期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：47
页码：E7483-E7489
DOI：10.1073/pnas.1611581113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceMolecular motors organize cells by hauling molecular cargoes along polymer tracks (actin filaments or microtubules). Until recently, the stepping direction of each motor was thought to be fixed; however, it now emerges that yeast kinesin-5 motors can reverse their stepping direction. How does this work? We show that the stepping direction of Cut7, a yeast kinesin-5 motor, depends on the level of motor crowding on the microtubule, and that crowding of Cut7 by non-Cut7 proteins also can drive reversal. To explain this, we propose that stepping of Cut7 in one direction is blocked by collisions with neighbors, whereas stepping in the other direction, being less space-hungry, is not. Crowding-dependent directional reversal is a hitherto-unsuspected aspect of motor-driven self-organization in cells. Cut7, the sole kinesin-5 in Schizosaccharomyces pombe, is essential for mitosis. Like other yeast kinesin-5 motors, Cut7 can reverse its stepping direction, by mechanisms that are currently unclear. Here we show that for full-length Cut7, the key determinant of stepping direction is the degree of motor crowding on the microtubule lattice, with greater crowding converting the motor from minus end-directed to plus end-directed stepping. To explain how high Cut7 occupancy causes this reversal, we postulate a simple proximity sensing mechanism that operates via steric blocking. We propose that the minus end-directed stepping action of Cut7 is selectively inhibited by collisions with neighbors under crowded conditions, whereas its plus end-directed action, being less space-hungry, is not. In support of this idea, we show that the direction of Cut7-driven microtubule sliding can be reversed by crowding it with non-Cut7 proteins. Thus, crowding by either dynein microtubule binding domain or Klp2, a kinesin-14, converts Cut7 from net minus end-directed to net plus end-directed stepping. Biochemical assays confirm that the Cut7 N terminus increases Cut7 occupancy by binding directly to microtubules. Direct observation by cryoEM reveals that this occupancy-enhancing N-terminal domain is partially ordered. Overall, our data point to a steric blocking mechanism for directional reversal through which collisions of Cut7 motor domains with their neighbors inhibit their minus end-directed stepping action, but not their plus end-directed stepping action. Our model can potentially reconcile a number of previous, apparently conflicting, observations and proposals for the reversal mechanism of yeast kinesins-5.
