Light-triggered protein secretion. (A) Schematic for UVR8-dependent ER retention. Tandem copies of UVR8 fused to VSVG results in oligomers that are retained in the ER. Exposure to UV-B triggers dissociation of these clusters, allowing forward trafficking through the secretory pathway. (B) Comparison of light- and chemical-mediated ER release. VSVG-YFP-2×UVR8 (top panels) and VSVG-4×FKBPF36M (bottom panels) were expressed in COS7 cells. Note the similar clustered appearance of both fusion proteins in the left panels. The right panels show the distribution of fusion proteins 5 min after UV-B exposure (top right) or addition of 1 µM AP21998 (bottom right). Note that UV-B treatment completely dissociates VSVG clusters, revealing the reticular structure of the ER (inset, top right), but AP21988 did not completely dissociate VSVG-4×FKBPF36M clusters. Bar, 10 µm (inset, 2.5 µm). (C) Trafficking of VSVG-YFP-2×UVR8 (green) from ER to Golgi membranes labeled with TGN38-RFP (red). VSVG-YFP-2×UVR8 was nearly completely localized to the Golgi 30 min after UV-B treatment. Bar, 10 µm. (D) Trafficking of UVR8 to the plasma membrane after UV-B treatment. Cos7 cells expressing mCh (red) and VSVG-YFP-2×UVR8 (green) were treated with UV-B for 7 s and imaged periodically over the course of 2 h. The bottom panels show the periphery of the cell with the green channel adjusted so that the plasma membrane signal is visible. Bars: (top panels) 10 µm; (bottom panels) 2.5 µm. (E) Kinetics of Golgi complex accumulation and release after UV-B treatment. Shown is a representative example from one cell out of ten cells imaged from five independent experiments. (F) Distribution of sizes of post-Golgi carriers. Full width at half maximal (FWHM) values for mobile post-Golgi carriers were determined by a Gaussian fit of line scans across the carriers (see Video 4). The inset shows a representative Gaussian fit (black line) to the line scan data (red circles). 93 carriers were quantified from 3 different cells from a single experiment. (G) Cells expressing mCh (red) and VSVG-YFP-2×UVR8 (green, top panels) were surface labeled with anti-VSVG at various times after UV-B treatment (bottom panels). Bar, 10 µm. (H) Quantification of the surface label at different time points is shown in the bar graph to the right. Data are expressed as a normalized ratio of surface (anti-VSVG) signal to the total YFP signal. Error bars represent the standard deviation of the mean from three different experiments. At least four cells were measured for each time point. (I) TIRF microscopy visualizing fusion of post-Golgi carriers with the plasma membrane. A wide-field fluorescent image of a COS7 cell expressing mCherry (red) and VSVG-YFP-3×UVR8 (green) is shown in the left panel and the TIRFM image of the same cell is in the middle panel. Bar, 10 µm. The image sequence to the right shows TIRFM imaging of post-Golgi carriers fusing with the plasma membrane. Bar, 1 µm.
