Tight attachment of the IM to the mitochondrial surface. (A) EM images of CCCP-treated HeLa cells expressing mCherry-Parkin (mCh-Parkin HeLa) and WT HEK293 cells (HEK), DFP-treated WT and DRP1 KO HeLa cells, and rapalog-treated B/N DKO HeLa cells expressing FKBP-LC3B (FKBP-LC3B B/N DKO). Boxed areas are enlarged and shown below. The arrows indicate OMM and IM. mt, mitochondria. Scale bars: 200 nm (upper row) and 100 nm (lower row). (B) Box and whisker plot of the mean distance (open circles) between the OMM and the IM under CCCP-induced mitophagy (mCherry-Parkin HeLa, n = 16; HEK, n = 11), DFP-induced mitophagy (WT HeLa, n = 6; DRP1 KO HeLa, n = 10), and rapalog-induced mitophagy (FKBP-LC3B B/N DKO, n = 11); *P < 0.05, **P < 0.01, ****P < 0.0001, determined by a Tukey–Kramer test. (C) Model of receptor-mediated mitophagy. At the initiation of mitophagy, ULK1 complex and nascent IM are recruited to the mitochondrial surface independently of BNIP3/NIX (B/N). The IM is tethered to the mitochondrial surface, where it elongates and ultimately forms a mitophagosome, depending on B/N. During elongation of the IM, B/N accumulate, promoting tight attachment of the IM to the mitochondria, while ER contacts the IM rim through linear structures.