The proteasome and the lysosome are the two major routes for cellular digestion of macromolecules. The ubiquitin-proteasome system is highly regulated, specific, and energy-intensive, and biophysical constraints limit the proteasome to degradation of individual proteins (reviewed in 
). In contrast, the lysosome, an acidic membrane-bound organelle containing a broad spectrum of acid hydrolases, degrades substrates non-specifically, and direct regulation of individual steps is minimal. The large size of lysosomes allows degradation not only of individual proteins, but of large complexes, lipids, and whole organelles, and enables recycling of the resulting raw materials (reviewed in 
). Defects in lysosomal function cause upwards of 40 lysosomal storage disorders that result in a buildup of undigested material and a wide spectrum of often organ- or cell type-specific secondary effects including neuronal damage or degeneration, and mild to severe developmental impairment (reviewed in 
). Bulk cargo for degradation is delivered to the lysosome by macroautophagy, a cellular recycling process conserved from yeast to man (hereafter referred to simply as autophagy). Autophagy enables cellular survival in nutrient-poor environments, removal of old or damaged organelles and macromolecules, and is implicated as a protective factor in a wide variety of human disease, from cancer to neurodegeneration to pathogen defense (reviewed in 
Initially characterized genetically and biochemically in yeast 
, autophagy is a multi-step process. The “core” machinery of autophagy involves nearly 20 proteins, and much progress has been made in understanding such unique features as ubiquitin-like conjugation reactions involving both proteins and lipids, and the formation and enlargement of the unusual double membrane enclosure 
. Fusion of the autophagosome to the lysosome releases the cargo-containing inner membrane into the lysosome for degradation by the more than 50 resident acid hydrolases. After degradation, effluxors mediate the release of nutrients to the cytoplasm for reuse 
, a key step in autophagy's primal function as a response to nutrient deprivation.
A high degree of homology between yeast and metazoans has enabled the study of the autophagy-lysosome pathway in multicellular animals. In both yeast and metazoans, target of rapamycin (TOR) signaling suppresses autophagy in nutrient-replete environments as part of its growth-promoting function, and permits autophagy when cells are starved. In higher organisms, TOR not only responds to nutrients but is also a major effector of growth factor signaling through the PI3K pathway, thus tying autophagy to the regulation of cell growth and cancer 
and to the coordination of organismal growth and feeding behavior (reviewed in 
Autophagy is also regulated by developmental signals. In Drosophila
, autophagy is induced during the late larval stages by the steroid hormone ecdysone 
, allowing the animal to break down and recycle larval material in the course of metamorphosis. While autophagy is not strictly required for embryonic development in flies 
or mice 
, it appears to be necessary for embryonic implantation 
and perinatal survival in mice 
. Loss of autophagy also impairs T- and B-cell development, proliferation, and function 
. Autophagy declines with age, and this decline has long been thought to play a role in aging-related cellular damage and senescence; in support of a role for autophagy in aging, it has recently been shown to be required (though not sufficient) for dietary restriction-induced lifespan extension in worms 
. Autophagy thus functions not only in cell autonomous responses to nutrient depletion as in yeast, but in regulatory and disease processes unique to multicellular organisms.
Because the bulk of our current genetic and biochemical knowledge of autophagy comes from seminal early genetic screens in yeast, it is not clear to what degree newer functions of autophagy rely on metazoan-specific signaling pathways. We therefore designed a genetic screen to search for novel cytoprotective features of the autophagy-lysosome pathway by looking for mutations that activate the pathway as a signaling or defense response. Using existing genetic tools in Drosophila we screened mitotic mosaic clones homozygous for lethal P-element insertions that caused enlargement of the lysosomal compartment as measured by LysoTracker Red, a fluorescent dye. Experiments were performed using collections of FRT-linked recombinogenic P-element insertions on Drosophila chromosome 2 L, and were carried out in the larval fat body, the primary nutrient storage organ of the developing larva, and one in which background lysosomal and autophagic activity are extremely low.
The screen revealed a large number of gene disruptions which amplify LysoTracker activity (which we termed LT+), typically in concert with decreased cell and nuclear size, and which were enriched for genes involved in protein synthesis, folding, transport, and degradation, and those involved in mitochondrial function and morphology. The former group included tRNA charging, translocation into the ER, glycosylation, and the unfolded protein response (UPR), while the latter included electron transport and mitochondrial fusion and fission. Because of the large size and monolayer tissue architecture of fat body cells and the sophisticated recombination and gene expression tools generated by the Drosophila community, the mosaic approach in fat body allows for the visualization of cell biological phenotypes in live cells in the context of a fully developed wild type animal organ and provides a useful set of tools for investigating a wide range of cellular phenotypes including cell size and shape, intracellular trafficking, and organellar dynamics.