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How and why to study autophagy in Drosophila: It’s more than just a garbage chute

Nagy, Péter, Varga, Ágnes, Kovács, Attila L., Takáts, Szabolcs, Juhász, Gábor·2015·Methods
DOI 10.1016/j.ymeth.2014.11.016PMC PMC4358840
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Graphical abstract CC BY · PubMed Central
Fig. 1 The process and molecular mechanisms of autophagy. The sequential and coordinated action of Atg protein complexes mediates the formation of phagophores and double-membrane autophagosomes. Fusion of autophagosomes with lysosomes (or with late endosomes) requires a Syntaxin 17-containing SNARE complex and the HOPS tethering complex. Autophagic cargo is degraded in autolysosomes, which is followed by recycling of degradation products in synthetic and energy producing pathways.
Fig. 1 The process and molecular mechanisms of autophagy. The sequential and coordinated action of Atg protein complexes mediates the formation of phagophores and double-membrane autophagosomes. Fusion of autophagosomes with lysosomes (or with late endosomes) requires a Syntaxin 17-containing SNARE complex and the HOPS tethering complex. Autophagic cargo is degraded in autolysosomes, which is followed by recycling of degradation products in synthetic and energy producing pathways.CC BY · PubMed Central
Fig. 2 Ultrastructural analysis of autophagy. (A) Transmission electron microscopy image of fat body from a starved larva. Autophagosomes (ap) bordered by a double membrane (with the often seen characteristic cleft between them) contain undegraded cargo, which appears largely similar to the surrounding cytoplasm. In contrast, the content of the single-membrane bound autolysosome (al) is typically dark and condensed due to ongoing breakdown, although remnants of cytoplasmic material can often be recognized. (B) Autophagic structures are practically never seen in ultrastructural images of wild type adult brains. (C) Double-membrane autophagosomes accumulate in large numbers in neurons of Syntaxin 17 mutant adult flies. (D) Apoptotic bodies (apopt) induced by clonal overexpression of Hid are engulfed by neighboring eye imaginal disc cells, and contain cytoplasmic remnants of the fragmented dying cell, but these are not of autophagic origin. Their recognition is facilitated by the presence of the condensed nucleus in some of the engulfed cell fragments, and apoptotic bodies may be found outside of healthy cells as well, as illustrated by the one situated between two neighboring cells in the bottom right corner of this panel. Note that apoptotic cells usually appear darker than healthy cells in ultrastructural images even before being engulfed, due to ongoing protein degradation by caspases and acidification of the cytoplasm. (E) Large protein aggregates form in neurons of Atg mutant adult flies. The aggregate (arrowhead) can be easily recognized by its homogenous appearance, as they mostly exclude cytoplasmic structures, for example vesicles and ribosomes. This image shows a single protein aggregate with a rarely seen phagophore (p) attached to its surface in an Atg2 mutant brain. (F) Protein aggregates (arrowheads) accumulating in Atg mutant neurons contain ubiquitinated proteins and their selective autophagic receptor p62 (also known as Ref(2)P in flies). This image shows immunogold labeling (black dots) of p62 in Atg8a mutant neurons. Abbreviations: ap, autophagosome; apopt, apoptotic cell fragment; al, autolysosome; p, phagophore; m, mitochondrion. Bars equal 1 μm in all panels.
Fig. 2 Ultrastructural analysis of autophagy. (A) Transmission electron microscopy image of fat body from a starved larva. Autophagosomes (ap) bordered by a double membrane (with the often seen characteristic cleft between them) contain undegraded cargo, which appears largely similar to the surrounding cytoplasm. In contrast, the content of the single-membrane bound autolysosome (al) is typically dark and condensed due to ongoing breakdown, although remnants of cytoplasmic material can often be recognized. (B) Autophagic structures are practically never seen in ultrastructural images of wild type adult brains. (C) Double-membrane autophagosomes accumulate in large numbers in neurons of Syntaxin 17 mutant adult flies. (D) Apoptotic bodies (apopt) induced by clonal overexpression of Hid are engulfed by neighboring eye imaginal disc cells, and contain cytoplasmic remnants of the fragmented dying cell, but these are not of autophagic origin. Their recognition is facilitated by the presence of the condensed nucleus in some of the engulfed cell fragments, and apoptotic bodies may be found outside of healthy cells as well, as illustrated by the one situated between two neighboring cells in the bottom right corner of this panel. Note that apoptotic cells usually appear darker than healthy cells in ultrastructural images even before being engulfed, due to ongoing protein degradation by caspases and acidification of the cytoplasm. (E) Large protein aggregates form in neurons of Atg mutant adult flies. The aggregate (arrowhead) can be easily recognized by its homogenous appearance, as they mostly exclude cytoplasmic structures, for example vesicles and ribosomes. This image shows a single protein aggregate with a rarely seen phagophore (p) attached to its surface in an Atg2 mutant brain. (F) Protein aggregates (arrowheads) accumulating in Atg mutant neurons contain ubiquitinated proteins and their selective autophagic receptor p62 (also known as Ref(2)P in flies). This image shows immunogold labeling (black dots) of p62 in Atg8a mutant neurons. Abbreviations: ap, autophagosome; apopt, apoptotic cell fragment; al, autolysosome; p, phagophore; m, mitochondrion. Bars equal 1 μm in all panels.CC BY · PubMed Central
Fig. 3 Specific markers of autophagy. (A) The most popular markers of autophagy are fluorescently tagged Atg8a reporters, such as mCherry-Atg8a. Atg8a is delivered to autolysosomes, where mCherry retains its fluorescence and accumulates to high levels. Thus, autolysosomes are labeled bright red in GFP-negative control cells, whereas autophagosomes appear fainter and smaller, as seen in the two GFP-positive cells expressing Syntaxin 17 RNAi, which silences the SNARE required for autophagosome-lysosome fusion. (B) Inhibition of Tor kinase increases autophagic flux, resulting in enhanced delivery of mCherry-Atg8a to lysosomes. Thus, autolysosomes are labeled much brighter with mCherry in GFP-positive cells in which Raptor (encoding an essential subunit of Tor kinase complex 1) is knocked down than in surrounding control cells. Note that mCherry signal in control cells looks fainter than in panel A because this image was taken using a much shorter exposure time. (C) and (D) Atg1 RNAi in GFP-positive cells inhibits puncta formation of both mCherry-Atg8a (C) and mCherry-Atg18 (D), as Atg1 acts upstream of Atg8a and Atg18 in the hierarchy of Atg proteins. (E) Knockdown of Atg12 blocks the generation of mCherry-Atg8a dots. (F) Small mCherry-Atg18 dots likely representing stalled phagophore assembly sites (PASs) accumulate in GFP-positive cells undergoing Atg12 RNAi, as Atg18 acts upstream of Atg12. (G) and (H) Protein aggregates containing p62 (green) accumulate in large numbers in the brain of Atg7 mutant flies compared to controls. Note that association of the upstream-acting Atg1 kinase subunit FIP200 (red) with p62 is also increased in Atg7 mutants, suggesting that such aggregates might represent stalled phagophore assembly sites. (I) Phagophores that are positive for endogenous Atg8a associate with large protein aggregates formed upon high-level overexpression of GFP-p62. (J) Ultrastructural analysis demonstrates an enlarged phagophore (P) attached to the surface of a protein aggregate in a fat body cell of a starved larva as in panel I. ap: autophagosome; m: mitochondrion. Panels A–F, I, J show fat body cells of starved L3 stage larvae, and panels G, H show adult brains. Bars equal 20 μm in panels A (for A–F), G, H. Bar in I equals 4 μm, and bar in J represents 1 μm, respectively.
Fig. 3 Specific markers of autophagy. (A) The most popular markers of autophagy are fluorescently tagged Atg8a reporters, such as mCherry-Atg8a. Atg8a is delivered to autolysosomes, where mCherry retains its fluorescence and accumulates to high levels. Thus, autolysosomes are labeled bright red in GFP-negative control cells, whereas autophagosomes appear fainter and smaller, as seen in the two GFP-positive cells expressing Syntaxin 17 RNAi, which silences the SNARE required for autophagosome-lysosome fusion. (B) Inhibition of Tor kinase increases autophagic flux, resulting in enhanced delivery of mCherry-Atg8a to lysosomes. Thus, autolysosomes are labeled much brighter with mCherry in GFP-positive cells in which Raptor (encoding an essential subunit of Tor kinase complex 1) is knocked down than in surrounding control cells. Note that mCherry signal in control cells looks fainter than in panel A because this image was taken using a much shorter exposure time. (C) and (D) Atg1 RNAi in GFP-positive cells inhibits puncta formation of both mCherry-Atg8a (C) and mCherry-Atg18 (D), as Atg1 acts upstream of Atg8a and Atg18 in the hierarchy of Atg proteins. (E) Knockdown of Atg12 blocks the generation of mCherry-Atg8a dots. (F) Small mCherry-Atg18 dots likely representing stalled phagophore assembly sites (PASs) accumulate in GFP-positive cells undergoing Atg12 RNAi, as Atg18 acts upstream of Atg12. (G) and (H) Protein aggregates containing p62 (green) accumulate in large numbers in the brain of Atg7 mutant flies compared to controls. Note that association of the upstream-acting Atg1 kinase subunit FIP200 (red) with p62 is also increased in Atg7 mutants, suggesting that such aggregates might represent stalled phagophore assembly sites. (I) Phagophores that are positive for endogenous Atg8a associate with large protein aggregates formed upon high-level overexpression of GFP-p62. (J) Ultrastructural analysis demonstrates an enlarged phagophore (P) attached to the surface of a protein aggregate in a fat body cell of a starved larva as in panel I. ap: autophagosome; m: mitochondrion. Panels A–F, I, J show fat body cells of starved L3 stage larvae, and panels G, H show adult brains. Bars equal 20 μm in panels A (for A–F), G, H. Bar in I equals 4 μm, and bar in J represents 1 μm, respectively.CC BY · PubMed Central
Fig. 4 Western blot analysis of autophagy. (A) The autophagic cargo p62 accumulates in starved larvae lacking the core autophagy gene Atg7, and also in animals that are mutant for Vps16A or Vps11, encoding subunits of the HOPS tethering complex. Autophagosome-associated, lipidated Atg8a-II is missing in Atg7 mutants but accumulates in HOPS loss-of-function (mutant for Vps16A or Vps11) larvae, as this tethering complex is required for the fusion of autophagosomes with lysosomes (compare Atg8a-II levels to tubulin in controls and mutants, respectively). (B) Various Atg mutants all accumulate p62, whereas the levels of Atg8a-II are increased in starved larvae lacking Atg1, Atg13, Atg18 and Atg2. Atg7 and Atg3 encode the E1 and E2 enzymes required for Atg8a lipidation, respectively, so the generation of Atg8a-II is blocked in these mutants. Both forms of Atg8a are missing in Atg8a mutants, as expected.
Fig. 4 Western blot analysis of autophagy. (A) The autophagic cargo p62 accumulates in starved larvae lacking the core autophagy gene Atg7, and also in animals that are mutant for Vps16A or Vps11, encoding subunits of the HOPS tethering complex. Autophagosome-associated, lipidated Atg8a-II is missing in Atg7 mutants but accumulates in HOPS loss-of-function (mutant for Vps16A or Vps11) larvae, as this tethering complex is required for the fusion of autophagosomes with lysosomes (compare Atg8a-II levels to tubulin in controls and mutants, respectively). (B) Various Atg mutants all accumulate p62, whereas the levels of Atg8a-II are increased in starved larvae lacking Atg1, Atg13, Atg18 and Atg2. Atg7 and Atg3 encode the E1 and E2 enzymes required for Atg8a lipidation, respectively, so the generation of Atg8a-II is blocked in these mutants. Both forms of Atg8a are missing in Atg8a mutants, as expected.CC BY · PubMed Central
Fig. 5 Assays for autophagic flux. (A) and (B) The tandem tagged mCherry-GFP-Atg8a reporter labels autophagosomes as yellow (positive for both GFP and mCherry), while autolysosomes appear mostly red due to faster lysosomal quenching of GFP than mCherry. Knockdown of Vps16A (encoding a HOPS tethering complex subunit) prevents autophagosome-lysosome fusion, so all dots are double positive for GFP and mCherry in fat body cells of a starved larva (panel B), compared to the larger dots that are only positive for mCherry in control cells (panel A). Note that this change is also obvious in the pixel intensity correlation profiles calculated from these images (left panels) and in Pearson correlation coefficients (R values). (C) Overexpression of the transcription factor Myc promotes autophagic degradation both under well-fed and starved conditions, as shown here by the increased conversion of mCherry-GFP-Atg8a into free mCherry in larval fat body lysates. Note that the level of the full-length protein is reduced accordingly. Vice versa, inhibition of Myc activity by overexpression of Mad, or by silencing Myc using either of two independent RNAi lines, reduces the autophagic degradation-dependent conversion of mCherry-GFP-Atg8a into free mCherry. Bar in panel A equals 20 μm for A, B.
Fig. 5 Assays for autophagic flux. (A) and (B) The tandem tagged mCherry-GFP-Atg8a reporter labels autophagosomes as yellow (positive for both GFP and mCherry), while autolysosomes appear mostly red due to faster lysosomal quenching of GFP than mCherry. Knockdown of Vps16A (encoding a HOPS tethering complex subunit) prevents autophagosome-lysosome fusion, so all dots are double positive for GFP and mCherry in fat body cells of a starved larva (panel B), compared to the larger dots that are only positive for mCherry in control cells (panel A). Note that this change is also obvious in the pixel intensity correlation profiles calculated from these images (left panels) and in Pearson correlation coefficients (R values). (C) Overexpression of the transcription factor Myc promotes autophagic degradation both under well-fed and starved conditions, as shown here by the increased conversion of mCherry-GFP-Atg8a into free mCherry in larval fat body lysates. Note that the level of the full-length protein is reduced accordingly. Vice versa, inhibition of Myc activity by overexpression of Mad, or by silencing Myc using either of two independent RNAi lines, reduces the autophagic degradation-dependent conversion of mCherry-GFP-Atg8a into free mCherry. Bar in panel A equals 20 μm for A, B.CC BY · PubMed Central

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Metadata

Authors
Nagy, P., Varga, Á., Kovács, A. L., Takáts, S., & Juhász, G.
Venue
Methods
Publisher
Elsevier BV
Published
2015-03
Volume
75
Pages
151-161
ISSN
1046-2023
DOI
10.1016/j.ymeth.2014.11.016
PMC
PMC4358840
Cited by
115
References
13
Language
en
Licence
Funders
  • Hungarian Scientific Research Fund (K83509)
  • Wellcome Trust (087518/Z/08/Z)

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