pink1 mutant cells have autophagy and mitochondrial phenotypes that are similar to vps13d mutant cells. (A) pink1^B9 loss-of-function mutant (−/−) intestine cells (nonred) fail to reduce in size and have increased Ref2p puncta (green) compared with neighboring control pink1^B9/+ heterozygous (+/−) cells (red) 2 h after pupariation. (B) Quantification of Ref2p puncta in pink1^B9 mutant (−/−) cells (n = 6) compared with heterozygous control (+/−) cells (n = 15). (C) Quantification of cell size in pink1^B9 mutant (−/−) cells (n = 9) compared with control (+/−) cells (n = 11). (D)pink1^B9 loss-of-function mutant (−/−) intestine cells (nonred) have increased mitochondrial ATP5a (purple) puncta and exhibit enlarged Atg8a puncta (green) compared with heterozygous (+/−) control cells (red) 2 h after pupariation (top panels). Atg8a puncta in mutant cells tend to encircle mitochondrial ATP5a puncta (bottom panels). (E) Quantification of the amount of ATP5a puncta in pink1^B9 mutant (−/−) cells (n = 9) compared with neighboring control (+/−) cells (n = 11). (F) Quantification of the size of Atg8a puncta in pink1^B9 mutant (−/−) cells (n = 7) compared with neighboring control (+/−) cells (n = 13). (G) Quantification of the number of ATP5a puncta having at least 50% of the perimeter encircled by Atg8a in pink1^B9/pink1^B9 mutant (−/−) cells (n = 7) compared with vps13d (MiMic) mutant intestine cells (n = 6). (H)pink1^B9/pink1^B9 loss-of-function mutant (−/−) intestine cells (nonred) have enlarged conjugated ubiquitin (FK2) puncta (purple) compared with heterozygous neighboring control cells (red) 2 h after pupariation (top panels). These enlarged puncta do not seem to encircle mitochondria labeled by mito-GFP (green; bottom panels). (I) Quantification of conjugated ubiquitin (FK2) puncta size in pink1^B9/pink1^B9 mutant (−/−) cells (n = 9) compared with control (+/−) cells (n = 11). (J) Quantification of the number of mito-GFP puncta having a perimeter at least 50% encircled by conjugated ubiquitin in pink1^B9/pink1^B9 mutant (−/−) cells (n = 9) compared with vps13d (MiMic) mutant cells (n = 6). (K) Representative TEM images from sections of control w¹¹¹⁸, pink1^B9 (-), and vps13d (ΔUBA) male larval intestine cells 2 h after pupariation. Images on top and bottom are lower and higher resolution images, respectively. (L) Quantification of mitochondria area in w¹¹¹⁸ (n = 202), pink1^B9 (n = 188), and vps13d (ΔUBA) (n = 185) intestine cells 2 h after pupariation. (M) Quantification of the percentage of mitochondria <0.1 μm or ≥0.1 μm in w¹¹¹⁸ (n = 202), pink1^B9 (-) (n = 188), and vps13d (ΔUBA) (n = 185) intestine cells 2 h after pupariation. Fisher’s exact test values are as follows: w¹¹¹⁸ vs. pink1^B9, P < 0.0001; w¹¹¹⁸ vs. vps13d (ΔUBA), P < 0.0001; and pink1^B9 vs. vps13d (ΔUBA), P < 0.0001. Scale bars in A, D, and H are 40 μm with the exception of the enlarged images in D and H, which are 5 μm. Scale bars in K are 2.0 μm (top panels) and 0.5 μm (bottom panels). Columns in B, C, E–G, I, and J were compared using two-tailed t test without Welch’s correlation. Columns in L were compared using one-way ANOVA with Tukey’s post hoc analysis. Error bars are SEM. Representative of three or more independent biological experiments.