Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes1,2. Accurate segregation depends on selective stabilization of correct ‘bi-oriented’ kinetochore-microtubule attachments, which come under tension due to opposing forces exerted by microtubules3. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B4,5. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules6. Moreover, tension increases the lifetimes of the reconstituted attachments directly, via a catch bond-like mechanism that does not require Aurora B7-10. Based on these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.

During vertebrate cell division, chromosomes oscillate with periods of smooth motion interrupted by abrupt reversals in direction. These oscillations must be spatially constrained in order to align and segregate chromosomes with high fidelity, but the molecular mechanism for this activity is uncertain. We report here that the human kinesin-8 Kif18A has a primary role in the control of chromosome oscillations. Kif18A accumulates as a gradient on kinetochore microtubules in a manner dependent on its motor activity. Quantitative analyses of kinetochore movements reveal that Kif18A reduces the amplitude of preanaphase oscillations and slows poleward movement during anaphase. Thus, the microtubule-depolymerizing kinesin Kif18A has the unexpected function of suppressing chromosome movements. Based on these findings, we propose a molecular model in which Kif18A regulates kinetochore microtubule dynamics to control mitotic chromosome positioning.

Kinesin is a double-headed motor protein that moves along microtubules in 8-nanometer steps. Two broad classes of model have been invoked to explain kinesin movement: hand-over-hand and inchworm. In hand-over-hand models, the heads exchange leading and trailing roles with every step, whereas no such exchange is postulated for inchworm models, where one head always leads. By measuring the stepwise motion of individual enzymes, we find that some kinesin molecules exhibit a marked alternation in the dwell times between sequential steps, causing these motors to "limp" along the microtubule. Limping implies that kinesin molecules strictly alternate between two different conformations as they step, indicative of an asymmetric, hand-over-hand mechanism.

SUMMARY Kinetochores couple chromosomes to the assembling and disassembling tips of microtubules, a dynamic behavior that is fundamental to mitosis in all eukaryotes but poorly understood. Genetic, biochemical and structural studies implicate the Ndc80 complex as a direct point of contact between kinetochores and microtubules, but these approaches provide only a static view. Here, using techniques for manipulating and tracking individual molecules in vitro, we demonstrate that the Ndc80 complex is capable of forming the dynamic, load-bearing attachments to assembling and disassembling tips required for coupling in vivo. We also establish that Ndc80-based coupling likely occurs through a biased diffusion mechanism, and that this activity is conserved from yeast to humans. Our findings demonstrate how an ensemble of Ndc80 complexes may provide a ‘slip clutch’ that allows the kinetochore to maintain a load-bearing tip attachment during both microtubule assembly and disassembly.