| The microtubule depolymerase, MCAK, is a member of the kinesin-13 family that can catalytically remove tubulin subunits from the ends of microtubules (also known as depolymerization) in the presence of ATP. Unlike its more familiar cousins, the motile kinesins, the mechanism of MCAK's function is still unclear. I have used fluorescent single molecule tracking techniques and real time imaging of microtubules undergoing the process of depolymerization to elucidate the role of two key characteristics of the MCAK molecule: dimerization and the positively charged neck. MCAK's positively charged neck region is known to be critical in MCAK's ability to depolymerize microtubules. However, prior to the research presented here, the mode of action of MCAK's neck has been unclear. In addition, the role of MCAK's natural dimeric quaternary structure has remained even more ambiguous. I have found that the major benefit of the positively charged neck is to improve the association rates of the MCAK molecule with the microtubule lattice. The positive charges in the neck, however, are a hindrance to other aspects of the molecule's function, leading to more rapid dissociation from the microtubule lattice and reduced efficiency of tubulin subunit removal from the microtubule end. Dimerization was found to have nearly the opposite effect, reducing both the association and dissociation rates with the microtubule lattice while improving the ability of the molecule to remove tubulin subunits upon arrival at the microtubule end. In addition to these findings, I have also made the observation that mutations to the motor affect association and dissociation rates in the equivalent direction (i.e. increases in association rate correspond with increases in dissociation rate and vice versa). This implies the existence of an energy barrier between bound and unbound states, the height of which can be affected by key mutations to the motor. For this research, I designed and constructed a custom two-color Total Internal Reflection Fluorescence (TIRF) microscope. In addition, this research required the development of advanced coverslip surface preparation techniques in order to prevent non-specific surface adsorption. Thorough details of the instrument design and the development of other advanced techniques are presented here. |
