Summary Mitotic chromosome segregation requires that kinetochores attach to microtubule polymers and harness microtubule dynamics to drive chromosome movement. In budding yeast, the Dam1 complex couples kinetochores with microtubule depolymerization. However, a metazoan homologue of the Dam1 complex has not been identified. To identify proteins that play a corresponding role at the vertebrate kinetochore-microtubule interface, we isolated a 3-subunit human Ska1 complex, including the previously uncharacterized protein Rama1, that localizes to the outer kinetochore and spindle microtubules. Depletion of Ska1 complex subunits severely compromises proper chromosome segregation. Reconstituted Ska1 complex possesses two separable biochemical activities; direct microtubule-binding through the Ska1 subunit, and microtubule-stimulated oligomerization imparted by the Rama1 subunit. The full Ska1 complex forms assemblies on microtubules that can facilitate the processive movement of microspheres along depolymerizing microtubules. In total, these results demonstrate a critical role for the Ska1 complex in interacting with dynamic microtubules at the outer kinetochore.

Summary To ensure equal chromosome segregation during mitosis, the macromolecular kinetochore must remain attached to depolymerizing microtubules, which drive chromosome movements. How kinetochores associate with depolymerizing microtubules, which undergo dramatic structural changes forming curved protofilaments, has yet to be defined in vertebrates. Here, we demonstrate that the conserved kinetochore-localized Ska1 complex tracks with depolymerizing microtubule ends and associates with both the microtubule lattice and curved protofilaments. In contrast, the Ndc80 complex, a central player in the kinetochore-microtubule interface, binds only to the straight microtubule lattice and lacks tracking activity. We demonstrate that the Ska1 complex imparts its tracking capability to the Ndc80 complex. Finally, we present a structure of the Ska1 microtubule binding domain that reveals its interaction with microtubules and its regulation by Aurora B. This work defines an integrated kinetochore-microtubule interface formed by the Ska1 and Ndc80 complexes that associates with depolymerizing microtubules, potentially by interacting with curved microtubule protofilaments.

Microtubules (MTs) are important components of the eukaryotic cytoskeleton: they contribute to cell shape and movement, as well as to the motions of organelles including mitotic chromosomes. MTs bind motor enzymes that drive many such movements, but MT dynamics can also contribute to organelle motility. Each MT polymer is a store of chemical energy that can be used to do mechanical work, but how this energy is converted to motility remains unknown. Here we show, by conjugating glass microbeads to tubulin polymers through strong inert linkages, such as biotin-avidin, that depolymerizing MTs exert a brief tug on the beads, as measured with laser tweezers. Analysis of these interactions with a molecular-mechanical model of MT structure and force production shows that a single depolymerizing MT can generate about ten times the force that is developed by a motor enzyme; thus, this mechanism might be the primary driving force for chromosome motion. Because even the simple coupler used here slows MT disassembly, physiological couplers may modulate MT dynamics in vivo.

Summary Kinetochores of mitotic chromosomes are coupled to spindle microtubules in ways that allow the energy from tubulin dynamics to drive chromosome motion. Most kinetochore-associated microtubule ends display curving “protofilaments,” strands of tubulin dimers that bend away from the microtubule axis. Both a kinetochore “plate” and an encircling, ring-shaped protein complex have been proposed to link protofilament bending to poleward chromosome motion. Here we show by electron tomography that slender fibrils connect curved protofilaments directly to the inner kinetochore. Fibril-protofilament associations correlate with a local straightening of the flared protofilaments. Theoretical analysis reveals that protofilament-fibril connections would be efficient couplers for chromosome motion, and experimental work on two very different kinetochore components suggests that filamentous proteins can couple shortening microtubules to cargo movements. These analyses define a ring-independent mechanism for harnessing microtubule dynamics directly to chromosome movement.