Larvae lacking buffy are sensitive to acute nutrient deprivation

Our study began by assessing the ability of buffy mutant larvae to survive acute nutrient starvation. When larvae are starved prior to 70 h after egg laying (AEL), they die within several days. Larvae that are starved beyond this time point pupate early, but undergo the normal developmental program of metamorphosis and produce viable, fertile adult flies [32]. We utilized this fact to test whether mutant larvae maintained normal energy stores and mobilized them when deprived of nutrients. buffy mutant and wild-type larvae, reared in nutrient rich conditions (grown without crowding and fed complete medium) were moved at 73 h AEL into vials containing water-moistened filter paper. For both genotypes, the majority of the larvae pupated. Animals deficient in buffy were twofold more sensitive to nutrient starvation: 65% of the starved wild-type larvae developed into flies, in comparison with 29% of the buffy mutant larvae (Figure 1A, dark bars). Most of this difference in survivorship was due to death during metamorphosis (Figure 1A, light gray bars).

The Drosophila Bcl-2 proteins play a role in stress-induced apoptosis [11–13]; therefore, we addressed the possibility of an apoptotic response to starvation. There was no evidence of caspase activation (Figure 1B) or terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL; data not shown) in buffy mutant or wild-type larvae. We conclude that protein starvation does not elicit an apoptotic response.

buffy mutant larvae store less lipid and glycogen in the fat body and mobilize lipids upon starvation

When nutrients and sugar were reduced (20% cornmeal/yeast/agar food (CY), 1.8% sucrose; see Methods for media description), less lipid was stored in larval fat bodies, reflecting reduced fat intake and carbon availability. Because the fat bodies stained much less intensely with Nile Red, visualization of lipid-storage droplets was enabled (Figure 1D). Quantification of Nile Red-stained droplets demonstrated that larvae lacking buffy stored less lipid in fewer lipid droplets of smaller size in comparison to wild-type controls (Table 1). buffy knockdown larvae, in which ubiquitous expression of a hairpin RNA targeting buffy significantly reduced buffy expression, also displayed reduced Nile Red staining (see Additional file 1, Figure S1). Supplementation of the growth medium with excess sucrose (100% CY, 20% sucrose) led to an increase in TAG storage (TAG generated through sugar metabolism) for both mutant and wild-type larvae, although the buffy mutant still stored less TAG relative to wild-type (Table 1). We investigated whether continual breakdown of lipids contributed to the small reduction in fat storage in the buffy mutant. In fasting Drosophila larvae, fat that is released by the fat body is metabolized through β oxidation in specialized hepatocyte-like cells called oenocytes [33]. Oenocytes are clustered along the larval body wall and, in feeding animals, contain little to no fat; however, upon nutrient withdrawal, oenocytes from starving animals stain robustly with Nile Red as lipids are catabolized [33]. buffy does not influence the process of fat catabolism since oenocytes from buffy mutant larvae were indistinguishable from oenocytes in wild-type larvae, whether feeding or following starvation (Figure 1E).

Another stored source of energy for the larva is glycogen. The concentration of glycogen in buffy mutant larvae, grown in complete medium, followed the same trend as lipid concentration (Table 1). Taken together, these data demonstrate that larvae lacking buffy are able to generate and store lipid and glycogen and mobilize lipids upon starvation. However, buffy mutants maintain a reduced steady-state level of energy sources in the fat body, which likely contributes to the nutrient starvation phenotype.

Larvae lacking buffy display metabolic changes similar to wild-type larvae following starvation, but have distinct metabolic characteristics in fed conditions

Energy metabolism also affects the ability of an animal to survive nutrient stress. When animals are subjected to nutrient stress, their metabolism shifts from favoring energy-requiring processes, such as protein synthesis and growth, to energy-generating processes such as glycolysis and oxidative phosphorylation. We investigated whether an inappropriate metabolic response to starvation also contributed to the inability of the buffy mutant to survive nutrient starvation.

A very early response to acute starvation is activation of AMPK. Phosphorylated AMPK was detected within 30 minutes of starvation in both buffy mutant and wild-type larval extracts (data not shown). AMPK activates catabolic pathways that generate ATP while at the same time switching off ATP-consuming anabolic pathways. Reflecting AMPK activation, ATP concentrations increased in both mutant and wild-type larvae following starvation as expected (Figure 2A, light gray and black bars). This was not due to upregulation of glycolysis since the effect was the same whether the larvae were fed sucrose or water. Instead, this likely reflects reduced ATP consumption. The fact that ATP concentrations increased to roughly similar levels in buffy mutant and wild-type larvae demonstrates that buffy is not required for this metabolic response to starvation. But we noticed a curiosity in the control, fed larvae: fed buffy larvae maintained a reduced pool of ATP relative to wild-type (Figure 2A, dark gray bars).

Both glycolysis and the more efficient mechanism of oxidative phosphorylation contribute to ATP generation in the Drosophila larva. Because the ATP concentration was similar in starved buffy mutant and wild-type larvae, we expected that changes in the relative contributions of glycolysis and oxidative phosphorylation to energy generation following starvation were also similar. To examine this, the concentration of lactate was determined as an indication of glycolytic rate. Upon protein starvation, the lactate concentration dropped in both mutant and wild-type larvae, confirming that buffy mutant larvae were comparable to wild-type in their starvation response (Figure 2B, light gray bars). However, we noted a distinction between fed wild-type and buffy larvae: fed larvae lacking buffy maintained a higher steady-state concentration of lactate (Figure 2B, dark gray bars). It should be noted that the experiments presented in this section were performed on larval extracts in which the major component is fat body (roughly 80% of the animal's weight). Muscles were removed with the cuticle (no detergent was used during lysis) making the muscle contribution small.

We conclude that, with regard to energy generation and the parameters tested here, buffy mutant larvae responded normally to starvation. However, our experiments unexpectedly uncovered differences between fed mutant and wild-type larvae. At this stage of development, larvae feed virtually continuously in order to develop and grow adult structures and store enough nutrients to survive the non-feeding period of metamorphosis. Larvae lacking buffy maintain low ATP concentration, a high concentration of lactate, and smaller glycogen and lipid stores. All of these are consistent with an abnormal basal metabolism that places the animal closer to an energetic breakpoint, thus rendering the buffy mutant unusually sensitive to nutrient stress.

Increased phosphorylated S6K, indicative of increased Tor signaling, is observed in buffy larvae

We next investigated survival and growth signaling that mediates metabolic responses to nutrient status. Signaling through the PI3K/Tor pathway promotes growth when nutrients are present, but is quickly inactivated when nutrients are withdrawn [22, 34–37]. In nutrient-rich conditions, PI3K signaling is active. Loss of signaling through PI3K results in Forkhead box O (FOXO) activation and subsequent transcription of FOXO targets, one of which is 4E-binding protein (4E-BP) [38, 39]. 4E-BP protein was readily detected following protein starvation of buffy mutant larvae (Figure 3A), demonstrating that the loss of nutrients was properly relayed to stop growth signaling through the PI3K pathway.

Tor, a downstream effector of PI3K signaling, is a Ser-Thr kinase that positively regulates growth by promoting ribosome biogenesis, protein synthesis, and nutrient import while at the same time negatively regulating the catabolic process of autophagy. Following loss of a positive nutrient signal, Tor signaling is inactivated resulting in inhibition of energy-requiring processes such as protein synthesis [40]. Tor signaling was monitored through S6K phosphorylation as another indicator of the response to starvation. Reflecting the loss of the positive nutrient signal, the amount of phosphorylated S6K dropped in starved buffy mutant larvae (Figure 3B; see also Figure six). Intriguingly, more phosphorylated S6K was detected in fed buffy mutant larvae indicating that the basal level of signaling through Tor was high in the buffy mutant (Figure 3B, quantification of five independent experiments shown in Figure 3C). Tor phosphorylates S6K on T398 (equivalent to T389 in mammalian S6K) and subsequent phosphorylation of S6K on T238 (T229 in mammalian S6K) by 3-phosphoinositide-dependent kinase 1 (PDK1) is required for full activation of S6K [41, 42]. Increased S6K T238-phosphorylation was also observed in buffy mutant larvae (Figure 3C). Furthermore, phosphorylated S6K (T398) was higher in fed buffy knockdown larvae relative to control larvae (see Additional file 2, Figure S2). Lastly, S6K phosphorylation in buffy mutant larvae was sensitive to Tor gene dosage (see Figure six). We conclude that larvae lacking buffy maintain hyperactive Tor signaling in their basal feeding state.

Clearly, the inability of the buffy mutant to survive nutrient withdrawal was not due to inappropriate signaling or metabolic responses to starvation. Instead, our study uncovered differences in basal energy storage and metabolism, as well as increased basal signaling through Tor that positively regulates growth.

Lack of buffy results in precocious onset of autophagy following amino-acid withdrawal

Autophagy is a well-characterized response to starvation and is activated in the Drosophila larval fat body hours after larvae are protein starved [22, 43]. This catabolic process involves the non-selective engulfment of cytoplasm, containing organelles and proteins, in double-membrane vesicles called autophagosomes [44, 45]. Autophagosomes move to and fuse with a lysosome, forming an autolysosome, where breakdown of organelles and cytoplasmic proteins occurs to allow recycling of these critical building blocks when nutrients are unavailable. Since one of the roles played by Tor is to inhibit autophagy when nutrients are plentiful, we reasoned that buffy larvae with increased Tor signaling might fail to activate autophagy following protein withdrawal. Loss of autophagy could also contribute to starvation sensitivity.

The initial assessment of protein starvation-induced autophagy utilized LysoTracker Red (LTR), a stain that marks acidic vesicles such as autolysosomes and lysosomes. Fat bodies from feeding larvae have very few lysosomes, and thus very little LTR staining, but upon transfer to protein-free media, (20% sucrose or water only), abundant autolysosomes are visualized as punctate LTR staining ([22, 43] and Figure 4A). In wild-type larvae, a small number of LTR puncta are visible after 2 h of amino-acid withdrawal and robust LTR staining is observed after 4 h ([22, 43] and Figure 4A, B and see Additional file 3, Figure S3). buffy null animals were competent in initiating autophagy; however, the autophagic response was faster with strong LTR staining visible after 2 h of protein starvation (Figure 4A, B, and Additional file 3, Figure S3). Similarly robust LTR staining after 2 h of starvation was also observed in buffy knockdown larvae (Additional file 1, Figure S1). Protein starvation-induced autophagy in the larval fat body is transient, dropping after 12 to 24 h [22, 43] and LTR staining in the starved buffy mutant followed a similar timecourse (data not shown).

Because LTR is not specific for autolysosomes, other methods of marking autophagic structures were employed to corroborate the LTR data. LC3-green fluorescent protein (GFP) and mCherry-ATG8 identify autophagosomes and early autolysosomes [43, 46]. Both markers confirmed that larvae lacking buffy activate the autophagic pathway faster than wild-type larvae (Figure 4C, and Additional file 3, Figure S3). Transmission electron microscopy (TEM) demonstrated that no autophagic structures were observed in fat bodies of mutant and wild-type larvae grown in complete medium and that lysosomes were rarely seen (Figure 4D). After 2 h of starvation, micrographs revealed many autolysosomes in the buffy mutant fat body whereas an autolysosome was rarely observed in the wild-type fat body (Figure 4D, quantified in Figure 4E).

Buffy does not directly inhibit formation and maturation of autophagic vesicles

Faster autophagy in response to starvation was the opposite of the expected outcome of high Tor signaling. A possible explanation is that Buffy negatively regulates autophagy, downstream of Tor signaling, to delay onset following starvation. 'AuTophaGy' related 1 (ATG1) is a Ser-Thr kinase that controls the autophagic machinery in response to Tor signaling and mutation of Atg1 blocked starvation-induced autophagy in Drosophila larvae [22, 47]. We generated the Atg1 buffy double mutant. No LTR staining was observed in the fat bodies of fed or starved double mutant larvae (data not shown), indicating that loss of buffy was unable to derepress autophagy downstream of Tor and Atg1.

We investigated whether ectopic Buffy inhibited starvation-induced autophagy. Indeed, overexpression of Buffy in the Drosophila larva delayed the autophagic response to starvation. After 4 h of starvation, LTR staining of fat bodies from animals with ectopic Buffy was dramatically reduced (Figure 5A, B; quantification shown in Figure 4B). Due to non-uniform expression of Gal4 in fat body, some regions had no LTR staining and other regions had a small number of LTR puncta; however robust LTR staining was observed throughout the control fat body at this timepoint. LTR staining was confirmed by EM analysis (Figure 5C, compare to Figure 4D). Ectopic Buffy, however, did not simply block the autophagic response, but rather delayed the response: extensive autophagy was apparent in fat bodies with ectopic Buffy after an extended period of protein starvation (data not shown).

Prior to larval pupation, the fat body undergoes autophagy in response to hormonal changes [43]. Ectopic Buffy did not inhibit programmed autophagy, as demonstrated by LTR staining, mCherry-ATG8a and EM analysis (Figure 5D-F). These data, together with the Atg1 genetic interaction data, demonstrate that Buffy does not inhibit formation and maturation of autophagic vesicles, but instead blocks the induction signal that is specific to the protein-starvation response upstream of the autophagic machinery.

Thus far, our study demonstrated that larvae lacking buffy were unable to survive acute nutrient starvation. Loss of buffy resulted in increased basal Tor signaling and altered energy storage and metabolism, but did not change the way the animal responded to protein starvation, with the exception of faster autophagy. This last phenotype was the opposite expected for increased Tor signaling (Tor inhibits autophagy and thus should slow the response). The next series of experiments were designed to understand the consequence of increased Tor signaling in the buffy mutant.

buffy null animals do not rely on increased Tor signaling for viability

We hypothesized that buffy larvae might rely on excess Tor signaling for viability and that this would render them sensitive to severe reduction in Tor signaling. To test this, we generated animals that were buffy null and Tor null double mutants. No genetic interaction was observed; buffy Tor double mutant larvae hatched in equal numbers and grew at the same rate as Tor single mutant larvae (data not shown). We confirmed that phosphorylated S6K was no longer increased in larvae that were doubly deficient for buffy and Tor (Figure 6A). Clearly, increased phosphorylated S6K is not required for survival of larvae lacking buffy. Further interpretation of this experiment is confounded by two points: firstly, even though phosphorylated S6K is reduced, it is still detectable and secondly, larvae of the Tor null mutant have a severely altered metabolism and maintain a phenotype of constitutive starvation in which autophagy is activated and nutrient intake is drastically reduced (due to lack of a positive input into insulin signaling by Tor [48, 49]). An expected consequence of increased Tor signaling is growth [50–54]. We did not observe any increase in cell size in buffy mutant fat body, the buffy mutant animals grew at normal rate [11], fat body cells were of the normal size distribution and no difference in pupal mass was observed (Figure 6B). Furthermore, if a consequence of Tor signaling were increased growth (and increased rate of energy consumption), precocious autophagy could occur because starvation causes Tor signaling to be shut off more rapidly in the buffy mutant than wild-type controls. However, this is not the case as the level of phosphorylated S6K in 2 h-starved buffy mutant larvae was equal to or higher than starved control larvae in several experiments (Figure 6A; ratio of phosphorylated S6K in buffy mutant relative to wild-type in four separate experiments: 1.1, 4.4, 4.0, 1.6).

Increased phosphorylated S6K in the buffy mutant poises the animal for starvation-induced autophagy

We investigated the possibility that activated S6K was sufficient to induce precocious starvation-induced autophagy in wild-type larvae. Ectopic expression of activated S6K was not sufficient to initiate autophagy in fat bodies of fed larvae [22]. Ectopic activated S6K in the wild-type fat body also did not affect the timing of initiation of protein starvation-induced autophagy or the density of LTR-positive puncta (Figure 6C-E).

To investigate whether the increased phosphorylated S6K was necessary for precocious starvation-induced autophagy in the absence of buffy, we wished to reduce the intracellular pool of phosphorylated S6K in buffy null larvae. Removing one genomic copy of S6K, by crossing in a null S6K allele, has been successfully used in other studies to drop active S6K levels (for example, see [52, 55–57]). Heterozygous control larvae (buffy ^H37 /buffy ⁺ ; s6k ^l-1 /s6k ⁺ ) had little to no LTR staining after 2 h of protein starvation (Figure 6F), whereas robust LTR staining was observed in the buffy mutant fat body (Figure 6G). In contrast, no LTR-positive puncta were visible in the experimental buffy mutant larvae with only one copy of wild-type S6K (buffy ^H37 /buffy ^H37 ; s6k ^l-1 /s6k ⁺ ; Figure 6H). These results demonstrate that when larvae lack buffy, an increased pool of phosphorylated S6K is sufficient to induce precocious autophagy in fat body cells upon protein starvation. When Buffy protein is present, increased phosphorylated S6K is not sufficient for the precocious autophagic response to nutrient withdrawal.

The next series of experiments made use of starvation-induced autophagy as an indicator of Tor signaling in order to investigate the interaction between nutrient stress and Tor.

Nutrient withdrawal activates autophagy in the buffy mutant faster than rapamycin-mediated inhibition of Tor in contrast to wild-type

Because reduction in S6K abolished precocious initiation of starvation-induced autophagy in the buffy mutant, we wished to downregulate Tor signaling in order to directly test the contribution of Tor to the phenotype. Ectopic expression of a dominant-negative truncated Tor protein in single cells within the fat body is sufficient to activate autophagy, even in fed animals [22]. Downregulation of Tor through expression of the dominant-negative protein also induced autophagy in the buffy mutant, preventing experimental determination of the timing of autophagy (see Additional file 4, Figure S4). LTR staining in cells expressing dominant-negative Tor was indistinguishable from cells with normal Tor signaling in the buffy mutant fat body following protein starvation (Additional file 4, Figure S4).

To more directly investigate the timing of autophagy initiation in the buffy mutant, we employed the drug rapamycin to temporally inhibit Tor signaling. After 2 h of rapamycin feeding, moderate to robust LTR staining was already visible in wild-type larval fat bodies (Figure 7A), demonstrating that direct inhibition of Tor signaling activates autophagy faster than nutrient deprivation in wild-type larvae. LTR-stained fat bodies from numerous individuals were examined at time points earlier than 2 h and classified as either 'zero to minor response' or 'moderate to robust response' to rapamycin. No LTR staining was visible in fat bodies after 40 minutes of rapamycin exposure, but after 80 minutes, 'moderate to robust' staining was evident in over 80% of wild-type fat bodies (Figure 7B). Unlike wild-type and in striking contrast to nutrient starvation, rapamycin-fed buffy mutant animals did not initiate autophagy faster than wild-type, but were instead slower: fat bodies from buffy mutant larvae displayed 'zero to minor' LTR staining even after 80 minutes of rapamycin feeding (Figure 7B). By 2 h of rapamycin exposure, LTR staining was fairly equivalent in both genetic backgrounds and declined in later time points (data not shown). The requirement for longer rapamycin exposure to initiate autophagy in the buffy mutant is a likely consequence of the observed higher basal Tor signaling. This is in contrast to the observation that starvation initiated autophagy faster in the buffy mutant and high basal Tor signaling was required for this phenotype. To confirm that nutrient withdrawal is more efficient at activating autophagy in the buffy mutant in comparison to rapamycin treatment, larvae were fed a combined rapamycin/protein-starvation media for 2 h. LTR stained fat bodies from wild-type larvae fed this media were similar to rapamycin-fed wild-type larvae (Figure 7C). Fat bodies from the buffy mutant, however, were more intensely stained with LTR after 2 h of rapamycin/protein-starvation treatment (Figure 7C). The increased autophagic response in the buffy mutant was confirmed by quantification of LTR-positive punctae (Figure 7D). To sum this data up (Figure 7E), rapamycin induced autophagy within 80 minutes in wild-type larvae, but protein-starvation took roughly three times longer to induce autophagy (4 h). In contrast, autophagy in the buffy mutant in response to protein starvation was faster than wild-type (2 h), even though rapamycin-induced autophagy was slower (2 h). Although it is formally possible that the nutrient-withdrawal signal inactivates Tor much faster than rapamycin in the buffy mutant (this possibility is not supported by the western blot analysis of phosphorylated S6K in starved larvae; Figures 3B and 6A), a simpler explanation is that the loss of the positive nutrient signal has an input into autophagy that is not Tor dependent.

Lack of buffy is permissive for autophagy even when PI3K signaling is artificially maintained

To further test how nutrient withdrawal initiates autophagy in the buffy mutant, we investigated the effect of enforcing Tor signaling through ectopic expression of class I PI3K. Ectopic PI3K signaling overrides loss of a positive nutrient signal and suppresses starvation-induced autophagy [22, 43]. Fat body cell clones were generated that express ectopic dp110, the catalytic subunit of class I PI3K, to activate PI3K signaling in larvae prior to protein starvation [22, 43, 58]. Cells with ectopic dp110 in both buffy mutant and wild-type fat bodies grew larger than neighboring cells as is expected for ectopic activation of PI3K signaling (data not shown). As shown previously, LTR-positive punctae in response to starvation were not visible in wild-type fat body cells with ectopic PI3K signaling (Figure 7F). In contrast, the majority of PI3K-expressing cell clones in starved buffy mutant fat body contained LTR-positive punctae (Figure 7F; quantified in Figure 7G). Several conclusions can be made from this experiment. First, Tor is known to suppress autophagy independently of S6K and this mechanism is ineffective in starved buffy mutant larvae. Second, the inability of rapamycin to phenocopy protein starvation in the buffy mutant is not due to a molecule competing with rapamycin for Tor binding as has been shown for phosphatidic acid [59]. Third, as suggested by the western blot analysis and rapamycin experiments and confirmed in this experiment, the protein-starvation input into autophagy in buffy larvae is either downstream of Tor or in a separate pathway from Tor. The finding that precocious initiation of starvation-induced autophagy requires augmented phosphorylated S6K, is strong evidence that S6K or a molecule downstream of S6K transmits the nutrient-withdrawal signal.

buffy is required for adaptation to nutrient restriction

Taken together, the data presented thus far indicate that buffy is required for maintaining normal basal energy metabolic homeostasis and normal Tor signaling. This study was initiated by an observation that buffy mutants are sensitive to poor growing conditions. The findings of inappropriate energy metabolism, Tor signaling and reduced energy storage all could contribute to this. Because the acute starvation experiment reported in Figure 1A is likely influenced by nutrient storage, we designed a second assay to test survival following nutrient stress in which energy storage was expected to have less of an impact. The assay involved growing larvae from first instar on food that contained a reduced concentration of nutrients (20% CY, 9% sucrose). Fed this nutrient-restriction diet, wild-type larvae develop and molt albeit with a reduced growth rate and minimal mass accumulation. After 6 days of feeding on the reduced nutrient medium, half of the wild-type larvae reached the third instar developmental stage (Figure 7H). In stark contrast, larvae lacking buffy were unable to metabolically adapt to the nutrient stress and only 5% of the buffy mutant first instar larvae developed into third instar larvae (Figure 7H). Instead, despite continual foraging, buffy mutant larvae remained small without noticeable growth or development: a phenotype similar to Tor-deficient or protein-starved larvae [60, 61]. Thus, even in the experimental setting in which utilization of stored energy sources is not required, buffy mutant animals were sensitive to nutrient stress. We conclude that buffy is required for either the attenuation of metabolic activity in response to nutrient stress or to maintain the basal metabolic state that is a requisite for adaptation.

Putting the metabolic experiments together with phosphorylated S6K, we propose that Buffy plays a role in maintaining optimal metabolic conditions and adapting metabolism to nutrient stress. Concomitant with this, Tor signaling, as evidenced by phosphorylated S6K, is increased in buffy mutant larvae. Although this affects precocious starvation-induced autophagy, it does not appear to be the only input into precocious autophagy as a second nutrient-withdrawal input is required. Our experiments suggest that the second input is downstream of Tor or Tor independent and is related to the altered energy homeostasis in the buffy mutant (Figure 8).

Drosophila strains and cultures

All flies were raised on standard CY containing the standard 9% sucrose concentration (complete medium) at 25°C unless otherwise noted. For acute starvation conditions, larvae raised at low density in nutrient rich media were aged until early third instars then transferred to vials containing water soaked filter paper only. Protein-starvation media refers to 20% sucrose in agar. Larvae reared on limited nutrients were picked just after hatching and placed onto food containing 20% of the standard cornmeal/yeast media with normal agar concentration and either the standard concentration of sucrose (20% CY, 9% sucrose) or a reduced concentration of sucrose (20% CY, 1.8% sucrose). For rapamycin feeding, larvae were placed into petri dishes that contained the indicated concentrations of rapamycin mixed with standard food and food coloring. Food coloring was used to ensure larval uptake and mechanical mixing of rapamycin with food was performed to avoid chemical degradation. Somatic overexpression clones were generated in larvae with a 30 to 60 minute 37°C heat shock, 10 to 20 h prior to starvation and dissection.

Stocks used were: daughterless-Gal4 (da-Gal4) (Bloomington Stock Center, Indiana University, Bloomington, IN, USA), UAS-TOR ^TED ([82]; Bloomington Stock Center), UAS-S6K ^STDE [50]; BSC), buffy ^H37 [11]; BSC), Tor^ΔPP{neoFRT}[36]40A/CyO ([60]; BSC), Cg-Gal4, UAS-GFP-LC3 ([43]; gift of Kim Finley, San Diego State University, San Diego, CA, USA), Act > CD2 > Gal4, UAS-GFP III (gift of T. Neufeld), S6K ^l-1 ([51]; gift of T Neufeld), Atg1^Δ3D([22]; gift of T Neufeld), UAS-mCherry-Atg8a [83]; gift of Thomas Neufeld, University of Minnesota, Minneapolis, MN, USA), and buffy ^H37 [11].

The Atg1^Δ3D;buffy ^H37 double mutant had a synthetic lethal phenotype: homozygous double mutant embryos hatched but died within 24 h of hatching. The very few double mutant larvae that survived past 48 h (5 larvae survived of 800 hatched larvae) were significantly smaller in size in comparison to their heterozygous siblings.

Nile Red stain

Fat bodies from indicated larvae were dissected and fixed with 4% paraformaldehyde for 1 h. Fat bodies were washed three times for five minutes in phosphate-buffered saline (PBS) and incubated in 5 μM Nile Red in PBS for 1 h (with gentle agitation). After staining, fat bodies were washed and mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA). For measurements of mean luminosity, care was taken to stain and observe dissected fat bodies identically. Measurements were made on images taken with identical exposure and camera settings and in which all staining was in the linear range. Note that these settings were necessarily different for Nile Red-stained fat bodies from 100% food-fed animals relative to animals fed on 20% food owing to the overall brightness of the stain in the fed animals. In order to keep the stains from fed animals in the linear range, a neutral density filter was used. Therefore, the luminosity data cannot be compared between 100% food and 20% food. Two identically-sized regions, encompassing only stained tissue, were chosen at random and the mean luminosity determined using the histogram function of Photoshop. The two regions were averaged and average data is presented for three individual animals. Lipid storage droplets (stained with Nile Red) were manually counted in 10 to 12 fat body cells for three individual animals and lipid droplet size in 33 to 43 fat body cells was determined using Photoshop. Nile Red staining of lysates was performed on pools of five third instar larvae lysed in radioimmunoprecipitation assay (RIPA) buffer and stained with Nile Red. Several dilutions were read at optical density (OD)_{600 nm}.

For oenocyte staining, third instar larvae were pinned down onto a Sylgard (Corning Inc. Corning, NY, USA) dish, at their anterior and posterior ends in a droplet of PBS. Using a surgical razor and insect pins, the larvae were filleted such that the inner side of the integument was exposed. Filleted larvae with all internal organs removed were fixed in a droplet of 4% paraformaldehyde in PBS for 1 h. Fillets were then washed in PBS followed by a 30 minute incubation with 5 μM Nile Red in PBS.

Caspase activity assay

Larvae were homogenized in a chilled glass mortar and pestle in a buffer containing 20 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES)-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl₂, 1 mM sodium ethylenediaminetetra-acetic acid (EDTA), 1 mM sodium ethylene glycol tetra-acetic acid (EGTA), 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5% Triton-X 100. Homogenates were spun at max speed for 10 minutes. A total of 50 μg of protein extract from the aqueous fraction was incubated with 50 μM Z-DEVD-AMC (Calbiochem EMD Millipore) substrate in a final volume of 100 μl in a 96-well plate. Fluorescence was monitored over time with excitation of 375 nm and emission of 450 nm using a SpectraMax GemimiXS (Molecular Devices, Sunnyvale, CA, USA) plate reader.

Lysotracker staining

Larvae were washed in wells containing H₂O, 96% ethanol and H₂O, in that order. Clean larvae were then dissected and inverted, exposing fat body, in PBS and then incubated in 100 μM LysoTracker Red DND-99 or LysoTracker Green DND-25 (Molecular Probes/Life Technologies, Grand Island, NY, USA) as a whole carcass for 10 to 20 s. Inverted carcasses were then washed in PBS and placed onto a droplet of Vectashield (Vector Laboratories) containing 4',6-diamidino-2-phenylindole (DAPI), for fat body separation and mounting. Samples were imaged immediately.

Fluorescence and transmission electron microscopy

All fluorescence microscopy (except mCherry-Atg8a) was performed on a Zeiss Axiovert 200 widefield microscope equipped with a mercury bulb, relevant filters and a Hamamatsu ORCA-ER CCD camera. Images were cropped and levels adjusted using Photoshop. Genotypes containing the UAS-mCherry-Atg8a transgene were treated as described above except imaged on a Leica SP5 confocal microscope using a 20 × 0.7 objective and 561 (mCherry) and 496 (LysoTracker Green DND 26, Life Technologies) laser lines for excitation. Emission bandwidths from beginning to end were: 493 nm to 520 nm (LTR-Green) and 566 nm to 700 nm (mCherry). Final images scanned using a line average of four.

For TEM, larvae were dissected and inverted to expose fat body and fixed overnight in 2% paraformaldehyde, 4% glutaraldehyde, 2% sucrose and PBS at 4°C. Fixed tissue was then rinsed in PBS and post fixed in 2% osmium tetroxide and PBS overnight at 4°C. Carcasses were dehydrated with ethanol and dissected to obtain fat body. Fat body was then embedded in epoxy resin, incubated in propylene oxide 2 × for 20 minutes, followed by a 50:50 propylene oxide and epoxy resin incubation overnight in a vacuum. The following day samples were treated with 100% epoxy resin for 2 h in a vacuum, and transferred to a capsule filled with resin followed by a 24 to 48 h incubation at 60°C. Tissue blocks were then trimmed using a single-edged razor blade under a dissecting microscope (Nikon Instruments). A short series of ultrathin (60 to 80 nm) sections containing fat body were cut from each block with an ultramicrotome (Reichert-Jung) and sequential sections were collected on mesh and formvar-coated slot grids. The sections were stained with uranylacetate and lead citrate to enhance contrast. Sections were examined with a Philips CM-10 transmission electron microscope and images were captured with a Gatan digital camera.

Quantification of autophagic structures, cell size and pupal mass

For TEM images, autolysosomes were quantified from over 60 images, each with a field view of about 40 μm² at a magnification of 2950 ×. A total of five or six larvae are represented per genotype or nutrient condition. For lysotracker staining, punctae were counted using MetaMorph from at least five representative images from five to seven different animals. For measurement of fat body cell size, fat bodies from random positions within the larva were stained with Nile Red and imaged. Individual fat body cells were outlined manually and the perimeter and area were measured using Photoshop. The perimeter is reported. To determine pupal mass, clean tan pupae (within a 24 h age window) were picked from the side of vials and weighed in groups of 5.

Immunoblot analysis

Pooled larvae were homogenized in a chilled buffer containing 1% NP-40, 150 mM sodium chloride, 10 mM sodium phosphate pH 7.2, 50 mM sodium fluoride, with freshly added 100 μM PMSF, 100 nM okadaic acid, 0.2 mM sodium vanadate and protease inhibitors. An aliquot was removed for bicinchoninic acid (BCA) analysis (Pierce, Thermo Scientific, Rockford, IL, USA), while the remaining homogenate was treated with gel loading buffer, heated at 90°C for 5 minutes, spun and loaded. For the detection of S6K, a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel was used, transferred onto a polyvinylidene fluoride (PVDF) membrane (EMD Millipore) and probed with either rabbit S6K-phospho-T398 S6K (Cell Signaling Technology, Danvers, MA, USA), rabbit S6K-phospho-T229 (mammalian) (Cell Signaling Technology) or rabbit anti-S6K (gift from T Neufeld) all at 1:1,000. β-Tubulin (E7) antibody (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA) was used at 1:4,000 and served as our loading control. For the detection of 4E-BP, a 15% SDS-PAGE gel was used and transferred onto a nitrocellulose membrane (Whatman, GE) with a 0.2 μm pore size. Antibody 1868 to 4E-BP was a gift from Nahum Sonenberg (McGill University, Montreal, Quebec, Canada) [84] and used at 1:1,000. Rabbit secondary antibodies conjugated to horseradish peroxidase (Vector Laboratories) were used at 1:10,000. For quantification, film was scanned and bands quantified using ImageJ. For quantification of anti-tubulin loading control, mouse AlexaFluor-800 nm secondary conjugates were used at 1:10,000 and an Odyssey InfraRed Imaging System and software (Li-Cor BioSciences, Lincoln, NB, USA) were used to analyze the intensity of bands according to manufacturer's instructions.

Biochemical assays

Total triacylglycerides were measured from 20 staged larvae. Pooled larvae were homogenized in PBT (0.05% Tween-20 in PBS) using a Kontes pestle (30 strokes). An aliquot was taken for protein determination (BCA assay, Thermo) while the rest was incubated at 70°C for 5 minutes. Heat-treated samples were spun at max speed in a table-top centrifuge. A fraction of the homogenate was used for the Thermo Infinity Triglyceride Kit (Thermo) following the manufacturer's protocol. Readings were normalized to protein content. For lactate determination, 30 staged larvae were homogenized in 100 μl of PBS (no detergent) using a Kontes pestle (30 strokes). A sample aliquot was taken for protein determination and the rest subjected to heat treatment (60°C for 15 minutes) and spun. A total of 10 μl of homogenate was used for Lactate Assay Kit (Biovision cat no. K607-100, Milpitas, CA, USA) following the manufacturer's protocol. Readings were normalized to protein content. For glycogen determination, 10 larvae were homogenized on ice with 200 μl of chilled ultra-pure water. An aliquot was quickly removed and flash frozen while the rest of the lysates was immediately placed in a boiling water bath for 5 minutes (all steps occurring within a minute from first stroke of homogenization). Samples were then spun for 10 minutes at high speed and supernatant was used for Glycogen Assay Kit (Biovision cat no. K646-100) at a tenfold dilution following the manufacturer's protocol. For ATP analysis, pooled larvae (10 larvae/200 μl buffer) were homogenized in 1 × Reporter lysis buffer (Promega, Madison, WI, USA), and immediately flash frozen. The frozen samples were boiled for 15 minutes to destroy ATPase activity, then spun at 17 800 g for 5 minutes and the supernatant was diluted 100-fold with the same buffer. The cellular ATP content was quantified by a luciferin/luciferase-based assay using an ATP Determination Kit (Sigma-Aldrich). Luminofluorescence was measured using the Wallac ARVO SX 1420 Multilabel Counter (Perkin Elmer Life Sciences, Waltham, MA, USA) and the data were normalized to the protein content.