Transport of GFP–COL to the Golgi occurs without the use of large carriers. Image stills from confocal live-cell imaging of RPE-1 cells stably expressing GFP–COL (green; GFP–COL–RPE) cotransfected with the trans-Golgi marker ST-Cherry (red). Time points indicate minutes after addition of asc/biotin. (A) A whole cell imaged at one frame every 20 s (derived from Video 6). Probable post-Golgi structures are indicated by arrows. (B) Corresponding enlargements of A shown with channels for GFP–COL and ST-Cherry in gray scale, as well as the overlay image. Large circular GFP–COL-positive structures appear negative for the trans-Golgi (B; circles). Similar smaller structures are positive for GFP and the Golgi marker (asterisks) and follow the previously described phenotype of concentration of GFP–COL at the edge of the Golgi (t = 14–18 min), followed by filling of the Golgi (t = 23 min). (C) Image stills of a whole cell taken at one frame every 30 s (derived from Video 7). Accumulation and filling of the Golgi with GFP–COL occurs within t = 10.5 min. (D) Corresponding enlargements of the Golgi area in C. Channels are displayed separately in gray scale followed by a merge image. A total of n = 4 sets was acquired. For each set of live imaging experiments, three cells from the same dish were imaged simultaneously. Scale bars, 10 µm; in enlargements, 1 µm.