Mitotic Kinesin CENP-E is a Robust Tracker of Dynamic Microtubule ends

Accurate chromosome segregation during mitosis requires durable linking between kinetochores and plus ends of spindle microtubules. During metaphase the ends of kinetochore microtubules continuously assemble and disassemble but the chromosomes remain stably attached and move concomitantly with microtubule dynamics. So far only Dam1 kinetochore complex has been reported to be able to maintain stable association with dynamic microtubule ends in vitro. The homologous genes, however, are present only in fungi, so the molecular identities of kinetochore-microtubule couplers in other cells are not known. Here, we report that a conserved kinesin-like CENP-E, playing an essential role in chromosome segregation, can maintain long-lasting association with the ends of dynamic microtubules in vitro and couple microtubule depolymerization to the motion of a microbead cargo. Using purified recombinant CENP-E from Xenopus laevis we show that a truncated dimeric motor, which lacks a stalk and a tail, fails to track the dynamic microtubule ends. However, the rate and processivity of the plus-end-directed motility and the response to applied force are similar for the full length and truncated proteins. Interestingly, a walking full length CENP-E has a folded configuration and remains compact even under tension, suggesting that such conformation is important for CENP-E's functioning at the kinetochore. To examine whether the 230nm-long CENP-E stalk is important for kinetochore-microtubule interactions, we used siRNA-mediated depletion of endogenous CENP-E in cells with stable expression of a truncated “bonsai” version of CENP-E, which localizes normally to kinetochores. In cells with “bonsai” CENP-E the chromosome congression was delayed. Furthermore, the aligned chromosomes showed marked instability and frequently moved away from the metaphase plate, implying defects in kinetochore-microtubule attachment. We propose that CENP-E exerts some of its mitotic functions via its non-motor domains, which enable it to track dynamic microtubule ends.