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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 mitochondria-associated genes leads to increased LysoTracker and autophagy.Clones of cells homozygous for P-element insertions in ab, nmd, RISP, and Drp1 display punctate LysoTracker staining (A, D, F, H, unfixed tissue). Ab, nmd, and Drp1 mutations also lead to redistribution of fluorescent Atg8a into autophagosomes (B, E, I, fixed tissue, mutant clones are indicated here and elsewhere by solid white arrows; representative Atg8a punctae in B are indicated by arrowheads). Cells homozygous for the nmd insertion also show aberrant mitochondrial morphology as visualized by MitoTracker Red dye (C, fixed tissue) compared to neighboring cells. Cells expressing RNAi against RISP (indicated by GFP expression, G, unfixed tissue) have punctate and perinuclear LysoTracker staining. Inset panels indicate mutant clones which lack GFP or RFP, or Gal4-activated cells which express GFP, as appropriate. Alleles used for mitotic clones: abk02807, nmdk10909, RFeSPk11704, Drp1KG03815.
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pone-0006068-g004: Disruption of mitochondria-associated genes leads to increased LysoTracker and autophagy.Clones of cells homozygous for P-element insertions in ab, nmd, RISP, and Drp1 display punctate LysoTracker staining (A, D, F, H, unfixed tissue). Ab, nmd, and Drp1 mutations also lead to redistribution of fluorescent Atg8a into autophagosomes (B, E, I, fixed tissue, mutant clones are indicated here and elsewhere by solid white arrows; representative Atg8a punctae in B are indicated by arrowheads). Cells homozygous for the nmd insertion also show aberrant mitochondrial morphology as visualized by MitoTracker Red dye (C, fixed tissue) compared to neighboring cells. Cells expressing RNAi against RISP (indicated by GFP expression, G, unfixed tissue) have punctate and perinuclear LysoTracker staining. Inset panels indicate mutant clones which lack GFP or RFP, or Gal4-activated cells which express GFP, as appropriate. Alleles used for mitotic clones: abk02807, nmdk10909, RFeSPk11704, Drp1KG03815.

Mentions: Selected results are shown in Figure 4, including P-element insertions in ab, Drp1, no mitochondrial derivative (nmd), and Rieske iron sulfur protein (RISP; FlyBase symbol RFeSP, Fig. 4A, D, F, H). While all 4 mutants induce a typical LT+ phenotype, RISP disruption additionally leads to an unusually perinuclear concentration of LysoTracker punctae, which was replicated by expression of RNAi against RISP (Fig. 4G). To discern whether the increased lysosomal volume correlated with an increase in autophagy, we expressed either GFP or mCherry fusion proteins of Atg8a/LC3, an essential autophagy protein which localizes to autophagosomal membranes—punctate subcellular localization of fluorescent Atg8 fusions is commonly used as an indicator of active autophagy. Atg8a-labeled vesicles can also accumulate due to a block in autophagy at a post-induction step, but the dual observation of both Atg8a and LysoTracker punctae strongly supports an increase in the overall autophagic flux. Ab, nmd, and Drp1 mutation all caused formation of Atg8a punctae (Figs. 4B, E, I). Ab mutant cells also had reduced overall GFP-Atg8a levels compared to neighboring wild type cells, suggesting that some mutations may lead to enhanced autophagosome-lysosome fusion and increased autophagic flux, causing a high rate of turnover of the fusion protein. To ascertain if the mutations in these mitochondria-associated genes caused obvious differences in the volume or the spatial distribution of mitochondria, we visualized mitochondria with MitoTracker Red. While most of the mutants did not show obvious differences in mitochondrial phenotypes, ab caused mitochondria to aggregate in large clumps (Fig. 4C) in mutant cells. This is consistent with recent results suggesting that ab plays a role in mitochondrial sorting and that mutations in this gene cause a Parkinson's-like neurodegenerative syndrome in flies (R. Cox and A. Spradling, personal communication).


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

Arsham AM, Neufeld TP - PLoS ONE (2009)

Disruption of mitochondria-associated genes leads to increased LysoTracker and autophagy.Clones of cells homozygous for P-element insertions in ab, nmd, RISP, and Drp1 display punctate LysoTracker staining (A, D, F, H, unfixed tissue). Ab, nmd, and Drp1 mutations also lead to redistribution of fluorescent Atg8a into autophagosomes (B, E, I, fixed tissue, mutant clones are indicated here and elsewhere by solid white arrows; representative Atg8a punctae in B are indicated by arrowheads). Cells homozygous for the nmd insertion also show aberrant mitochondrial morphology as visualized by MitoTracker Red dye (C, fixed tissue) compared to neighboring cells. Cells expressing RNAi against RISP (indicated by GFP expression, G, unfixed tissue) have punctate and perinuclear LysoTracker staining. Inset panels indicate mutant clones which lack GFP or RFP, or Gal4-activated cells which express GFP, as appropriate. Alleles used for mitotic clones: abk02807, nmdk10909, RFeSPk11704, Drp1KG03815.
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Related In: Results  -  Collection

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pone-0006068-g004: Disruption of mitochondria-associated genes leads to increased LysoTracker and autophagy.Clones of cells homozygous for P-element insertions in ab, nmd, RISP, and Drp1 display punctate LysoTracker staining (A, D, F, H, unfixed tissue). Ab, nmd, and Drp1 mutations also lead to redistribution of fluorescent Atg8a into autophagosomes (B, E, I, fixed tissue, mutant clones are indicated here and elsewhere by solid white arrows; representative Atg8a punctae in B are indicated by arrowheads). Cells homozygous for the nmd insertion also show aberrant mitochondrial morphology as visualized by MitoTracker Red dye (C, fixed tissue) compared to neighboring cells. Cells expressing RNAi against RISP (indicated by GFP expression, G, unfixed tissue) have punctate and perinuclear LysoTracker staining. Inset panels indicate mutant clones which lack GFP or RFP, or Gal4-activated cells which express GFP, as appropriate. Alleles used for mitotic clones: abk02807, nmdk10909, RFeSPk11704, Drp1KG03815.
Mentions: Selected results are shown in Figure 4, including P-element insertions in ab, Drp1, no mitochondrial derivative (nmd), and Rieske iron sulfur protein (RISP; FlyBase symbol RFeSP, Fig. 4A, D, F, H). While all 4 mutants induce a typical LT+ phenotype, RISP disruption additionally leads to an unusually perinuclear concentration of LysoTracker punctae, which was replicated by expression of RNAi against RISP (Fig. 4G). To discern whether the increased lysosomal volume correlated with an increase in autophagy, we expressed either GFP or mCherry fusion proteins of Atg8a/LC3, an essential autophagy protein which localizes to autophagosomal membranes—punctate subcellular localization of fluorescent Atg8 fusions is commonly used as an indicator of active autophagy. Atg8a-labeled vesicles can also accumulate due to a block in autophagy at a post-induction step, but the dual observation of both Atg8a and LysoTracker punctae strongly supports an increase in the overall autophagic flux. Ab, nmd, and Drp1 mutation all caused formation of Atg8a punctae (Figs. 4B, E, I). Ab mutant cells also had reduced overall GFP-Atg8a levels compared to neighboring wild type cells, suggesting that some mutations may lead to enhanced autophagosome-lysosome fusion and increased autophagic flux, causing a high rate of turnover of the fusion protein. To ascertain if the mutations in these mitochondria-associated genes caused obvious differences in the volume or the spatial distribution of mitochondria, we visualized mitochondria with MitoTracker Red. While most of the mutants did not show obvious differences in mitochondrial phenotypes, ab caused mitochondria to aggregate in large clumps (Fig. 4C) in mutant cells. This is consistent with recent results suggesting that ab plays a role in mitochondrial sorting and that mutations in this gene cause a Parkinson's-like neurodegenerative syndrome in flies (R. Cox and A. Spradling, personal communication).

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