Requirement for Aurora A predominates over Plk4 to initiate microtubule nucleation. (A–G) Time-lapse series of oocytes injected with mRNA encoding EGFP-EB3 (green, top; inverted, bottom) and H2B-mRFP (red, top). Progression through meiosis I was followed in controls (A) and under simultaneous inhibition of Aurora A and Plk4 by a combination of Aurora A inhibitor 1 and centrinone (B) and treatment with 1 µM VX-680 (C). To assess reversion of the defects in microtubule growth caused by VX-680, oocytes were coinjected with mRNAs for the Plk4 point mutant G95R (D), Aurora A G229L (E), kinase-dead Aurora A G229 (F), and both Aurora A G229L and Plk4 G95R (G). (H–J) Quantifications of the size of the projected spindle area over time. (H) Compared with single kinase inhibition (AurA Inh1, n = 33; centrinone, n = 22), treatment with Aurora A inhibitor 1 and centrinone in combination (AurA Inh1 + centrinone, n = 10) and VX-680 (n = 26) further depresses microtubule growth. (I) Defects caused by VX-680 are not restored by kinase-dead Aurora A G229L (n = 9), partially rescued by expression of Plk4 G95R (n = 10), and significantly restored by expression of inhibitor-resistant Aurora A G229L (n = 15). (J) Rescue by Aurora A G229L is enhanced when Plk4 G95R is coexpressed (n = 14). Error bars indicate SEM (***, P < 0.001; **P < 0.01; *P < 0.05). See also Tables S1 and S2 and Video 3.