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.

The Dam1 complex, regulated by aurora B phosphorylation, confers a more stable microtubule association for the Ndc80 complex at kinetochores (see also related paper by Lampert et al. in this issue).

Summary Alignment of chromosomes at the metaphase plate is a signature of cell division in metazoan cells, yet the mechanisms controlling this process remain ambiguous. Here we use a combination of quantitative live cell imaging and reconstituted dynamic microtubule assays to investigate the molecular control of mitotic centromere movements. We establish that Kif18A (kinesin-8) attenuates centromere movement by directly promoting microtubule pausing in a concentration-dependent manner. This activity provides the dominant mechanism for restricting centromere movement to the spindle midzone. Furthermore, polar ejection forces spatially confine chromosomes via position-dependent regulation of kinetochore tension and centromere switch rates. We demonstrate that polar ejection forces are antagonistically modulated by chromokinesins. These pushing forces depend on Kid (kinesin-10) activity and are antagonized by Kif4A (kinesin-4), which functions to directly suppress microtubule growth. These data support a model in which Kif18A and polar ejection forces synergistically promote centromere alignment via spatial control of kinetochore-microtubule dynamics.

Kinetochores remain attached to microtubule (MT) tips during mitosis even as the tips assemble and disassemble under their grip, allowing filament dynamics to produce force and move chromosomes. The specific proteins that mediate tip attachment are uncertain, and the mechanism of MT-dependent force production is unknown. Recent work suggests that the Dam1 complex, an essential component of kinetochores in yeast, may contribute directly to kinetochore-MT attachment and force production, perhaps by forming a sliding ring encircling the MT. To test these hypotheses, we developed an in vitro motility assay where beads coated with pure recombinant Dam1 complex were bound to the tips of individual dynamic MTs. The Dam1-coated beads remained tip-bound and underwent assembly-and disassembly-driven movement over Ϸ3 m, comparable to chromosome displacements in vivo. Dam1-based attachments to assembling tips were robust, supporting 0.5-3 pN of tension applied with a feedback-controlled optical trap as the MTs lengthened Ϸ1 m. The attachments also harnessed energy from MT disassembly to generate movement against tension. Reversing the direction of force (i.e., switching to compressive force) caused the attachments to disengage the tip and slide over the filament, but sliding was blocked by areas where the MT was anchored to a coverslip, consistent with a coupling structure encircling the filament. Our findings demonstrate how the Dam1 complex may contribute directly to MT-driven chromosome movement.cytoskeleton ͉ DASH ͉ disassembly ͉ mitosis ͉ motility