PROJECT SUMMARY “Molecular friction” between kinetochore proteins and microtubule walls sustains inter-kinetochore tension and prevents mitotic kinetochores from slipping from the microtubule ends. This phenomenon is particularly important in human cells during oscillations of metaphase chromosomes, which move repeatedly toward the plus- and minus-ends of the microtubules under significant forces. The microtubule wall–binding protein, Ndc80, is thought to serve as the primary molecular glue connecting the gliding kinetochores to microtubule surface. However, it remained unclear whether Ndc80 can form mobile diffusive bonds that are capable of generating frictional resistance. To bridge this gap in our knowledge, we developed a highly sensitive dual- trap, three-bead assay employing ultrafast force-clamp spectroscopy. Using this instrument, we pulled on the microtubule wall–bound Ndc80 protein complex in vitro, imitating the forces it experiences during metaphase chromosome oscillations. Strikingly, under dragging force, Ndc80 glides on the microtubule wall in both directions, but it generates stronger friction and exhibits catch-bond-like behavior only when pulled towards the microtubule plus-end. Thus, Ndc80 can serve as an intrinsic regulator of the direction-dependent molecular friction at mitotic kinetochores. To capitalize on this novel finding, in Aim 1 we will use this powerful technology in combination with targeted mutations in the calponin-homology domains of the Hec1 and Nuf2 subunits of the Ndc80 complex to investigate the specific molecular features that are responsible for direction- dependent friction generation at a single molecule level. In Aim 2, we will examine microtubule- wall gliding and friction generation by multiple Ndc80 molecules, characterizing their ensemble properties and emergent behaviors. With phosphomimetic substitutions in the disordered tail of the Hec1 subunit, we will determine how these behaviors are regulated by phosphorylation at the sites that strongly affect chromosome oscillations. In Aim 3 we will increase the molecular complexity of our reconstitutions to investigate how other kinetochore-associated microtubule- binding proteins modify friction generation by the gliding Ndc80. The results from these studies will provide an unprecedented molecular-mechanical insights into the key microtubule-binding components of human kinetochores, enabling us to construct a highly quantitative model of the frictional interface between microtubules and chromosomes in dividing human cells.