. Chromosome segregation thus depends on the mechanical integrity of k-fibers. Here, we investigate the physical and molecular basis of k-fiber bundle cohesion. We detach k-fibers from poles by laser ablation-based cutting, thus revealing the contribution of pole-localized forces to k-fiber cohesion. We then measure the physical response of the remaining kinetochore-bound segments of the k-fibers. We observe that microtubules within ablated k-fibers often splay apart from their minus-ends. Furthermore, we find that minus-end clustering forces induced by ablation seem at least partially responsible for k-fiber splaying. We also investigate the role of the k-fiber-binding kinesin-12 Kif15. We find that pharmacological inhibition of Kif15-microtubule binding reduces the mechanical integrity of k-fibers. In contrast, inhibition of its motor activity but not its microtubule binding ability, i.e., locking Kif15 into a rigor state, does not greatly affect splaying. Altogether, the data suggest that forces holding k-fibers together are of similar magnitude to other spindle forces, and that Kif15, acting as a microtubule crosslinker, helps fortify and repair k-fibers. This feature of Kif15 may help support robust k-fiber function and prevent chromosome segregation errors. [Media: see text] [Media: see text] [Media: see text]

AbstractThe mitotic spindle, a self-constructed microtubule-based machine, segregates chromosomes into two eventual daughter nuclei. In mammalian cells, microtubule bundles called kinetochore-fibers (k-fibers) anchor chromosomes within the spindle. Chromosome segregation thus depends on the mechanical integrity of k-fibers. Here, we investigate the physical and molecular basis of k-fiber bundle cohesion. We sever k-fibers using laser ablation, thereby detaching them from poles and testing the contribution of pole-localized force generation to k-fiber cohesion. We then measure the physical response of the remaining kinetochore-bound segments of the k-fibers. We observe that microtubules within ablated k-fibers often, but not always, splay apart from their minus-ends. Furthermore, we find that minus-end clustering forces induced in response to ablation seem at least partially responsible for k-fiber splaying. We also investigate the role of the putative k-fiber-binding kinesin-12 Kif15. We find that pharmacological inhibition of Kif15 microtubule binding reduces k-fiber mechanical integrity. In contrast, inhibition of its motor activity but not its microtubule binding does not greatly affect splaying. Altogether, the data suggest that forces holding k-fibers together are of similar magnitude to other spindle forces, and that Kif15, acting as a microtubule crosslinker, helps fortify and repair k-fibers. This feature of Kif15 may help support robust k-fiber function and prevent chromosome segregation errors.

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