1. Role and Regulation of Starvation-Induced Autophagy in the Drosophila Fat Body 
Ryan C. Scott, Oren Schuldiner, Thomas P. Neufeld 
Developmental Cell 
Volume 7, Issue 2, Pages (August 2004)
DOI: /j.devcel

2. Figure 1
Autophagy Is Rapidly Induced in the Larval Fat Body in Response to Nutrient Withdrawal
(A–D) TEM images of fat body cells from wild-type animals that were well fed (A) or starved of dietary protein by incubating in 20% sucrose for 3 hr (B–D). Starvation results in accumulation of numerous large autolysosomes (arrows in [B]). In (C), several autophagosomes containing undigested cytoplasmic material (arrowheads) are adjacent to an autolysosome. The autolysosome shown in (D) contains an intact mitochondrion (arrowhead). Scale bars represent 1 μm.
(E) and (F) show live fat body tissues stained with the lysosome-specific fluorescent dye LysoTracker Red (red) and the nuclear dye Höechst (blue). In fed control animals (E), lysotracker staining is weak and diffuse. Fat body tissues from animals starved for 3 hr (F) display intense, punctate lysotracker staining. Scale bar in (E) represents 25 μm.
Developmental Cell 2004 7, DOI: ( /j.devcel )

3. Figure 2 Quantitation of Autophagic Structures
(A) Quantitation of autophagosomes and autolysosomes from TEM images of fat body from fed and starved animals. For induction of RNAi against ATG5, UAS-ATG5IR24-1 was driven by AdhE2-GAL4. (B) Quantitation of lysotracker staining. The number of lysotracker-positive spots per unit area under fed (left) and starved (right) conditions are shown for each genotype, normalized to the value for starved wild-type cells processed in parallel. For UAS-regulated transgenes, clonal expression was induced through flippase-driven activation of Act>CD2>GAL4. For both (A) and (B), early third instar larvae were either cultured continuously on standard fly food (fed) or placed on 20% sucrose (starved) for 3 hr prior to dissection.
Developmental Cell 2004 7, DOI: ( /j.devcel )

4. Figure 3 TOR and PI3K Signaling Inhibit Starvation-Induced Autophagy
(A–C) Loss of TOR activity causes induction of autophagy in normally fed animals. Lysotracker staining (A) and TEM (B) of fat body from a TOR null homozygote showing accumulation of autophagic lysosomes. Scale bar in (B) represents 1 μm. For all other panels, scale bar in (A) represents 25 μm. (C) Clonal co-overexpression of TSC1 and TSC2 (GFP-positive cells) in fed animals causes cell-autonomous induction of autophagy.
(D) Activation of autophagy by TSC1/2 overexpression can nonautonomously suppress starvation-induced autophagy in neighboring cells. Following 3 hr starvation, heatshock-induced clones of cells coexpressing TSC1 and TSC2 (marked with GFP) display lysotracker staining, whereas staining in some adjacent wild-type cells (outlined in yellow in inset) is reduced or undetectable.
(E and F) Constitutive activation of TOR prevents starvation-induced autophagy. Shown are lysotracker-stained fat bodies from starved animals homozygous mutant for TSC2 (E) or clonally overexpressing Rheb and GFP (F).
(G–I) Class I PI3K signaling suppresses starvation-induced autophagy. Loss-of-function mutations in PI3K/p110 result in lysotracker staining under fed conditions (G), whereas clonal expression of PI3K prevents lysotracker staining induced by starvation (H). Expression of PI3K does not inhibit the lysotracker staining observed in TOR mutant animals (I).
Insets show the lysotracker channel of the respective images, with clonal boundaries outlined in yellow.
Genotypes: (A and B) TORΔP/TORΔP. (C and D) hs-flp/+; UAS-TSC1 UAS-TSC2/Act>CD2>GAL4 UAS-GFP. (E) TSC2109/TSC2192. (F) hs-flp/+; UAS-RhebEP50.084/+; Act>CD2>GAL4 UAS-GFP/+. (G) p110A/p110A. (H) hs-flp/+; UAS-PI3K/Act>CD2>GAL4 UAS-GFP. (I) hs-flp/+; TORΔP UAS-PI3K/TORΔP tub>CD2>GAL4 UAS-GFP.
Developmental Cell 2004 7, DOI: ( /j.devcel )

5. Figure 4 S6 Kinase Has a Positive Role in Autophagy Induction
(A–C) Animals lacking S6K fail to induce autophagy under fed or starvation conditions or in response to loss of TOR. The images show lysotracker-stained fat body from an S6K null animal either fed (A), starved 3 hr prior to dissection and staining (B), or null for TOR (C). Scale bar in (A) represents 25 μm for all panels.
(D–F) Activation of S6K does not alter autophagy induction in wild-type animals, but increases the level of autophagy in TOR mutants. GFP-marked clones of cells expressing activated S6K were induced in wild-type (D and E) or TOR mutant (F) larvae. Insets depict lysotracker staining with clonal boundaries outlined in yellow.
(G) TOR suppresses autophagy independently of S6K, by inhibiting the activity of ATG1 or other regulators or effectors of autophagy. In contrast, induction of autophagy requires S6K function, either directly or due to its effects on protein synthesis.
Genotypes: (A and B) S6Kl1/S6Kl1. (C) TORΔP/TORΔP; S6Kl1/S6Kl1. (D and E) hs-flp/+; UAS-S6KT398E/+; Act>CD2>GAL4 UAS-GFP/+. (F) hs-flp/+; TORΔP tub>CD2>GAL4 UAS-GFP/TORΔP UAS-S6KT398E.
Developmental Cell 2004 7, DOI: ( /j.devcel )

6. Figure 5 Drosophila ATG Homologs Promote Starvation-Induced Autophagy
(A and B) Genetic disruption of ATG1 or ATG5 prevents induction of autophagy in response to starvation. Lysotracker stainings of ATG1 null fat body (A) and fat body containing clones of cells in which RNA-mediated interference against ATG5 has been induced ([B]; GFP-positive cells) are shown. The inset in (B) shows lysotracker staining with the ATG5-RNAi clones outlined in yellow. Scale bar in (A) represents 25 μm for (A) and (B), and scale bar in (C) represents 25 μm for (C) and (D).
(C and D) Drosophila homologs of ATG8/LC3 are redistributed from a uniform distribution in fed animals (C) to a punctate pattern in response to starvation (4 hr starvation shown in [D]). Most GFP-ATG8b punctae are found either adjacent to (small arrowheads) or overlapping with (large arrowheads) lysotracker-labeled structures.
Genotypes: (A) ATG1Δ3D/ATG1Δ3D. (B) UAS-ATG5IR24-1/hs-flp; Act>CD2>GAL4 UAS-GFP/+. (C and D) hs-GFP-ATG8b6B/hs-GFP-ATG8b6B.
Developmental Cell 2004 7, DOI: ( /j.devcel )

7. Figure 6
Inhibition of Autophagy Increases the Severity of TOR Mutant Phenotypes
(A) Inhibition of ATG5 increases the severity of the larval growth arrest of TOR mutants. RNAi-mediated suppression of ATG5 does not alter growth of wild-type larvae, but causes a more severe growth arrest in TOR null animals. Average calculated volume (v, mm3) of 15 larvae of each genotype is shown. Scale bar represents 1 mm. Genotypes (left to right): UAS-ATG5IR24-1/+, UAS-ATG5IR24-1/+; tub-GAL4/+, UAS-ATG5IR24-1/+; TORΔP/TORΔP, UAS-ATG5IR24-1/+; TORΔP tub-GAL4/TORΔP.
(B) Inhibition of ATG5 causes increased rapamycin sensitivity. Under normal conditions, the growth rate of animals with ubiquitous induction of RNA interference against ATG5 (UAS-ATG5IR/+; Act-GAL4/+) is indistinguishable from wild-type. Growth on fly media containing 1 μM rapamycin delays development by ∼4 days in wild-type, and by ∼5 days in ATG5IR animals. Three sets of 60 larvae of each genotype were scored for time of development to adult stage.
(C and D) Cell size in TOR mutant fat body is altered in response to inactivation of ATG5 or activation of S6K. Clonally induced RNAi against ATG5 further reduces the size of TOR mutant cells (C). Clonal expression of activated S6K suppresses the small size phenotype of TOR mutant cells (D). In these images, clones are marked by coexpression of GFP, and cell outlines (cortical actin-phalloidin staining) are shown in red. Scale bar in (C) represents 25 μm for (C) and (D). Genotypes: (C) hs-flp UAS-ATG5IR24-1/UAS-ATG5IR24-1; TORΔP tub>CD2>GAL4 UAS-GFP/TORΔP. (D) hs-flp/+; TORΔP tub>CD2>GAL4 UAS-GFP/TORΔP UAS-S6KTE1.
(E) Simultaneous loss of ATG1 and TOR causes embryonic lethality. The graph shows hatching rates of wild-type, ATG1EP3348, TORΔP, and ATG1EP3348 TORΔP homozygous mutant embryos. n = number embryos scored per genotype.
(F and G) Genetic disruption of ATG1 or ATG5 results in hypersensitivity to starvation. Animals with organism-wide loss of ATG1 (F) or with fat body-specific induction of RNAi against ATG5 using the FB-GAL4 driver (G) display accelerated death under starvation conditions. The graphs show percent surviving wild-type and mutant larvae at the indicated days of starvation. The data in (F) are normalized to percent survival of fed larvae. Three sets of 30–50 larvae of each genotype were scored. Genotypes: (F) ATGΔ3D/ATGΔ3D. (G) UAS-ATG5IR24-1/UAS-ATG5IR24-1; FB-GAL4/FB-GAL4. Control: FB-GAL4/FB-GAL4.
Developmental Cell 2004 7, DOI: ( /j.devcel )

8. Figure 7 Regulation of Cellular Nutrient Flux by TOR
In cells with active TOR signaling, stimulation of nutrient import and protein synthesis causes accumulation of mass and hence cell growth. Disruption of TOR inhibits these processes, thereby causing reduced growth, and leads to induction of autophagy, which recycles nutrients to provide energy necessary for normal cell function and survival.
Developmental Cell 2004 7, DOI: ( /j.devcel )