BNIP3/NIX are required for tight attachment of the IM to the mitochondrial surface. (A) CLEM images of MitoTracker Deep Red (Mito) and GFP-ULK1 fluorescence (left panels) and EM (right panels) of WT and B/N DKO cells expressing GFP-ULK1 and treated with DFP for 16 h. Boxed areas of EM images are enlarged and shown at the right. (B) Colored circles at the upper right corner outside each image correspond to the colors in the graph in B. mt, mitochondria; yellow arrow, IM; green arrowhead, IMAT; red arrowhead, IM-like structure; orange arrowhead, ER structure. Scale bars: 500 and 100 nm (EM, rightmost). (B) Distance between the OMM and IMs or nascent IMs in 10 profiles of WT and B/N DKO cells. EM profiles are classified into three patterns: IM attached to the mitochondrial surface (IM on mito, blue); IM not covering the mitochondrial surface (IM not covering mito, white); and IM-like structures or ER attached to the mitochondrial surface (nascent IM on mito, orange). Horizontal bars indicate the median; **P < 0.005 determined by Mann–Whitney U test. IMAT, IM-associated tubules.