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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 7. — Vps13D and Park function in parallel Pink1-regulated mitophagy pathways. (A) Mito-QC was expressed in intestine cells from park²⁵/park²⁵ (−/−) prepupae with and without expression of vps13d RNAi. park²⁵/+ (+/−) heterozygous control cells expressing control uas-flp (to account for off-target effects of UAS expression) possessed mostly red puncta without corresponding green puncta, reflecting mitochondria in autolysomes (mitolysosomes). By contrast, park²⁵/park²⁵ (−/−) expressing flp, park²⁵/+ (+/−) expressing vps13d RNAi, and park²⁵/park²⁵ (−/−) expressing vps13d RNAi intestine cells all exhibited red and green structures 2 h after pupariation, reflecting mitochondria that failed to be cleared by mitophagy. park²⁵/park²⁵ (−/−) cells had almost exclusively filamentous structures (arrowheads), park²⁵/+ (+/−) expressing vps13d RNAi cells had almost exclusively enlarged punctate structures (arrows), and park²⁵/park²⁵ (−/−) expressing vps13d RNAi intestine cells had both filamentous and enlarged punctate structures. (B) Quantification of the number of enlarged (>1.0 μm²) punctate structures in (left to right) park²⁵/+ (+/−) heterozygous control (n = 24), park²⁵/park²⁵ (−/−) expressing flp (n = 16), park²⁵/+ (+/−) expressing vps13d RNAi (n = 15), and park²⁵/park²⁵ (−/−) expressing vps13d RNAi (n = 15) intestine cells. (C) Number of mitolysosomes (mCherry-only puncta) were quantified in the following intestine cell genotypes 2 h after pupariation (left to right): park25/+ (+), pink1 (+), luc RNA-expressing (+) intestine cells (n = 14); park25/+ (+), pink1 (+), vps13d RNAi–expressing (-) intestine cells (n = 13); park25/park25 (-), pink1 (+) mutant, luc RNAi–expressing (+) intestine cells (n = 13); park25/park25 (-), pink1 (+), vps13d RNAi–expressing (-) intestine cells (n = 13); and pink1 (-), luc RNAi–expressing (+) intestine cells (n = 11). P < 0.0001 for quantification of mitolysosomes in park25/+ (+), pink1 (+), luc RNAi–expressing (+) intestine cells with all other genotypes. (D) pink1^B9/pink1^B9 (−/−) loss-of-function mutant intestine cells (nonred) have reduced Park protein puncta (green) compared with neighboring pink1^B9/+ (+/−) heterozygous control cells (red) 2 h after pupariation. (E) Quantification of Park protein puncta in pink1^B9/pink1^B9 (−/−) intestine cells (n = 9) compared with neighboring pink1^B9/+ (+/−) control cells (n = 15). (F) vps13d (MiMic) loss-of-function mutant intestine cells (nonred) have similar levels of Park protein puncta (green) compared with neighboring heterozygous control (red) cells 2 h after pupariation. (G) Quantification of Park protein puncta in vps13d (MiMic) intestine cells (n = 9) compared with heterozygous control cells (n = 11). Scale bars in A, D, and F are 40 μm with the exception of the enlarged images in A, which are 5 μm. Columns in B and C were compared using one-sided ANOVA test with Tukey’s post hoc analysis, while E and G were compared using two-tailed t test without Welch’s correlation. Error bars are SEM. Representative of three or more independent biological experiments.