Abstract
The Aurora B kinase-containing Chromosomal Passenger Complex (CPC) is an essential regulator of cytokinesis, the final and irreversible step of cell division. During anaphase, the CPC concentrates at the spindle midzone - a bundle of overlapping antiparallel microtubules organized at the cell center. How CPC is selectively enriched at the midzone within the dense, heterogeneous, and dynamic spindle microtubule network is unknown. Here, we define a minimal CPC midzone enrichment module. We show that the maximal enrichment of CPC at antiparallel microtubule overlaps requires PRC1-crosslinked microtubules and the interaction of CPC with two mitotic kinesins, KIF4A and KIF20A. We find that the two motors exhibit a division of labor: KIF20A delivers CPC from non-overlapping microtubules, and KIF4A retains CPC at PRC1-crosslinked overlaps. Conditional depletion of KIF4A in mitotic cells reveals that it is required for CPC localization at the spindle midzone in anaphase. Taken together, our findings reveal how the collective activity of two kinesins enables navigation through the complex microtubule network of the spindle to organize kinase signaling at the cell center for cytokinesis.