Figure 6 | Cell Death & Differentiation

Figure 6

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 6

mSigR1 impairs ER to Golgi transport. (a) Cos7 cells showing abnormal SigR1 accumulations (arrows) after mSigR1 transfection and normal ER localization of SigR1 (right) after wtSigR1 transfection. Scale bar, 15 μm. (b) Cos7 cells were co-transfected with VSVG-GFP together with pcDNA, wtSigR1 or mSigR1. 48 h after transfection, fluorescence associated with the Golgi complex was photobleached (FRAP, see Materials and Methods) with a high-intensity laser beam. Subsequently, the inward delivery of VSVG-GFP from pre-Golgi intermediates was monitored for the indicated periods of time. Scale bar, 10 μm. (c) Fluorescence recoveries after photobleaching curves of mSigR1-transfected cells show a clear decrease of the mobile fraction as compared to cell transfected with pcDNA or wtSigR1. Error bars indicate the S.E.M.; (right) the comparison of the mobile fractions in pcDNA, wtSigR1 and mSigR1 expressing cells. In the box plots, the line in the middle of the box indicates the median; the top line indicates the 75th quartile, whereas the bottom line indicates the 25th quartile. Whiskers represent the 10th and 90th (upper) percentile, respectively. (d) mSigR1 aggregates (arrowheads) co-localize with the endosomal markers Rab5 (upper) and Rab7 (lower) in Cos-7 cells transfected with wtSigR1 and mSigR1, respectively. Scale bar, 10 μm. (e) Co-localization of mSigR1 aggregates with the endosomal markers EEA1 and Rab7 in E102Q-SigR1 fALS patients' lymphoblastoid cells compared to healthy controls. Scale bars, 15 μm. (f) Immunoblot analysis of Cos-7 cells transfected with pcDNA, wtSigR1 and mSigR1. (g) Quantification of band intensities normalized against α-tubulin depicted in (f). Values are expressed as mean±S.D. from three independent experiments. *P<0.05

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