Figure 1.
Developing zapERtrap for light-triggered protein secretion. (A) Schematic of zapERtrap strategy. DHFR is fused to the lumenal domain of a membrane protein cargo (TfR shown here). An ER-retained version of FKBP (C-terminal KDEL tag) is expressed in the lumen of the ER. The zapalog compound bridges these domains, retaining the DHFR-fused protein in the ER. 405-nm light disrupts the zapalog tether between DHFR and FKBP-KDEL, allowing forward trafficking to proceed. (B) A pulse of 405-nm light triggers redistribution of TfR-GFP-DHFR (green) from the ER to the GA (arrowhead). Magnified images (taken from the region marked by the white boxes) before (top) and 12 min following (bottom) ER release are shown on the right. Scale bar, 10 µm. Inset scale bar, 5 µm. (C) Live cell antibody surface labeling following ER release. Top left: Schematic of strategy visualizing surface accumulation using extracellular Alexa647-anti-GFP. Right: Image time series of TfR-GFP-DHFR accumulation in the GA (top; arrowhead) and on the cell surface (middle) following ER release. Scale bar, 7 µm. Bottom left: Kinetics of TfR-GFP-DHFR accumulation in the GA (blue) and on the surface (red) following release; mean ± SEM (n = 6 cells from two independent experiments). (D) Focal 405-nm illumination (left; pink dashed line) triggers TfR-GFP-DHFR (green) trafficking to the GA (arrowhead) and the surface (right, Alexa647-anti-GFP puncta shown in red) only in the photoactivated cell. Magnified images show TfR-GFP-DHFR surface label (arrowheads) in the photoactivated cell (stim.; top) and control cell (bottom). Scale bar, 10 µm. Inset, 5 µm. The average time courses of TfR-GFP-DHFR surface accumulation for photoactivated cells (pink line) and neighboring control cells (gray line) are plotted; mean ± SEM (n = 6–8 cells from two independent experiments). cntrl, control; surf, surface.

Developing zapERtrap for light-triggered protein secretion. (A) Schematic of zapERtrap strategy. DHFR is fused to the lumenal domain of a membrane protein cargo (TfR shown here). An ER-retained version of FKBP (C-terminal KDEL tag) is expressed in the lumen of the ER. The zapalog compound bridges these domains, retaining the DHFR-fused protein in the ER. 405-nm light disrupts the zapalog tether between DHFR and FKBP-KDEL, allowing forward trafficking to proceed. (B) A pulse of 405-nm light triggers redistribution of TfR-GFP-DHFR (green) from the ER to the GA (arrowhead). Magnified images (taken from the region marked by the white boxes) before (top) and 12 min following (bottom) ER release are shown on the right. Scale bar, 10 µm. Inset scale bar, 5 µm. (C) Live cell antibody surface labeling following ER release. Top left: Schematic of strategy visualizing surface accumulation using extracellular Alexa647-anti-GFP. Right: Image time series of TfR-GFP-DHFR accumulation in the GA (top; arrowhead) and on the cell surface (middle) following ER release. Scale bar, 7 µm. Bottom left: Kinetics of TfR-GFP-DHFR accumulation in the GA (blue) and on the surface (red) following release; mean ± SEM (n = 6 cells from two independent experiments). (D) Focal 405-nm illumination (left; pink dashed line) triggers TfR-GFP-DHFR (green) trafficking to the GA (arrowhead) and the surface (right, Alexa647-anti-GFP puncta shown in red) only in the photoactivated cell. Magnified images show TfR-GFP-DHFR surface label (arrowheads) in the photoactivated cell (stim.; top) and control cell (bottom). Scale bar, 10 µm. Inset, 5 µm. The average time courses of TfR-GFP-DHFR surface accumulation for photoactivated cells (pink line) and neighboring control cells (gray line) are plotted; mean ± SEM (n = 6–8 cells from two independent experiments). cntrl, control; surf, surface.

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