Ultrafast Force-Clamp Studies of the Diffusing Microtubule-Binding Proteins

Abstract

Chromosome segregation during cell division crucially depends on the activities of the microtubule associated proteins (MAPs) that couple chromosomal kinetochores to spindle microtubules. When a kinetochore-bound microtubule shortens, it pulls the coupled kinetochore toward the spindle pole-associated microtubule minus-end, whereas the sister kinetochore is dragged toward the microtubule plus-end. However, there is little understanding of how the coupling kinetochore MAPs translocate along microtubule in different directions under the dragging force. To bridge this gap, we applied a highly sensitive ultrafast force-clamp spectroscopy to investigate directional mobility of key kinetochore components, including human Ndc80 and Ska complexes, and the ring-forming Dam1 complex from yeast. Single molecules of these proteins were conjugated to the coverslip-immobilized bead pedestals and the microtubule “dumbbell” was oscillated near the pedestal's surface under a continuously operating force clamp. Surprisingly, the character of the force-induced translocation was dramatically different for these MAPs, even though they undergo thermal diffusion along microtubules at highly similar rates. Under the dragging force, the Ska complex glides smoothly in both microtubule directions, whereas the Ndc80 exhibits differential gliding and the Dam1 shows asymmetric force-induced static bindings. These results strongly support the model in which the translocating kinetochore MAPs function as integral components of the molecular machine powered by microtubule depolymerization, rather than by biased diffusion. We propose that the asymmetric behavior of Ndc80 and Dam1 complexes underlies differential internal friction at moving kinetochores, which enables their poleward-directed gliding, while reducing the microtubule end slippage under tension.