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. 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.
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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 and Atg9 mutants was quantified. n ≥ 5, data are mean ±s.e.m., ***p<0.001. Scale bar: 5 μm. (B) Atg9 mutants exhibit delayed degradation of larval midgut as indicated by the gastric cecum structure (arrowheads) and the overall size of midgut. Quantification of gastric caeca size in control and Atg9 mutants at 8 hr after puparium formation (8h-APF). n = 23, data are mean ±s.e.m., ***p<0.001.
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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. (B) Climbing analysis showed that Atg9 mutant exhibit decreased climbing activity in both male and female flies, compared with controls (***p<0.001). The locomotion defects can be rescued by expressing the Atg9 genomic rescue construct. (C–D) Both male and female Atg9 mutant flies die faster than control flies under complete starvation conditions (Log-rank test, p<0.001) or on paraquat treatment (Log-rank test, p<0.001).
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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 mutant flies. n = 10, **p<0.01. (B) Phalloidin staining of midgut visceral muscles revealed that loss of Atg9 leads to disruption of actin filaments. Scale bar: 20 μm. (C–D) Optical sections of control and the Atg9 mutant midgut epithelium layer stained with anti-Dlg showing that Atg9 mutants display abnormally enlarged cells with apical protrusions (arrowheads) into the lumen. Scale bar: 20 μm. (E) Quantification of cell size (shown in panel C) in control and Atg9 mutant posterior midgut. n ≥ 25, *p<0.05, **p<0.01. (F) Quantification of phospho-Histone3 positive (PH3+) cells per midgut of control and Atg9 mutant flies at 5 days and 30 days of age. n ≥ 8. (G) Quantification of total midgut cell numbers, posterior midgut ISC (Delta+) and EE (Pros+) cell numbers of 5-day-old control and Atg9 mutant adults. n ≥ 10. Data are mean ±s.e.m. ns, not statistically significant.
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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. (B) Quantification of Smurf flies showing loss of intestinal integrity in 30-day-old Atg9 mutants compared with control and Atg9 genomic rescue flies. n > 100, data are mean ±s.e.m., ***p<0.001.
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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) Temporal control of Atg9RNAi expression using the Gal80ts; Tub-Gal4 inducible system. The flies were either maintained at 18°C throughout development or shifted to 29°C after eclosion for 5 days to inactivate Gal80ts and enable expression of the RNAi targeting Atg9. (C) Clonal analysis in adult midgut using Flp-FRT-mediated recombination revealed that Atg9d51 mutant cells (marked by lack of GFP and Atg9 expression) are larger than the controls (GFP-positive cells). n ≥ 17, ***p<0.001. (D) MARCM analysis showed that the enlarged Atg9d51 mutant cells (marked by GFP) are Pdm1 positive EC cells. n ≥ 21, ***p<0.001. Scale bar: 20 μm. Genotypes: (C) hsFLP; FRT42D Ubi-GFP/FRT42D Atg9d51 (D) hsFLP; FRT42D tubGal80/FRT42D Atg9d51; Tub-Gal4/UAS-mCD8GFP.
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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 eclosion for 5 days to inactivate Gal80ts and enable expression of the RNAi targeting Atg9. Scale bar: 20 µm.
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. Genotypes: hsflp; Act-CD2-Gal4-UAS-GFP-Atg8a/UAS-Atg9RNAi.
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 18°C throughout development or shifted to 29°C after eclosion for 5 days to inactivate Gal80ts and enable expression of the RNAi targeting Atg genes. Ablation of Atg1, Atg13, and Atg17, but not other Atg genes, caused increased cell size. (B) Quantification of posterior midgut cell size (shown in A) of Atg depleted flies. n ≥ 15, data are mean ±s.e.m. **p<0.01, ***p<0.001. ns, not statistically significant. (C) Overexpression of Atg1 suppressed Atg9RNAi-induced adult midgut defects. n ≥ 30, ***p<0.001. Scale bar: 20 μm.
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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 are mean ±s.e.m., ***p<0.001. (B) Temporal knockdown of Atg1, Atg9, Atg13 and Atg17 with Gal80ts; Tub-Gal4 led to shortened lifespan, compared with the control (Long-rank test, p<0.001).
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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 laying for inactivate Gal80ts and enable expression of the RNAi. The larval fat bodies from well fed or starved animals were stained with DAPI (blue) and anti-Atg8 antibody. Scale bar: 20 µm. (B) Quantification of Atg8-positive dots of denoted genotypes. n ≥ 4, data are mean ±s.e.m., ***p<0.001. ns, not statistically significant.
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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) Inhibition of TOR activity by feeding flies with rapamycin rescued Atg9 mutant midgut defects. (C) Quantification of posterior midgut cell size shown in (B). n ≥ 17, data are mean ±s.e.m. ***p<0.001. (D–K) Atg9 genetically interacts with components of the TOR signaling pathway. The Atg9RNAi-induced midgut defects (D) could be suppressed by the coexpression of TSC1-TSC2 (E), RhebRNAi (F), dominant-negative TOR (TORTED) (G), or dominant-negative S6K (S6KKQ) (H), whereas coexpression of TOR activator Rheb (I) or knock-down of TSC1 (J) or TSC2 (K) could not rescue the Atg9RNAi-induced midgut defects. Genetic analyses were performed for three times with 100% penetrance of the phenotype. (L) Quantification of posterior midgut cell size shown in (D–K). n ≥ 18, data are mean ±s.e.m. ***p<0.001. ns, not statistically significant. Scale bar: 20 μm.
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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 statistically significant. (B) Inhibition of TOR activity by feeding flies with rapamycin could not rescue the short lifespan of Atg9 mutants, compared with controls. (Long-rank test, p<0.001).
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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 Atg9RNAi-induced midgut defect. Genetic analyses were performed for three times with 100% penetrance of the phenotype. The cell size of posterior midgut ECs of each genotype was quantified. n ≥ 16, data are mean ±s.e.m. ***p<0.001, *p<0.05. ns, not statistically significant. Scale bar: 20 μm.
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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 with the anti-Myc antibody. (B–C) HEK293T cells transfected with Flag-Atg9 and Myc-Patj were subjected to immunoprecipitation with anti-Flag (B) or anti-Myc (C) antibody. The immunoprecipitates and total cell lysates (TCL) were analyzed by Western blot with antibodies as indicated. (D) S2 cells transfected with pWA-Gal4, pUAS-Flag-TSC2 and pUAS-Myc-Patj were subjected to immunoprecipitation with anti-Flag or anti-Myc antibody. The immunoprecipitates and total cell lysates were analyzed by Western blot with antibodies as indicated. (E) Depletion of Patj with NP1-Gal4 resulted in aberrant midgut epithelium and increased EC cell size. n ≥ 34, data are mean ±s.e.m. ***p<0.001. (F) Patj genetically interacts with Atg9. Overexpression of Patj suppressed the Atg9 depletion-induced midgut defects (60% penetrance, n = 24). The cell size of posterior midgut ECs of each genotype was quantified. n ≥ 20, data are mean ±s.e.m. ***p<0.001. Scale bar: 20 μm.
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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 midgut ECs of each genotype was quantified. n ≥ 12, data are mean ±s.e.m. ns, not statistically significant. Scale bar: 20 µm. (C) Overexpression of Patj rescued the paraquat-induced lethality of Atg9 knockdown animals, compared with controls. (Long-rank test, p<0.001).
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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 analyzed by Western blot with antibodies as indicated. (B) Clonal expression of Atg9RNAi (marked in Red) in the adult midgut resulted in marked decrease in TSC2 levels (green). Line scan across the Atg9RNAi clone to show the relative fluorescent intensities of TSC2 in control (dsRed-negative) and Atg9 depletion (dsRed-positive) cells. Scale bar: 5 μm. (C) The adult midguts of denoted genotypes were dissected, lysed, and subjected to Western blot analysis with anti-Atg9, anti-TSC2 and anti-tubulin antibodies. N = 3, data are mean ±s.e.m. *p<0.05. (D) Model for the antagonistic effect of Atg9 on TOR signaling in the regulation of intestinal cell growth and midgut homeostasis in Drosophila. Genotypes: (G) hsflp; UAS-Atg9RNAi/+; Act-CD2-Gal4-UAS-dsRed..
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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 anti-Flag antibody. The immunoprecipitates and total cell lysates were analyzed by Western blot with antibodies as indicated. (B) Clonal analysis showed that TSC2 levels were not affect in Atg1 and Atg17 knockdown cells. Line scan across the TSC2RNAi, Atg1RNAi and Atg17RNAi clones to show the relative fluorescent intensities of TSC2 in control (dsRed-negative) and RNAi-knockdown (dsRed-positive) cells. Scale bar: 5 μm. Genotypes: (B) hsflp; UAS-TSC2RNAi/+; Act-CD2-Gal4-UAS-dsRed, hsflp; UAS-Atg1RNAi/+; Act-CD2-Gal4-UAS-dsRed, and hsflp; UAS-Atg17RNAi/+; Act-CD2-Gal4-UAS-dsRed..
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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.
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- https://doi.org/10.7554/eLife.29338.036
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Atg9 antagonizes TOR signaling to regulate intestinal cell growth and epithelial homeostasis in Drosophila
eLife 6:e29338.
https://doi.org/10.7554/eLife.29338
