Three interacting regions of the Ndc80 and Dam1 complexes support microtubule tip-coupling under load

Abstract

Accurate mitosis requires kinetochores to make persistent, load-bearing attachments to dynamic microtubule tips, thereby coupling chromosome movements to tip growth and shortening. This tip-coupling behavior depends on the conserved Ndc80 complex and, in budding yeast, on the Dam1 complex, which bind each other directly via three distinct interacting regions. The functional relevance of these multiple interactions was mysterious. Here we show that interactions between two of these regions support the high rupture strengths that occur when applied force is rapidly increased and also support the stability of tip-coupling when force is held constant over longer durations. The contribution of either of these two regions to tip-coupling is reduced by phosphorylation by Aurora B kinase. The third interaction region makes no apparent contribution to rupture strength, but its phosphorylation by Aurora B kinase specifically decreases the long-term stability of tip-coupling. The specific reduction of long-term stability relative to short-term strength might have important implications for mitotic error correction.

DASH/Dam1 complex mutants stabilize ploidy by weakening kinetochore-microtubule attachments

“…Saccharomyces cerevisiae DASH complex was expressed in Escherichia coli using a single polycistronic vector, as previously described (Flores et al, 2022). The Spc34p component of the DASH/Dam1 complex contained either a C-terminal FLAG-tag or a His6-tag.…”

“…Data collection was performed as previously described (Flores et al, 2022). Briefly, data was collected for a total of 1 h after the addition of tubulin into the reaction mixture.…”

Structures of the Ndc80 complex and its interactions at the yeast kinetochore-microtubule interface

“…Ndc80:Nuf2:Dam1 interaction Genetic, biochemical, and crosslinking data suggest that a segment of the extended C-terminus of the Dam1 subunit binds with a region of Ndc80:Nuf2 near the position at which the Ndc80 and Nuf2 polypeptide chains transition from their paired, CH domains into a helical coiled-coil (Flores et al, 2022;Kim et al, 2017;Lampert et al, 2013). Various trial calculations in multi-chain AF2, in which we varied the details of the Dam1 segment we included, led to a self-consistent prediction for binding, in which two stretches of conserved Dam1 residues (252-270 and 290-305) (Fig.…”

“…Fig. 1 shows positions of the DASH/Dam1c interactions, as mapped by genetic and biochemical studies including mass spectrometric analysis of cross-linked peptides (Flores et al, 2022; Kim et al, 2017; Lampert et al, 2013). The Dam1 subunit binds at the base of the Ndc80:Nuf2 globular “head”, Ask1 along the shaft between the globular head and the hinge, and Spc19:Spc34 between the loop and the four-way junction, respectively.…”

“…The Dam1 subunit binds at the base of the Ndc80:Nuf2 globular “head”, Ask1 along the shaft between the globular head and the hinge, and Spc19:Spc34 between the loop and the four-way junction, respectively. All these interactions between Ndc80c and DASH/Dam1c are regulated by phosphorylation of certain serine or threonine residues (Cheeseman et al, 2002; Flores et al, 2022). The Stu2 interaction, visualized by x-ray crystallography, is with the four-way junction (Zahm et al, 2021).…”