A Genetic Screen in Drosophila Reveals Novel Cytoprotective Functions of the Autophagy-Lysosome Pathway

Neufeld TP (2009) A Genetic Screen in Drosophila Reveals Novel Cytoprotective Functions of the Autophagy-Lysosome Pathway. PLoS ONE 4(6): e6068. doi:10.1371/journal.pone.0006068 A Genetic Screen in Drosophila Reveals Novel Cytoprotective Functions of the Autophagy-Lysosome Pathway Andrew M. Arsham 0 Thomas P. Neufeld 0 Andreas Bergmann, University of Texas MD Anderson Cancer Center, United States of America 0 Department of Genetics, Cell Biology, and Development, University of Minnesota , Minneapolis, Minnesota , United States of America The highly conserved autophagy-lysosome pathway is the primary mechanism for breakdown and recycling of macromolecular and organellar cargo in the eukaryotic cell. Autophagy has recently been implicated in protection against cancer, neurodegeneration, and infection, and interest is increasing in additional roles of autophagy in human health, disease, and aging. To search for novel cytoprotective features of this pathway, we carried out a genetic mosaic screen for mutations causing increased lysosomal and/or autophagic activity in the Drosophila melanogaster larval fat body. By combining Drosophila genetics with live-cell imaging of the fluorescent dye LysoTracker Red and fixed-cell imaging of autophagy-specific fluorescent protein markers, the screen was designed to identify essential metazoan genes whose disruption causes increased flux through the autophagy-lysosome pathway. The screen identified a large number of genes associated with the protein synthesis and ER-secretory pathways (e.g. aminoacyl tRNA synthetases, Oligosaccharyl transferase, Sec61a), and with mitochondrial function and dynamics (e.g. Rieske iron-sulfur protein, Dynamin-related protein 1). We also observed that increased lysosomal and autophagic activity were consistently associated with decreased cell size. Our work demonstrates that disruption of the synthesis, transport, folding, or glycosylation of ER-targeted proteins at any of multiple steps leads to autophagy induction. In addition to illuminating cytoprotective features of autophagy in response to cellular damage, this screen establishes a genetic methodology for investigating cell biological phenotypes in live cells, in the context of viable wild type organisms. - Funding: This work was supported by postdoctoral fellowship #PF-05-114-01-CS from the American Cancer Society (AMA; http://www.cancer.org) and grant GM62509 from the NIH (TPN; http://www.nih.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 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 [1]). 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 [2]). 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 [3]). 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 [4]). Initially characterized genetically and biochemically in yeast [5], 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 [2]. 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 [6], a key step in autophagys 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 nutrientreplete 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 [7] and to the coordination of organismal growth and feeding behavior (reviewed in [8]). Autophagy is also regulated by developmental signals. In Drosophila, autophagy is induced during the late larval stages by the steroid hormone ecdysone [9], 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 [10] or mice [11,12], it appears to be necessary for embryonic implantation [13] and perinatal survival in mice [12]. Loss of autophagy also impairs T- and B-cell development, proliferation, and function [14,15]. Autophagy declines with age, and this decline has long been thought to play a role in agingrelated 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 [16,17]. 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 (...truncated)