Atg9 antagonizes TOR signaling to regulate intestinal cell growth and epithelial homeostasis in Drosophila
- Academia Sinica, Taiwan
- College of Life Science, National Taiwan University, Taiwan
- National Taiwan University College of Medicine, Taiwan
Figures
Figure 1 with 1 supplement
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. …
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Figure 1—source data 1
Quantification of lysotracker dots.
- https://doi.org/10.7554/eLife.29338.004
Figure 1—figure supplement 1
Loss of Atg9 leads to impaired developmental autophagy and delayed degradation of larval midgut.
(A) LysoTracker Green staining reveals that developmental autophagy is strongly reduced in Atg9 mutant larval fat bodies, compared with controls. The number of lysotracker-positive dots in control …
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Figure 1—figure supplement 1—source data 1
Quantification of lysotracker dots and gastric caeca size.
- https://doi.org/10.7554/eLife.29338.005
Figure 2
The Atg9 mutant flies display shortened lifespan, locomotor defects and decreased stress tolerance.
(A) Both Atg7 and Atg9 mutant flies showed shortened lifespan compared with control (Log-rank test, p<0.001). Comparing with Atg7 mutant, Atg9 mutant flies exhibited dramatically shortened lifespan. …
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Figure 2—source data 1
Survival rate and climbing activity of control and Atg9 mutants.
- https://doi.org/10.7554/eLife.29338.007
Figure 3 with 1 supplement
Atg9 is required for adult Drosophila midgut morphogenesis.
(A) Atg9 mutant midguts are shortened and display enlargement in the posterior region (arrow). Scale bar: 500 μm. Quantification of adult midgut length and posterior midgut width of control and Atg9 …
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Figure 3—source data 1
Quantification of midgut length and width, cell size, total cell number, and PH3+, Delta+, Pros+ cells per gut.
- https://doi.org/10.7554/eLife.29338.010
Figure 3—figure supplement 1
Loss of intestinal integrity in aged Atg9 mutant flies.
(A) Representative images of Smurf flies. Smurf-, the blue dye is restricted to the proboscis and digestive tract. Smurf+, the dye is seen throughout the body due to intestinal barrier dysfunction. …
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Figure 3—figure supplement 1—source data 1
Percentage of Smurfs.
- https://doi.org/10.7554/eLife.29338.011
Figure 4 with 2 supplements
Loss of Atg9 leads to enlarged enterocytes.
(A) Expression Atg9RNAi in ISCs, EBs and ECs with Dl-Gal4, Su(H)GBE-Gal4, or NP1-Gal4, respectively. Ablation of Atg9 in ECs, but not ISCs or EBs, caused enlarged cell size. n ≥ 25, ***p<0.001. (B) …
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Figure 4—source data 1
Quantification of cell size.
- https://doi.org/10.7554/eLife.29338.015
Figure 4—figure supplement 1
Atg9 depletion in visceral muscle does not cause any observable defects in the midgut.
Temporal control of Atg9RNAi expression using the muscle-specific how-Gal4; Gal80ts inducible system. The flies were either maintained at 18°C throughout development or shifted to 29°C after …
Figure 4—figure supplement 2
Atg9 depletion does not affect cell size of larval imaginal discs.
Clonal depletion of Atg9RNAi (GFP positive cells) in larval wing and eye discs showed that Atg9 depletion did not affect cell size, compared with controls (GFP negative cells). Scale bar: 20 μm. …
Figure 5 with 2 supplements
Components of Atg1 kinase complex are required for adult midgut epithelium homeostasis.
(A) Systematic knock-down of Drosophila Atg1, Atg7, Atg9, Atg12, Atg13, Atg16, Atg17, Atg18 and Vps34 in adult midgut with the EC-specific driver NP1-Gal4; Gal80ts. The flies were either kept at …
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Figure 5—source data 1
Quantification of cell size.
- https://doi.org/10.7554/eLife.29338.019
Figure 5—figure supplement 1
Depletion of Atg1, Atg13, or Atg17 in adult fly causes intestinal barrier dysfunction and shortened lifespan.
(A) Quantification of Smurf flies showing that temporal knockdown of Atg1, Atg9, Atg13 and Atg17 resulted in intestinal barrier dysfunction in 30-day-old flies, compared with controls. n ≥ 40, data …
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Figure 5—figure supplement 1—source data 1
Survival rate and percentage of Smurfs.
- https://doi.org/10.7554/eLife.29338.020
Figure 5—figure supplement 2
Temporal knockdown of Atg genes impairs autophagy.
(A) Temporal control of Atg genes RNAi expression using the Tub-Gal80ts; Tub-Ga4 inducible system. The flies were either maintained at 18°C throughout development or shifted to 29°C 1 day after egg …
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Figure 5—figure supplement 2—source data 1
Quantification of Atg8 dots.
- https://doi.org/10.7554/eLife.29338.021
Figure 6 with 2 supplements
Loss of Atg9 enhances TOR activity in Drosophila adult midgut.
(A) The adult midguts of denoted genotypes were dissected, lysed, and subjected to Western blot analysis with anti-Atg9, anti-Ref(2)P, anti-p-S6K, anti-p-4EBP and anti-tubulin antibodies. (B) …
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Figure 6—source data 1
Quantification of cell size.
- https://doi.org/10.7554/eLife.29338.025
Figure 6—figure supplement 1
Rapamycin treatment rescues the intestinal barrier dysfunction of Atg9 mutants.
(A) Quantification of Smurf flies showing that rapamycin treatment significantly decreased the number of Smurf-positive flies in aged Atg9 mutants. n ≥ 120, data are mean ±s.e.m., **p<0.01. ns, not …
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Figure 6—figure supplement 1—source data 1
Survival rate and percentage of Smurfs.
- https://doi.org/10.7554/eLife.29338.026
Figure 6—figure supplement 2
Atg9 genetically interacts with components of the insulin receptor/phosphoinositide 3-kinase (InR/PI3K) signaling pathway.
The Atg9RNAi-induced midgut defects (A) could be suppressed by the coexpression of InRRNAi (B), PI3KRNAi (C), or AktRNAi (D), whereas coexpression of PI3K (E) or Akt (F) dramatically enhanced Atg9RNA…
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Figure 6—figure supplement 2—source data 1
Quantification of cell size.
- https://doi.org/10.7554/eLife.29338.027
Figure 7 with 1 supplement
Atg9 interacts with Patj to regulate midgut cell growth.
(A) Lysates of cells expressing Myc-Patj were incubated with GST or GST–Atg9-C (amino acids 668–845) in GST pull-down assays. The pull-down products and input Myc-Patj were analyzed by Western blots …
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Figure 7—source data 1
Quantification of cell size.
- https://doi.org/10.7554/eLife.29338.030
Figure 7—figure supplement 1
Atg9 genetically interacts with Patj.
(A) Overexpression Atg9 or Patj could not suppress the Atg1RNAi-induced midgut defects. (B) Overexpression of Patj could not suppress the Atg17RNAi-induced midgut defects. The cell size of posterior …
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Figure 7—figure supplement 1—source data 1
Survival rate and quantification of cell size.
- https://doi.org/10.7554/eLife.29338.031
Figure 8 with 1 supplement
Atg9 interacts with TSC2 to regulate midgut cell growth.
(A) S2 cells transfected with pWA-Gal4, pUAS-Flag-TSC2 and pUAS-GFP-Atg9 were subjected to immunoprecipitation with anti-Flag or anti-GFP antibody. The immunoprecipitates and total cell lysates were …
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Figure 8—source data 1
Quantification of fluorescent intensity and Western blots.
- https://doi.org/10.7554/eLife.29338.034
Figure 8—figure supplement 1
Atg9 interacts with Patj and TSC2 independent of Atg1.
(A) S2 cells treated with dsRNAs targeting luciferase (Luc) or Atg1 were transfected with pWA-Gal4, pUAS-GFP-Atg9, pUAS-Myc-Patj, and pUAS-Flag-TSC2 and subjected to immunoprecipitation with …
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Figure 8—figure supplement 1—source data 1
Quantification of fluorescent intensity.
- https://doi.org/10.7554/eLife.29338.035
Author response image 1
Loss of Atg7 impairs paraquat-induced autophagy.
Adult flies fed with paraquat caused a marked increase of Atg8 puncta formation in midgut cells. Loss of Atg7 or Atg9 resulted in a marked decrease of paraquat-induced Atg8 puncta formation.
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.29338.036
