Ran, Plk4, and Aurora A cooperate to promote bipolar spindle assembly. (A–D) Time-lapse series of oocytes injected with mRNA encoding EGFP-EB3 (green, top; inverted monochrome, bottom) and H2B-mRFP (red, top). Progression through meiosis I is shown for controls (A), oocytes treated with 1 µM Aurora A inhibitor 1 (AurA Inh 1; B), oocytes imaged in the presence of 2 µM of the Plk4 inhibitor centrinone (C), and oocytes coinjected with RanT24N-mRFP (D). (E) Quantification of the size of the projected spindle area over time. RanT24N, Plk4, and Aurora A alone each contribute to meiotic spindle assembly, because their inhibition causes significantly reduced microtubule growth. Error bars indicate SEM. For p-values, see Table S1. (F) Stills of time-lapse movies showing metaphase spindles in oocytes undergoing meiosis I in conditions described in A–D. RanT24N, as opposed to Aurora A or Plk4 inhibition, affects spindle pole integrity (arrowheads). (G) Compared with Aurora A and Plk4 inhibition, dominant-negative Ran (RanT24N) significantly increases the time taken to establish bipolarity. Error bars indicate SEM.