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. Author manuscript; available in PMC: 2014 Jan 21.
Published in final edited form as: Curr Top Dev Biol. 2013;103:101–125. doi: 10.1016/B978-0-12-385979-2.00004-6

The Role of Autophagy in Drosophila Metamorphosis

Kirsten Tracy 1, Eric H Baehrecke 1,1
PMCID: PMC3896998  NIHMSID: NIHMS544486  PMID: 23347517

Abstract

Macroautophagy (autophagy) is a conserved catabolic process that targets cytoplasmic components to lysosomes for degradation. Autophagy is required for cellular homeostasis and cell survival in response to starvation and stress, and paradoxically, it also plays a role in programmed cell death during development. The mechanisms that regulate the relationship between autophagy, cell survival, and cell death are poorly understood. Here we review research in Drosophila that has provided insights into the regulation of autophagy by steroid hormones and nutrient restriction and discuss how autophagy influences cell growth, nutrient utilization, cell survival, and cell death.

1. INTRODUCTION

All animals transition through several different stages during their development. The first stage is embryonic development, followed by a juvenile growth phase, then sexual maturation, and finally reproductive adult-hood. Some animals, such as mammals, exhibit few changes in their body plan during development except for growth; human babies look like miniature adults. Other animals go through drastic changes, such as the frog, starting its juvenile phase as a tadpole and developing into a frog. Progression through the developmental stages requires the coordination of cell and tissue growth, cell survival, and cell death. In the example of the frog, cell growth allows the limbs to develop, and cell death causes the regression of the tadpole’s tail. The balance of cell growth, survival, and death is critical to maintaining homeostasis of the organism.

Autophagy is a catabolic process that functions at the crossroads of the different cell fates. Autophagy is predominately associated with cell survival in response to cellular stress; however, mounting evidence suggests that it also plays a role in programmed cell death. Additionally, autophagy is regulated by the same pathways that control cell growth. Autophagy and the pathways that regulate it have been studied extensively in the fruit fly Drosophila melanogaster. These studies have provided insights into the relationship between cell growth, cell death, and autophagy, but the questions of how and why these signals are integrated remain poorly understood.

2. AUTOPHAGY

Autophagy is an important catabolic process in all eukaryotic cells. There are three known types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy (Klionsky, 2005). Macroautophagy (hereafter referred to as autophagy) is the best characterized of the three types, and it involves the sequestration of cytoplasmic components and long-lived proteins into lysosomes for degradation. During autophagy, an isolation membrane sequesters cytoplasmic material, and it elongates to form a double-membrane vesicle, the autophagosome (Fig. 4.1). The autophagosome traffics to the lysosomal compartment where its outer membrane fuses with lysosomes and releases the inner cargo for degradation. Lysosomal permeases then recycle the degradation products back to the cytoplasm (Mizushima & Komatsu, 2011). Autophagy is an important process for maintaining cell homeostasis, responding to stress, and surviving nutrient starvation.

Figure 4.1.

Figure 4.1

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Diagram of the steps in autophagy. Autophagy is a catabolic process by which organelles and cytoplasmic proteins are degraded. Induction of autophagy results in the formation of an isolation membrane, which expands and closes around cytoplasmic material, forming the double-membraned autophagosome. The autophagosome traffics to the lysosome where it docks and fuses, releasing its inner membrane and its contents. The autophagosome contents are degraded by lysosomal enzymes and are recycled back to the cytoplasm through permeases.

2.1. Regulatory pathways

Several metabolic regulatory factors affect autophagy induction, including nutrient availability, insulin signaling, and ATP levels (Meijer & Codogno, 2004). The mechanistic target of rapamycin (TOR) plays a central role in autophagy by integrating the class I phosphatidylinositol-3-kinase (PI3K) and amino acid signaling pathways (Wullschleger, Loewith, & Hall, 2006). When nutrients are available, class I PI3K activates TOR, which represses autophagy by phosphorylating Atg13. This hyperphosphorylation reduces the affinity of Atg13 for Atg1, decreasing the kinase activity of Atg1 and inhibiting autophagy (Kamada et al., 2000; Noda & Ohsumi, 1998). During nutrient starvation, TOR activity is reduced, relieving its repression of Atg1, and autophagy is induced. Increased autophagy contributes to cell survival by producing amino acids and fatty acids that are used by the tricarboxylic acid cycle to generate ATP (Lum et al., 2005).

The origin of the autophagic membrane is not completely understood and remains a subject of debate (Juhasz & Neufeld, 2006). In yeast, autophagy proteins gather at the pre-autophagosomal structure (PAS) near the vacuole (Mizushima, 2007). In animal cells, a PAS-like structure has never been observed. Some studies suggest that in mammalian cells, the autophagosomal membrane originates from the endoplasmic reticulum (Axe et al., 2008; Dunn, 1990). In addition, more recent research suggests that autophagosome formation involves membrane derived from the mitochondria or the plasma membrane (Hailey et al., 2010; Ravikumar, Moreau, Jahreiss, Puri, & Rubinsztein, 2010).

Formation of the autophagosomal membrane requires phosphorylation of phosphatidylinositol. In yeast, this is accomplished by a class III PI3K complex consisting of Vps30/Atg6 /Beclin1, Vps34/ class III PI3K, Atg14, and Vps15 (Kametaka, Okano, Ohsumi, & Ohsumi, 1998; Kihara, Noda, Ishihara, &Ohsumi, 2001; Suzuki et al., 2001). Atg6 also forms a complex required for the vacuolar protein sorting (VPS) pathway in yeast, which consists of Atg6, Vps35, Vps15, and Vps38(Kihara et al., 2001).The Beclin 1–Vps34 complex is similar in mammalian cells; however, it contains additional regulators, including UVRAG, Bif1, Ambra1, and Barkor (Fimia et al., 2007; Liang et al., 2006; Sun et al., 2008; Takahashi et al., 2007). As in yeast, it has been suggested that Beclin1 forms at least two distinct complexes in animal cells that play different roles in membrane trafficking (Itakura, Kishi, Inoue, & Mizushima, 2008).

2.2. Autophagosome formation

Genetic studies in yeast have identified several Atg genes that are required for autophagy (Harding, Hefner-Gravink, Thumm, & Klionsky, 1996; Harding, Morano, Scott, & Klionsky, 1995; Klionsky et al., 2003; Thumm et al., 1994; Tsukada&Ohsumi, 1993). Many of these genes are involved in two conserved ubiquitin-like conjugation systems that are required for autophagosome formation, Atg12 and Atg8 (LC3 in mammals) (Klionsky & Emr, 2000; Ohsumi, 2001). Atg12 and Atg8 are both activated by the E1-like enzyme Atg7. Atg12 is then transferred to the E2-like enzyme Atg10. Finally, Atg12 is conjugated to Atg5 and forms a complex with Atg16 on the isolation membrane (Kuma, Mizushima, Ishihara,&Ohsumi, 2002;Mizushima, Noda,&Ohsumi, 1999;Mizushima et al., 1998; Shintani et al., 1999; Tanida et al., 1999). Atg8 is transferred to the E2-like enzyme Atg3 and is then conjugated to the phospholipid anchor phosphatidylethanolamine (PE) (Ichimura et al., 2000). This final conjugation results in the anchoring of Atg8-PE to the isolation membrane and is thought to regulate the elongation of the isolation membrane (Nakatogawa, Ichimura, &Ohsumi, 2007). In addition to Atg7 and Atg3, Atg8 modification requires Atg4, a cysteine protease that processes Atg8 before conjugation and cleaves Atg8 from PE once the autophagosome has fused with the lysosome (Ichimura et al., 2000). Since Atg8 remains on the membrane throughout autophagosome maturation, it is a useful marker of autophagosomes (Klionsky et al., 2008).

3. DROSOPHILA AS A MODEL FOR STUDYING THE INTERFACE BETWEEN STEROID SIGNALING, NUTRITION, AND GROWTH DURING DEVELOPMENT

Drosophila development provides a useful system for studying the coordination of cell growth, division, and death that is necessary for the animal to reach its proper size. Fly development is regulated by the steroid 20-hydroxyecdysone (ecdysone), and insulin and insulin-like growth factor signaling. These pathways are also known to regulate autophagy in different contexts; however, the coordination of steroid, insulin signaling, and autophagy is poorly understood. Recent studies have investigated the relationship between ecdysone and growth factor signaling in flies (Colombani et al., 2005; Layalle, Arquier, & Léopold, 2008), and understanding how these two pathways coordinate with each other may provide insight into how autophagy fits into this dynamic to facilitate animal homeostasis.

3.1. Steroid signaling

During development, Drosophila transitions through many different stages, and these transitions are signaled by pulses of the steroid hormone, ecdysone (Riddiford, Cherbas, & Truman, 2000; Thummel, 2001). Drosophila begins life as an embryo, and approximately 1 day after egg lay, they hatch as first instar larvae. The larvae feed and grow for approximately 3.5 days, and they molt twice during this period to become second instar larvae 24 h after hatching and third instar larvae 48 h after hatching. After the larval period, the animal stops feeding and a high titer pulse of ecdysone triggers puparium formation. This ecdysone pulse also induces the programmed cell death of the larval midgut (Lee, Cooksey, & Baehrecke, 2002). Prepupal development lasts for 12 h, and another peak in ecdysone titer triggers the prepupal–pupal transition and initiates programmed cell death of the larval salivary glands (Lee et al., 2003). Pupal development lasts for 3.5 days, after which the adult animal ecloses. A remarkable transformation occurs during this final developmental period; the tissues necessary to the feeding larva degrade through histolysis and are replaced by growing tissues that will be necessary to the walking, flying, and reproducing adult.

Ecdysone signaling has been studied extensively in the larval salivary glands of Drosophila. The pulses of ecdysone regulate stage- and tissue-specific developmental pathways through a transcriptional hierarchy (Thummel, 1995) (Fig. 4.2). Ecdysone signals by binding its receptor which is a heterodimer of two nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP) (Koelle et al., 1991; Thomas, Stunnenberg, & Stewart, 1993; Yao, Segraves, Oro, McKeown, & Evans, 1992). The EcR complex activates transcription of the early genes; these include Broad Complex (BR-C), E74A, E75, and E93 (Baehrecke & Thummel, 1995; Burtis, Thummel, Jones, Karim, & Hogness, 1990; DiBello, Withers, Bayer, Fristrom, & Guild, 1991; Segraves & Hogness, 1990). The early genes then activate transcription of the late genes, which are thought to function more directly in the regulation of developmental processes. In the salivary glands, the βFTZ-F1 orphan nuclear receptor is expressed during the mid-prepupal dip in ecdysone titer (Lavorgna, Karim, Thummel, &Wu, 1993). During the ecdysone peak that triggers salivary gland degradation, the EcR complex and βFTZ-F1 function together to reinduce transcription of BR-C, E74A, and E75 and to activate transcription of the stage-specific early gene, E93 (Baehrecke & Thummel, 1995;Broadus, McCabe, Endrizzi, Thummel, & Woodard, 1999; Woodard, Baehrecke, & Thummel, 1994). βFTZ-F1, BR-C, E74A, and E93 are all necessary for the proper degradation of larval salivary glands (Broadus et al., 1999; Jiang, Lamblin, Steller, & Thummel, 2000; Lee et al., 2000; Restifo and White, 1991). E93 may have a more prominent role in autophagic cell death than the other early genes as it also appears to be required for autophagosome formation in the dying larval midgut (Lee, Cooksey, & Baehrecke, 2002).

Figure 4.2.

Figure 4.2

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Genetic regulation of ecdysone-induced autophagy in Drosophila salivary glands. At 10 h after puparium formation, there is a rise in ecdysone titer, and ecdysone binds to its heterodimeric receptor which consists of EcR and USP. The ecdysone receptor complex functions together with βFTZ-F1 to induce transcription of the early genes; BR-C, E74A, and E93. The early genes activate transcription of many late genes involved in signaling, cellular organization, apoptosis, and autophagy.

3.2. Growth and nutrient utilization

Growth regulation at the cellular, tissue, and organismal level is critical for proper size development in all multicellular organisms, and it is affected by several environmental factors including nutrient availability (Mirth & Riddiford, 2007). In Drosophila, the feeding larva grows an astounding amount, increasing its size by ~200-fold during the 3.5-day period (Church & Robertson, 1966). Without this accumulation of body mass, the fly may have reduced reproductive success as an adult or it may not even be able to survive metamorphosis from the larva to adult.

For the adult fly to reach its proper size, the larva must pass three weight checkpoints. The first checkpoint occurs near the second instar to third instar molt and is called the threshold size for metamorphosis (Zhou, Zhou, Truman, & Riddiford, 2004). This size assessment determines whether the next molt will be a larval or metamorphic molt (Nijhout, 1975). The second checkpoint is the minimal viable weight which is the minimum body mass that is necessary to complete larval and pupal development in the absence of nutrients (Bakker, 1959). The final checkpoint, critical weight, occurs during the last larval stage (Nijhout, 2003; Nijhout &Williams, 1974). Reaching critical weight ensures that the animal will pupate within a certain amount of time regardless of nutrient availability (Bakker, 1959; Mirth & Riddiford, 2007;Nijhout, 2003;Robertson, 1963).Of these three size assessment check-points, critical weight is the most studied and best understood in Drosophila.

Once larvae reach their critical weight, environmental factors have a large impact on adult size. Larvae that starve before they achieve critical weight will delay their development until the nutrient supply improves. If nutrients are still abundant after larvae reach critical weight, they will continue to accumulate body mass (Mirth & Riddiford, 2007; Tennessen & Thummel, 2011). On the other hand, if postcritical weight larvae starve, they will stop growing in size. Since these starved larvae have reached their critical weight, they will enter metamorphosis within a similar time frame as fed larvae, but they will be smaller and will mature into smaller adults than the fed animals. This suggests that the mechanisms that regulate development and puparium formation must coordinate with nutrient utilization.

The endocrine cascade that follows critical weight achievement was originally described in the tobacco hookworm, Manduca sexta (Nijhout & Williams, 1974; Truman & Riddiford, 1974). Briefly, once larvae reach critical weight, juvenile hormone (JH) titers drop, causing a release of prothoracicotropic hormone, which signals to the prothoracic gland (PG) to produce ecdysone. However, this function of JH does not seem to be conserved in Drosophila, suggesting that critical weight is determined through another mechanism (Nijhout, 2003; Stern & Emlen, 1999).

Recent studies have elucidated some of the mechanisms required for critical weight assessment in Drosophila. One study showed that the Drosophila insulin receptor (InR), which has a conserved role in nutrition-dependent growth in animals, affects growth differently in precritical weight and postcritical weight larvae (Shingleton, Das, Vinicius, & Stern, 2005). Before larvae reach critical weight, InR signaling influences developmental timing but not larval growth. In contrast, InR activity affects final body size but not developmental timing in postcritical weight larvae. This is consistent with the observations in starved larvae discussed above. Several other studies showed that in Drosophila the size of the PG affects developmental rate and body size (Caldwell, Walkiewicz, & Stern, 2005; Colombani et al., 2005; Mirth, Truman, & Riddiford, 2005). They did this by manipulating insulin-dependent growth in the PG. When PG growth was suppressed by the expression of PTEN, a phosphatase that antagonizes class I PI3K activity, dominant negative class I PI3K, or dominant negative Ras, the larvae were larger than controls and had a longer developmental period. Conversely, larvae with an enlarged PG due to either class I PI3K or Ras activation initiated metamorphosis earlier than controls and thus the adults were smaller. Interestingly, the effects of growth in the PG appear to be specific to the insulin signaling pathway and not to cell size increase in general. In the study done by Colombani et al., they increased PG size by manipulating two other growth pathways in addition to PI3K: Myc and cyclin D/Cdk4. Although activation of these two genes increased the size of the PG, they had no effect on pupal or adult size (Colombani et al., 2005).

It is clear from these studies that tissue growth coordinates with developmental timing through InR signaling; however, the signals that regulate this have not been well studied. Recently, two independent groups performed screens to identify molecules that couple tissue growth with developmental timing and identified a novel Drosophila insulin-like peptide (dilp), dilp8 (Colombani, Andersen, & Léopold, 2012; Garelli, Gontijo, Miguela, Caparros, & Dominguez, 2012). Perturbing growth of larval imaginal disks through either damage or tumor promotion causes a delay in the time to pupariation, allowing the imaginal disks to reach their correct size (Menut et al., 2007; Poodry & Woods, 1990; Simpson, Berreur, & Berreur-Bonnenfant, 1980; Smith-Bolton, Worley, Kanda, & Hariharan, 2009). dilp8 is highly induced in imaginal disks with growth perturbations (Colombani et al., 2012; Garelli et al., 2012). Importantly, knockdown of dilp8 in tissues with abnormal growth prevents the delay in pupariation, suggesting that it is required for the coupling of tissue growth and developmental timing. Expression of dilp8 in imaginal disks is also sufficient to delay the onset of metamorphosis, which can be overcome by feeding larvae ecdysone (Garelli et al., 2012). Additionally, coculture experiments reveal that ecdysone production in the ring gland is suppressed in response to Dilp8 produced by imaginal disks (Colombani et al., 2012). Taken together, these results suggest that Dilp8 is secreted by the imaginal disks and remotely acts on the ring gland to suppress ecdysone production and delay development. How Dilp8 suppresses ecdysone is not known, but it may signal through the InR pathway.

It has been shown that insulin signaling and ecdysone regulate each other antagonistically (Caldwell et al., 2005; Colombani et al., 2005; Mirth et al., 2005). A recent study has demonstrated a role for the nuclear cofactor, dDOR, in the relationship between insulin signaling and ecdysone. They show that dDOR is a coactivator of EcR, and that its expression is down-regulated by insulin signaling via the inhibition of FOXO activity (Francis, Zorzano, & Teleman, 2010). In addition, ecdysone induces translocation of dFOXO into the nucleus, promoting dDOR expression, which further activates EcR and initiates a feed-forward loop. Intriguingly, dDOR knockout flies have a salivary gland degradation defect, and DOR has been shown to regulate autophagy in both mammalian and Drosophila cells (Francis et al., 2010; Mauvezin et al., 2010). These results provide one of the few clues to how the relationship between insulin signaling, ecdysone, and autophagy functions (Fig. 4.3).

Figure 4.3.

Figure 4.3

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Relationship between ecdysone, autophagy, and insulin signaling and growth. Ecdysone and insulin have opposing effects on autophagy; ecdysone induces autophagy, and insulin inhibits autophagy through class I PI3K and TOR signaling. Ecdysone and insulin also antagonize each other, suggesting that a balance between these two hormones may be required to regulate autophagy. For example, increased ecdysone would inhibit insulin signaling, releasing insulin's inhibition on autophagy and further inducing autophagy. Whether autophagy itself regulates ecdysone and insulin is unclear; however, in some contexts autophagy may be a negative regulator of growth.

4. AUTOPHAGY AND DROSOPHILA DEVELOPMENT

Most autophagy studies have been done using either yeast or mammalian cell culture. While these studies have been essential to our understanding of the genetic mechanisms that regulate autophagy, there is little known about the impact of autophagy on the homeostasis of multicellular organisms. It would be interesting to understand how autophagy in different cell contexts, such as cell growth, cell survival, and cell death, affects the organism as a whole.

Drosophila is an ideal system for studying autophagy in a multicellular organism. The steroid and growth factor signaling pathways that regulate autophagy are similar in flies and humans. Importantly, Atg genes and their regulators are highly conserved between flies and humans (Baehrecke, 2003). In contrast to mammalian systems, Drosophila has little genetic redundancy and has single copies for most genes in the autophagic pathway and its regulatory pathways. In addition, autophagy is induced in Drosophila tissues in response to either nutrient starvation or the steroid hormone ecdysone (Lee & Baehrecke, 2001; Lee, Cooksey, & Baehrecke, 2002; Rusten et al., 2004).

4.1. Autophagy in growth and nutrient utilization

Autophagy is critical for proper nutrient utilization during Drosophila larval development. In the fly, the major storage site for glycogen, lipids, and proteins is the fat body, an organ that shares attributes with both mammalian adipose tissue and liver. The fat body provides an excellent model for studying the mechanisms that regulate autophagy. When larvae are deprived of amino acids, autophagy is induced in the fat body, and this starvation-induced autophagy is regulated by TOR signaling (Scott, Schuldiner, & Neufeld, 2004). It has been shown that inactivation of TOR signaling either by a TOR null mutant or by manipulating upstream regulators of TOR induces autophagy in the fat body of feeding larvae. On the other hand, activation of either TOR or class I PI3K suppresses starvation-induced autophagy in the fat body (Scott et al., 2004). These results, taken together with the result that constitutive expression of PI3K in the fat body causes reduced viability during starvation (Britton, Lockwood, Li, Cohen, & Edgar, 2002), suggest that proper regulation of the class I PI3K signaling pathway is necessary for autophagy to promote survival during starvation.

In addition to being necessary for survival during starvation, autophagy may have a critical role in lipid metabolism of the Drosophila fat body. In mammalian cells, it has been shown that there is a connection between autophagy and lipolysis as well as lipid storage. Singh et al. demonstrated that triglycerides (TGs) and lipid droplet (LD) proteins were associated with both autophagosomes and lysosomes. Moreover, inhibition of autophagy in mouse liver cells led to increased TGs and LDs in vitro and in vivo, while increased autophagy led to decreased TGs and LDs in vitro (Singh et al., 2009). Their data suggests that lipid accumulation during autophagy inhibition is a result of blocked lipolysis. By contrast, it has been shown that loss of either Atg5 or Atg7 in mouse adipocytes leads to reduced lipid accumulation and impaired adipocyte differentiation (Baerga, Zhang, Chen, Goldman, & Jin, 2009; Zhang et al., 2009). Similar results were obtained in a recent study of Drosophila larval fat body. Atg7 loss-of-function mutants had smaller LDs in the fat body, indicating a lipid accumulation defect (Wang et al., 2012). One possible explanation for the discrepancies between these studies is that autophagy may affect lipid metabolism in a tissue-specific manner. It would be interesting to further investigate the relationship between autophagy and lipid metabolism and how it is regulated in different tissues.

Wang et al. (2012) provided insight into the relationship between lipid metabolism and autophagy. Members of the Rab small GTPase family have been associated with LDs, and are known to participate in many cellular processes, including endocytosis, exocytosis, autophagosome formation, lysosome formation, and signaling transduction (Liu et al., 2007; Stenmark, 2009; Zehmer et al., 2009). In a screen for Rab proteins that affect LD size, Wang et al. found 18 Rab proteins that either increased or decreased LD size (Wang et al., 2012).They focused onRab32 and showed that as well as having smaller LDs, Rab32 mutants have impaired autophagy in the fat body. Importantly, Rab32 localized on autophagosomes, but not LDs, suggesting that its effect on LD size is due to regulation of autophagy rather than a direct effect on LDs. Since different Rab proteins have different effects on LD size, investigating the remaining Rab proteins might shed some light on the regulation of the relationship between autophagy and lipid metabolism.

Autophagy is also induced in the fat body and other tissues, including the salivary glands and mid gut during development in response to rises in ecdysone titer. This developmental autophagy is induced during the wandering larval stage and metamorphosis at times when the animal is not feeding, suggesting that autophagy may play an important role in survival and even tissue growth during nonfeeding periods (Lee & Baehrecke, 2001; Lee, Cooksey, & Baehrecke, 2002; Rusten et al., 2004). In the fat body, programmed autophagy is induced in response to ecdysone late during the third larval stage. This induction requires the downregulation of class I PI3K signaling (Rusten et al., 2004), suggesting that regulation of the class I PI3K pathway is involved in both starvation-induced autophagy and developmental autophagy.

Studies in the Drosophila fat body have identified other genes that are necessary for autophagy induced in response to ecdysone. SNF4Aγ, the Drosophila homologue of the AMP-activated protein kinase (AMPK) γ sub-unit, was identified in a fat body screen for mutants that fail to induce autophagy in response to ecdysone (Lippai et al., 2008). AMPK is an evolutionarily conserved enzyme that maintains cellular energy balance and is an inhibitor of TOR signaling (Shaw, 2009). In mammalian cells, AMPK has been implicated in the induction of autophagy in response to stimuli other than starvation, including growth factor withdrawal and increased calcium signaling (Hoyer-Hansen et al., 2007; Liang et al., 2007). Importantly, several recent studies in mammalian cells have shown that AMPK may directly control ULK1, the mammalian homologue of Atg1, via phosphorylation; however, the exact sites of phosphorylation are still debated (Egan et al., 2011; Kim, Kundu, Viollet, & Guan, 2011; Lee, Park, Takahashi, & Wang, 2010; Shang et al., 2011). AMPK has also been shown to suppress cell proliferation in Drosophila (Mandal, Guptan, Owusu-Ansah, & Banerjee, 2005). Taken together, these studies suggest that AMPK is an important regulator of the relationship between autophagy and growth.

Studies in Drosophila have further investigated the relationship between autophagy and growth.TOR is a key regulator of cell growth that was first implicated in the regulation of autophagy when rapamycin, a TOR inhibitor, was shown to induce autophagy (Blommaart, Luiken, Blommaart, vanWoerkom, &Meijer, 1995). TOR represses autophagy through phosphorylation of Atg1 (Kamada et al., 2000; Scott, Juhász, & Neufeld, 2007). In Drosophila larval fat body, overexpression of Atg1 inhibits cell growth through a negative feedback mechanism on TOR. Conversely, Atg1 mutant cells with reduced TOR signaling have increased growth (Scott et al., 2007). These results suggest that autophagy is a negative regulator of cell growth. Interestingly, it has been shown that inhibiting autophagy in a TOR null background enhances the TOR mutant phenotypes, including reduced growth rate, smaller cell size, and decreased survival (Scott et al., 2004).This suggests that under these conditions, in contradiction to its role as a negative regulator of growth, autophagy is necessary to promote cell survival and maintain growth.

The relationship between autophagy and growth signaling has also been studied in the context of degrading tissues during Drosophila metamorphosis. Growth arrest is required for the induction of autophagy in degrading salivary glands (Berry & Baehrecke, 2007). This growth arrest is regulated by the class I PI3K pathway. Maintaining growth in the salivary glands through expression of activated Ras, Akt, or the class I PI3K catalytic subunit Dp110, inhibits autophagy and gland degradation. In addition, coexpression of a dominant negative TOR with either Ras or Dp110 partially suppresses the overgrowth phenotypes and the salivary gland degradation defects (Berry & Baehrecke, 2007). These data suggest that cell growth regulators signal through TOR to inhibit autophagy and prevent salivary gland degradation. Further, overexpression of Atg1, which induces autophagy, suppresses the Dp110 persistent salivary gland phenotype, while Atg loss-of-function mutations cause persistent salivary glands (Berry & Baehrecke, 2007), indicating that both growth arrest and autophagy are required for proper salivary gland degradation.

A recent study has observed a similar relationship between growth arrest and autophagy during midgut programmed cell death in Drosophila. In the midgut, as in the salivary glands, growth arrest occurs before programmed cell death induction (Denton, Chang, et al., 2012). When cell growth in the midgut is maintained by expression of either activated Ras or Dp110, autophagy is suppressed and midgut degradation is delayed (Denton, Chang, et al., 2012). These results indicate a role for growth arrest in midgut programmed cell death. In contrast, inhibition of growth by the expression of PTEN or TSC1/TSC2, negative regulators of class I PI3K signaling, results in smaller midguts and premature autophagy induction. This growth inhibition can be suppressed by knockdown of either Atg1 or Atg18 in a PTEN or TSC1/TSC2 expressing background (Denton, Chang, et al., 2012). Interestingly, knockdown of Atg genes alone in the midgut causes persistent PI3K growth signaling and a significant delay in midgut degradation. These results suggest that in the midgut, growth and autophagy have a reciprocal relationship as in the salivary glands; however, there is also a feedback mechanism by which autophagy downregulates class I PI3K signaling. The nature of this feedback mechanism is unknown and deserves future investigation.

There has been some recent progress on the study of how cell growth arrest is regulated in dying salivary glands. The evolutionarily conserved Warts (Wts)/Hippo (Hpo) signaling pathway is an important negative regulator of cell growth that functions through the inactivation of Yorkie (Yki), a transcriptional coactivator and positive regulator of growth (Huang, Wu, Barrera, Matthews, & Pan, 2005). Loss-of-function mutations in the Wts pathway or overexpression of Yki lead to tissue overgrowth (Huang et al., 2005). Importantly, wts is required for growth arrest and autophagy induction in degrading salivary glands (Dutta & Baehrecke, 2008). Disruption of this pathway by mutations in wts and hpo or knockdown of sav and mats prevents salivary gland degradation (Dutta & Baehrecke, 2008). Surprisingly, overexpression of Yki fails to inhibit salivary gland degradation, suggesting that Wts regulates salivary gland growth in a Yki-independent manner. Significantly, wts mutants cause persistent class I PI3K signaling in salivary glands, and knockdown of chico or expression of dominant-negative TOR suppresses the wts cell death defects (Dutta & Baehrecke, 2008). These data suggest that Wts regulates salivary gland cell growth in a class I PI3K-dependent manner. However, Wts does not have a common role in programmed cell death. Despite the clear requirement for class I PI3K signaling in the regulation of cell growth and cell death in the midgut, knockdown of wts does not affect midgut morphology or degradation (Denton, Chang, et al., 2012).

4.2. Autophagy and cell death

Programmed cell death is a highly conserved and genetically regulated fundamental biological process. During development, cell death is required for tissue pattern formation and to maintain tissue homeostasis. Cell death also functions to remove abnormal or damaged cells. Schweichel and Merker (1973) described three major types of cell death during mammalian development based on morphology and involvement of the lysosomal compartment. Type I cell death, or apoptosis, is characterized by caspase activation, cell shrinkage, cytoplasmic blebbing, nuclear and DNA fragmentation, and engulfment by a phagocyte where the lysosome of the engulfing cell degrades the dying cell (Kerr, Wyllie, & Currie, 1972). In contrast to apoptosis, type II cell death, or autophagic cell death, requires little or no help from phagocytes, and the dying cell is degraded by its own lysosome. Type III cell death, or necrosis, is the least common form of cell death, and it has no known lysosomal involvement.

Type II cell death is observed in a variety of organisms. The plant, Arabidopsis, requires type II cell death for the formation of tracheary elements (Kwon et al., 2010). Type II cell death has also been observed in several tissues during mammalian development, including regression of the corpus luteum and involution of mammary and prostate glands (Clarke, 1990). Type II cell death is best characterized in insects and has been observed in several tissues during development, including dying flight muscles of the Hawkmoth Manduca sexta (Lockshin & Williams, 1965), and degrading salivary glands and midgut in Drosophila (Lee & Baehrecke, 2001; Lee, Cooksey, & Baehrecke, 2002). Although autophagosomes are present in dying cells with type II morphology, the role of autophagy in cell death remains controversial (Denton, Nicolson, et al., 2012; Levine&Yuan, 2005).

Studies of dying larval tissues during Drosophila metamorphosis have provided evidence for a role of autophagy in programmed cell death. As described above, a peak in ecdysone titer triggers salivary gland degradation during metamorphosis. Several Atg genes exhibit increased transcription in salivary glands in response to the rise in ecdysone, including Atg2, Atg3, Atg4, Atg5, Atg7, and Atg18 (Gorski et al., 2003; Lee et al., 2003). Additionally, mutations in transcription factors downstream of the EcR inhibit transcription of Atg-related genes and prevent proper salivary gland cell death (Lee et al., 2003), suggesting that ecdysone-induced autophagy promotes cell death. It was not until recently though that the function of autophagy in cell death was rigorously tested in vivo. Mutations in Atg8, Atg18, Atg2, or Atg3 or decreased function of Atg1 all result in incomplete degradation of the larval salivary glands (Berry & Baehrecke, 2007). In addition, knockdown of Atg3, Atg6, Atg7, or Atg12 specifically in the salivary glands leads to incomplete gland destruction, suggesting that autophagy functions in a tissue-autonomous manner in these dying cells (Berry & Baehrecke, 2007). Moreover, misexpression of Atg1 in the salivary glands induces autophagy and leads to premature gland degradation in a caspase-independent manner (Berry & Baehrecke, 2007). This is in contrast to previous work which showed that overexpression of Atg1 in the fat body induces cell death that depends on caspase function (Scott et al., 2007).

There is also mounting evidence for a role of autophagy during programmed cell death of the larval midgut. Similar to salivary glands, larval midgut destruction is triggered by a peak in ecdysone titer at the end of larval development. The dying midguts have increased autophagosome formation, and inhibition of autophagy by loss-of-function mutations in Atg2 or Atg18 or knockdown of either Atg1 or Atg18 severely delays midgut removal (Denton et al., 2009). Additionally, overexpression of Atg1 in the larval midgut is sufficient to induce autophagy and premature degradation (Denton, Chang, et al., 2012). Surprisingly, caspases are active, but they are not required for removal of the midgut (Denton et al., 2009; Denton, Shravage, Simin, Baehrecke, & Kumar, 2010), indicating that there is a complex relationship between autophagy and caspases in this tissue.

Autophagy and caspases have a complex relationship that may be context dependent. During salivary gland degradation, the rise in ecdysone titer triggers increased transcription of not only Atg genes but also the proapoptotic genes, rpr and hid, caspases, the BCL-2 family member buffy, and ark, the fly Apaf-1 homologue (Dorstyn, Colussi, Quinn, Richardson, & Kumar, 1999; Jiang, Baehrecke, & Thummel, 1997; Lee, Simon, Woodard, & Baehrecke, 2002). Caspase activation occurs in the glands, but expression of the caspase inhibitor p35 only partially inhibits salivary gland degradation (Lee & Baehrecke, 2001). Additionally, ark mutants have a partial salivary gland degradation defect, but autophagy occurs normally, suggesting that ark may function downstream or parallel to autophagy in programmed cell death (Akdemir et al., 2006; Mills et al., 2006). Significantly, inhibiting both caspases and autophagy by expressing p35 in salivary glands of Atg18 loss-of-function mutants or with dominant negative Atg1 results in increased persistence of the salivary glands (Berry & Baehrecke, 2007). These results suggest that autophagy and caspases function in parallel during salivary gland cell death. Many of the components of the apoptotic machinery are also upregulated in dying midguts. Despite the presence of high levels of caspase activity, p35 expression or genetic ablation of the canonical caspase activation pathway has no effect on midgut degradation (Denton et al., 2009). This is in contrast to what has been observed in salivary glands, and it would be interesting to study what causes these distinct differences between how programmed cell death is executed in these two tissues.

Although these in vivo studies indicate a role for autophagy in programmed cell death, the mechanistic differences that determine whether autophagy will support cell survival or cell death are not clear. Recently, Draper (Drpr), the Drosophila homologue of Caenorhabditis elegans engulfment receptor CED-1, and other components of the engulfment pathway were shown to be required for induction of autophagy during cell death (McPhee, Logan, Freeman, & Baehrecke, 2010). Null mutations in drpr and salivary gland-specific knockdown of drpr prevent induction of autophagy and cause persistent salivary glands. Expression of Atg1 in drpr mutants is sufficient to rescue the salivary gland degradation defect, indicating that Drpr functions upstream of autophagy. Surprisingly, clonal analysis of degrading glands reveals that Draper functions in a cell-autonomous manner, as there is only a reduction of autophagy in the drpr mutant cells. Interestingly, knockdown of drpr in the fat body does not affect starvation-induced autophagy, implicating drpr as the first known factor to regulate autophagy’s role in cell death but not cell survival (McPhee et al., 2010). It would be interesting to further investigate how Drpr is regulated in salivary glands and why an engulfment receptor is functioning cell-autonomously.

5. CONCLUSIONS

Organisms require a balance between cell survival and cell death to maintain homeostasis, and although in vivo evidence supports a role for autophagy in both cell survival and cell death, many fundamental questions remain. Since autophagy is involved in both protecting and killing the cell, it is important to determine the mechanisms that decide between these cell fates. One possibility is that autophagy selectively depletes a cell survival factor or an essential organelle, which leads to cell death (Abeliovich, 2007; Nezis et al., 2010; Yu et al., 2006). Another possibility is that there is a threshold of autophagic flux that is crossed to promote cell death. Extended growth factor withdrawal in apoptotic-resistant mouse cells leads to stress-induced autophagy and eventual death by depletion of cellular resources (Lum et al., 2005). Under more physiological conditions, degradation of the Drosophila salivary glands and midgut is preceded by an increase in both transcription of the Atg genes and autophagosome levels. Additionally, mis-expression of Atg1 in several tissues promotes cell demise, supporting the idea that excessive autophagy leads to cell death. However, excessive autophagy might not always be enough to kill, and other death factors may be required in addition to autophagy. Cell death induced by Atg1 mis-expression in the fat body is caspase dependent. Further, salivary glands require caspases and autophagy, functioning in parallel, to fully degrade (Berry & Baehrecke, 2007).

Autophagy has been shown to be both an alternative form of cell death in nonphysiological conditions and a necessary component of cell death in physiological contexts; however, why cells die by autophagy is not understood. Apoptosis requires a phagocyte to engulf the dying cell, while autophagic cell death has little or no phagocyte involvement. One possibility is that phagocytes have restricted access to the dying cells. In Drosophila, the adult midgut forms around the degrading larval midgut isolating the dying cells from the rest of the tissues. Similarly, in vitro models of mammary lumen formation, where the dying cells are isolated from phagocytes, implicate the necessity of both caspases and autophagy for elimination of the dying cells (Debnath et al., 2002; Mills, Reginato, Debnath, Queenan, & Brugge, 2004). Alternatively, large cells and tissues, such as the giant larval salivary glands, may be too big to degrade by phagocytosis alone, and they require autophagy for the bulk degradation of their cytoplasm. Finally, autophagy may contribute to nutrient resource reallocation and survival in multicellular organisms. In yeast and mammalian cell culture, autophagy degrades cellular content to produce ATP and resources to protect the cell during starvation. Interestingly, autophagic cell death of tissues in Drosophila occurs during a time when the animal receives no external nutrients and must rely on its nutrient stores for survival and development of adult structures. Further, the majority of Atg mutants are pupal lethal, suggesting that autophagy is necessary to survive metamorphosis. Thus, although autophagy is killing individual cells and tissues, this form of cell death could be promoting organism survival. Future studies in Drosophila will hopefully lead to a better understanding of autophagy’s dual roles in life and death.

ACKNOWLEDGMENTS

We thank the Baehrecke lab for constructive comments. Work on this subject is supported by NIH grants GM079431 and CA159314, and the Ellison Medical Foundation to E. H. B. E. H. B. is a member of the UMass DERC (DK32520).

REFERENCES

  1. Abeliovich H. Mitophagy: The life-or-death dichotomy includes yeast. Autophagy. 2007;3:275–277. doi: 10.4161/auto.3915. [DOI] [PubMed] [Google Scholar]
  2. Akdemir F, Farkas R, Chen P, Juhasz G, Medved’ová L, Sass M, et al. Autophagy occurs upstream or parallel to the apoptosome during histolytic cell death. Development (Cambridge, England) 2006;133:1457–1465. doi: 10.1242/dev.02332. [DOI] [PubMed] [Google Scholar]
  3. Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. The Journal of Cell Biology. 2008;182:685–701. doi: 10.1083/jcb.200803137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baehrecke EH. Autophagic programmed cell death in Drosophila. Cell Death and Differentiation. 2003;10:940–945. doi: 10.1038/sj.cdd.4401280. [DOI] [PubMed] [Google Scholar]
  5. Baehrecke EH, Thummel CS. The Drosophila E93 gene from the 93F early puff displays stage- and tissue-specific regulation by 20-hydroxyecdysone. Developmental Biology. 1995;171:85–97. doi: 10.1006/dbio.1995.1262. [DOI] [PubMed] [Google Scholar]
  6. Baerga R, Zhang Y, Chen P-H, Goldman S, Jin S. Targeted deletion of autophagy-related 5 (atg5) impairs adipogenesis in a cellular model and in mice. Autophagy. 2009;5:1118–1130. doi: 10.4161/auto.5.8.9991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bakker K. Feeding period, growth, and pupation in larvae of Drosophila melanogaster. Entomologia Experimentalis et Applicata. 1959;2:171–186. [Google Scholar]
  8. Berry DL, Baehrecke EH. Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila. Cell. 2007;131:1137–1148. doi: 10.1016/j.cell.2007.10.048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Blommaart EF, Luiken JJ, Blommaart PJ, van Woerkom GM, Meijer AJ. Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes. The Journal of Biological Chemistry. 1995;270:2320–2326. doi: 10.1074/jbc.270.5.2320. [DOI] [PubMed] [Google Scholar]
  10. Britton JS, Lockwood WK, Li L, Cohen SM, Edgar BA. Drosophila’s insulin/PI3-kinase pathway coordinates cellular metabolism with nutritional conditions. Developmental Cell. 2002;2:239–249. doi: 10.1016/s1534-5807(02)00117-x. [DOI] [PubMed] [Google Scholar]
  11. Broadus J, McCabe JR, Endrizzi B, Thummel CS, Woodard CT. The Drosophila beta FTZ-F1 orphan nuclear receptor provides competence for stage-specific responses to the steroid hormone ecdysone. Molecular Cell. 1999;3:143–149. doi: 10.1016/s1097-2765(00)80305-6. [DOI] [PubMed] [Google Scholar]
  12. Burtis KC, Thummel CS, Jones CW, Karim FD, Hogness DS. The Drosophila 74EF early puff contains E74, a complex ecdysone-inducible gene that encodes two ets-related proteins. Cell. 1990;61:85–99. doi: 10.1016/0092-8674(90)90217-3. [DOI] [PubMed] [Google Scholar]
  13. Caldwell PE, Walkiewicz M, Stern M. Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release. Current Biology. 2005;15:1785–1795. doi: 10.1016/j.cub.2005.09.011. [DOI] [PubMed] [Google Scholar]
  14. Church RB, Robertson FW. Biochemical analysis of genetic differences in the growth of Drosophila. Genetical Research. 1966;7:383–407. doi: 10.1017/s0016672300009836. [DOI] [PubMed] [Google Scholar]
  15. Clarke PG. Developmental cell death: Morphological diversity and multiple mechanisms. Anatomy and Embryology. 1990;181:195–213. doi: 10.1007/BF00174615. [DOI] [PubMed] [Google Scholar]
  16. Colombani J, Andersen DS, Léopold P. Secreted peptide Dilp8 coordinates Drosophila tissue growth with developmental timing. Science. 2012;336:582–585. doi: 10.1126/science.1216689. [DOI] [PubMed] [Google Scholar]
  17. Colombani J, Bianchini L, Layalle S, Pondeville E, Dauphin-Villemant C, Antoniewski C, et al. Antagonistic actions of ecdysone and insulins determine final size in Drosophila. Science. 2005;310:667–670. doi: 10.1126/science.1119432. [DOI] [PubMed] [Google Scholar]
  18. Debnath J, Mills KR, Collins NL, Reginato MJ, Muthuswamy SK, Brugge JS. The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini. Cell. 2002;111:29–40. doi: 10.1016/s0092-8674(02)01001-2. [DOI] [PubMed] [Google Scholar]
  19. Denton D, Chang T-K, Nicolson S, Shravage B, Simin R, Baehrecke EH, et al. Relationship between growth arrest and autophagy in midgut programmed cell death in Drosophila. Cell Death and Differentiation. 2012;19:1299–1307. doi: 10.1038/cdd.2012.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Denton D, Nicolson S, Kumar S. Cell death by autophagy: Facts and apparent artefacts. Cell Death and Differentiation. 2012;19:87–95. doi: 10.1038/cdd.2011.146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Denton D, Shravage B, Simin R, Baehrecke EH, Kumar S. Larval midgut destruction in Drosophila: Not dependent on caspases but suppressed by the loss of autophagy. Autophagy. 2010;6:163–165. doi: 10.4161/auto.6.1.10601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Denton D, Shravage B, Simin R, Mills K, Berry DL, Baehrecke EH, et al. Autophagy, not apoptosis, is essential for midgut cell death in Drosophila. Current Biology. 2009;19:1741–1746. doi: 10.1016/j.cub.2009.08.042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. DiBello PR, Withers DA, Bayer CA, Fristrom JW, Guild GM. The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers. Genetics. 1991;129:385–397. doi: 10.1093/genetics/129.2.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Dorstyn L, Colussi PA, Quinn LM, Richardson H, Kumar S. DRONC, an ecdysone-inducible Drosophila caspase. Proceedings of the National Academy of Sciences of the United States of America. 1999;96:4307–4312. doi: 10.1073/pnas.96.8.4307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Dunn WA., Jr Studies on the mechanisms of autophagy: Formation of the autophagic vacuole. The Journal of Cell Biology. 1990;110:1923–1933. doi: 10.1083/jcb.110.6.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Dutta S, Baehrecke EH. Warts is required for PI3K-regulated growth arrest, autophagy, and autophagic cell death in Drosophila. Current Biology. 2008;18:1466–1475. doi: 10.1016/j.cub.2008.08.052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Egan DF, Shackelford DB, Mihaylova MM, Gelino S, Kohnz RA, Mair W, et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science. 2011;331:456–461. doi: 10.1126/science.1196371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Fimia GM, Stoykova A, Romagnoli A, Giunta L, Di Bartolomeo S, Nardacci R, et al. Ambra1 regulates autophagy and development of the nervous system. Nature. 2007;447:1121–1125. doi: 10.1038/nature05925. [DOI] [PubMed] [Google Scholar]
  29. Francis VA, Zorzano A, Teleman AA. dDOR is an EcR coactivator that forms a feed-forward loop connecting insulin and ecdysone signaling. Current Biology. 2010;20:1799–1808. doi: 10.1016/j.cub.2010.08.055. [DOI] [PubMed] [Google Scholar]
  30. Garelli A, Gontijo AM, Miguela V, Caparros E, Dominguez M. Imaginal discs secrete insulin-like peptide 8 to mediate plasticity of growth and maturation. Science. 2012;336:579–582. doi: 10.1126/science.1216735. [DOI] [PubMed] [Google Scholar]
  31. Gorski SM, Chittaranjan S, Pleasance ED, Freeman JD, Anderson CL, Varhol RJ, et al. A SAGE approach to discovery of genes involved in autophagic cell death. Current Biology. 2003;13:358–363. doi: 10.1016/s0960-9822(03)00082-4. [DOI] [PubMed] [Google Scholar]
  32. Hailey DW, Rambold AS, Satpute-Krishnan P, Mitra K, Sougrat R, Kim PK, et al. Mitochondria supply membranes for autophagosome biogenesis during starvation. Cell. 2010;141:656–667. doi: 10.1016/j.cell.2010.04.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Harding TM, Hefner-Gravink A, Thumm M, Klionsky DJ. Genetic and phenotypic overlap between autophagy and the cytoplasm to vacuole protein targeting pathway. The Journal of Biological Chemistry. 1996;271:17621–17624. doi: 10.1074/jbc.271.30.17621. [DOI] [PubMed] [Google Scholar]
  34. Harding TM, Morano KA, Scott SV, Klionsky DJ. Isolation and characterization of yeast mutants in the cytoplasm to vacuole protein targeting pathway. The Journal of Cell Biology. 1995;131:591–602. doi: 10.1083/jcb.131.3.591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Hoyer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai G, Farkas T, et al. Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Molecular Cell. 2007;25:193–205. doi: 10.1016/j.molcel.2006.12.009. [DOI] [PubMed] [Google Scholar]
  36. Huang J, Wu S, Barrera J, Matthews K, Pan D. The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila Homolog of YAP. Cell. 2005;122:421–434. doi: 10.1016/j.cell.2005.06.007. [DOI] [PubMed] [Google Scholar]
  37. Ichimura Y, Kirisako T, Takao T, Satomi Y, Shimonishi Y, Ishihara N, et al. A ubiquitin-like system mediates protein lipidation. Nature. 2000;408:488–492. doi: 10.1038/35044114. [DOI] [PubMed] [Google Scholar]
  38. Itakura E, Kishi C, Inoue K, Mizushima N. Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG. Molecular Biology of the Cell. 2008;19:5360–5372. doi: 10.1091/mbc.E08-01-0080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Jiang C, Baehrecke EH, Thummel CS. Steroid regulated programmed cell death during Drosophila metamorphosis. Development (Cambridge, England) 1997;124:4673–4683. doi: 10.1242/dev.124.22.4673. [DOI] [PubMed] [Google Scholar]
  40. Jiang C, Lamblin AF, Steller H, Thummel CS. A steroid-triggered transcriptional hierarchy controls salivary gland cell death during Drosophila metamorphosis. Molecular Cell. 2000;5:445–455. doi: 10.1016/s1097-2765(00)80439-6. [DOI] [PubMed] [Google Scholar]
  41. Juhasz G, Neufeld TP. Autophagy: A forty-year search for a missing membrane source. PLoS Biology. 2006;4:e36. doi: 10.1371/journal.pbio.0040036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Kamada Y, Funakoshi T, Shintani T, Nagano K, Ohsumi M, Ohsumi Y. Tor-mediated induction of autophagy via an Apg1 protein kinase complex. The Journal of Cell Biology. 2000;150:1507–1513. doi: 10.1083/jcb.150.6.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Kametaka S, Okano T, Ohsumi M, Ohsumi Y. Apg14p and Apg6/Vps30p form a protein complex essential for autophagy in the yeast, Saccharomyces cerevisiae. The Journal of Biological Chemistry. 1998;273:22284–22291. doi: 10.1074/jbc.273.35.22284. [DOI] [PubMed] [Google Scholar]
  44. Kerr JF, Wyllie AH, Currie AR. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer. 1972;26:239–257. doi: 10.1038/bjc.1972.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Kihara A, Noda T, Ishihara N, Ohsumi Y. Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae. The Journal of Cell Biology. 2001;152:519–530. doi: 10.1083/jcb.152.3.519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Kim J, Kundu M, Viollet B, Guan K-L. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nature Cell Biology. 2011;13:132–141. doi: 10.1038/ncb2152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Klionsky DJ. The molecular machinery of autophagy: Unanswered questions. Journal of Cell Science. 2005;118:7–18. doi: 10.1242/jcs.01620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy. 2008;4:151–175. doi: 10.4161/auto.5338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Klionsky DJ, Cregg JM, Dunn WA, Jr, Emr SD, Sakai Y, Sandoval IV, et al. A unified nomenclature for yeast autophagy-related genes. Developmental Cell. 2003;5:539–545. doi: 10.1016/s1534-5807(03)00296-x. [DOI] [PubMed] [Google Scholar]
  50. Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science. 2000;290:1717–1721. doi: 10.1126/science.290.5497.1717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Koelle MR, Talbot WS, Segraves WA, Bender MT, Cherbas P, Hogness DS. The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell. 1991;67:59–77. doi: 10.1016/0092-8674(91)90572-g. [DOI] [PubMed] [Google Scholar]
  52. Kuma A, Mizushima N, Ishihara N, Ohsumi Y. Formation of the approximately 350-kDa Apg12-Apg5.Apg16 multimeric complex, mediated by Apg16 oligomerization, is essential for autophagy in yeast. The Journal of Biological Chemistry. 2002;277:18619–18625. doi: 10.1074/jbc.M111889200. [DOI] [PubMed] [Google Scholar]
  53. Kwon SI, Cho HJ, Jung JH, Yoshimoto K, Shirasu K, Park OK. The Rab GTPase RabG3b functions in autophagy and contributes to tracheary element differentiation in Arabidopsis. The Plant Journal. 2010;64:151–164. doi: 10.1111/j.1365-313X.2010.04315.x. [DOI] [PubMed] [Google Scholar]
  54. Lavorgna G, Karim FD, Thummel CS, Wu C. Potential role for a FTZ-F1 steroid receptor superfamily member in the control of Drosophila metamorphosis. Proceedings of the National Academy of Sciences of the United States of America. 1993;90:3004–3008. doi: 10.1073/pnas.90.7.3004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Layalle S, Arquier N, Léopold P. The TOR pathway couples nutrition and developmental timing in Drosophila. Developmental Cell. 2008;15:568–577. doi: 10.1016/j.devcel.2008.08.003. [DOI] [PubMed] [Google Scholar]
  56. Lee CY, Baehrecke EH. Steroid regulation of autophagic programmed cell death during development. Development (Cambridge, England) 2001;128:1443–1455. doi: 10.1242/dev.128.8.1443. [DOI] [PubMed] [Google Scholar]
  57. Lee C-Y, Clough EA, Yellon P, Teslovich TM, Stephan DA, Baehrecke EH. Genome-wide analyses of steroid- and radiation-triggered programmed cell death in Drosophila. Current Biology. 2003;13:350–357. doi: 10.1016/s0960-9822(03)00085-x. [DOI] [PubMed] [Google Scholar]
  58. Lee C-Y, Cooksey BAK, Baehrecke EH. Steroid regulation of midgut cell death during Drosophila development. Developmental Biology. 2002;250:101–111. doi: 10.1006/dbio.2002.0784. [DOI] [PubMed] [Google Scholar]
  59. Lee JW, Park S, Takahashi Y, Wang H-G. The association of AMPK with ULK1 regulates autophagy. PLoS One. 2010;5:e15394. doi: 10.1371/journal.pone.0015394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Lee C-Y, Simon CR, Woodard CT, Baehrecke EH. Genetic mechanism for the stage- and tissue-specific regulation of steroid triggered programmed cell death in Drosophila. Developmental Biology. 2002;252:138–148. doi: 10.1006/dbio.2002.0838. [DOI] [PubMed] [Google Scholar]
  61. Lee CY, Wendel DP, Reid P, Lam G, Thummel CS, Baehrecke EH. E93 directs steroid-triggered programmed cell death in Drosophila. Molecular Cell. 2000;6:433–443. doi: 10.1016/s1097-2765(00)00042-3. [DOI] [PubMed] [Google Scholar]
  62. Levine B, Yuan J. Autophagy in cell death: An innocent convict? The Journal of Clinical Investigation. 2005;115:2679–2688. doi: 10.1172/JCI26390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Liang C, Feng P, Ku B, Dotan I, Canaani D, Oh B-H, et al. Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG. Nature Cell Biology. 2006;8:688–699. doi: 10.1038/ncb1426. [DOI] [PubMed] [Google Scholar]
  64. Liang J, Shao SH, Xu Z-X, Hennessy B, Ding Z, Larrea M, et al. The energy sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to enter autophagy or apoptosis. Nature Cell Biology. 2007;9:218–224. doi: 10.1038/ncb1537. [DOI] [PubMed] [Google Scholar]
  65. Lippai M, Csikós G, Maróy P, Lukácsovich T, Juhász G, Sass M. SNF4Agamma, the Drosophila AMPK gamma subunit is required for regulation of developmental and stress-induced autophagy. Autophagy. 2008;4:476–486. doi: 10.4161/auto.5719. [DOI] [PubMed] [Google Scholar]
  66. Liu P, Bartz R, Zehmer JK, Ying YS, Zhu M, Serrero G, et al. Rabregulated interaction of early endosomes with lipid droplets. Biochimica et Biophysica Acta. 2007;1773:784–793. doi: 10.1016/j.bbamcr.2007.02.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Lockshin RA, Williams CM. Programmed cell death I. Cytology of degeneration in the intersegmental muscles of the pernyi silkmoth. Journal of Insect Physiology. 1965;11:123–133. doi: 10.1016/0022-1910(65)90099-5. [DOI] [PubMed] [Google Scholar]
  68. Lum JJ, Bauer DE, Kong M, Harris MH, Li C, Lindsten T, et al. Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell. 2005;120:237–248. doi: 10.1016/j.cell.2004.11.046. [DOI] [PubMed] [Google Scholar]
  69. Mandal S, Guptan P, Owusu-Ansah E, Banerjee U. Mitochondrial regulation of cell cycle progression during development as revealed by the tenured mutation in Drosophila. Developmental Cell. 2005;9:843–854. doi: 10.1016/j.devcel.2005.11.006. [DOI] [PubMed] [Google Scholar]
  70. Mauvezin C, Orpinell M, Francis VA, Mansilla F, Duran J, Ribas V, et al. The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells. EMBO Reports. 2010;11:37–44. doi: 10.1038/embor.2009.242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. McPhee CK, Logan MA, Freeman MR, Baehrecke EH. Activation of autophagy during cell death requires the engulfment receptor Draper. Nature. 2010;465:1093–1096. doi: 10.1038/nature09127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Meijer AJ, Codogno P. Regulation and role of autophagy in mammalian cells. The International Journal of Biochemistry & Cell Biology. 2004;36:2445–2462. doi: 10.1016/j.biocel.2004.02.002. [DOI] [PubMed] [Google Scholar]
  73. Menut L, Vaccari T, Dionne H, Hill J, Wu G, Bilder D. A mosaic genetic screen for Drosophila neoplastic tumor suppressor genes based on defective pupation. Genetics. 2007;177:1667–1677. doi: 10.1534/genetics.107.078360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Mills K, Daish T, Harvey KF, Pfleger CM, Hariharan IK, Kumar S. The Drosophila melanogaster Apaf-1 homologue ARK is required for most, but not all programmed cell death. The Journal of Cell Biology. 2006;172:809–815. doi: 10.1083/jcb.200512126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Mills KR, Reginato M, Debnath J, Queenan B, Brugge JS. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is required for induction of autophagy during lumen formation in vitro. Proceedings of the National Academy of Sciences of the United States of America. 2004;101:3438–3443. doi: 10.1073/pnas.0400443101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Mirth CK, Riddiford LM. Size assessment and growth control: How adult size is determined in insects. Bioessays. 2007;29:344–355. doi: 10.1002/bies.20552. [DOI] [PubMed] [Google Scholar]
  77. Mirth C, Truman JW, Riddiford LM. The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster. Current Biology. 2005;15:1796–1807. doi: 10.1016/j.cub.2005.09.017. [DOI] [PubMed] [Google Scholar]
  78. Mizushima N. Autophagy: Process and function. Genes&Development. 2007;21:2861–2873. doi: 10.1101/gad.1599207. [DOI] [PubMed] [Google Scholar]
  79. Mizushima N, Komatsu M. Autophagy: Renovation of cells and tissues. Cell. 2011;147:728–741. doi: 10.1016/j.cell.2011.10.026. [DOI] [PubMed] [Google Scholar]
  80. Mizushima N, Noda T, Ohsumi Y. Apg16p is required for the function of the Apg12p-Apg5p conjugate in the yeast autophagy pathway. The EMBO Journal. 1999;18:3888–3896. doi: 10.1093/emboj/18.14.3888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Mizushima N, Noda T, Yoshimori T, Tanaka Y, Ishii T, George MD, et al. A protein conjugation system essential for autophagy. Nature. 1998;395:395–398. doi: 10.1038/26506. [DOI] [PubMed] [Google Scholar]
  82. Nakatogawa H, Ichimura Y, Ohsumi Y. Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion. Cell. 2007;130:165–178. doi: 10.1016/j.cell.2007.05.021. [DOI] [PubMed] [Google Scholar]
  83. Nezis IP, Shravage BV, Sagona AP, Lamark T, Bjørkøy G, Johansen T, et al. Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis. The Journal of Cell Biology. 2010;190:523–531. doi: 10.1083/jcb.201002035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  84. Nijhout HF. A threshold size for metamorphosis in the tobacco hornworm, Manduca sexta (L.) The Biological Bulletin. 1975;149:214–225. doi: 10.2307/1540491. [DOI] [PubMed] [Google Scholar]
  85. Nijhout HF. The control of body size in insects. Developmental Biology. 2003;261:1–9. doi: 10.1016/s0012-1606(03)00276-8. [DOI] [PubMed] [Google Scholar]
  86. Nijhout HF, Williams CM. Control of moulting and metamorphosis in the tobacco hornworm, Manduca sexta (L.): Growth of the last-instar larva and the decision to pupate. The Journal of Experimental Biology. 1974;61:481–491. doi: 10.1242/jeb.61.2.481. [DOI] [PubMed] [Google Scholar]
  87. Noda T, Ohsumi Y. Tor a phosphatidylinositol kinase homologue, controls autophagy in yeast. The Journal of Biological Chemistry. 1998;273:3963–3966. doi: 10.1074/jbc.273.7.3963. [DOI] [PubMed] [Google Scholar]
  88. Ohsumi Y. Molecular dissection of autophagy: Two ubiquitin-like systems. Nature Reviews. Molecular Cell Biology. 2001;2:211–216. doi: 10.1038/35056522. [DOI] [PubMed] [Google Scholar]
  89. Poodry CA, Woods DF. Control of the developmental timer for Drosophila pupariation. Roux’s Archives of Developmental Biology. 1990;199:219–227. doi: 10.1007/BF01682081. [DOI] [PubMed] [Google Scholar]
  90. Ravikumar B, Moreau K, Jahreiss L, Puri C, Rubinsztein DC. Plasma membrane contributes to the formation of pre-autophagosomal structures. Nature Cell Biology. 2010;12:747–757. doi: 10.1038/ncb2078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  91. Restifo LL, White K. Mutations in a steroid hormone-regulated gene disrupt the metamorphosis of the central nervous system in Drosophila. Developmental Biology. 1991;148:174–194. doi: 10.1016/0012-1606(91)90328-z. [DOI] [PubMed] [Google Scholar]
  92. Riddiford LM, Cherbas P, Truman JW. Ecdysone receptors and their biological actions. Vitamins and Hormones. 2000;60:1–73. doi: 10.1016/s0083-6729(00)60016-x. [DOI] [PubMed] [Google Scholar]
  93. Robertson F. The ecological genetics of growth in Drosophila. 6. The genetic correlation between the duration of the larval period and body size in relation to larval diet. Genetical Research. 1963;4:74–92. [Google Scholar]
  94. Rusten TE, Lindmo K, Juhász G, Sass M, Seglen PO, Brech A, et al. Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway. Developmental Cell. 2004;7:179–192. doi: 10.1016/j.devcel.2004.07.005. [DOI] [PubMed] [Google Scholar]
  95. Schweichel JU, Merker HJ. The morphology of various types of cell death in prenatal tissues. Teratology. 1973;7:253–266. doi: 10.1002/tera.1420070306. [DOI] [PubMed] [Google Scholar]
  96. Scott RC, Juhász G, Neufeld TP. Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death. Current Biology. 2007;17:1–11. doi: 10.1016/j.cub.2006.10.053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  97. Scott RC, Schuldiner O, Neufeld TP. Role and regulation of starvation-induced autophagy in the Drosophila fat body. Developmental Cell. 2004;7:167–178. doi: 10.1016/j.devcel.2004.07.009. [DOI] [PubMed] [Google Scholar]
  98. Segraves WA, Hogness DS. The E75 ecdysone-inducible gene responsible for the 75B early puff in Drosophila encodes two new members of the steroid receptor superfamily. Genes & Development. 1990;4:204–219. doi: 10.1101/gad.4.2.204. [DOI] [PubMed] [Google Scholar]
  99. Shang L, Chen S, Du F, Li S, Zhao L, Wang X. Nutrient starvation elicits an acute autophagic response mediated by Ulk1 dephosphorylation and its subsequent dissociation from AMPK. Proceedings of the National Academy of Sciences of the United States of America. 2011;108:4788–4793. doi: 10.1073/pnas.1100844108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  100. Shaw RJ. LKB1 and AMP-activated protein kinase control of mTOR signalling and growth. Acta Physiologica (Oxford, England) 2009;196:65–80. doi: 10.1111/j.1748-1716.2009.01972.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  101. Shingleton AW, Das J, Vinicius L, Stern DL. The temporal requirements for insulin signaling during development in Drosophila. PLoS Biology. 2005;3:e289. doi: 10.1371/journal.pbio.0030289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  102. Shintani T, Mizushima N, Ogawa Y, Matsuura A, Noda T, Ohsumi Y. Apg10p, a novel protein-conjugating enzyme essential for autophagy in yeast. The EMBO Journal. 1999;18:5234–5241. doi: 10.1093/emboj/18.19.5234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  103. Simpson P, Berreur P, Berreur-Bonnenfant J. The initiation of pupariation in Drosophila: Dependence on growth of the imaginal discs. Journal of Embryology and Experimental Morphology. 1980;57:155–165. [PubMed] [Google Scholar]
  104. Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, Komatsu M, et al. Autophagy regulates lipid metabolism. Nature. 2009;458:1131–1135. doi: 10.1038/nature07976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  105. Smith-Bolton RK, Worley MI, Kanda H, Hariharan IK. Regenerative growth in Drosophila imaginal discs is regulated by Wingless and Myc. Developmental Cell. 2009;16:797–809. doi: 10.1016/j.devcel.2009.04.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  106. Stenmark H. Rab GTPases as coordinators of vesicle traffic. Nature Reviews. Molecular Cell Biology. 2009;10:513–525. doi: 10.1038/nrm2728. [DOI] [PubMed] [Google Scholar]
  107. Stern DL, Emlen DJ. The developmental basis for allometry in insects. Development (Cambridge, England) 1999;126:1091–1101. doi: 10.1242/dev.126.6.1091. [DOI] [PubMed] [Google Scholar]
  108. Sun Q, Fan W, Chen K, Ding X, Chen S, Zhong Q. Identification of Barkor as a mammalian autophagy-specific factor for Beclin 1 and class III phosphatidylinositol 3-kinase. Proceedings of the National Academy of Sciences of the United States of America. 2008;105:19211–19216. doi: 10.1073/pnas.0810452105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  109. Suzuki K, Kirisako T, Kamada Y, Mizushima N, Noda T, Ohsumi Y. The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation. The EMBO Journal. 2001;20:5971–5981. doi: 10.1093/emboj/20.21.5971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  110. Takahashi Y, Coppola D, Matsushita N, Cualing HD, Sun M, Sato Y, et al. Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis. Nature Cell Biology. 2007;9:1142–1151. doi: 10.1038/ncb1634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  111. Tanida I, Mizushima N, Kiyooka M, Ohsumi M, Ueno T, Ohsumi Y, et al. Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy. Molecular Biology of the Cell. 1999;10:1367–1379. doi: 10.1091/mbc.10.5.1367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  112. Tennessen JM, Thummel CS. Coordinating growth and maturation—Insights from Drosophila. Current Biology. 2011;21:R750–R757. doi: 10.1016/j.cub.2011.06.033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  113. Thomas HE, Stunnenberg HG, Stewart AF. Heterodimerization of the Drosophila ecdysone receptor with retinoid X receptor and ultraspiracle. Nature. 1993;362:471–475. doi: 10.1038/362471a0. [DOI] [PubMed] [Google Scholar]
  114. Thumm M, Egner R, Koch B, Schlumpberger M, Straub M, Veenhuis M, et al. Isolation of autophagocytosis mutants of Saccharomyces cerevisiae. FEBS Letters. 1994;349:275–280. doi: 10.1016/0014-5793(94)00672-5. [DOI] [PubMed] [Google Scholar]
  115. Thummel CS. From embryogenesis to metamorphosis: The regulation and function of drosophila nuclear receptor superfamily members. Cell. 1995;83:871–877. doi: 10.1016/0092-8674(95)90203-1. [DOI] [PubMed] [Google Scholar]
  116. Thummel CS. Molecular mechanisms of developmental timing in C. elegans and Drosophila. Developmental Cell. 2001;1:453–465. doi: 10.1016/s1534-5807(01)00060-0. [DOI] [PubMed] [Google Scholar]
  117. Truman JW, Riddiford LM. Physiology of insect rhythms. 3. The temporal organization of the endocrine events underlying pupation of the tobacco hornworm. The Journal of Experimental Biology. 1974;60:371–382. doi: 10.1242/jeb.60.2.371. [DOI] [PubMed] [Google Scholar]
  118. Tsukada M, Ohsumi Y. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Letters. 1993;333:169–174. doi: 10.1016/0014-5793(93)80398-e. [DOI] [PubMed] [Google Scholar]
  119. Wang C, Liu Z, Huang X. Rab32 is important for autophagy and lipid storage in Drosophila. PLoS One. 2012;7:e32086. doi: 10.1371/journal.pone.0032086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  120. Woodard CT, Baehrecke EH, Thummel CS. A molecular mechanism for the stage specificity of the Drosophila prepupal genetic response to ecdysone. Cell. 1994;79:607–615. doi: 10.1016/0092-8674(94)90546-0. [DOI] [PubMed] [Google Scholar]
  121. Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell. 2006;124:471–484. doi: 10.1016/j.cell.2006.01.016. [DOI] [PubMed] [Google Scholar]
  122. Yao TP, Segraves WA, Oro AE, McKeown M, Evans RM. Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation. Cell. 1992;71:63–72. doi: 10.1016/0092-8674(92)90266-f. [DOI] [PubMed] [Google Scholar]
  123. Yu L, Wan F, Dutta S, Welsh S, Liu Z, Freundt E, et al. Autophagic programmed cell death by selective catalase degradation. Proceedings of the National Academy of Sciences of the United States of America. 2006;103:4952–4957. doi: 10.1073/pnas.0511288103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  124. Zehmer JK, Huang Y, Peng G, Pu J, Anderson RGW, Liu P. A role for lipid droplets in inter-membrane lipid traffic. Proteomics. 2009;9:914–921. doi: 10.1002/pmic.200800584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  125. Zhang Y, Goldman S, Baerga R, Zhao Y, Komatsu M, Jin S. Adipose-specific deletion of autophagy-related gene 7 (atg7) in mice reveals a role in adipogenesis. Proceedings of the National Academy of Sciences of the United States of America. 2009;106:19860–19865. doi: 10.1073/pnas.0906048106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  126. Zhou X, Zhou B, Truman JW, Riddiford LM. Overexpression of broad: A new insight into its role in the Drosophila prothoracic gland cells. The Journal of Experimental Biology. 2004;207:1151–1161. doi: 10.1242/jeb.00855. [DOI] [PubMed] [Google Scholar]

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