Loss of <i>Drosophila</i> Vps16A enhances autophagosome formation through reduced Tor activity

<p>The HOPS tethering complex facilitates autophagosome-lysosome fusion by binding to Syx17 (Syntaxin 17), the autophagosomal SNARE. Here we show that loss of the core HOPS complex subunit Vps16A enhances autophagosome formation and slows down <i>Drosophila</i> development. Mechanistically, Tor kinase is less active in <i>Vps16A</i> mutants likely due to impaired endocytic and biosynthetic transport to the lysosome, a site of its activation. Tor reactivation by overexpression of Rheb suppresses autophagosome formation and restores growth and developmental timing in these animals. Thus, Vps16A reduces autophagosome numbers both by indirectly restricting their formation rate and by directly promoting their clearance. In contrast, the loss of Syx17 blocks autophagic flux without affecting the induction step in <i>Drosophila</i>.</p

Myc is necessary for starvation-induced autophagy in Drosophila.

<p>(A) <i>Myc</i> depletion in LAMP1-GFP marked fat body cell clones inhibits starvation-induced punctate Lysotracker staining, compared to surrounding control cells that do not express GFP. Mid-L3 stage larvae were analyzed in all fat body experiments unless noted otherwise. (B) Overexpression of Mad also blocks Lysotracker dot formation in cells marked by LAMP1-GFP. (C) Quantification of data presented in panels A and B, n = 7 for all genotypes. (D) Starvation-induced punctate endogenous Atg8a staining is inhibited in <i>Myc</i> RNAi cells marked by LAMP1-GFP expression, compared to neighboring control tissue. (E) Forced expression of Mad also decreases Atg8a dot formation. (F) Quantification of data presented in panels D and E, n = 6 for all genotypes. (G) Starvation leads to the formation of Lysotracker-positive autolysosomes in fat body cells of control L1 stage larvae. (H) A near-complete block of Lysotracker puncta formation is seen in <i>Myc</i> null mutant L1 stage larvae. (I) Quantification of data presented in panels G and H. (J) Double-membrane autophagosomes (ap) and dense autolysosomes (al) degrading sequestered cytoplasmic cargo are observed in midgut cells of control L1 stage larvae. (K) No autophagic structures are seen in midgut cells of L1 stage <i>Myc</i> null mutant larvae. (L) The selective autophagy cargo p62 accumulates in <i>Myc</i> RNAi cells (marked by LAMP1-GFP expression) compared to surrounding control tissue in well-fed animals. (M) Mad overexpression also leads to large-scale accumulation of p62 aggregates. (N) Statistical evaluation of data presented in panels M and N, n = 7 for all genotypes. Scalebars in A, B, D, E, G, H, L, M equal 20 µm. Statistically significant differences are indicated, ** p<0.01.</p

Summary of genetic interactions in the context of Myc-induced overgrowth.

<p>(A) Overexpression of Myc in GFP-positive cells leads to a cell-autonomous increase of size. (B) Genetic inhibition of PERK signaling, autophagy, p62 or Nrf2/cnc signaling prevents Myc-induced overgrowth, as GFP-positive cells are now unable to overgrow neighboring cells. (C) Overexpression of Nrf2 restores Myc-induced overgrowth in the absence of p62, while it has no effect in autophagy-deficient cells, indicating that Nrf2 acts downstream of p62 but in parallel to autophagy. (D) Overexpression of Myc induces UPR, leading to activation of PERK in the ER that triggers autophagy. At the same time, cytoplasmic UPR presumably results in accumulation of p62, which then activates Nrf2.</p

Myc overexpression induces autophagy in Drosophila.

<p>(A) Myc overexpression in GFP-marked fat body cell clones leads to cell-autonomous overgrowth. (B–D) Enhanced punctate Lysotracker staining (B), mCherry-Atg8a dot formation (C) and endogenous Atg8a labeling (D) is observed in Myc-overexpressing fat body cell clones (marked by expression of LAMP1-GFP in panels B and D and by GFP in panel C), when compared to surrounding control tissue in well-fed larvae. (E) Quantification of data presented in B–D, n = 7 for all genotypes. (F) Fat body-specific expression of Myc from transgenes located on chromosome 2 or 3 increase the amount of activated Atg8a-II compared to control fat bodies of well-fed larvae. Numbers indicate the level of lipidated Atg8a-II relative to the loading control Tubulin in different genotypes, as determined by densitometric evaluation. (G) Fat body-specific overexpression of Myc induces formation of typical double-membrane autophagosomes (ap) and autolysosomes that contain cytoplasmic material in various stages of degradation (al), n = 5 for all genotypes. (H, I) Myc overexpression leads to punctate mCherry-Atg8a labeling (I) and increased expression area in the <i>patched</i> domain of wing discs compared to control discs (H). Scalebars in A–D and H–I equal 20 µm. Statistically significant differences are indicated, ** p<0.01.</p

Activation of cnc/Nrf2 is required for Myc-induced overgrowth.

<p>(A) p62 aggregates accumulate in Myc-expressing cells (marked by LAMP1-GFP) relative to neighboring control cells, n = 7 for both genotypes. (B) Overexpression of Myc upregulates p62 and increases ubiquitinated protein profiles in fat bodies of well-fed larvae. Numbers indicate the level of p62 relative to Tubulin in different genotypes, as determined by densitometry. (C) HA-Keap1 strongly binds to FLAG-tagged Drosophila p62 in immunoprecipitation (IP) experiments in cultured cells, unlike HA-Atg9. WB, western blot. (D) Myc overexpression in GFP-positive cells activates the Nrf2/cnc-dependent transcriptional reporter gstD-LacZ. (E, F) Depletion of <i>p62</i> (E) or overexpression of Keap1 (F) inhibits Myc-induced overgrowth of GFP-positive cells relative to neighboring control cells. (G) Co-overexpression of cnc restores Myc-induced overgrowth of GFP-positive <i>p62</i> RNAi cells compared to surrounding cells. (H) Knockdown of <i>p62, cnc, Maf</i>, or overexpression of Keap1 prevents Myc-induced overgrowth relative to neighboring control cells. Co-overexpression of cnc or <i>Keap1</i> depletion has little effect on Myc-expressing cell size. Depletion of Keap1 restores Myc-induced overgrowth in <i>p62</i> RNAi cells, similar to overexpression of cnc. Myc-induced overgrowth is not restored by cnc overexpression in <i>FIP200</i> RNAi or dominant-negative Vps34 expressing cells. n = 8–11 per genotype. (I–K) Knockdown of <i>cnc</i> (I), <i>p62</i> or overexpression of Keap1 prevents Myc-induced increases in mCherry-Atg8a expression area of wing discs (quantifications shown in panel J; n = 10–25 per genotype) without affecting punctate mCherry-Atg8a labeling (K; n = 10–13 per genotype). (L) Myc overexpression in the <i>patched</i> domain increases L3–L4 vein distance, which is inhibited by knockdown of <i>p62</i> or <i>cnc</i>, or by co-overexpression of Keap1. <i>Keap1</i> depletion or cnc co-overexpression restores Myc-induced increased vein distance in <i>p62</i> RNAi wings. n = 8–25 per genotype. Scalebars in A, E–G, I equal 20 µm, and 40 µm in D. Statistically significant differences are indicated, ** p<0.01.</p

Myc-induced UPR is required for increased cell growth and autophagy.

<p>(A) Overexpression of Myc in larval fat bodies increases the level of phosphorylated eIF2α compared to controls. Numbers indicate the level of phospho- eIF2α relative to the loading control Tubulin in different genotypes, as determined by densitometric evaluation. (B) Forced expression of Myc in GFP-marked fat body cell clones increases the levels of phospho- eIF2α in a cell-autonomous manner. (C, D) Overexpression of PERK induces punctate mCherry-Atg8a (C) and Lysotracker (D) labeling in GFP-marked fat body cell clones of well-fed larvae. (E) Quantification of data presented in panels C and D, n = 7–8 per genotype. (F, G) Depletion of <i>PERK</i> (F) or co-overexpression of Gadd34 (G) prevents Myc-induced overgrowth of GFP-marked fat body cell clones relative to surrounding control cells. (H) Quantification of data presented in panels F and G, n = 9–11 per genotype. (I–L) Knockdown of <i>PERK</i> (I) or overexpression of Gadd34 (J) prevents Myc-induced increases in the mCherry-Atg8a expression area of wing discs (K; n = 10–25 per genotype) and inhibits punctate mCherry-Atg8a labeling (L; n = 10–13 per genotype). (M) Myc overexpression in the <i>patched</i> domain increases L3–L4 vein distance relative to L4–L5 vein distance in adult wings, which is inhibited by knockdown of <i>PERK</i> or by overexpression of Gadd34, n = 19–25 per genotype. Scalebars in B–D, F, G, I, J equal 20 µm. Statistically significant differences are indicated, ** p<0.01.</p

Myc-Driven Overgrowth Requires Unfolded Protein Response-Mediated Induction of Autophagy and Antioxidant Responses in <i>Drosophila melanogaster</i>

<div><p>Autophagy, a lysosomal self-degradation and recycling pathway, plays dual roles in tumorigenesis. Autophagy deficiency predisposes to cancer, at least in part, through accumulation of the selective autophagy cargo p62, leading to activation of antioxidant responses and tumor formation. While cell growth and autophagy are inversely regulated in most cells, elevated levels of autophagy are observed in many established tumors, presumably mediating survival of cancer cells. Still, the relationship of autophagy and oncogenic signaling is poorly characterized. Here we show that the evolutionarily conserved transcription factor Myc (dm), a proto-oncogene involved in cell growth and proliferation, is also a physiological regulator of autophagy in <i>Drosophila melanogaster</i>. Loss of Myc activity in null mutants or in somatic clones of cells inhibits autophagy. Forced expression of Myc results in cell-autonomous increases in cell growth, autophagy induction, and p62 (Ref2P)-mediated activation of Nrf2 (cnc), a transcription factor promoting antioxidant responses. Mechanistically, Myc overexpression increases unfolded protein response (UPR), which leads to PERK-dependent autophagy induction and may be responsible for p62 accumulation. Genetic or pharmacological inhibition of UPR, autophagy or p62/Nrf2 signaling prevents Myc-induced overgrowth, while these pathways are dispensable for proper growth of control cells. In addition, we show that the autophagy and antioxidant pathways are required in parallel for excess cell growth driven by Myc. Deregulated expression of Myc drives tumor progression in most human cancers, and UPR and autophagy have been implicated in the survival of Myc-dependent cancer cells. Our data obtained in a complete animal show that UPR, autophagy and p62/Nrf2 signaling are required for Myc-dependent cell growth. These novel results give additional support for finding future approaches to specifically inhibit the growth of cancer cells addicted to oncogenic Myc.</p></div

Myc increases autophagic flux.

<p>(A) Practically no mCherry-GFP-Atg8a dots are seen in control fat body cells of well-fed larvae. (B) Dots positive for both mCherry and GFP appear in control cells upon feeding larvae chloroquine, a drug that blocks lysosomal degradation. (C) Overexpression of Myc results in the formation of puncta that are mostly mCherry-positive. (D) Chloroquine treatment strongly increases the number of mCherry punctae in Myc overexpressing cells. Note that dots are now positive for GFP as well. (A′–D′) Fluorescent intensity and colocalization profiles of mCherry and GFP channels from panels A–D. Pearson correlation coefficients shown in A′–D′ indicate that the correlation of the mCherry and GFP pixel intensity is low in panel C but high in panels A, B, and D. (E) Quantification of data in panels A–D, n = 15–30 per genotype/treatment. Myc expression in fat body cells strongly induces mCherry puncta formation compared to control cells. Chloroquine treatment increases dot numbers both in control and Myc overexpressing cells. (F) Autophagic degradation-dependent conversion of mCherry-GFP-Atg8a to free mCherry is increased by overexpression of Myc in fat body cells. Numbers indicate the level of free mCherry relative to the loading control Tubulin in different genotypes, as determined by densitometric evaluation. Scalebars in A–D equal 20 µm. Statistically significant differences are indicated, ** p<0.01.</p

Normal mitochondrial development before spermatid elongation.

<p>(A-B) Primary spermatocytes have normal mitochondria, stained with Mitotracker in WT (A) and <i>bb8</i>^<i>ms</i> (B) mutants. (C-F’) Development of Nebenkern in <i>bb8</i>^<i>ms</i> mutant (D, F) is normal, similar to WT (C, E) by phase contrast microscopy and mitochondrial sensitive Mitotracker staining in post-meiotic onion stage spermatids (E’, F’). Scale bars: 5 μm. (G-H) Mitochondria of elongated spermatids are decorated by DJ-GFP in WT (G), but not in <i>bb8</i>^<i>ms</i> mutants (H). Nuclei stained with DAPI. Scale bars: 200 μm.</p

Bb8 is a testis-specific glutamate dehydrogenase.

<p>(A) Genomic organization of the <i>bb8</i> locus and the <i>Mi{ET1}CG4434</i>^{<i>MB10362</i>} transposon insertion in the first exon of <i>bb8</i>. Thick grey bars represent UTR sequences, thick blue bars the coding sequences and thin black bars the intronic and non-coding regions. Schematic shows the genomic rescue construct (<i>bb8</i>^<i>gr</i>) (brown bar) and the genomic region (dark green bar) used to make the GFP reporter construct (<i>bb8</i>^{<i>N100aa</i>}). Arrows indicate primer pairs used in the cloning of transgenes (black) and in the Q-RT-PCR (blue). (B-C) Evolutionary conservation showed of Bb8 in <i>Drosophila</i> species (B) and higher organism (C). Maximum-likelihood (B, C) method was used to construct the bootstrap consensus protein phylogeny. Bootstrap values are indicated next to the relevant nodes. (D) The expression of <i>bb8</i> mRNA is restricted to testis and show a strong reduction in <i>bb8</i>^<i>ms</i> mutant testis samples. Relative <i>bb8</i> expression was measured by Q-RT-PCR from wild type (WT) and <i>bb8</i>^<i>ms</i> mutant, from isolated head, carcass and testis samples using <i>bb8</i> and <i>rp49</i> specific primers. Measurements were made in triplicates, <i>rp49</i> was used as reference.</p