Kinase Cdk1 and kinesin Kar3 regulate the Dam1 complex in its role to strengthen kinetochore-microtubule attachment

Abstract

Cell division is successful when each new daughter cell ends up with the correct complement of DNA. To obtain this outcome, a macromolecular protein complex known as the kinetochore is required to connect chromosomes to microtubules. The inner portion of the kinetochore binds to chromosomes at a region known as the centromere while the outer structure of the kinetochore fastens onto the microtubule. Sustained attachments of kinetochores to microtubules for accurate chromosome segregation depends on a kinetochore component, the ten-member Dam1 complex (Dam1c), in budding yeast. The Dam1c oligomerizes to encircle the microtubule and can harness the forces generated by shortening microtubules to power chromosome segregation. Previous work found that Dam1c undergoes phosphor-regulation, in which phosphorylation by the Ipl1 kinase (Aurora-B) predominantly destabilizes erroneous kinetochore-microtubule interactions, a critical step to ensure proper segregation of genetic material. I, along with collaborators, used an optical trap-based assay to test purified native kinetochores, or its subcomplexes, from yeast or from bacterially expressed recombinant protein to examine attachment strength of these various protein complexes to microtubules. Surprisingly, I found that kinetochore-microtubule coupling is strengthened when the native Dam1c is phosphorylated. The kinase responsible for the increased in strength is Cdk1 and the key substrate is the Ask1 component of the Dam1c. To determine how Cdk1-mediated phosphorylation strengthens attachments I tested the ability of Dam1c to: (1) bind kinetochores, (2) bind microtubules, and (3) form higher order oligomeric rings. This revealed that Cdk1 phosphorylation had no influence in microtubule binding or enhance Dam1c retention at the kinetochore, but it did enhance Dam1c function in oligomerizing around microtubules ensuring stronger kinetochore-microtubule attachments. In addition, preliminary data suggests nuclear motor proteins, Cin8 and Kar3, have a role in kinetochore-microtubule coupling. To determine how motor proteins strengthen kinetochore-microtubule attachment, I tested whether Dam1c localization at the kinetochores required motor proteins. Indeed, we found that Kar3 had a role in retaining Dam1c at the kinetochore. Together, my data has identified additional regulatory mechanisms that tune kinetochore-microtubule attachment strength.