Drp1 functions to prevent wholesale mitophagy by restricting PINK1–Parkin activity to mitochondrial subdomains. (A) Tet ON: WT OTC or ΔOTC-expressing HeLa cells expressing Drp1 K38A were treated with DOX for 48 h and then processed for indirect immunofluorescence with an antibody to OTC. (B) Quantification of the percentage of cells with Parkin recruitment in control and Drp1 K38A expressing Tet ON: WT OTC or ΔOTC HeLa cells that also express YFP-Parkin. n = 2; n ≥ 50. (C) Tet-ON: ΔOTC-expressing cells expressing YFP-Parkin (green) with or without a Drp1 KO background with or without treatment with DOX for 48 h were labeled with TMRM (red) and imaged live. (D) HeLa cells expressing Tet ON: WT OTC and ΔOTC in the same cell expressing YFP-Parkin with or without a Drp1 KO background were treated with DOX for 48 h or 48 h with a 24- or 48-h washout of DOX and then processed for Western blot analysis. (E) Quantification of Western blots as described in D expressed as the percentage of OTC levels relative to OTC levels after 48 h DOX treatment normalized to Hsp90 levels. n = 3. *, P < 0.05; **, P < 0.01. Error bars indicate SD. (F) WT HCT116 or HeLa cells expressing YFP-Parkin (green) with or without a Drp1 KO background were labeled with TMRM (red) and imaged live. Bars, 10 µm. (G) Tet ON: ΔOTC-expressing cells also expressing YFP-Parkin with or without a Drp1 KO background were infected with Cell Light mito-GFP virus overnight and then treated with DOX for 48 h or 48 h with a 24-h washout of DOX and processed for Western blot analysis. (H) Model depicting the role of PINK, Parkin, and Drp1 in the selective mitophagy of protein aggregates from mitochondria.