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

Li Tao,Barbara Fasulo,William Sullivan,Brandt Warecki

doi:10.1038/ncomms11182

Abstract

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.

Highlights

Centralspindlin is essential for central spindle and cleavage furrow formation
Centralspindlin is a heterotetrameric protein complex consisting of dimeric kinesin-6 (Pavarotti or Pav-KLP in Drosophila melanogaster (Pav/kinesin-6), ZEN-4 in Caenorhabditis elegans and CHO1/MKLP1 or MKLP2 in mammals) and the dimeric Rho-family GTPase-activating protein RacGAP (Tumbleweed or RacGAP50C in D. melanogaster (Tum/RacGAP), CYK-4 in C. elegans and MgcRacGAP in mammals)[1,2,3,4]
Ni-NTA affinity chromatography pulled down the centralspindlin complex (Fig. 1a), which suggests that Tum/RacGAP and Pav/kinesin-6 assemble into a centralspindlin complex inside sf[9] cells

Summary

Introduction

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). 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. In vivo studies of Drosophila cytokinesis reveal that Pav/kinesin-6 fails to localize to anaphase midzone MT bundles in Tum/RacGAP mutants[11]. This suggests that Tum/RacGAP may promote Pav/kinesin-6 motor activity. By developing conditions to stably express full-length Drosophila Pav/kinesin-6 and Tum/RacGAP, we demonstrate that Tum/RacGAP is required for the plus-enddirected motor activity, but not the bundling activity of Pav/kinesin-6. In Tum/RacGAP RNA interference (RNAi)-depleted S2 cells, Pav/kinesin-6 fails to concentrate at the midzone microtubules and instead maintains an unusually pronounced concentration at the centrosomes

Methods

Results

Conclusion