FIGURE 4.

Proteasome inhibition suppresses OMM rupture but not mitophagy. A, wild-type (panels a–g) and FIP200 KO (panels h–k) MEFs stably expressing Parkin were incubated with 20 μm CCCP for 12 h with (panels c–g, j, and k) or without (panels a, b, h, and i) 5 μm lactacystin (Lac) and then subjected to electron microscopy. Rectangles show enlarged areas in panels b, d, i, and k. Autophagosomes without mitochondria (black arrowheads) and with mitochondria (white arrowheads) are shown. Single stars indicate mitochondria with ruptured OMM (panels b and i). In panel g, double stars indicate a degrading mitochondrion inside an autophagic vacuole. In panels b and i, arrows indicate residual OMM. Scale bars, 1 μm (panels a, c, h, and j) and 500 nm (panels b, d, e–g, i, and k). B, the ratio of ruptured and unruptured mitochondria (mt) in isolation membrane (IM)/autophagosome (AP)-like structures or in the cytoplasm was calculated from at least eight randomly selected cells. Lac, lactacystin. C, the number of IM/AP-like structures with (white columns) and without (black columns) mitochondria was counted from at least eight randomly selected cells. For bafilomycin A₁ (Baf) treatment, cells were incubated with 20 μm CCCP and 0.2 μm bafilomycin A₁ for 12 h. Selected images of wild-type cells are shown in supplemental Fig. S4.