Drp1 is required for fission of subdomains harboring ΔOTC but not ΔOTC clearance. (A) Tet ON: ΔOTC-expressing HeLa cells expressing YFP-Parkin (green) and mito-RFP (red) were imaged live over the indicated time points. Yellow arrows mark a Parkin focus that fragmented and trafficked away from a mitochondrion. (B) Tet-ON: ΔOTC-expressing HeLa cells expressing YFP-Parkin (green), mito-RFP (red), and Drp1K38A were imaged live over the indicated time points. Yellow arrows denote the location of immobile Parkin foci on the intact mitochondrial network. Bars, 10 µm. (C) Quantification of the percentage of cells with mobile Parkin foci that underwent fission events from mitochondrial subdomains in Tet-ON: ΔOTC-expressing HeLa cells expressing YFP-Parkin with or without Drp1 inhibition after 48 h DOX treatment. For control and Drp1 KO, n ≥ 3; n ≥ 20. For Drp1 K38A, n = 2; n ≥ 10. (D) Tet-ON: ΔOTC-expressing HeLa cells with or without a Drp1 KO background expressing YFP-Parkin were treated with DOX for 48 h or 48 h with a 24-h washout of DOX with or without treatment with 200 nM bafilomycin and 20 µM QVD and then processed for Western blot analysis. (E) Quantification of Western blots as described in D and expressed as the percentage of ΔOTC levels after 48 h DOX treatment after normalization to Hsp90 levels. n = 4. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars indicate SD.