Figure 5 | Cell Death & Differentiation

Figure 5

From: The ALS-linked E102Q mutation in Sigma receptor-1 leads to ER stress-mediated defects in protein homeostasis and dysregulation of RNA-binding proteins

Figure 5

mSigR1 leads to defective autophagy and autophagosome-lysosome fusion. (a) Immunoblot analysis for autophagy markers in A431 cells transiently transfected with pcDNA, wtSigR1 and mSigR1. Note the increased levels of autophagy markers in mSigR1 expressing cells. (b–d) MCF-7 cells were transiently transfected with wtSigR1 or mSigR1 and then processed for (b) co-immunolabelling using SigR1 and LC3 antibodies, (c) co-immunolabelling using SigR1 and Cyto-ID dye (for labelling autophagosomes) and (d) co-immunolabelling using SigR1 and p62 antibodies. Scale, 10 μm. (e) Increased accumulation of autophagosomes in the stable autophagy reporter cell line NIH3T3-GFP transfected with mSigR1. Green: GFP-LC3; red: SigR1; scale bar, 10 μm. (f) Immunoblot analysis of NIH3T3-GFP-LC3 cells transfected with wtSigR1 or mSigR1 and additionally treated with the autophagy inhibitor Bafilomycin A for 2 h. Note the unchanged LC3-II levels in mSigR1-transfected cells after Bafilomycin A treatment. Corresponding densitometric data are shown at the bottom; where the upper number represents the relative LC3II/Tub levels and the lower numbers are the S.D. The asterisks (*) denote significant differences (*P<0.05), while # denotes absence of a significant difference. (g and h) Primary fibroblasts isolated from autophagy reporter GFP-LC3 transgenic mice were transfected with wtSigR1 or mSigR1 and immuno-labelled by SigR1 (g) and p62 (h) antibody (merge image for GFP-LC3, green, and SigR1/p62, red). Note the localization of SigR1 at the periphery of this particular cell (g, arrowheads) and the co-localization of GFP-LC3 with globular p62 accumulations. Scale bar, 10 μm. (i) Co-localization of SigR1 with p62 and LC3 in E102Q-SigR1 fALS lymphoblastoid cells compared to healthy control lymphoblastoid cells. (j) Cyto-ID (green) staining (right) showing the accumulation of autophagosomes in E102Q-SigR1 fALS lymphoblastoid cells. (k) Immunoblot analysis of established autophagy markers in E102Q-SigR1 fALS lymphoblastoid cells in comparison to healthy controls. (l) A431 cells were transfected as described above. Forty-eight hours later, transfected cells were processed for the EGFR degradation assay as described in Materials and Methods and analyzed by immunoblotting with the EGFR antibody. Note the delayed EGFR degradation in mSigR1 expressing cells. (lower) Quantification of immunoblot analysis. Values are expressed as mean±S.D. from three independent experiments. *P<0.05. (m) NIH3T3 cells expressing RFP-GFP-LC3 were transfected with pcDNA, wtSigR1 or mSigR1. Forty-eight hours later the fusion of autophagosomes with lysosomes was measured by live cell imaging. Scale bar, 25 μm. (lower) The rate of autophagosome maturation reflected by the Pearson coefficient (green/red fluorescence ratio) at each time point indicated. Values are represented as means±S.E.M. of triplicate experiments *P<<0.0001. (n) EM picture of NIH-3T3 cells stably expressing RFP-GFP-LC3 transfected with mSigR1. Note the autophagosome (white arrow) and lysosomes (gray arrow) without any fusion

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