Kinetochore and aurora A activities cooperate to drive centrosome separation. (A) Percentage of cells that fail to establish a bipolar spindle within 15 min after NEBD using either the prophase or prometaphase pathway. Cells were treated with siControl (n = 73), siMcm21R (n = 73), siaurora A + siControl (n = 40), siaurora A + siMcm21R (n = 38), siCENP-E (n = 42), siaurora A + siCENP-E (n = 32), siNuf2R (n = 99), siaurora A + siNuf2R (n = 20), siMCAK + siKif2A (n = 79), siaurora A + siKif2A + siMCAK (n = 52), siControl + ZM1 (n = 33), or siMcm21R + ZM1 (n = 46). (B) Composite images of HeLa H2B-EGFP (green) and α-tubulin–mRFP (red) cells treated with siaurora A + siCENP-E or siaurora A + siKif2A + siMCAK in the prophase or prometaphase pathways. (C) Proposed model for the function of kinetochores in bipolar spindle formation during prometaphase. Just after NEBD, centrosomes are positioned on the same side of the chromatin (blue). Sister kinetochores are in close proximity, face the centrosomes, and are connected to centrosomes via k-fibers, thereby establishing a triangular geometry. A pushing force from kinetochores (F1) based on plus end MT polymerization (poleward MT flux; white arrows) drives centrosomes apart from each other. F1 is combined with additional forces (F2), which include astral MT–cortex interactions (mediated by actin-myosin) and extensile MT sliding (mediated by Eg5), which are two processes under the control of the aurora A kinase activity. The resultant force (F3) has an altered magnitude and directionality, which allows more efficient centrosome separation. Note that the centrosome–kinetochore geometry, and therefore the magnitude and direction of forces, will evolve dynamically as the spindle forms into a structure under force balance. Error bars represent SEM. Bar, 10 µm.