Tum/RacGAP functions as a switch activating the Pav/kinesin-6 motor

Centralspindlin is essential for central spindle and cleavage furrow formation. Drosophila centralspindlin consists of a kinesin-6 motor (Pav/kinesin-6) and a GTPase-activating protein (Tum/RacGAP). Centralspindlin localization to the central spindle is mediated by Pav/kinesin-6. While Tum/RacGAP has well-documented scaffolding functions, whether it influences Pav/kinesin-6 function is less well-explored. Here we demonstrate that both Pav/kinesin-6 and the centralspindlin complex (co-expressed Pav/Tum) have strong microtubule bundling activity. Centralspindlin also has robust plus-end-directed motility. In contrast, Pav/kinesin-6 alone cannot move microtubules. However, the addition of Tum/RacGAP or a 65 amino acid Tum/RacGAP fragment to Pav/kinesin-6 restores microtubule motility. Further, ATPase assays reveal that microtubule-stimulated ATPase activity of centralspindlin is seven times higher than that of Pav/kinesin-6. These findings are supported by in vivo studies demonstrating that in Tum/RacGAP-depleted S2 Drosophila cells, Pav/kinesin-6 exhibits severely reduced localization to the central spindle and an abnormal concentration at the centrosomes.