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. 2024 Mar 19;13:e92900. doi: 10.7554/eLife.92900

Figure 5. Effect of brefeldin A (BFA) on endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and Golgi proteins.

(A) Epifluorescence images of yeast cells expressing Grh1-2xmCherry, EGFP-Rer1, EGFP-Sed5, GFP-Vrg4, or Mnn2-EGFP treated with 0.4% dimethyl sulfoxide (DMSO) (control) (upper panels) or 200 μM BFA (lower panels). The blue broken lines indicate the edge of the cells. (B) Epifluorescence images of yeast cells co-expressing GFP-Vrg4 and mCherry-HDEL treated with 0.4% DMSO (left panels) or 200 μM BFA (right panels). Insets: merged images in the white dashed boxes. (C and D) Dual-color 3D SCLIM images of yeast cells co-expressing EGFP-Rer1 and Grh1-2xmCherry (C) or EGFP-Sed5 and Grh1-2xmCherry (D) treated with DMSO or BFA. Left, center, and right panels show merged, green, and red channels, respectively. The white broken lines indicate the edge of the cells. The graphs show Pearson’s correlation coefficient (r) for co-localization. Numbers in parentheses indicate the number of cells examined. Each value represents mean ± SEM. ****p<0.0001; ***p<0.0005, unpaired t-test with Welch’s correction. (E–G) BFA perfusion experiments. (E) Time schedule of drug perfusion. Time 0 indicates the onset of time-lapse imaging. The cells were originally exposed to 0.4% DMSO (light blue) solution in a perfusion chamber with a flow pressure of 2 psi. The perfusion solution was changed to 200 μM BFA (orange) at 5 min, and then returned to 0.4% DMSO (light blue) at 65 min to wash out BFA. (F and G) Time-lapse epifluorescence images of yeast cells expressing EGFP-Sed5 (F) or GFP-Vrg4 (G) treated sequentially with DMSO (0 min), BFA (10–60 min), and then DMSO again (70–120 min). The blue broken lines indicate the edge of the cells. Scale bars: 5 μm (A, B, and G) and 3 μm (D).

Figure 5—source data 1. Data used for graphs presented in Figure 5C, D, and Figure 5-supplement 1B.

Figure 5.

Figure 5—figure supplement 1. Effect of brefeldin A (BFA) on endoplasmic reticulum exit sites (ERES).

Figure 5—figure supplement 1.

(A) Dual-color three-dimensional (3D) super-resolution confocal live imaging microscopy (SCLIM) images of yeast cells co-expressing EGFP-Rer1 and Sec13-mCherry treated with dimethyl sulfoxide (DMSO) (upper panels) or BFA (lower panels). Left, center, and right panels show merged, green, and red channels, respectively. The white broken lines indicate the edge of the cells. (B) Averaged number of Sec13-positive puncta in a cell. Each value was normalized by cell volume (μm3). Numbers in parentheses indicate the number of cells examined. Each value represents mean ± SEM. ns, not significant, unpaired t-test with Welch’s correction.