Load-Bearing Interactions between the NDC80 and DAM1 Complexes Differ on Growing and Shortening Microtubule Tips

Abstract

Interaction between the Ndc80 and Dam1 complexes enhance the load-bearing ability of the kinetochore. We previously found that three different components in the Dam1 complex each binds to a different site in the Ndc80 protein. Mutations in any of these three sites disrupt the ability of the Ndc80 complex to bridge two rings in vitro and disrupt proper Dam1 complex localization to the rest of the kinetochore in vivo. However, which interaction sites between the Ndc80 and Dam1 complexes play a role in load-bearing is still unknown. To test whether regions in the Ndc80p play a role in establishing load-bearing interactions with the Dam1 complex, I generated three lethal insertion mutations along the Ndc80p (ANdc80p, BNdc80p, and CNdc80p) and utilized the optical trap to measure the strength of the Ndc80 complex attachment to an assembling microtubule in the presence of Dam1 complex. Mutation at region ANdc80p conferred a complete defect in the ability of the Ndc80 complex to form a load-bearing interaction with the Dam1 complex, while mutation in region BNdc80p conferred a partial defect and a mutation in region CNdc80p did not confer any defect.I next tested the role of different regions of Ndc80p to form load-bearing interactions on disassembling microtubules in the presence of the Dam1 complex. insertion mutations in either region BNdc80p or region CNdc80p led to a 12-15 fold increase in detachment rate compared to wild-type. in conclusion, region A forms a load-bearing interaction between the Ndc80 and Dam1 complexes on an assembling microtubule tip. Region B forms load-bearing interactions between the Ndc80 and Dam1 complexes on both assembling and disassembling microtubule tips. Region C only forms a load-bearing interaction between the Ndc80 and Dam1 complexes on a disassembling microtubule tip.