Fig. 7. Trafficking of transferrin in compartments marked by GFP-SCAMP1 at early and late times after uptake. (A) Overall image of a cell expressing GFP-SCAMP1. (B) Portion of a plasma membrane sheet from a GFP-SCAMP1 expressing cell labeled with anti-clathrin antibody (clat). (C,D) transferrin uptake in NRK cells stably expressing GFP-SCAMP1 was performed as in Fig. 6E-L and the internalization stopped after 20 seconds (C) and 1 minute (D). Cells were fixed and observed by fluorescence, and deconvolved sections are shown. (E,F) Cells expressing GFP-SCAMP1 were labeled for 10 minutes with Alexa 594-transferrin and chased for 90 seconds. (E) Image of a live cell. (F) Selected frames from supplementary material Movie 1 of the cell shown in (E). The open arrowheads track the appearance (upper left panel) and movement of both transferrin and GFP-SCAMP1. The apparent separation of transferrin and GFP-SCAMP1 is due to sequential capture of images from the two different channels. (G-J) Live imaging of transferrin and GFP-SCAMP1 in carriers recycling transferrin to the surface. (G) Live cell expressing GFP-SCAMP1 imaged at 40 minutes of chase following a 15-minute labeling with Alexa 594-transferrin. (H) Selected images from Movie 2 in supplementary material showing transferrin movement (arrowhead) while GFP-SCAMP1 remains stationary. (I) Three panels from supplementary material Movie 3 showing movement of GFP-SCAMP1 (arrowhead) and not transferrin. (J) Selected images of supplementary material Movie 4 showing that distinct puncta of transferrin and GFP-SCAMP1 are tethered to one another and appear to be trying to separate during relocation (arrowhead). Bars, 10 µm (A,E,G); 2 µm (D,J).