Figure 5.
Characterization and localization of COPII in ERESs in BFA/Noc-treated intact living cells. (A) 3D confocal analysis of ERESs in BFA/Noc-treated living cells. A single confocal image from a z-section stack of a COS7 cell cotransfected with VSVG-YFP (green) and Sec24C-mCherry (red) transferred to permissive temperature (32°C) after overnight incubation at 39.5°C and treated with BFA/Noc as described in Materials and methods. The top left insert is a fivefold enlargement of a single ERES. The top right insert is a 3D reconstruction of the same ERES. Bar and white arrows are 1 µm in the x, y, and z directions. Scale bar = 10 µm. (B) Colocalization of ERGIC53 and COPII in ERESs under BFA/Noc treatment. COS7 cells expressing ERGIC53-YFP and the COPII subunit Sec24C-mCherry were treated with BFA/Noc as described in Materials and methods section. Inset (scale bar = 2 µm) is a fivefold enlargement of a single representative ERES. Scale bar = 10 µm. (C) COPII-labeled membranes separate ER and ERES membranes. Confocal deconvolved images of a COS7 cell were transfected and treated as in A. Six representative fourfold enlarged images of ERESs are shown on the right. Yellow/blue arrows point to COPII-coated collars (Sec24C-mCherry, red) intersecting between ER and ERES. The VSVG (green) channel is shown inverted on the right. See Video 11 and Video 12. Scale bar = 10 µm. (D) ERESs are connected to the ER in BFA/Noc-treated cells. Confocal image of a living COS7 cell cotransfected with VSVG-YFP (green) and the soluble secreted luminal marker signal sequence-mCherry (red) under BFA/Noc treatment. See Video 13. Bar = 1 µm. (E) Immunogold EM analysis demonstrates the transformation of ERES membranes under BFA/Noc treatment to spherical-tubular membranes known as glumerolini. Left: Confocal image of an ERES of a fixed COS7 cell expressing Sec23-GFP (green) and VSVG-Scarlet (red) under BFA/Noc treatment after 30 min at the permissive temperature 32°C. Right: Immunogold EM serial sections labeled with anti-GFP antibodies of ERESs in BFA/Noc-treated cells. Yellow arrowheads point to sites of contact to ER with increased labeling of COPII. Scale bar = 200 nm. Bottom: A 3D reconstruction of the membrane structure from serial sections. Bottom right: A scheme depicting the positioning of the section relative to the ERESs under BFA/Noc treatment.

Characterization and localization of COPII in ERESs in BFA/Noc-treated intact living cells. (A) 3D confocal analysis of ERESs in BFA/Noc-treated living cells. A single confocal image from a z-section stack of a COS7 cell cotransfected with VSVG-YFP (green) and Sec24C-mCherry (red) transferred to permissive temperature (32°C) after overnight incubation at 39.5°C and treated with BFA/Noc as described in Materials and methods. The top left insert is a fivefold enlargement of a single ERES. The top right insert is a 3D reconstruction of the same ERES. Bar and white arrows are 1 µm in the x, y, and z directions. Scale bar = 10 µm. (B) Colocalization of ERGIC53 and COPII in ERESs under BFA/Noc treatment. COS7 cells expressing ERGIC53-YFP and the COPII subunit Sec24C-mCherry were treated with BFA/Noc as described in Materials and methods section. Inset (scale bar = 2 µm) is a fivefold enlargement of a single representative ERES. Scale bar = 10 µm. (C) COPII-labeled membranes separate ER and ERES membranes. Confocal deconvolved images of a COS7 cell were transfected and treated as in A. Six representative fourfold enlarged images of ERESs are shown on the right. Yellow/blue arrows point to COPII-coated collars (Sec24C-mCherry, red) intersecting between ER and ERES. The VSVG (green) channel is shown inverted on the right. See Video 11 and Video 12. Scale bar = 10 µm. (D) ERESs are connected to the ER in BFA/Noc-treated cells. Confocal image of a living COS7 cell cotransfected with VSVG-YFP (green) and the soluble secreted luminal marker signal sequence-mCherry (red) under BFA/Noc treatment. See Video 13. Bar = 1 µm. (E) Immunogold EM analysis demonstrates the transformation of ERES membranes under BFA/Noc treatment to spherical-tubular membranes known as glumerolini. Left: Confocal image of an ERES of a fixed COS7 cell expressing Sec23-GFP (green) and VSVG-Scarlet (red) under BFA/Noc treatment after 30 min at the permissive temperature 32°C. Right: Immunogold EM serial sections labeled with anti-GFP antibodies of ERESs in BFA/Noc-treated cells. Yellow arrowheads point to sites of contact to ER with increased labeling of COPII. Scale bar = 200 nm. Bottom: A 3D reconstruction of the membrane structure from serial sections. Bottom right: A scheme depicting the positioning of the section relative to the ERESs under BFA/Noc treatment.

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