Synthesis of Novel Photochromic Inhibitors of Mitotic Kinesins Eg5 and Kif18a and their Phtoresponsive Interaction with the Kinesins

Abstract

that CENP-E plays an important mitotic role at the kinetochore-associated microtubule tips. To determine the molecular mechanism of CENP-E tip-tracking, we characterized two purified recombinant fragments of CENP-E: one containing the motor and neck domains and the second with the dimeric C-terminal tails. The motor-containing truncated protein walked on the microtubule wall in essentially the same manner as the full length CENP-E, while the C-terminal tail exhibited rapid diffusion. Neither of these fragments showed the tiptracking, however, this activity was recapitulated by artificially joining these two proteins by conjugating to Qdots. A computational model of CENP-E motility successfully described the tip-tracking ability by repeating the cycles of plus-end-directed walking and the tail-mediated diffusion of the microtubule wall-tethered motor. This novel ''tethered motor'' mechanism of tip-tracking does not rely on the specific properties of the assembling or disassembling microtubule tips, explaining why CENP-E can tip-track bi-directionally, i.e. with the growing and shortening microtubule ends. Together, these results establish the requirement for CENP-E in stably linking the kinetochores to dynamic microtubule tips, and provide a detailed molecular mechanism to explain how CENP-E can achieve this function.