Kinesins are molecular motors that move along tracks - microtubules - in the course of carrying out their many critical functions in the cell. Different classes of kinesins move towards the plus end or the minus end of microtubules and some kinesins depolymerize microtubules. High-resolution structures of both plus-end directed and minus-end directed kinesins and of the tubulin protofilament have been available for some time, but there has been no success in preparing crystals of the track-motor complexes for atomic level analyses. Combining the high resolution structures of the components with lower resolution (20-30 Angstroms) three-dimensional data obtained by cryo-electron microscopy and image analysis is the only way to understand the detailed workings of the track-motor complexes at various stages in the cycle of interaction. In the previous grant period, we used this methodology to determine the mechanism of processivity and directionality that operates in conventional human brain kinesin - a Kin N class, plus-end directed motor. Building on these findings, as well as work from many other laboratories, we outlined design principles that unify our thinking about kinesins and myosins and provided a general framework for a mechanistic understanding of these two superfamilies of molecular motors. We also carried out preliminary experiments which suggest a working hypothesis for microtubule depolymerization by members of the Kin I class of kinesins. Finally, we determined how MAP2c / tau proteins bind to microtubules - data which suggest how members of this class of proteins increase microtubule stability. These results set the stage for the current application, which will address fundamental questions concerning the molecular basis for (I) minus-end directed movement by Ncd, a Kin C motor, (ii) the action of Unc104, a Kin N neuronal motor, (iii), microtubule depolymerization by MCAK, a Kin I motor, and (iv) the binding of a number of Microtubule Associated Proteins to microtubules and/or actin filaments.
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