Figure 7.
Mitochondria are taken up into early endosomes in response to the mitochondrial recruitment of ATG16L1[100-250]. (A) ARPE-19 cells stably expressing HA-RAB5AQ79L and mitochondrially localized mCherry-FIS1[101-152] were transduced to express the indicated proteins, or were treated with DFP (1 mM), for 48 h before immunofluorescence staining with anti-HA and confocal microscopy analysis. Representative images are shown. Scale bar: 5 µm. Arrows: mitochondrial material inside of early endosomes. Arrowheads: mitochondrial material at the edge of early endosomes (possibly mitochondria in the process of being taken up). (B) Quantification of the percentage of endosomes containing mitochondrial material from at least 3 independent experiments with a minimum of 10 cells being analyzed per condition in each experiment. Statistics: One-way ANOVA + Tukey’s multiple comparisons test. (C) ARPE-19 cells, transduced as in A, were stained using antibodies against HA and the endogenous mitochondrial protein COX4. Shown are representative confocal images. Scale bar: 5 µm. (D) ARPE-19 cells, as in A, were processed for transmission electron microscopy analysis. Shown is a representative image of an enlarged early endosome in anti-mCherry-ATG16L1[100-250] treated cells. See also Fig. S7C. Scale bar: 1 µm, or 0.5 µm for insets i. And ii. Arrow: mitochondrial structures inside of early endosomes; large arrowhead: mitochondria close to early endosomes; small double arrowheads: limiting single endosomal membrane.