Biophysical Study of Native Yeast Kinetochores Indicates Distinct Roles for Phospho-Regulation of Core Microtubule-Binding Subcomplexes

Abstract

Chromosome segregation is orchestrated by multi-protein complexes called kinetochores, which initially bind microtubules and later form persistent load-bearing tip attachments. Accurate segregation requires regulation of kinetochores by Aurora B kinase. One popular view is that Aurora acts on kinetochores that have already established erroneous attachments to microtubule tips, phosphorylating two major microtubule-binding subcomplexes, Ndc80 and Dam1, and thereby triggering detachment. It is unknown whether phosphorylation affects initial binding of kinetochores to microtubules. Moreover, the relative importance of phosphoregulation of these subcomplexes in the context of whole kinetochores is unclear. To address these uncertainties, we relied on recently developed laser trap and fluorescence-based assays (Akiyoshi et al. Nature 2010) in which individual native kinetochore particles purified from budding yeast are coupled to single microtubules. Neither phospho-mimetic mutations at kinase target sites on Dam1 nor a mutation that disrupts the Dam1 complex had a strong effect on the initial binding of kinetochore-coated beads to microtubules. By contrast, phospho-mimetic mutations on Ndc80 reduced binding 3-fold. In force-ramp assays with tip-attached kinetochores, phospho-mimetic mutations on either Ndc80 or Dam1 reduced rupture forces roughly 1.5-fold relative to wild type and phospho-deficient controls. Likewise, in force-clamp experiments, phospho-mimetic kinetochores detached up to 8-fold more quickly, depending on the level of force. Taken together, these results indicate that Ndc80 and Dam1 play distinct roles in kinetochore function. Initial binding is dependent mainly on Ndc80 and modulated strongly by phosphorylation of Ndc80. Tip coupling depends on both Ndc80 and Dam1 and it is modulated by phosphorylation of both.