Temporal and spatial analysis of Aurora, Plk4 and RAN in the early steps of microtubule growth and dynamics. (A) Representative time-lapse images of oocytes injected with EGFP-EB3 (green) and histone H2B-mRFP (red) mRNAs showing the three different stages after oocyte meiosis I resumption under four experimental conditions: standard control conditions, Plk4 inhibition by addition of 2 µM centrinone, Aurora A inhibition by addition of 1 µM Aurora A inhibitor I, and Ran pathway inhibition by coexpression of the RanDN-negative form RanT24N. Regions of interest (ROIs) are indicated in the image, where ROI 1 is marking a cytoplasmic MTOC and ROI 2 a chromosomal MTOC typically bleached and analyzed by FRAP. Stage 1 occurs immediately after NEBD for a period of 30–45 min, during which time microtubules emanating from cytoplasmic MTOCs and around chromosomes are observed. Stage 2 comprises the time when microtubule density increases adopting a round ball-like shape. Stage 3 is the early phase of bipolarization when the spindle begins to elongate and MTOCs cluster toward the poles. Bars, 10 µm. (B) Graphs show the nonlinear fit curve for EB3-EGFP fluorescence intensity after bleaching (see Materials and methods for details) in oocytes after meiosis resumption under the different experimental conditions through the three stages described in A. Time after photobleaching is indicated as t = 0 s Fluorescence recovery and t1/2 were determined by applying a single exponential fitting to the recovery curve. Time is given in seconds. Each curve represents the mean value and 95% confidence interval. The 95% confidence interval for mobile fraction and t1/2 are shown below each graph. Plot of mean of values for each dataset at the three different stages are also shown. Error bar represents SEM. See also Table S4. These samples were both tested for normally distributed (D’Agostino–Pearson) and nonparametric tests (Wilcoxon matched pair test). In both cases, the null hypothesis was rejected (***, P < 0.001).
