UC San Diego

The mitotic kinesin CENP-E is an essential kinetochore motor that directly contributes to the capture and stabilization of spindle microtubules by kinetochores. Although it has been well established that CENP-E is essential for metaphase chromosome alignment and reduction of CENP-E leads to high rates of whole chromosome missegregation in cells, its properties as a microtubule- dependent motor, the mechanism by which CENP-E contributes to the dynamic linkage between kinetochores and spindle microtubules, and how its motility is regulated are unknown. In this dissertation, using single molecule assays, CENP-E has been shown to be a processive, plus end -directed motor with one-dimensional diffusion, which allows microtubule attachment for long periods. Direct visualization of recombinant full-length Xenopus CENP-E by electron microscopy reveals a highly flexible, 230 nm-long coiled-coil separating its kinesin motor and kinetochore targeting domains. Furthermore, full-length CENP-E is a plus end-directed motor whose activity is essential for metaphase chromosome alignment. Based on these findings, I propose that the highly processive microtubule-dependent motor activity of CENP-E serves not only to power chromosome congression, but also to provide a flexible, motile tether linking kinetochores to dynamic spindle microtubules. CENP-E is highly phosphorylated during mitosis, which raises the possibility that phosphorylation may regulate the function of CENP-E at individual kinetochores. Strikingly, blocking phosphorylation of a single conserved site close to the motor domain of CENP-E prevents chromosome alignment with few chromosomes remaining close to spindle poles. Using purified components and a phospho-specific antibody, I demonstrate that Aurora kinases, both A and B, phosphorylate this site both in vitro and in vivo. Phosphorylation occurs while CENP-E is bound to the kinetochore and reduces the affinity of CENP-E for microtubules in vitro. Moreover, CENP-E phosphorylation is shown to be required, downstream of Aurora activation, for the congression of incorrectly attached chromosomes that are moved to the spindle pole. My findings demonstrate that CENP-E is under direct control of Aurora kinases to promote chromosome biorientation