Kinesin-5 Acts as a Microtubule Stabilizer, Polymerase and Plus-Tip Tracker

Abstract

Kinesin-5 slides antiparallel microtubules apart during mitosis and is necessary for bipolar spindle formation. Besides its unique homotetrameric configuration, determined by the coiled-coil domain, kinesin-5 motor domains also possess specific properties optimal for their spindle organizing function. To study properties intrinsic to the kinesin-5 motor domain, we generated functional kinesin-5 dimers by fusing the kinesin-5 head and 18-residue neck linker to the coiled-coil rod of kinesin-1. In ATP this kinesin-5 dimer decorated plus-ends of taxol-stabilized microtubules and slowed depolymerization of GMPCPP microtubules. On dynamic microtubules, kinesin-5 dimer increased the microtubule growth rate by more than a factor of two and reduced the catastrophe frequency three-fold. These findings are consistent with kinesin-5 acting as a microtubule polymerase. To understand this polymerase mechanism, TIRF microscopy was used to visualize individual GFP-labeled kinesin-5 dimers on immobilized microtubules. Motors walked to the ends of microtubules and remained bound there for 7 seconds, much longer than the 0.1 s motor step time. We hypothesize that kinesin-5 promotes microtubule polymerization by stabilizing longitudinal interactions between tubulin subunits on a single protofilament. Consistent with this protofilament stabilization hypothesis, fluorescence analysis suggests that microtubule plus-ends are more tapered in the presence of kinesin-5. Furthermore, curved and looped ends were observed, which occasionally resolved into straight microtubules, consistent with kinesin-5 stabilizing long protofilament bundles. In cells, these end-binding and polymerase activities should enhance the ability of kinesin-5 to establish and maintain spindle pole separation during mitosis.