Phosphorylation controls spatial and temporal activities of motor‐PRC1 complexes to complete mitosis

During mitosis, spindle architecture alters as chromosomes segregate into daughter cells. The microtubule crosslinker protein regulator of cytokinesis 1 (PRC1) is essential for spindle stability, chromosome segregation and completion of cytokinesis, but how it recruits motors to the central spindle to coordinate the segregation of chromosomes is unknown. Here, we combine structural and cell biology approaches to show that the human CENP‐E motor, which is essential for chromosome capture and alignment by microtubules, binds to PRC1 through a conserved hydrophobic motif. This binding mechanism is also used by Kinesin‐4 Kif4A:PRC1. Using in vitro reconstitution, we demonstrate that CENP‐E slides antiparallel PRC1‐crosslinked microtubules. We find that the regulation of CENP‐E ‐PRC1 interaction is spatially and temporally coupled with relocalization to overlapping microtubules in anaphase. Finally, we demonstrate that the PRC1–microtubule motor interaction is essential in anaphase to control chromosome partitioning, retain central spindle integrity and ensure cytokinesis. Taken together our findings reveal the molecular basis for the cell cycle regulation of motor‐PRC1 complexes to couple chromosome segregation and cytokinesis. Synopsis PRC1 associates with microtubule motors to stabilize the anaphase central spindle, however how they interact remains elusive. This study reveals the molecular basis for the cell cycle regulation of mitotic motor‐PRC1 complexes to organize antiparallel microtubule bundle, and to ensure central spindle integrity, midbody assembly and cytokinesis. Kinesin motors Kif4A and CENP‐E bind PRC1 using a bipartite hydrophobic motif. Phosphorylation of CENP‐E controls its affinity for PRC1 to provide temporal and spatial control for the interaction. CENP‐E slides antiparallel microtubules in the presence of PRC1. Disruption of the PRC1:motor interaction disrupts central spindle integrity and midbody assembly. Kinesin motors Kif4A and CENP‐E bind PRC1 via a bipartite hydrophobic motif to ensure central spindle integrity and midbody assembly.