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A genetic screen in Drosophila reveals novel cytoprotective functions of the autophagy-lysosome pathway.

Arsham AM, Neufeld TP - PLoS ONE (2009)

Bottom Line: The highly conserved autophagy-lysosome pathway is the primary mechanism for breakdown and recycling of macromolecular and organellar cargo in the eukaryotic cell.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA.

ABSTRACT
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, Sec61alpha), 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.

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Related in: MedlinePlus

Disruption of tRNA synthetase genes increases LysoTracker staining.(A) Clones of cells homozygous for P-element insertion KG03126 in the seryl tRNA synthetase CG17259 (indicated by white arrows and by lack of GFP in the inset panel) display punctate LysoTracker staining under fed conditions. (B and C) Cell clones expressing UAS-driven RNAi (indicated by GFP expression in inset panels) against phenylanalyl (B) and leucyl tRNA synthetases (C) display punctate LysoTracker staining. Allele used for mitotic clones: CG17259KG03126.
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pone-0006068-g003: Disruption of tRNA synthetase genes increases LysoTracker staining.(A) Clones of cells homozygous for P-element insertion KG03126 in the seryl tRNA synthetase CG17259 (indicated by white arrows and by lack of GFP in the inset panel) display punctate LysoTracker staining under fed conditions. (B and C) Cell clones expressing UAS-driven RNAi (indicated by GFP expression in inset panels) against phenylanalyl (B) and leucyl tRNA synthetases (C) display punctate LysoTracker staining. Allele used for mitotic clones: CG17259KG03126.

Mentions: Aminoacyl tRNA synthetases (AARS) catalyze the loading of tRNAs with their cognate amino acid. P-element insertion in the 5′ UTR of the gene CG17259, the seryl-tRNA synthetase (SerRS), caused elevated LysoTracker staining (Fig. 3A), while a P-element insertion in the first intron of threonyl-tRNA synthetase (ThrRS, FlyBase symbol Aats-thr) showed weak and intermittent LT+ phenotypes (data not shown). To extend these results, we used a clonal RNAi approach to inhibit expression of a panel of AARS genes. Briefly, stochastic FLP-FRT recombination events cause the looping out of a transcription stop signal embedded in a GAL4 expression construct driven by the constitutive Act5c promoter. The resulting clonal expression of GAL4 then activates transcription of stably integrated RNAi and GFP expression constructs driven by similar GAL4-responsive promoters. This system enables the analysis of RNAi effects on small numbers of GFP-marked cells in otherwise healthy organisms.


A genetic screen in Drosophila reveals novel cytoprotective functions of the autophagy-lysosome pathway.

Arsham AM, Neufeld TP - PLoS ONE (2009)

Disruption of tRNA synthetase genes increases LysoTracker staining.(A) Clones of cells homozygous for P-element insertion KG03126 in the seryl tRNA synthetase CG17259 (indicated by white arrows and by lack of GFP in the inset panel) display punctate LysoTracker staining under fed conditions. (B and C) Cell clones expressing UAS-driven RNAi (indicated by GFP expression in inset panels) against phenylanalyl (B) and leucyl tRNA synthetases (C) display punctate LysoTracker staining. Allele used for mitotic clones: CG17259KG03126.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2698153&req=5

pone-0006068-g003: Disruption of tRNA synthetase genes increases LysoTracker staining.(A) Clones of cells homozygous for P-element insertion KG03126 in the seryl tRNA synthetase CG17259 (indicated by white arrows and by lack of GFP in the inset panel) display punctate LysoTracker staining under fed conditions. (B and C) Cell clones expressing UAS-driven RNAi (indicated by GFP expression in inset panels) against phenylanalyl (B) and leucyl tRNA synthetases (C) display punctate LysoTracker staining. Allele used for mitotic clones: CG17259KG03126.
Mentions: Aminoacyl tRNA synthetases (AARS) catalyze the loading of tRNAs with their cognate amino acid. P-element insertion in the 5′ UTR of the gene CG17259, the seryl-tRNA synthetase (SerRS), caused elevated LysoTracker staining (Fig. 3A), while a P-element insertion in the first intron of threonyl-tRNA synthetase (ThrRS, FlyBase symbol Aats-thr) showed weak and intermittent LT+ phenotypes (data not shown). To extend these results, we used a clonal RNAi approach to inhibit expression of a panel of AARS genes. Briefly, stochastic FLP-FRT recombination events cause the looping out of a transcription stop signal embedded in a GAL4 expression construct driven by the constitutive Act5c promoter. The resulting clonal expression of GAL4 then activates transcription of stably integrated RNAi and GFP expression constructs driven by similar GAL4-responsive promoters. This system enables the analysis of RNAi effects on small numbers of GFP-marked cells in otherwise healthy organisms.

Bottom Line: The highly conserved autophagy-lysosome pathway is the primary mechanism for breakdown and recycling of macromolecular and organellar cargo in the eukaryotic cell.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA.

ABSTRACT
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, Sec61alpha), 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.

Show MeSH
Related in: MedlinePlus