Fig 1: Exogenous SM phenocopies NPA-associated lysosomal and autophagic pathologies. (A) Representative SQSTM1, MAP1LCB3-I and MAP1LCB3-II immunoblots of protein lysates from the indicated fibroblasts left untreated (UT) or treated with DMSO vehicle (V), 3 µM C16-ceramide (Cer) or 39 µM C12-SM (SM) for the indicated times. HSP90 served as a loading control. SQSTM1:HSP90 (upper row) and MAP1LCB3-II:HSP90 (lower row) ratios as a percentage of the ratio in vehicle-treated cells from the same individual are shown below. (B) Representative confocal images of the HC-3 (top) and HC-1 (bottom) fibroblasts treated with 39 µM of BODIPY® C12-SM (green) for 12 h and stained for LAMP2 and WIPI2 (red), respectively. Note juxtanuclear mainly accumulation of BODIPY-FL® C12-SM and lack of colocalization with either LAMP2 or WIPI2. Step size, 1 µm. (C) Lysosomal areas in the indicated fibroblasts treated for 12 h with vehicle (V) or 19.5 or 39 µM C12-SM (SM) were measured with Celigo® adherent cell cytometer after staining with LysoTracker®Red and Hoechst. Bars, SEM for a representative duplicate experiment (n = 3). (D) Quantification of the indicated autophagic membranes in HC-1 cells treated with vehicle or 39 µM C12-SM for 24 h with or without 10 µg/ml bSMase for the last 12 h, stained for WIPI2 and LC3, and analyzed by super resolution 3D-SIM. Bars, SEM for a representative experiment with 4 fields analyzed (n = 3). (E) Representative confocal images of HC-3 control fibroblasts treated and stained as in (D) (left), and quantification of WIPI2 and MAP1LC3B puncta (right; n = 20). HC-3 fibroblasts starved for amino acids by 2 h treatment with HBSS plus 1 g/L glucose served as a positive control. White arrows and arrowheads indicate WIPI2-positive/MAP1LC3B-negative (red) and double-positive (yellow) structures, respectively. Similar data were obtained with fibroblasts from another healthy individual. (F) Representative confocal images of HC-3 fibroblasts treated with 39 µM C12-SM for 24 h and stained for WIPI2 and MAP1LC3B. Close-ups show a typical omegasome- (1) and phagophore membrane (2). (G) Representative confocal images of HC-1 fibroblasts treated with vehicle or 39 µM C12-SM for 12 h and stained for ATG9A and TFRC. Close-ups demonstrate the perinuclear colocalization of ATG9A and TFRC and the histogram shows Manders coefficients for the colocalization. Bars, SEM for minimum 20 randomly chosen cells. C16-SM induced a similar increase in the colocalization of ATG9A and TFRC (Fig. S3). Scale bars: 20 µm (white) or 2 µm (yellow). *, P < 0.05; **, P < 0.01; ***, P < 0.001 by the Student t test.
Fig 2: Monensin induces endosomal autophagy dependent on Rabaptin5 and ATG16L1-binding just like chloroquine AHEK+Rbpt5 cells, 24 h after transfection with mCherry-ATG16L1, were treated with 100 µM monensin for 0, 15, and 30 min and stained for Rabaptin5, WIPI2, and mCherry-ATG16L1 to assess their colocalization on swollen early endosomes. Scale bar, 10 µm.B, CManders’ colocalization coefficients were determined, showing the fraction of Rabaptin5-positive structures also positive for mCherry-ATG16L1 (B) or for WIPI2 (C) (mean ± SD of three independent experiments, quantifying ~40 cells for each sample; ANOVA for Rbpt5-KO+AAA versus HEK+Rbpt5 cells : *P < 0.05, **P < 0.01).D, EWild-type HEK293A cells, HEK+Rbpt5 cells, and Rabaptin5-knockout cells without (Rbpt5-KO) or with stable re-expression of wild-type (Rbpt5-KO+wt) or AAA mutant Rabaptin5 (Rbpt5-KO+AAA) were treated without (–Mon) or with 100 µM monensin for 150 min (+Mon), and analyzed by immunofluorescence microscopy for WIPI2 or LC3B. WIPI2 (D) of LC3B (E) puncta per cell were quantified for each condition (mean ± SD of four independent experiments; ANOVA: *P < 0.05, **P < 0.01, ****P < 0.0001).
Fig 3: Autophagy proteins are targeted to Rabaptin5-positive endosomes upon chloroquine treatment A–ITo more easily visualize Rabaptin5, a stable HEK293A cell line overexpressing Rabaptin5 (HEK+Rbpt5) was generated. Immunofluorescence microscopy of Rabaptin5 and transferrin receptor (TfR) showed swelling of early endosomes upon treatment with 60 µM chloroquine for 30 min (+CQ, panel A) compared with untreated cells (–CQ, A'). Rabaptin5 levels were analyzed by immunoblotting in comparison with parental HEK293A cells (panel B). HEK+Rbpt5 cells, untransfected or 24 h after transfection with mCherry-galectin 3 (mCh-Gal3), mRuby3-galectin 8 (mRuby-Gal8), mCherry-FIP200, or mCherry-ATG16L1 were analyzed upon chloroquine treatment by immunofluorescence microscopy for Rabaptin5 and mCherry-galectin 3 or mRuby3-galectin 8 (C), ubiquitin (Ub; D), p62 (E), mCherry-FIP200 (F), WIPI2 (G), mCherry-ATG16L1 (H), or LC3B (I). Scale bar, 10 µm. In the enlarged insets, arrowheads point out chloroquine-induced enlarged, ring-like early endosomes. Rabaptin5-positive enlarged endosomes positive for mCherry-galectin 3 (Gal3) or mRuby3-galectin 8 (Gal8) were quantified (C'; mean ± SD and individual values of three independent experiments counting > 45 structures each).J, KHEK+Rbpt5 cells, untransfected or 24 h after transfection with mCherry-ATG16L1, were treated with 60 µM chloroquine for 0, 15, and 30 min and stained for Rabaptin5 and either WIPI2 or mCherry-ATG16L1. Manders’ colocalization coefficients were determined, M1 showing the fraction of Rabaptin5-positive structures also positive for WIPI2 (J) or mCherry-ATG16L1 (K), and M2 showing the respective inverse (mean ± SD of three independent experiments; ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001).
Fig 4: Phenotypic comparison between WIPI2, WIPI3 and WIPI4. (A) Frequency of symptoms. (B) Frequency of neuroradiological findings. (C) Range of phenotype severity. The actual numbers leading to the displayed percentages are provided in Supplementary Table 1.
Fig 5: TRPML1-mediated lysosomal Ca2+ release induces AV biogenesis through CaMKKß.a Representative images of HEK-293 overexpressing DFCP1-GFP treated with DMSO and MK6-83 ± BAPTA-AM. b Representative images of WIPI2 in ARPE-19 treated with DMSO and MK6-83 ± BAPTA-AM. c Representative images of WIPI2 in ARPE-19 treated with DMSO and MK6-83 alone or in co-treatment with STO-609. d Representative images of WIPI2 in ARPE-19 treated with a pool of three siRNAs targeting CaMKKß or CaMKI ± MK6-83. e Representative image of immunoblot analysis of LC3 (LC3I-II) in HeLa cells silenced with siRNAs targeting CaMKI and treated with MK6-83 ± bafilomycin A1 (BafA1). Plot shows the densitometry of LC3II normalized to actin as fold induction to DMSO. The data in the graphs on the right are mean values ± SD, n = 4 lysates per condition pooled from four independent experiments. f Representative images of WIPI2 in ARPE-19 treated with DMSO and MK6-83 ± dorsomorphin. g Representative images of HEK-293 overexpressing DFCP1-GFP treated with DMSO and MK6-83 ± dorsomorphin (DM). h Representative image of immunoblot analysis of phosphorylation AMPK on T172 and ULK1 on S555, Acetyl-CoA Carboxylase (ACC) on S79, upon MK6-83 treatment. Plot shows the densitometry of p-AMPK T172, p-ULK1 S555, and p-ACC S79 on AMPK, ULK1, and ACC normalized on Actin as fold induction to DMSO. The data in the graphs are mean values ± SD, n = 3 lysates per condition pooled from three independent experiments. i Representative image of immunoblot analysis of phosphorylation of AMPK on T172 and its substrate ULK1 on S555 upon MK6-83 treatment ± STO-609 or dorsomorphin. Plot shows the densitometry of p-AMPK T172 and p-ULK1 S555 on AMPK and ULK1 normalized on actin as fold induction to DMSO. The data in the graphs are mean values ± SD, n = 4 lysates pooled from four independent experiments. a–d, f, g Plot shows the quantification of -WIPI2 (b–d, f), -DFCP (a, g) positive puncta as fold induction to DMSO-treated cells. Values are means ± SD of n = 208 (a), n = 225 (b), n = 1169 (c), n = 610 (d), n = 385 (f), n = 687 (g) cells pooled from three independent experiments. Scale bar: 20 µm. *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001.
Supplier Page from Abcam for Anti-WIPI2 antibody [2A2]