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. 2021 Aug 30;220(11):e202104073. doi: 10.1083/jcb.202104073

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© 2021 Shen et al.

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Figure 4. — Vps13D functions in a mitophagy pathway with Pink1. (A) Representative TEM images of male pink1^B9 (−);vps13d (ΔUBA/+) single-mutant and pink1^B9 (−);vps13d (ΔUBA/ΔUBA) double-mutant intestine cells 2 h after pupariation. (B) Quantification of mitochondria area in pink1^B9 (−);vps13d (ΔUBA/+) single-mutant (n = 187) and pink1^B9;vps13d (ΔUBA/ΔUBA) double-mutant (n = 181) intestine cells 2 h after pupariation. (C) Quantification of the percentage of mitochondria <0.01 μm and ≥0.1 μm in pink1^B9 (−);vps13d (ΔUBA/+) single-mutant (n = 187) and pink1^B9 (−);vps13d (ΔUBA/ΔUBA) double-mutant (n = 181) intestine cells 2 h after pupariation using Fisher’s exact test (P = 0.7507). (D) Mito-QC was expressed using the Myo31DFNP0001 driver in pink1^B9 (−);vps13d (ΔUBA) mutant intestine cells from 2-h-old male prepupae. pink1 (+);vps13d (ΔUBA)/+ heterozygote control cells possessed mostly red puncta without corresponding green puncta, reflecting mitochondria in autolysomes (mitolysosomes). By contrast, pink1^B9 (−);vps13d (ΔUBA)/+ single-mutant, pink1 (+);vps13d (ΔUBA/ΔUBA) single-mutant, and pink1^B9 (−);vps13d (ΔUBA)/ΔUBA) double-mutant intestine cells all exhibited red and green structures, reflecting mitochondria that failed to be cleared by mitophagy. These structures were either filamentous (arrowheads) or enlarged and punctate (arrows). Enlarged punctate structures were defined as having an area of at least 1.0 μm². (E) Quantification of the amount of red and green structures that presented as enlarged puncta in pink1 (+);vps13d (ΔUBA)/+ heterozygote control (n = 17), pink1^B9 (−);vps13d (ΔUBA)/+ single-mutant (n = 14), pink1 (+);vps13d (ΔUBA/ΔUBA) single-mutant, and pink1^B9 (−);vps13d (ΔUBA)/ΔUBA) double-mutant (n = 14) cells 2 h after pupariation. (F) pink1^B9/pink1^B9 (−/−);vps13d (ΔUBA/ΔUBA) double-mutant intestine cells (nonred) have similar levels and size of Atg8a puncta (green) compared with pink1^B9/+ (+/−);vps13d (ΔUBA/ΔUBA) single-mutant cells (red) 2 h after pupariation. (G) Quantification of Atg8a puncta number in pink1^B9/pink1^B9 (−/−);vps13d (ΔUBA/ΔUBA) double-mutant cells (n = 11) compared with neighboring pink1^B9/+ (+/−);vps13d (ΔUBA/ΔUBA) single-mutant cells (n = 15). (H) Quantification of Atg8a puncta size in pink1^B9/pink1^B9 (−/−);vps13d (ΔUBA/ΔUBA) double-mutant cells (n = 11) compared with pink1^B9/+ (+/−);vps13d (ΔUBA/ΔUBA) single-mutant cells (n = 15). Scale bars in A are 0.5 μm. Scale bars in D and F are 40 μm with the exception of the enlarged images in D, which are 5 μm. Columns in B, G, and H were compared using two-tailed t test without Welch’s correlation. Columns in E were compared using one-way ANOVA with Tukey’s post hoc analysis. Error bars are SEM. Representative of three or more independent biological experiments.