Fig 2: Neurons depleted for Vps13D contain mitophagy intermediates lacking matrix.Representative images of motoneurons in the larval VNC which co-express UAS-mitoGFP (cyan) with UAS-luciferase RNAi (control) (BL# 31603) versus UAS-Vps13D RNAi (BL# 38320), via the D42-Gal4 driver. A) VNC tissue was stained for polyubiquitin (PolyUb, FK1, red) and Ref(2)p (p62 homolog, yellow). Dashed box outlines a single Gal4-expressing neuronal cell body that is shown in high magnification in the inset in the bottom right corner. Arrowheads indicate examples of PolyUb+ objects (PolyUb+/Ref(2)p+/mitoGFP-). Vps13D RNAi expressing neurons contained on average 1.88 PolyUb+ objects per cell body (n = 239 neurons from 5 ventral nerve cords (VNCs)), while such objects were never observed in neurons expressing control RNAi. 98.8% of PolyUb+ objects were Ref(2)p+, n = 485 PolyUb+ objects from 5 larval VNCs. Scale bars = 10μm, 2μm. B) Co-staining for mitoGFP (cyan), polyubiquitin (PolyUb, red) and endogenous IMM protein ATP5A (yellow). The arrowhead highlights an example PolyUb+ object, which contains ATP5A, but lacks mitoGFP. Scale bar = 2μm. C) Distribution of mean intensities for mitoGFP (top) and ATP5A (bottom) from mitochondria in motoneurons. The right column shows objects selected based on positivity for PolyUb. The left column shows the population of conventional (PolyUb-) mitochondria (selected by similar criteria). Based on the criteria of >2.5% of the intensity distribution of the PolyUb- mitochondria population (shaded boxes), PolyUb+ objects were classified as + for mitoGFP and/or ATP5A if they were within the shaded box. n = 809 mitochondria and 184 PolyUb+ objects from 5 larval VNCs.

Fig 3: Utility of transgenic reporter lines in RNAi based genetic screen. (A) Germaria of nosP GFP-Ref(2)P nos 3′UTR and nosP mCherry-Atg8a nos 3′UTR transgenic flies in background of either no knockdown (control) or knockdown of Atg5 by RNAi in the germline. Scale bar 10 μm. (B) Interleaved scatter graph showing the increase in GFP-Ref(2)P punctae upon Atg5 knockdown. Error bars represent SD in red and the mean is blue. n = 20 for both the data sets. ∗∗∗∗p < 0.0001. (C) Interleaved scatter graph showing the decrease in mCherry-Atg8a punctae upon Atg5 knockdown. Error bars represent SD in red and the mean is blue. n = 15 for both the data sets. ∗∗∗p < 0.001.

Fig 4: Generation of mutations in Drosophila Atg9.(A) Schematic view of Atg9Gal4KO and Atg9d51mutations relative to the Atg9 transcripts. For the Atg9Gal4KO mutation, the complete Atg9 open reading frame was replaced with a Gal4 knock-in cassette. For Atg9d51 mutation, the 52–102 bp after the Atg9 start codon was replaced with the attPX-3-frameStop-floxed 3xP3-RFP cassette. (B) RT-PCR analysis of Atg9 mRNA expression level in control, mutant and Atg9 genomic rescue adult flies. Atg9 mRNA levels were undetectable in the Atg9 mutant. (C) Western blots show the endogenous Atg9 protein in control and Atg9 genomic rescue flies but fail to detect the protein in mutants. (D) LysoTracker Green staining reveals that starvation-induced autophagy is strongly reduced in Atg9 mutant fat bodies, compared with controls. Scale bar: 5 μm. (E) Quantification of data shown in (D). n ≥ 10, data are mean ±s.e.m. *p<0.05, **p<0.01. ns, not statistically significant. (F) Western blots show markedly increased Ref(2)P and ubiquitinated protein levels in Atg9 mutants. (G) Immunostaining of Drosophila thoracic muscles with anti-Ub (FK2) and anti-Ref(2)p antibodies showed an accumulation and colocalization of polyubiquitin protein aggregates and Ref(2)p (arrowheads) in Atg9 mutant flies. Scale bar: 10 μm. Df refers to Df(2R)Exel7142, which removes Atg9 and flanking genes.10.7554/eLife.29338.004Figure 1—source data 1.Quantification of lysotracker dots.

Fig 5: New genes function downstream of Arf1 in regulating ISC survival(A–J) The genotypes of the flies in each panel were as follows: (A) Arf1RNAi, n = 37; (B) Arf1RNAi/Dcp-13, n = 33; (C) Arf1RNAi/Dcp-1Prev1, n = 35; (D) Arf1RNAi/LRRUM−8319−3, n= 31; (E) Arf1RNAi/CalrS114307, n = 29; (F) Arf1RNAi/prtpRNAi, n= 34; (G) Arf1RNAi/McrEY0742, n= 31; (H) Arf1RNAi/McrRNAi,n = 36; (I) Arf1RNAi/LRP1EY07878,n = 29; (J) Arf1RNAi/LRP1MI03128, n = 32. All RNAi was driven by esgts.(K) Quantification of Dcp1+ cells in the indicated panels.(L) Quantification of GFP+ cells in the indicated panels.(M) Quantification of Ref(2)P+ cells in the indicated panels.The posterior midgut of flies with the indicated genotypes was cultured for 4 days at 29°C, dissected, stained with the indicated antibodies, and analyzed by confocal microscopy. Data show the mean ± SEM. Statistical significance was determined by Student’s t test; ***p < 0.001. n indicates the number of midguts examined for each genotype. Scale bars, 10 μm.