Fig. 6: DRP1 inhibits APC/C activity by targeting APC2.

a Co-IP and immunofluorescence were performed to show the interaction between APC2 and DRP1. Chromosome spreads were stained with DRP1 (red) and APC2 (green). Chromosomes: Hoechst 33342 (blue), kinetochores: CREST (magenta). Enlarged images show representative results in the white dotted frames. Scale bar, 5 μm. b Inhibitory effect of the DRP1 on the ubiquitination mediated by APC2/11 and Ubc4 was confirmed with in vitro ubiquitination assay. Purified GST-DRP1 in dilutions of 0.0094, 0.0198, 0.0375, 0.0750, 0.1500, 0.3000 and 0.6000 μg/μl were used in the ubiquitination system. The blots were incubated with anti-FLAG (Ub) and anti-DRP1, respectively. c Inhibitory effect of the DRP1 on the ubiquitination mediated by APC2/11 and UbcH5 was confirmed with in vitro ubiquitination assay. Purified GST-DRP1 in dilutions of 0.0375, 0.0750, 0.1500, 0.3000 and 0.6000 μg/μl were used in the ubiquitination system. The blots were incubated with anti-FLAG (Ub) and anti-DRP1, respectively. d, e Live-cell imaging of eGFP-cyclinB1 and mCherry-Securin in oocytes injected with Apc2 mRNA or Apc2 + Drp1 mRNA. Control, APC2 or APC2 + DRP1 overexpressed oocytes were injected with eGFP-cyclinB1 (d) and mCherry-Securin (e) mRNA, respectively. Time points after NEBD are indicated as hours: minutes. Scale bar, 20 μm. See also in Supplementary Movie 13–18. f, g Time-lapse fluorescence intensities of eGFP-cyclinB1 (d) and mCherry-Securin (e) were measured, respectively. n refers to the number of oocytes from three independent replicates. Data are presented as the mean ± standard deviation (S.D.). h APC/C defects inhibit premature degradation of cyclinB1 and Securin in DRP1-depleted oocytes. Apc2 siRNA or proTAME was used to inhibit APC/C activity. Per lysate contains 150 oocytes (NEBD + 6 h). β-Tubulin was used as the loading control. i Ubiquitination levels in control, APC2 and APC2 + DRP1 overexpressed oocytes were detected with western blot. The blots were probed with anti-ubiquitin and anti-cMyc, respectively. β-Tubulin was used as the loading control. NEBD, nuclear envelope breakdown. Representative blots, stainings, or captures from at least three independent repeats are shown. Source data are provided as a Source Data file. See also in Supplementary Fig. 9.