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Autophagosome–lysosome fusion is independent of V-ATPase-mediated acidification

Mauvezin, Caroline, Nagy, Péter, Juhász, Gábor, Neufeld, Thomas P.·2015·Nature Communications
DOI 10.1038/ncomms8007PMC PMC4428688
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Abstract

The ATP-dependent proton pump V-ATPase ensures low intralysosomal pH, which is essential for lysosomal hydrolase activity. Based on studies with the V-ATPase inhibitor BafilomycinA1, lysosomal acidification is also thought to be required for fusion with incoming vesicles from the autophagic and endocytic pathways. Here we show that loss of V-ATPase subunits in the Drosophila fat body causes an accumulation of non-functional lysosomes, leading to a block in autophagic flux. However, V-ATPase-deficient lysosomes remain competent to fuse with autophagosomes and endosomes, resulting in a time-dependent formation of giant autolysosomes. In contrast, BafilomycinA1 prevents autophagosome–lysosome fusion in these cells, and this defect is phenocopied by depletion of the Ca 2+ pump SERCA, a secondary target of this drug. Moreover, activation of SERCA promotes fusion in a BafilomycinA1-sensitive manner. Collectively, our results indicate that lysosomal acidification is not a prerequisite for fusion, and that BafilomycinA1 inhibits fusion independent of its effect on lysosomal pH.

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Figure 1 Depletion of V-ATPase subunits leads to accumulation of enlarged, defective autophagic structures. (a) Representative confocal images of larval fat body expressing mCh-Atg8a (red), which localizes to autophagosomes and autolysosomes in control cells (GFP negative) under 4 h starvation conditions. Cells expressing the transgenic double-stranded RNA that depletes the V-ATPase subunit V1C (GFP-positive clones) accumulate autophagic structures in their cytosol under fed conditions (left) and display enlarged vesicles in starved animals (right). Greyscale images at right show mCh-Atg8a. (b) Loss of V-ATPase subunits inhibits vesicle acidification. Fat body cells were stained with LysoTracker Red, which localizes to starvation-induced acidified autolysosomes in control cells (GFP negative). Cells depleted for V1H (GFP-positive clones) fail to accumulate acidic organelles. The LysoTracker signal alone is shown in the greyscale image at right. (c) Lysosomal enzymes are inhibited in cells depleted for V-ATPase subunits. All cells are stained with Magic Red cresyl violet-(RR)2, which monitors lysosomal Cathepsin B enzyme activity (red). In wild-type cells (GFP negative), Cathepsin B is activated under starvation conditions. Depletion of V1H (GFP-positive clone) results in failure to activate Cathepsin B. Magic Red signal alone is shown in greyscale at right. Scale bar, 10 μm. Nuclei are labelled with 4,6-diamidino-2-phenylindole (DAPI; blue) in a and b. N≥6 larvae tested for each genotype or experimental condition. (d) Representative confocal images of tandem mCherry-GFP-Atg8a expression in fat body cells. In control animals, starvation leads to the formation of acidic autolysosomes, which can be visualized only in the red channel due to quenching of GFP. In cells depleted for V1H, non-acidified (red and green fluorescent) autophagic structures are evident under both nutrient-rich and starvation conditions. Genotypes: control: Cg-GAL4 UAS-mCherry-GFP-Atg8a/+. V1H RNAi: Cg-GAL4 UAS-mCherry-GFP-Atg8a/ UAS-RNAi-V1H. Scale bar, 5 μm. (e) Quantification of number of acidified (red only) and non-acidified (red and green) vesicles from images shown in d. Error bars mark s.e.m., N≥7 larvae per experimental condition. ***P<0.001, Student’s t-test.
Figure 1 Depletion of V-ATPase subunits leads to accumulation of enlarged, defective autophagic structures. (a) Representative confocal images of larval fat body expressing mCh-Atg8a (red), which localizes to autophagosomes and autolysosomes in control cells (GFP negative) under 4 h starvation conditions. Cells expressing the transgenic double-stranded RNA that depletes the V-ATPase subunit V1C (GFP-positive clones) accumulate autophagic structures in their cytosol under fed conditions (left) and display enlarged vesicles in starved animals (right). Greyscale images at right show mCh-Atg8a. (b) Loss of V-ATPase subunits inhibits vesicle acidification. Fat body cells were stained with LysoTracker Red, which localizes to starvation-induced acidified autolysosomes in control cells (GFP negative). Cells depleted for V1H (GFP-positive clones) fail to accumulate acidic organelles. The LysoTracker signal alone is shown in the greyscale image at right. (c) Lysosomal enzymes are inhibited in cells depleted for V-ATPase subunits. All cells are stained with Magic Red cresyl violet-(RR)2, which monitors lysosomal Cathepsin B enzyme activity (red). In wild-type cells (GFP negative), Cathepsin B is activated under starvation conditions. Depletion of V1H (GFP-positive clone) results in failure to activate Cathepsin B. Magic Red signal alone is shown in greyscale at right. Scale bar, 10 μm. Nuclei are labelled with 4,6-diamidino-2-phenylindole (DAPI; blue) in a and b. N≥6 larvae tested for each genotype or experimental condition. (d) Representative confocal images of tandem mCherry-GFP-Atg8a expression in fat body cells. In control animals, starvation leads to the formation of acidic autolysosomes, which can be visualized only in the red channel due to quenching of GFP. In cells depleted for V1H, non-acidified (red and green fluorescent) autophagic structures are evident under both nutrient-rich and starvation conditions. Genotypes: control: Cg-GAL4 UAS-mCherry-GFP-Atg8a/+. V1H RNAi: Cg-GAL4 UAS-mCherry-GFP-Atg8a/ UAS-RNAi-V1H. Scale bar, 5 μm. (e) Quantification of number of acidified (red only) and non-acidified (red and green) vesicles from images shown in d. Error bars mark s.e.m., N≥7 larvae per experimental condition. ***P<0.001, Student’s t-test.CC BY · PubMed Central
Figure 2 V-ATPase is required for autophagic degradation. (a) Representative confocal images showing GFP-Ref(2)P in fat body cells. Starvation-induced autophagy triggers degradation of Ref(2)P-positive aggregates in control animals, whereas Ref(2)P clearance is defective in cells expressing V1H RNAi. N≥10 for each genotype. (b) Confocal images showing accumulation of endogenous Ref(2)P in cells depleted for the V1H subunit (RNAi-expressing clone is outlined) under nutrient-rich conditions. Endogenous Ref(2)P was detected by immunofluorescence and labelled in red. Ref(2)P signal alone is shown in greyscale. In a and b, DAPI staining is shown in blue; scale bar, 10 μm. (c) TEM images of control (Cg-GAL4/+) and V1H-depleted (Cg-GAL4/UAS-V1H-dsRNA) fat body cells under starved conditions (4 h). Insets highlight individual autolysosomes (yellow arrowheads) and autophagosomes (white arrowheads). Scale bar, 1 μm. (d) Quantification of per cent cell area occupied by double-membrane autophagosomes and single-membrane autolysosomes in control and V1H-depleted cells under starvation conditions. Error bars mark s.e.m., N=3 animals, 15 images analysed for each genotype. ***P<0.001, Student’s t-test. (e) High-magnification TEM image of an enlarged autolysosome from V1H-depleted fat body. Note the intact mitochondria visible within this vesicle. Scale bar, 1 μm.
Figure 2 V-ATPase is required for autophagic degradation. (a) Representative confocal images showing GFP-Ref(2)P in fat body cells. Starvation-induced autophagy triggers degradation of Ref(2)P-positive aggregates in control animals, whereas Ref(2)P clearance is defective in cells expressing V1H RNAi. N≥10 for each genotype. (b) Confocal images showing accumulation of endogenous Ref(2)P in cells depleted for the V1H subunit (RNAi-expressing clone is outlined) under nutrient-rich conditions. Endogenous Ref(2)P was detected by immunofluorescence and labelled in red. Ref(2)P signal alone is shown in greyscale. In a and b, DAPI staining is shown in blue; scale bar, 10 μm. (c) TEM images of control (Cg-GAL4/+) and V1H-depleted (Cg-GAL4/UAS-V1H-dsRNA) fat body cells under starved conditions (4 h). Insets highlight individual autolysosomes (yellow arrowheads) and autophagosomes (white arrowheads). Scale bar, 1 μm. (d) Quantification of per cent cell area occupied by double-membrane autophagosomes and single-membrane autolysosomes in control and V1H-depleted cells under starvation conditions. Error bars mark s.e.m., N=3 animals, 15 images analysed for each genotype. ***P<0.001, Student’s t-test. (e) High-magnification TEM image of an enlarged autolysosome from V1H-depleted fat body. Note the intact mitochondria visible within this vesicle. Scale bar, 1 μm.CC BY · PubMed Central
Figure 3 Autophagosome–lysosome fusion is active in V-ATPase-depleted cells. (a) Confocal images of larval fat body cells co-expressing mCherry-Atg8a (red), the lysosomal marker LAMP1-GFP (green) and the indicated RNAi constructs under fed or 4 h starved conditions. Insets illustrate the presence of autolysosomes indicating fusion between autophagosomes and lysosomes. Autolysosomes can be identified as mCh-Atg8a-positive structures surrounded by LAMP1-GFP in starved controls and fed or starved V1H-depleted cells. Genotypes: control: Cg-GAL4 UAS-LAMP1-GFP UAS-mCh-Atg8a/+. Syx17 RNAi: Cg-GAL4 UAS-LAMP1-GFP UAS-mCh-Atg8a/ UAS-Syx17-dsRNA. V1H RNAi: Cg-GAL4 UAS-LAMP1-GFP UAS-mCh-Atg8a/ UAS-V1H-dsRNA. Nuclei are labelled in blue with DAPI; scale bars, 10 μm. (b) Quantitation of LAMP1-GFP and mCh-Atg8a co-localization as in a. Pearson’s correlation coefficient Rr was calculated using ≥10 independent samples for each genotype; error bars mark s.e.m. ***P<0.001, Student’s t-test.
Figure 3 Autophagosome–lysosome fusion is active in V-ATPase-depleted cells. (a) Confocal images of larval fat body cells co-expressing mCherry-Atg8a (red), the lysosomal marker LAMP1-GFP (green) and the indicated RNAi constructs under fed or 4 h starved conditions. Insets illustrate the presence of autolysosomes indicating fusion between autophagosomes and lysosomes. Autolysosomes can be identified as mCh-Atg8a-positive structures surrounded by LAMP1-GFP in starved controls and fed or starved V1H-depleted cells. Genotypes: control: Cg-GAL4 UAS-LAMP1-GFP UAS-mCh-Atg8a/+. Syx17 RNAi: Cg-GAL4 UAS-LAMP1-GFP UAS-mCh-Atg8a/ UAS-Syx17-dsRNA. V1H RNAi: Cg-GAL4 UAS-LAMP1-GFP UAS-mCh-Atg8a/ UAS-V1H-dsRNA. Nuclei are labelled in blue with DAPI; scale bars, 10 μm. (b) Quantitation of LAMP1-GFP and mCh-Atg8a co-localization as in a. Pearson’s correlation coefficient Rr was calculated using ≥10 independent samples for each genotype; error bars mark s.e.m. ***P<0.001, Student’s t-test.CC BY · PubMed Central
Figure 4 Autophagic vesicles expand under prolonged starvation in V-ATPase-depleted cells. (a) Representative images of LAMP1-GFP and mCh-Atg8a-positive vesicles in fat body cells depleted for Syx17 or V1H under fed, 4 h starvation or 24 h starvation conditions. Autophagic vesicles progressively expand in V-ATPase-depleted but not Syx17-depleted cells. (b) Quantification of LAMP1-positive vesicle diameter in cells depleted for Syx17 or V1H as shown in a. Error bars mark s.e.m.; fat body cells from a minimum of eight larvae were analysed for each genotype and time point. ***P<0.001, Student’s t-test. (c) TEM images showing representative autolysosomes in control and V1H-depleted fat body cells following 0, 4 and 24 h starvation. Scale bar, 1 μm. (d) Quantification of individual autolysosome area in control and V1H-depleted fat body cells as shown in c. Vesicles from 15 sections from three larvae were analysed for each genotype and time point. Error bars mark s.e.m.; ***P<0.001, Student’s t-test. (e) Mosaic fat body containing a GFP-marked clone of cells depleted for Syx17 and the V-ATPase subunit V0d, following 4 h starvation. mCh-Atg8a-marked autophagic vesicles in these cells are not enlarged relative to those in the surrounding GFP-negative control cells. N=7. In a and e, nuclei are labelled in blue with DAPI; scale bars, 10 μm. (f) High-magnification confocal images of mCh-Atg8a-positive vesicles in fat body cells depleted of V1H under starvation-induced autophagy (4 h). Variation of fluorescence intensity is observable within individual single vesicles. N≥10. Scale bar, 1 μm. (g) High-magnification TEM image illustrates a non-degradative autolysosome containing four autophagic bodies (1–4) in V0c-depleted fat body. Scale bar, 0.3 μm.
Figure 4 Autophagic vesicles expand under prolonged starvation in V-ATPase-depleted cells. (a) Representative images of LAMP1-GFP and mCh-Atg8a-positive vesicles in fat body cells depleted for Syx17 or V1H under fed, 4 h starvation or 24 h starvation conditions. Autophagic vesicles progressively expand in V-ATPase-depleted but not Syx17-depleted cells. (b) Quantification of LAMP1-positive vesicle diameter in cells depleted for Syx17 or V1H as shown in a. Error bars mark s.e.m.; fat body cells from a minimum of eight larvae were analysed for each genotype and time point. ***P<0.001, Student’s t-test. (c) TEM images showing representative autolysosomes in control and V1H-depleted fat body cells following 0, 4 and 24 h starvation. Scale bar, 1 μm. (d) Quantification of individual autolysosome area in control and V1H-depleted fat body cells as shown in c. Vesicles from 15 sections from three larvae were analysed for each genotype and time point. Error bars mark s.e.m.; ***P<0.001, Student’s t-test. (e) Mosaic fat body containing a GFP-marked clone of cells depleted for Syx17 and the V-ATPase subunit V0d, following 4 h starvation. mCh-Atg8a-marked autophagic vesicles in these cells are not enlarged relative to those in the surrounding GFP-negative control cells. N=7. In a and e, nuclei are labelled in blue with DAPI; scale bars, 10 μm. (f) High-magnification confocal images of mCh-Atg8a-positive vesicles in fat body cells depleted of V1H under starvation-induced autophagy (4 h). Variation of fluorescence intensity is observable within individual single vesicles. N≥10. Scale bar, 1 μm. (g) High-magnification TEM image illustrates a non-degradative autolysosome containing four autophagic bodies (1–4) in V0c-depleted fat body. Scale bar, 0.3 μm.CC BY · PubMed Central
Figure 5 BafA1 inhibits autolysosomal acidification and fusion. (a) Representative confocal images of fat body cells expressing the tandem-tagged mCherry-GFP-Atg8a reporter. Following 4 h starvation in vivo or 6 h incubation in the absence of BafA1 ex vivo, most mCherry-positive punctae exhibit quenching of the GFP signal, indicating its delivery to the acidic lumen of the lysosome. Cells treated with 200 nM BafA1 accumulate non-acidified autophagic structures positive for both GFP and mCherry. Nuclei are labelled with DAPI; blue. N≥15 for each genotype. Scale bar, 10 μm. Genotype: Cg-GAL4 UAS-mCherry-GFP-Atg8a/+. (b) Representative images of fat body cells co-expressing LAMP1-GFP and mCh-Atg8a incubated ex vivo for 6 h with 200 nM BafA1 in the presence (basal autophagy) or absence (induced autophagy) of 10 μg ml−1 insulin. Nuclei are labelled with DAPI (blue). N≥15 for each condition. Scale bar, 10 μm. Genotype: Cg-GAL4 UAS-mCherry-Atg8a UAS-LAMP1-GFP/+. (c) Quantification of co-localization between LAMP1-GFP and mCh-Atg8a as measured by Pearson’s correlation coefficient Rr, using a minimum of 15 images for each condition. Error bars mark s.d. ***P<0.001, Student’s t-test.
Figure 5 BafA1 inhibits autolysosomal acidification and fusion. (a) Representative confocal images of fat body cells expressing the tandem-tagged mCherry-GFP-Atg8a reporter. Following 4 h starvation in vivo or 6 h incubation in the absence of BafA1 ex vivo, most mCherry-positive punctae exhibit quenching of the GFP signal, indicating its delivery to the acidic lumen of the lysosome. Cells treated with 200 nM BafA1 accumulate non-acidified autophagic structures positive for both GFP and mCherry. Nuclei are labelled with DAPI; blue. N≥15 for each genotype. Scale bar, 10 μm. Genotype: Cg-GAL4 UAS-mCherry-GFP-Atg8a/+. (b) Representative images of fat body cells co-expressing LAMP1-GFP and mCh-Atg8a incubated ex vivo for 6 h with 200 nM BafA1 in the presence (basal autophagy) or absence (induced autophagy) of 10 μg ml−1 insulin. Nuclei are labelled with DAPI (blue). N≥15 for each condition. Scale bar, 10 μm. Genotype: Cg-GAL4 UAS-mCherry-Atg8a UAS-LAMP1-GFP/+. (c) Quantification of co-localization between LAMP1-GFP and mCh-Atg8a as measured by Pearson’s correlation coefficient Rr, using a minimum of 15 images for each condition. Error bars mark s.d. ***P<0.001, Student’s t-test.CC BY · PubMed Central
Figure 6 BafA1 blocks autophagosome–lysosome fusion through inhibition of dSERCA. (a) Representative confocal images of control (GFP negative) and dSERCA-depleted (GFP-positive clones) fat body cells from well-fed and 4 h starved larvae. Depletion of dSERCA leads to accumulation of mCh-Atg8a punctae (red, and shown in greyscale at right) under fed conditions with no further accumulation in starved samples. (b) Quantification of size of Atg8-positive vesicles in control and dSERCA-depleted fat body cells from experiment in a. Error bars mark s.e.m. for each genotype (N≥5). **P<0.05 and ***P<0.001, Student’s t-test. (c) Localization of LAMP1-GFP and mCh-Atg8a in control and dSERCA-depleted fat body from 4 h starved larvae. Insets illustrate the presence or absence of autolysosomes. Autolysosomes identified as mCh-Atg8a-positive structures surrounded by LAMP1-GFP were observed only in control. N≥9 for each genotype. (d) Quantification of co-localization between LAMP1-GFP and mCh-Atg8a. Pearson’s correlation coefficient Rr was calculated using a minimum of nine representative images for each genotype. Error bars mark s.e.m. for each condition. ***P<0.001, Student’s t-test. (e) Quantification of GCaMP3 fluorescent intensity relative to internal control DsRed. Fat body cells incubated ex vivo for 1 h with EGTA (10 mM) showed a significant reduction of GCaMP3/DsRed ratio, while cells treated with BafA1 (200 nM) or thapsigargin (200 nM) presented a strong increase compared with control. Error bars mark s.e.m. for each condition, N≥25. **P<0.005, ***P<0.001, Student’s t-test. (f) Morphology of LAMP1-GFP and mCh-Atg8a structures in control and Seipin-overexpressing cells. Fat body were dissected from larvae either starved for 4 h in vivo or incubated for 6 h ex vivo in M3 media in the presence or absence of BafA1 (200 nM). In a, c and f, nuclei are labelled with DAPI; blue; scale bars, 10 μm. (g) Quantification of the presence of elongated mCh-Atg8a- and LAMP1-GFP-positive tubular structures in fat body cells as shown in f. Error bars mark s.e.m. for each condition, N≥16. **P<0.05 ***P<0.005, Student’s t-test.
Figure 6 BafA1 blocks autophagosome–lysosome fusion through inhibition of dSERCA. (a) Representative confocal images of control (GFP negative) and dSERCA-depleted (GFP-positive clones) fat body cells from well-fed and 4 h starved larvae. Depletion of dSERCA leads to accumulation of mCh-Atg8a punctae (red, and shown in greyscale at right) under fed conditions with no further accumulation in starved samples. (b) Quantification of size of Atg8-positive vesicles in control and dSERCA-depleted fat body cells from experiment in a. Error bars mark s.e.m. for each genotype (N≥5). **P<0.05 and ***P<0.001, Student’s t-test. (c) Localization of LAMP1-GFP and mCh-Atg8a in control and dSERCA-depleted fat body from 4 h starved larvae. Insets illustrate the presence or absence of autolysosomes. Autolysosomes identified as mCh-Atg8a-positive structures surrounded by LAMP1-GFP were observed only in control. N≥9 for each genotype. (d) Quantification of co-localization between LAMP1-GFP and mCh-Atg8a. Pearson’s correlation coefficient Rr was calculated using a minimum of nine representative images for each genotype. Error bars mark s.e.m. for each condition. ***P<0.001, Student’s t-test. (e) Quantification of GCaMP3 fluorescent intensity relative to internal control DsRed. Fat body cells incubated ex vivo for 1 h with EGTA (10 mM) showed a significant reduction of GCaMP3/DsRed ratio, while cells treated with BafA1 (200 nM) or thapsigargin (200 nM) presented a strong increase compared with control. Error bars mark s.e.m. for each condition, N≥25. **P<0.005, ***P<0.001, Student’s t-test. (f) Morphology of LAMP1-GFP and mCh-Atg8a structures in control and Seipin-overexpressing cells. Fat body were dissected from larvae either starved for 4 h in vivo or incubated for 6 h ex vivo in M3 media in the presence or absence of BafA1 (200 nM). In a, c and f, nuclei are labelled with DAPI; blue; scale bars, 10 μm. (g) Quantification of the presence of elongated mCh-Atg8a- and LAMP1-GFP-positive tubular structures in fat body cells as shown in f. Error bars mark s.e.m. for each condition, N≥16. **P<0.05 ***P<0.005, Student’s t-test.CC BY · PubMed Central
Figure 7 Schematic model of fusion and acidification events. (a) Under normal conditions, SERCA generates a Ca2+ gradient with a high concentration of Ca2+ in the ER lumen. The role of SERCA in autophagosome–lysosome fusion may reflect its ability to maintain a relatively low/moderate cytoplasmic Ca2+ concentration, or to allow localized release of ER lumenal Ca2+ near sites of fusion. Pumping of protons into the lysosomal lumen by the V-ATPase leads to acidification, activation of hydrolytic enzymes and autophagic cargo degradation. The V-ATPase, along with amino acids released by autophagic breakdown, also promotes activation of mTOR (not shown), which is required for lysosomal reformation. (b) Genetic depletion of V-ATPase subunits results in a loss of lysosomal acidification, while Ca2+/SERCA-dependent fusion remains active. Continued autophagosome–lysosome fusion in the absence of lysosomal degradation and recycling disrupts lysosomal homeostasis, leading to an expansion of the autolysosomal compartment. (c) Inhibition of both the V-ATPase and SERCA by BafA1 blocks lysosomal acidification and dissipates the ER Ca2+ gradient. Either abnormally high Ca2+ concentration in the cytosol and/or lower Ca2+ concentration in the ER lumen may contribute to defective autophagosome–lysosome fusion.
Figure 7 Schematic model of fusion and acidification events. (a) Under normal conditions, SERCA generates a Ca2+ gradient with a high concentration of Ca2+ in the ER lumen. The role of SERCA in autophagosome–lysosome fusion may reflect its ability to maintain a relatively low/moderate cytoplasmic Ca2+ concentration, or to allow localized release of ER lumenal Ca2+ near sites of fusion. Pumping of protons into the lysosomal lumen by the V-ATPase leads to acidification, activation of hydrolytic enzymes and autophagic cargo degradation. The V-ATPase, along with amino acids released by autophagic breakdown, also promotes activation of mTOR (not shown), which is required for lysosomal reformation. (b) Genetic depletion of V-ATPase subunits results in a loss of lysosomal acidification, while Ca2+/SERCA-dependent fusion remains active. Continued autophagosome–lysosome fusion in the absence of lysosomal degradation and recycling disrupts lysosomal homeostasis, leading to an expansion of the autolysosomal compartment. (c) Inhibition of both the V-ATPase and SERCA by BafA1 blocks lysosomal acidification and dissipates the ER Ca2+ gradient. Either abnormally high Ca2+ concentration in the cytosol and/or lower Ca2+ concentration in the ER lumen may contribute to defective autophagosome–lysosome fusion.CC BY · PubMed Central

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Metadata

Authors
Mauvezin, C., Nagy, P., Juhász, G., & Neufeld, T. P.
ORCID
Mauvezin, Caroline
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2015-05-11
Volume
6
Issue
1
ISSN
2041-1723
DOI
10.1038/ncomms8007
PMC
PMC4428688
Cited by
424
References
6
Language
en
Licence

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