Kinesin-5/Cut7 C-terminal tail phosphorylation is essential for microtubule sliding force and bipolar mitotic spindle assembly.

Kinesin-5 motors play an essential role during mitotic spindle assembly in many organisms(1)(,)(2)(,)(3)(,)(4)(,)(5)(,)(6)(,)(7)(,)(8)(,)(9)(,)(10)(,)(11): they crosslink antiparallel spindle microtubules, step toward plus ends, and slide the microtubules apart.(12)(,)(13)(,)(14)(,)(15)(,)(16)(,)(17) This activity separates the spindle poles and chromosomes. Kinesin-5s are not only plus-end-directed but can walk or be carried toward MT minus ends,(18)(,)(19)(,)(20)(,)(21)(,)(22)(,)(23)(,)(24)(,)(25)(,)(26)(,)(27)(,)(28)(,)(29)(,)(30)(,)(31)(,)(32)(,)(33)(,)(34) where they show enhanced localization.(3)(,)(5)(,)(7)(,)(27)(,)(29)(,)(32) The kinesin-5 C-terminal tail interacts with and regulates the motor, affecting structure, motility, and sliding force of purified kinesin-5(35)(,)(36)(,)(37) along with motility and spindle assembly in cells.(27)(,)(38)(,)(39) The tail contains phosphorylation sites, particularly in the conserved BimC box.(6)(,)(7)(,)(40)(,)(41)(,)(42)(,)(43)(,)(44) Nine mitotic tail phosphorylation sites were identified in the kinesin-5 motor of the fission yeast Schizosaccharomyces pombe,(45)(,)(46)(,)(47)(,)(48) suggesting that multi-site phosphorylation may regulate kinesin-5s. Here, we show that mutating all nine sites to either alanine or glutamate causes temperature-sensitive lethality due to a failure of bipolar spindle assembly. We characterize kinesin-5 localization and sliding force in the spindle based on Cut7-dependent microtubule minus-end protrusions in cells lacking kinesin-14 motors.(39)(,)(49)(,)(50)(,)(51)(,)(52) Imaging and computational modeling show that Cut7p simultaneously moves toward the minus ends of protrusion MTs and the plus ends of spindle midzone MTs. Phosphorylation mutants show dramatic decreases in protrusions and sliding force. Comparison to a model of force to create protrusions suggests that tail truncation and phosphorylation mutants decrease Cut7p sliding force similarly to tail-truncated human Eg5.(36) Our results show that C-terminal tail phosphorylation is required for kinesin-5/Cut7 sliding force and bipolar spindle assembly in fission yeast.