Figure 3.
γ-Secretase subunits are present in minute amounts in p58-positive structures. (A and B) GFP-PSEN1 was retained in the ER in the absence of NCT or PEN-2. MEFs expressing GFP-PSEN1/γ-secretase (top, mimicking WT situation), lacking NCT (middle) or PEN-2 (bottom), were transfected with mCherry-SEC61B to visualize the ER. After 24 h, fixed cells were stained for Lamp1 or TfR (A) or p58 (B). When γ-secretase complex formation was restored, GFP-PSEN1 codistributed on Lamp1-positive organelles and at the cell surface (marked by TfR in A). p58 and GFP-PSEN1 colocalized in none of the conditions (B). Insets: Zooms of the squares on the merged panels. (C) Localization of GFP-PSEN1 in Noco/BFA-induced dilated ERES. PSEN1/PSEN2/NCT-tKO MEFs stably rescued with GFP-PSEN1 treated with 1 µg/ml Noco on ice (20 min) followed by 1 µg/ml Noco + 5 µg/ml BFA (37°C, 3 h), were fixed and immunostained for GFP (green) and p58 (red, identifying dilated ERES). Arrowheads: GFP-PSEN1 accumulation close to enlarged ERES. Right: Quantification of mean fluorescence intensity (MFI) of GFP in p58-positive enlarged structures showed increased colocalization of GFP-PSEN1 with p58 in NCT-KO and PEN-2-KO. The box and whisker graph shows all the data points. P values were determined by unpaired Student's t-test. (D) Airyscan imaging of NCT-KO MEFs treated as in C. Left: Merge of GFP (green) and p58 (red) with two regions of interest (ROIs 1 and 2). Middle and right: Zooms of ROIs as Z-stack images (0.2-µm steps), correlating frame-by-frame GFP-PSEN1 presence in p58-positive dilated ERES (arrowheads). (E) GFP-PSEN1 does not accumulate upon Arf1Q71L expression. MEFs expressing GFP-PSEN1/γ-secretase (top) or lacking PEN-2 (middle) or NCT (bottom) were transfected with HA-Arf1Q71L. After 24 h, cells were fixed and stained for GFP, HA, and p58. p58, but not GFP-PSEN1, is strongly enriched in Arf1-Q71L–positive compartments. Scale bar = 10 µm (A–C and E); 5 µm (D).

γ-Secretase subunits are present in minute amounts in p58-positive structures. (A and B) GFP-PSEN1 was retained in the ER in the absence of NCT or PEN-2. MEFs expressing GFP-PSEN1/γ-secretase (top, mimicking WT situation), lacking NCT (middle) or PEN-2 (bottom), were transfected with mCherry-SEC61B to visualize the ER. After 24 h, fixed cells were stained for Lamp1 or TfR (A) or p58 (B). When γ-secretase complex formation was restored, GFP-PSEN1 codistributed on Lamp1-positive organelles and at the cell surface (marked by TfR in A). p58 and GFP-PSEN1 colocalized in none of the conditions (B). Insets: Zooms of the squares on the merged panels. (C) Localization of GFP-PSEN1 in Noco/BFA-induced dilated ERES. PSEN1/PSEN2/NCT-tKO MEFs stably rescued with GFP-PSEN1 treated with 1 µg/ml Noco on ice (20 min) followed by 1 µg/ml Noco + 5 µg/ml BFA (37°C, 3 h), were fixed and immunostained for GFP (green) and p58 (red, identifying dilated ERES). Arrowheads: GFP-PSEN1 accumulation close to enlarged ERES. Right: Quantification of mean fluorescence intensity (MFI) of GFP in p58-positive enlarged structures showed increased colocalization of GFP-PSEN1 with p58 in NCT-KO and PEN-2-KO. The box and whisker graph shows all the data points. P values were determined by unpaired Student's t-test. (D) Airyscan imaging of NCT-KO MEFs treated as in C. Left: Merge of GFP (green) and p58 (red) with two regions of interest (ROIs 1 and 2). Middle and right: Zooms of ROIs as Z-stack images (0.2-µm steps), correlating frame-by-frame GFP-PSEN1 presence in p58-positive dilated ERES (arrowheads). (E) GFP-PSEN1 does not accumulate upon Arf1Q71L expression. MEFs expressing GFP-PSEN1/γ-secretase (top) or lacking PEN-2 (middle) or NCT (bottom) were transfected with HA-Arf1Q71L. After 24 h, cells were fixed and stained for GFP, HA, and p58. p58, but not GFP-PSEN1, is strongly enriched in Arf1-Q71L–positive compartments. Scale bar = 10 µm (A–C and E); 5 µm (D).

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