Autophagy is an evolutionarily conserved process to catabolize cytoplasmic proteins and organelles1, 2. During starvation, the target of rapamycin (TOR), a nutrient-responsive kinase, is inhibited, thereby inducing autophagy. In autophagy, double-membrane autophagosomes envelop and sequester intracellular components and then fuse with lysosomes to form autolysosomes which degrade their contents to regenerate nutrients. Current models of autophagy terminate with the degradation of autophagosome cargo in autolysosomes3-5, but the regulation of autophagy in response to nutrients and the subsequent fate of the autolysosome are poorly defined. Here we show that mTOR signaling is inhibited during autophagy initiation, but reactivated with prolonged starvation. mTOR reactivation is autophagy-dependent, and requires the degradation of autolysosomal products. Increased mTOR activity attenuates autophagy and generates proto-lysosomal tubules and vesicles that extrude from autolysosomes and ultimately mature into functional lysosomes, thereby restoring the full complement of lysosomes in the cell – a process we identify in multiple animal species. Thus, an evolutionarily-conserved cycle in autophagy governs nutrient sensing and lysosome homeostasis during starvation.

Autophagy is a conserved cellular process to degrade and recycle cytoplasmic components. During autophagy, lysosomes fuse with an autophagosome to form an autolysosome. Sequestered components are degraded by lysosomal hydrolases and presumably released into the cytosol by lysosomal efflux permeases. Following starvation-induced autophagy, lysosome homeostasis is restored by autophagic lysosome reformation (ALR) requiring activation of the “target of rapamycin” (TOR) kinase. Spinster (Spin) encodes a putative lysosomal efflux permease with the hallmarks of a sugar transporter. Drosophila spin mutants accumulate lysosomal carbohydrates and enlarged lysosomes. Here we show that defects in spin lead to the accumulation of enlarged autolysosomes. We find that spin is essential for mTOR reactivation and lysosome reformation following prolonged starvation. Further, we demonstrate that the sugar transporter activity of Spin is essential for ALR.

Autophagy degrades cytoplasmic components that are required for cell survival in response to starvation1. Autophagy has also been associated with cell death, but it is unclear what may distinguish autophagy during cell survival and death. Drosophila salivary glands undergo programmed cell death that requires autophagy genes2, and engulfment of salivary gland cells by phagocytes does not appear to occur3. Here we show that Draper (Drpr), the Drosophila orthologue of the C. elegans engulfment receptor CED-1, is required for autophagy during cell death. Null mutations in drpr, as well as salivary gland-specific knockdown of drpr, inhibits salivary gland degradation. drpr knockdown prevents the induction of autophagy in dying salivary glands, and Atg1 expression in drpr mutants suppresses the failure in salivary gland degradation. Surprisingly, drpr is cell-autonomously required for autophagy induction in dying salivary gland cells, while drpr knockdown does not prevent starvation-induced autophagy in the fatbody which is associated with survival. In addition, components of the conserved engulfment pathway are required for clearance of salivary glands. This is the first example of an engulfment factor that is autonomously required for self-clearance. Furthermore, Drpr is the first factor that distinguishes autophagy that is associated with cell death from cell survival.Macroautophagy (autophagy) delivers cytoplasmic components to the lysosome for degradation in eukaryotic cells. Autophagy is an important cellular response to stress that is required for survival in response to starvation1, and has also been associated with cell death Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the To identify genes that may regulate autophagy in cell-specific contexts, we queried genomewide DNA microarray data from dying salivary glands. Interestingly, several factors that have been implicated in the engulfment of apoptotic cells are induced in dying salivary glands7 (Supplementary Table 1), while there are no detectable changes in these genes after larval starvation8. Although many engulfment factors are pleiotropic through their regulation of the cytoskeleton and vesicular transport, the identification of the engulfment receptor drpr9,10 is intriguing, as salivary gland destruction is thought to be largely independent of phagocytes.We analyzed whether Drpr is present in dying salivary glands. Whereas no Drpr protein is present in drpr Δ5 null mutants, Drpr-I, II and/or III isoforms are present at low levels and localize to the luminal/apical region of salivary gland cells at 6 hours after puparium formation ( Fig. 1a and Supplementary Fig. 1). Following the rise in steroid that triggers cell death 12 hours after puparium formation11, Drpr-I, II and/or III levels increase (Fig. 1a). Drpr-I protein levels remain high through 14 hours after puparium formation when a portion of Drpr changes from apical to cytoplasmic in loca...

Macroautophagy (autophagy) is a bulk cytoplasmic degradation process that is conserved from yeast to mammals. Autophagy is an important cellular response to starvation and stress, and plays important roles in development, cell death, aging, immunity, and cancer. The fruit fly Drosophila melanogaster provides an excellent model system to study autophagy in vivo, in the context of a developing organism. Autophagy (atg) genes and their regulators are conserved in Drosophila, and autophagy is induced in response to nutrient starvation and hormones during development. In this review we provide an overview of how Drosophila research has contributed to our understanding of the role and regulation of autophagy in cell survival, growth, nutrient utilization, and cell death. Recent Drosophila research has also provided important mechanistic information about the role of autophagy in protein aggregation disorders, neurodegeneration, aging, and innate immunity. Differences in the role of autophagy in specific contexts and/or cell types suggest that there may be cell-context-specific regulators of autophagy, and studies in Drosophila are well-suited to yield discoveries about this specificity.

In this chapter we discuss methods to study autophagic cell death. A large body of evidence demonstrates that autophagy is a cell survival mechanism in response to starvation. The role of autophagy in cell death, however, has long been controversial. Recently, molecular approaches have provided direct evidence that autophagy contributes to cell death in certain contexts. We begin this chapter by outlining methods to quantify cell death, for example by assaying for cell viability. Next, we discuss methods to measure processes involved in cell death, such as caspase activation and autophagy. Finally, we discuss methods to genetically or chemically perturb autophagy in order to test whether autophagy is required for cell death. Together, these approaches provide a guide to investigate the relationship between autophagy and cell death.

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Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation. Programmed cell death is critical to multi-cellular animal development and homeostasis, and misregulation of cell death can lead to disorders including autoimmunity and cancer.

Most autophagy genes have been discovered in the single-celled yeast Saccharomyces cerevisiae, and little is known about autophagy genes that are specific to multicellular animals. In this issue, Tian et al. (2010) now identify four new autophagy genes: one specific to the nematode Caenorhabditis elegans and three conserved from worms to mammals.