Controllable ER-to-Golgi trafficking of GFP–COL in the absence of large carriers. (A) The GFP–COL fusion construct. It consists of a synthetic prosequence of human COL1A1 followed by an SBP, mGFP, the N-propeptide cleavage site (indicated by the arrowhead) within the nonhelical region, and the triple-helical domain with the C-terminal nonhelical region followed by the C-propeptide of human COL1A1. The signal sequence is indicated by the asterisk. (B and C) Widefield microscopy of cells transiently expressing GFP-COL (A). 16 h after transfection, GFP-COL (green) colocalized with Hsp47 (red) in the ER in IMR-90 (B, image i) and RPE-1 (B, image ii) cells. Large images show whole cells, while the small panels show the enlargements with the corresponding channels in gray scale and the merged image including nuclear DAPI staining (blue). (C) Same image setup as in B with GFP–COL (green) and cis-Golgi marker GM130 (red) labeling in transiently GFP–COL-expressing IMR-90 cells. Time points indicate length of incubation in the presence of 50 µg·ml⁻¹ ascorbate before fixation. A concentration of GFP–COL in the Golgi can be observed after both 30 (C, image ii) and 60 min (C, image iii) in the presence of ascorbate. Number of cells imaged n ≥ 10. (D) Confocal images from live-cell imaging of RPE-1 cells stably expressing GFP–COL (green; GFP–COL–RPE) cotransfected with the trans-Golgi marker ST-Cherry (magenta; using the RUSH system). Acquisition at one frame every 30 s. Image layout as in C, followed by line graphs showing the signal intensity (y axis) in arbitrary units for GFP–COL (green) and ST-Cherry (magenta) for the corresponding line with a 5-pixel width drawn through the Golgi in the enlarged overlays. The x axis shows the distance in micrometers. Image stills are derived from Video 1. Time points indicate minutes of incubation in presence of 500 µg·ml⁻¹ ascorbate and 400 µM biotin (asc/biotin). At time point 0 (t = 0 min), GFP–COL and ST-Cherry show distinct localization. Over time an accumulation of GFP–COL around the Golgi marker occurs and increases until t = 18 min, with subsequent filling of the trans-Golgi (t = 24 min). Later time points show the decrease in intensity of GFP–COL overall and in the Golgi area (t = 29, 40, and 51 min), implying functional transport to the Golgi, followed by secretion from the cell. For each set of live imaging experiments, three cells from the same dish were imaged simultaneously. A total of n = 4 sets was acquired. Scale bars, 10 µm; in enlargements, 1 µm.