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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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Disruption of the signal recognition particle and its receptor induce autophagosome formation.Cells expressing inducible RNAi against the indicated subunits of the SRP and SRP receptor, marked by GFP-Atg8a expression, have elevated numbers of autophagosomes as indicated by punctate localization of GFP fluorescence. A, B, C, D, fixed tissue.
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pone-0006068-g006: Disruption of the signal recognition particle and its receptor induce autophagosome formation.Cells expressing inducible RNAi against the indicated subunits of the SRP and SRP receptor, marked by GFP-Atg8a expression, have elevated numbers of autophagosomes as indicated by punctate localization of GFP fluorescence. A, B, C, D, fixed tissue.

Mentions: One function of the ER translocon is to co-translationally import nascent transmembrane and secretory proteins into the ER for glycosylation and other processing steps via the signal recognition particle (SRP) and its receptor in the ER membrane. Once docked at the ER, the nascent polypeptide is co-translationally fed through the translocon. Because the translocon supports bidirectional traffic (unfolded proteins out of the ER, membrane and secretory pathway proteins in), we attempted to separate the import function by disrupting the SRP receptor and individual SRP subunits by RNAi. Knockdown of SRP subunits Srp14, Srp54, and Srp68, as well as the β subunit of the SRP receptor (SrpRβ) all caused marked increases in both LysoTracker staining (data not shown) and GFP-Atg8a punctae (Fig. 6A–D). In keeping with the importance of lysosomal degradation as a defense against defects in protein folding, mutation of a putative GlcNAc-1 phosphotransferase (CG5287) and the homolog of mammalian oligosaccharyl transferase (CG13393) cause an LT+ cellular phenotype (Fig. 7).


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

Arsham AM, Neufeld TP - PLoS ONE (2009)

Disruption of the signal recognition particle and its receptor induce autophagosome formation.Cells expressing inducible RNAi against the indicated subunits of the SRP and SRP receptor, marked by GFP-Atg8a expression, have elevated numbers of autophagosomes as indicated by punctate localization of GFP fluorescence. A, B, C, D, fixed tissue.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006068-g006: Disruption of the signal recognition particle and its receptor induce autophagosome formation.Cells expressing inducible RNAi against the indicated subunits of the SRP and SRP receptor, marked by GFP-Atg8a expression, have elevated numbers of autophagosomes as indicated by punctate localization of GFP fluorescence. A, B, C, D, fixed tissue.
Mentions: One function of the ER translocon is to co-translationally import nascent transmembrane and secretory proteins into the ER for glycosylation and other processing steps via the signal recognition particle (SRP) and its receptor in the ER membrane. Once docked at the ER, the nascent polypeptide is co-translationally fed through the translocon. Because the translocon supports bidirectional traffic (unfolded proteins out of the ER, membrane and secretory pathway proteins in), we attempted to separate the import function by disrupting the SRP receptor and individual SRP subunits by RNAi. Knockdown of SRP subunits Srp14, Srp54, and Srp68, as well as the β subunit of the SRP receptor (SrpRβ) all caused marked increases in both LysoTracker staining (data not shown) and GFP-Atg8a punctae (Fig. 6A–D). In keeping with the importance of lysosomal degradation as a defense against defects in protein folding, mutation of a putative GlcNAc-1 phosphotransferase (CG5287) and the homolog of mammalian oligosaccharyl transferase (CG13393) cause an LT+ cellular phenotype (Fig. 7).

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