Target of rapamycin (Tor) inhibition and protein starvation are both required for precocious autophagy in buffy mutant fat body. (A) After 2 h of rapamycin treatment, LTR staining of wild-type and buffy fat bodies are roughly equivalent. Scale bar = 11.8 μm and is the same for all images. (B) Quantification of the percentage of larvae with moderate to robust fat body LTR stain after the indicated minutes of rapamycin exposure (number of larvae: 40 minutes: wild-type and buffyH37 n = 4; 80 minutes: wild-type n = 7, buffyH37 n = 5; 120 minutes: wild-type n = 16, buffyH37 n = 15). (C) The addition of 2 h of protein starvation to rapamycin treatment induces a robust LTR-positive response in the buffy mutant. (D) Quantification of the density of LTR-positive punctae in fat bodies from larvae treated with rapamycin and protein starved for 2 h (number of larvae: wild-type, n = 7; buffyH37, n = 5). (E) Schematics to describe data from (A-D). In the top panel depicting protein starvation, Tor activity drops from a greater level in the buffy mutant relative to wild-type following the timepoint (arrowheads) at which nutrient stress is sensed. Autophagy is initiated later in wild-type than in the buffy mutant (blue arrows). In the middle panel depicting rapamycin feeding, rapamycin inhibition of Tor begins at the same time (vertical black arrow) in both genetic backgrounds, but because there is more Tor activity in the buffy mutant, autophagy initiation is delayed (blue arrows). In the bottom panel depicting rapamycin plus protein starvation, two scenarios are presented. In (1), the nutrient stress input (arrowhead) is faster than rapamycin inhibition (vertical black arrow) in inactivating Tor activity. In (2), the nutrient stress input (arrowhead on Time axis) activates autophagy (blue arrow) before Tor is fully inhibited. Only the wild-type curve for rapamycin is shown since autophagy is activated faster than starvation in this condition. In this graph, autophagy is quicker in wild-type relative to the buffy mutant, but it could also be slower. (F) Ectopic phosphoinositide 3-kinase (PI3K) is inefficient at inhibiting starvation-induced autophagy when buffy is absent. Cells with ectopic PI3K signaling are indicated by GFP. For each genotype a representative image of the major LTR pattern observed is presented. The left panel shows the merge of GFP (green) and LTR (red) and the right panel shows the LTR pattern alone. Genotypes: y w hsFLP; buffyH37/+; actin > CD4 > Gal4, UAS-GFP/UAS-dp110 and y w hsFLP; buffyH37/buffyH37; actin > CD4 > Gal4, UAS-GFP/UAS-dp110. (G) Quantification of the percentage of clones with ectopic PI3K signaling that had no LTR-positive punctae. Clones (control, n = 123; buffyH37, n = 66) were from ten control larvae and seven buffyH37 larvae. In addition to the percentage of clones with no LTR-positive punctae, the number of clones with reduced number or intensity of LTR punctae was also quantified: control, 19%; buffyH37, 22%. The remaining clones (control, 6%; buffyH37, 52%) had an LTR response equivalent to neighboring cells. (H) buffy mutant larvae are unable to adapt to nutrient restriction. First instar larvae were transferred to nutrient-restriction medium (20% cornmeal/yeast/agar food (CY), 9% sucrose) and the percentage of these larvae that developed into third instar larvae after 6 days is presented (number of larvae: wild-type, n = 132; buffyH37 n = 231). Mean ± SEM.