Bif-1 plays a key role in the maturation of nascent autophagosomes during mitophagy. myc-Parkin–expressing mouse embryonic fibroblasts were treated with 30μM CCCP for 24 hours and subjected to electron microscopic analysis. (A) Representative images of Bif-1 wild-type and Bif-1–deficient cells are shown in subpanels i and ii. OMM-ruptured or OMM-preserved fragmented mitochondria that were closely associated with an isolation membrane are shown in subpanels iii and iv. An Avd-like structure observed in Bif-1–deficient cells is shown in subpanel v. Black arrowheads and arrows indicate endoplasmic reticulum (ER) membranes and ER-associated IMs, respectively. Open arrowheads, double-arrowheads, and arrows indicate Avd-like structures, fragmented mitochondria, and Avi-like structures, respectively. The scale bars represent 1 μm in subpanels i and ii and 0.5 μm in subpanels iii through v. The number of ruptured mitochondria and total mitochondria (OMM-ruptured and OMM-preserved; B), ER-associated IM/Avi-like structures and total IM/Avi-like structures (ER-associated and ER-nonassociated; C), and Avd-like structures (D) per cell were counted, normalized with cytoplasmic area (100 μm2) and are shown as box plots. The lines, boxes, and error bars represent median values, 25-75 percentiles, and 10-90 percentiles, respectively. Statistical significance was determined using a Wilcoxon rank-sum test with continuity correction. *P < .0001; **P = .0227 in panel C and P = .0018 in panel D.
