Atg9 antagonizes TOR signaling to regulate intestinal cell growth and epithelial homeostasis in Drosophila

Essential revisions:

1) It is not completely clear whether the two autophagy factors, Atg9 and Atg1, regulate mTOR independently as shown in Figure 7H. The following experiments would clarify this issue.

- Can the midgut phenotype of Atg1 or Atg17 knockdown flies be rescued by overexpression of Patj (Figure 7E)?

- Is TSC2 unstabilized in Atg1 or Atg17 knockdown flies as in Atg9 knockdown flies (Figure 7G)?

- Can the Atg9-Ptj-TSC2 form a complex normally in Atg1 knockdown flies?

We thank the reviewer for this comment. We have performed several new experiments to demonstrate that Atg9 and Atg1 regulate TOR activity independently. As shown in new Figure 7—figure supplement 1A-B, we found that overexpression of Patj could not rescue the midgut defects caused by Atg1 or Atg17 depletion. Moreover, clonal analysis showed that although the TSC2 level was markedly decreased in Atg9 knockdown cells, TSC2 levels were not affected in Atg1 or Atg17 knockdown cells, compared with controls (new data, Figure 8—figure supplement 1B). Finally, co-immunoprecipitation assays showed that Atg9 can still interact with Patj and TSC2 in Atg1 knockdown S2 cells (new data, Figure 8—figure supplement 1A). In mammals, it has been shown that Atg1/Ulk1 inhibits TOR signaling by phosphorylating Raptor and impairs substrate binding to Raptor (Dunlop et al. 2011, Autophagy and Jung et al. 2011,Autophagy), and the inhibitory effect of Ulk1 on TOR signaling occurred independently of TSC2 (Jung et al. 2011, Autophagy). Our new results and findings from mammalian cells together strongly indicate that Atg9 inhibits TOR activity independent of Atg1.

2) Figure 5A and 5B: Is autophagy efficiently blocked by temporal knockdown using the TARGET system. To strengthen the results, the authors should show that autophagic activity is indeed inhibited in these knockdown flies or examine adult midgut in pre-existing mutant flies such Atg7 mutants.

We thank the reviewer for this comment. To determine whether autophagy is efficiently blocked by temporal knockdown Atg genes using the TARGET system, we have knocked down Atg genes using Tub-Gal4^ts and checked their effects on starvation-induced autophagy in the larval fat body. The negative control was flies maintained at 18°C, and the positive control was flies shifted to 29°C to inactivate Gal80^ts and enable expression of the RNAi targeting Atg genes. As shown in new Figure 5—figure supplement 2A-B, depletion of Atg1, Atg13, Atg17, Atg7, Atg12, Atg16, Atg18, and Vps34 impaired starvation-induced autophagy. We have previously shown that depletion of Atg9 and Atg12 blocked ROS-induced autophagy in adult midgut (Tang et al. 2013, Dev Cell). Similarly, we found that ROS-induced autophagy is efficiently blocked in the adult midgut of Atg7 mutants (Author response image 1).

Author response image 1

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3) Figure 7G: Depletion of Atg9 in the adult midgut causes a marked decrease in the TSC2 signal level in immunostaining, but it is not very quantitative. The authors should perform immunoblotting to compare the TSC2 protein level in control and Atg9 mutants, as performed in Figure 6A.

We thank the reviewer for this comment. We have performed immunoblotting analysis to show that TSC2 level is markedly decreased in the midgut of Atg9 mutants, compared with controls (new data, Figure 8C).

4) It remains unclear whether the increased growth of EC in ATG9 mutants is really biologically important. When the increased EC size in ATG9 mutants is rescued (e.g. by TOR inhibition or Patj overexpression in EC cells), are the barrier function and the shortened lifespan restored? These will demonstrate that the increased EC size after a loss of ATG9 is functionally important in affecting the midgut epithelium and lifespan.

We thank the reviewer for this comment. Our new data showed that treating flies with TOR inhibitor rapamycin rescues the intestinal barrier defects of Atg9 mutant animals (new data, Figure 6—figure supplement 1A). Moreover, we have previously shown that ablation of Atg9 in EC cells leads to increased lethality in response to paraquat ingestion (Tang et al. 2013, Dev Cell). Strikingly, ectopic expression of Patj in EC cells significantly rescued the paraquat-induced lethality of Atg9 knockdown animals (new data, Figure 7—figure supplement 1C). These results together strongly suggest that Atg9 is critical for maintaining midgut epithelium function and integrity.

However, we found that rapamycin treatment could not fully rescue the lifespan defects of Atg9 mutants, compared with controls (new data, Figure 6—figure supplement 1B). It is likely that the additional function of Atg9 may contribute to the shortened lifespan in Atg9 mutant animals.

5) Some of the critical data should be quantified (e.g., Figure 1D, Figure 1—figure supplement 1A, Figure 5C, and Figure 6B-J) to strengthen this manuscript.

We have quantified the data in Figures 1D, Figure 1—figure supplement 1A, Figure 5C, and Figure 6B-J.