Figure 3. Lysosome damage activates CASM... | Rockefeller University Press

Figure 3.

$Lysosome damage activates CASM through the V-ATPase-ATG16L1 axis. (A) Western blotting and Coomassie staining of GFP-IPs from MCF10A GFP-LC3A expressing cells treated ± LLOMe (250 μM, 40 min). (B) Normalized mass spectrometry analysis of LC3A-PE and LC3A-PS in cells treated as in A. (C) Representative confocal images of GFP-LC3A in WT and ATG13 KO MCF10A cells treated with LLOMe (250 μM, 30 min) or PP242 (1 μM, 1 h), after pretreatment with BafA1 (100 nM). Scale bar, 10 μm. (D) Representative confocal images of endogenous LC3 in WT MCF10A cells treated as in C. Scale bar, 10 μm. (E) Confocal images of WT or ATG13 KO MCF10A cells expressing GFP-LC3A treated with LLOMe (250 μM, 20 min) ± BafA1 pretreatment (100 nM) and stained for Galectin-3. Inserts show single-channel cropped images. Scale bar, 5 μm. (F) Quantification of Gal3 overlap with GFP-LC3A from E. Eight fields of view from two experiments were analyzed; ****, P < 0.0001, ***, P < 0.0002, **, P < 0.0009, unpaired t test. (G) MCF10A cells were treated with monensin (100 μM, 40 min), SaliP (2.5 μM, 1 h), PP242 (1 μM, 1 h), LLOMe (250 μM, 30 min), or LLOMe + BafA1 (100 nM), 30 min. Following fractionation, membrane and cytosol fractions were probed for ATP6V1A and ATP6V0D1 by Western blotting. V1/V0 ratios are shown below. (H) Western blotting analysis of GFP-LC3A expressing WT MCF10A and ATG16L1 KO cells re-expressing ATG16L1 WT or K490A, treated with LLOMe (250 μM, 20 min). (I) Confocal images of GFP-LC3A in the MCF10A ATG16L1 cell line panel treated with LLOMe (250 μM, 20 min) or MSU crystals (200 μg/ml, 4 h) and costained for LAMP1. Arrows denote LAMP1 positive crystals, numbers show the percentage of GFP-LC3A recruitment to >100 LAMP1 positive crystals over two repeats. Scale bar, 10 μm. (J) Confocal images of GFP-LC3A expressing MCF10A cells cotransfected with mCherry-SopF. Outlined cells mark mCherry-SopF expressing cells. Scale bar, 20 μm. (K) Quantification of the area of thresholded GFP-LC3A intensity in SopF positive and negative cells from J. 18 cells from 2 experiments were analyzed; ****, P < 0.0001, unpaired t test. Source data are available for this figure: SourceData F3.$

Lysosome damage activates CASM through the V-ATPase-ATG16L1 axis. (A) Western blotting and Coomassie staining of GFP-IPs from MCF10A GFP-LC3A expressing cells treated ± LLOMe (250 μM, 40 min). (B) Normalized mass spectrometry analysis of LC3A-PE and LC3A-PS in cells treated as in A. (C) Representative confocal images of GFP-LC3A in WT and ATG13 KO MCF10A cells treated with LLOMe (250 μM, 30 min) or PP242 (1 μM, 1 h), after pretreatment with BafA1 (100 nM). Scale bar, 10 μm. (D) Representative confocal images of endogenous LC3 in WT MCF10A cells treated as in C. Scale bar, 10 μm. (E) Confocal images of WT or ATG13 KO MCF10A cells expressing GFP-LC3A treated with LLOMe (250 μM, 20 min) ± BafA1 pretreatment (100 nM) and stained for Galectin-3. Inserts show single-channel cropped images. Scale bar, 5 μm. (F) Quantification of Gal3 overlap with GFP-LC3A from E. Eight fields of view from two experiments were analyzed; ****, P < 0.0001, ***, P < 0.0002, **, P < 0.0009, unpaired t test. (G) MCF10A cells were treated with monensin (100 μM, 40 min), SaliP (2.5 μM, 1 h), PP242 (1 μM, 1 h), LLOMe (250 μM, 30 min), or LLOMe + BafA1 (100 nM), 30 min. Following fractionation, membrane and cytosol fractions were probed for ATP6V1A and ATP6V0D1 by Western blotting. V1/V0 ratios are shown below. (H) Western blotting analysis of GFP-LC3A expressing WT MCF10A and ATG16L1 KO cells re-expressing ATG16L1 WT or K490A, treated with LLOMe (250 μM, 20 min). (I) Confocal images of GFP-LC3A in the MCF10A ATG16L1 cell line panel treated with LLOMe (250 μM, 20 min) or MSU crystals (200 μg/ml, 4 h) and costained for LAMP1. Arrows denote LAMP1 positive crystals, numbers show the percentage of GFP-LC3A recruitment to >100 LAMP1 positive crystals over two repeats. Scale bar, 10 μm. (J) Confocal images of GFP-LC3A expressing MCF10A cells cotransfected with mCherry-SopF. Outlined cells mark mCherry-SopF expressing cells. Scale bar, 20 μm. (K) Quantification of the area of thresholded GFP-LC3A intensity in SopF positive and negative cells from J. 18 cells from 2 experiments were analyzed; ****, P < 0.0001, unpaired t test. Source data are available for this figure: SourceData F3.