| Unlike most kinesins which walk along the surface of microtubules, Mitotic Centromere-Associated Kinesin (MCAK) depolymerizes them. This observation raises the interesting possibility of a motor playing an unconventional role linking microtubule depolymerization with chromosome movement during mitosis. This thesis addresses this issue by analyzing the cellular function and regulation of MCAK. First, experiments analyzing the phosphorylation and protein associations of MCAK are described. These experiments were negative (although incomplete) with respect to the phosphorylative control of MCAK but a possible MCAK interactor, Ubc9, was identified. This protein was pulled out of a yeast two-hybrid screen and suggests a ubiquitin-related control mechanism for MCAK regulation. Next, the cellular function of MCAK was investigated by transfecting cells with a motorless MCAK construct. These cells display a marked increase in lagging chromosomes during anaphase while other chromosome movements appear normal. These results indicate that MCAK is important for the efficient segregation of chromosomes and that its function is critical during anaphase. Finally, the minimal MCAK structure necessary to elicit in vivo microtubule depolymerization is defined. These experiments reveal that the conserved kinesin-like motor domain of MCAK is insufficient for microtubule depolymerization and that the addition of 30 residues amino-terminal to the motor restores some depolymerization activity. In addition, it was determined that the smallest functional MCAK fragments that depolymerize microtubules act as monomers. The results described in this thesis provide support for the hypothesis that MCAK promotes the fidelity of chromosome segregation by depolymerizing microtubules at the kinetochore during the anaphase period of mitosis. |
