The mitotic kinesin-14 KlpA contains a context-dependent directionality switch

Kinesins are microtubule-based motor proteins that convert chemical energy from ATP hydrolysis into mechanical work for a variety of essential intracellular processes. Kinesin-14s (i.e. kinesins with a C-terminal motor domain) are commonly considered to be nonprocessive minus end-directed motors that mainly function for mitotic spindle assembly and maintenance. Here, we show that KlpA - a mitotic kinesin-14 motor from the filamentous fungus Aspergillus nidulans - contains a context-dependent directionality switch. KlpA exhibits canonical minus end-directed motility inside microtubule bundles, but on individual microtubules it unexpectedly moves processively toward the plus ends. Removal of the N-terminal nonmotor microtubule-binding domain renders KlpA diffusive on individual microtubules but does not abolish its minus end-directed motility to collectively glide microtubules, suggesting that the nonmotor microtubule-binding domain likely acts as a switch for controlling the direction of KlpA motility. Collectively, these findings provide important insights into the mechanism and regulation of KlpA functions inside the mitotic spindle.