Mechanistic studies of the role of MCAK dimerization and features of the microtubule important for the depolymerization activity of MCAK

Regulation of cellular microtubule (MT) dynamics is crucial for normal cell division. MCAK (Mitotic Centromere Associated Kinesin) is one such regulator of MT dynamics, and depolymerizes MTs by binding specifically to the MT end where it induces or stabilizes a curvature leading to destabilization of the MT. Although some aspects of the molecular mechanism of MCAK-induced microtubule destabilization are understood, many of the specific details are as yet unclear. To more accurately describe the mechanism of action of this depolymerase, we have completed work defining the minimal functional domain of the enzyme as well as elucidating the features of the MT important for the enzymatic activity of MCAK.;Additionally, ambiguity exists as to what features of the MT are important for MCAK activity. We used MTs lacking the C-termini of alpha- and beta-tubulin (SMTs) and alternative tubulin substrates to study which structural and geometrical features of the MT are critical for MCAK activity. We found that removal of the C-termini significantly decreased the efficiency of MCAK-induced depolymerization. Using alternative tubulin substrates with varying C-terminal and geometric statuses to study the depolymerization cycle, we found that geometry affected the ability of MCAK to depolymerize tubulin substrates and that MCAK can stabilize ring-like tubulin structures. We propose that the C-termini and substrate geometry play important roles in the efficient formation and release of depolymerization products.;We defined the minimal functional unit of MCAK, and using a series of biochemical assays we compared the activity of full-length dimeric MCAK with its minimal function unit, which is a monomer. Our studies suggest that MCAK dimerization is important for its catalytic cycle by promoting MCAK binding to microtubule ends, enhancing the ability of MCAK to recycle for multiple rounds of microtubule depolymerization, and preventing MCAK from being sequestered by tubulin heterodimers.