Large GFP–COL structures are positive for ER markers and Hsp47 but not COPII. Confocal imaging of RPE-1 cells stably expressing GFP–COL (green; GFP–COL–RPE) cotransfected with either ST-Cherry (red; A, B, D; 16–20 h after transfection) or the ER membrane marker ERM-mScarlet-i (red; C; 6–8 h after transfection). Cells were fixed after the given time points (minutes after addition of asc/biotin [500 µg·ml⁻¹ and 400 µM, respectively]) and labeled after fixation with antibodies against further proteins of interest. Large panels show whole cells, while smaller panels show corresponding enlargements of areas of interest with the separate channels in gray scale, followed by the merge image including DAPI (acquired as separate channel and displayed in magenta in A, B, and D or blue in C). Scale bars, 10 µm; in enlargements, 1 µm. n ≥ 10. (A) Maximum projection images of z stacks containing the Golgi apparatus. Duration of asc/biotin corresponds to duration of prior live imaging at approximately one image every 30 s until trafficking of GFP–COL to the Golgi was detectable by eye. Cells were labeled with the cis/medial Golgi marker Giantin (blue). Image i (after corresponding Video 8, i) shows an accumulation of GFP–COL at the edge of the Golgi, without visible large GFP-positive structures. Small puncta can be observed in the cell periphery and close to the Golgi (images i and ii, arrows). Large GFP–COL-positive structures are negative for both the trans- and cis/medial-Golgi markers (image ii, circles). The corresponding video of live imaging before fixation of image ii is shown in Video 6 with image stills shown in Fig. 2, A and B. (B) Duration of asc/biotin corresponds to duration of prior live imaging at approximately one image every 30 s until trafficking of GFP–COL to the Golgi was detectable by eye (Video 8, ii). The GFP–COL within the ER at 57 min asc/biotin colocalizes with Hsp47 (blue) in the ER (images i and ii). The GFP–COL puncta in the cell periphery and close to the Golgi are negative for Hsp47 and the trans-Golgi marker ST-Cherry (arrows). Cells that show large (>1-µm diameter) GFP–COL-positive structures appear negative for the trans-Golgi but positive for Hsp47 (image i, circles). (C) Time point indicates incubation with 400 µg·ml⁻¹ ascorbate before fixation. The GFP–COL colocalizes with the transiently expressed ERM-mScarlet-i (ERM; red) and Hsp47 (gray scale) in the ER, including large structures (images i and ii, circles). Small GFP-positive puncta, likely post-Golgi carriers, show no colocalization with ERM or Hsp47 (image i, arrows), while some small punctate GFP–COL structures are positive for Hsp47 but not ERM (image ii, square). (D) Maximum intensity projection images of z stacks of whole cells. Duration of asc/biotin corresponds to duration of prior live imaging at approximately one image every 30 s until trafficking of GFP–COL to the Golgi was detectable by eye (Video 8, iii and iv, respectively). The red channel marked as “Golgi” shows the combined signal from ST-Cherry and antibody-labeling for giantin in the same channel to enable complete visualization of the Golgi apparatus. Observed large GFP-positive structures do not colocalize with the COPII marker Sec31A (blue; image i, circles). Small GFP–COL puncta (<0.5 µm in diameter) close to the Golgi and in the cell periphery label for Sec31A (images i and ii, arrowheads). Most small punctate GFP-positive structures do not colocalize with Sec31A (images i and ii, arrows). (E) Size distribution of GFP-positive objects relative to colocalization with the COPII marker Sec31A from confocal z stacks with Δz = 0.29 µm and a sufficient number of slices to represent whole cells (as shown in D). The x axis shows the calculated object diameter of GFP-positive objects in micrometer, while the y axis shows the calculated percentage overlap of GFP-positive objects with Sec31A. Images for analysis were obtained after live-cell microscopy, as described in Figs. 1 and 2, and samples were fixed at the time when an accumulation of GFP–COL at the Golgi was visible by eye. In 11 of the 55 analyzed cells, no punctate or large GFP–COL objects were detected. These cells only show a GFP–COL accumulation around or in the Golgi. The remaining 44 cells show a total number of 1,149 detected objects in the GFP channel.