Characterization of the Microtubules Modified with Photochromic Molecules

Abstract

Previously we have introduced photochromic molecules into the functional region of kinesin and succeeded to regulate the ATPase activity of kinesin reversibly by light irradiation. However, the regulation efficiency of the ATPase activity was not significant. It is well known that the contraction of skeletal muscle is regulated by calcium binding to the rail protein actin filament. The regulating system achieves high efficient switching. Therefore, it is expected that introducing artificial switching system into the rail protein microtubules enables high efficient regulation of kinesin motor activity. In this study, we tried to introduce photochromic molecules into the microtubules to regulate kinesin movement. First we modified the microtubules composed of wild type tubulin and measured gliding speed for kinesin using invitro motility assay under UV or VIS light irradiation, respectively. Gliding speeds of the microtubules modified with 4-phenylazophenyl maleimide (PAM) or 3,3-dimethyl-6’-nitro −1-[2- [3-(3- pyrroline −2,5-dion) −1-yl] propanoyloxy] ethylindoline −2-spiro-2’ (2H)- chromene (MASP) were almost identical to intact microtubules and also were not influenced by UV or VIS light irradiation. This result suggested that that these photochromic molecules incorporated into intrinsic reactive cysteine residues on the surface of microtubules did not affect kinesin motor activity. Subsequently, we prepared the 4 types of α-tubulin mutants (V406C, V410C, M414C, G417C), which have additional cysteine residues in the kinesin binding region. Microtubule concentration dependent kinesin ATPase activity for the mutant microtubules, V406C, V410C and M414 showed almost similar values to that of WT microtubules. On the other hand, ATPase activity for G417C mutant microtubule drastically decreased. The mutant microtubules were modified with photochromic molecules and the effect upon UV-VIS light irradiation for gliding speed and kinesin ATPase activity were studied.