Autophagy machinery is recruited at focal sites on mitochondria containing misfolded protein aggregates. (A) Tet-ON: WT OTC or ΔOTC-expressing cells expressing YFP-Parkin (green) were treated with DOX for 48 h and processed for indirect immunofluorescence microscopy with antibodies against OTC (red) and ubiquitin (Ub; blue). (B) Tet ON: ΔOTC-expressing HeLa cells expressing YFP-Parkin (green) and mCherry-optineurin (mCh-OPTN; blue) were fixed and stained with an antibody against OTC (red). (C) Tet ON: ΔOTC-expressing HeLa cells expressing YFP-Parkin (green) and mCherry-optineurin (red) were fixed and stained with antibodies against TOM20 and PDH (blue). A superresolution Airyscan is depicted. (D) Tet ON: ΔOTC-expressing HeLa cells also expressing YFP-Parkin (green) and mCherry-LC3 (red) were fixed and stained with antibodies to TOM20 and PDH (blue). For A–D, blue arrows denote Parkin foci that colocalized with ΔOTC, optineurin, ubiquitin, or LC3 on mitochondria, respectively. Bars: (A, top, and B–D) 10 µm; (A, bottom) 5 µm.