Analysis of cargo accumulation and carrier formation in living cells. (A) Accumulation of VSVG-YFP cargo and fission of carriers from ERESs in living cells. Confocal images were captured after the shift to the permissive temperature of COS7 cells coexpressing COPII subunit Sec24C-mCherry (red) and the cargo protein VSVG-YFP (green). See Video 4. Scale bar = 2 µm. (B) Cargo accumulation in a single ERES and carrier fission. Confocal images captured at 2.6-s intervals of COS7 cells transfected and treated as in A. VSVG-YFP (green) accumulation downstream to Sec24C-mCherry (red) is shown in two top panels and fission of carriers in the two bottom panels. See Video 5, Video 6, Video 7, and Video 8. Scale bar = 1 µm. (C) Analysis of ER export using the RUSH system. Images were captured after the addition of biotin to living COS7 cells 48 h after cotransfection of Sec24C-mCherry (red) and the cargo protein RUSH-TNF-GFP (green). Yellow arrowheads point to carrier elongating and budding. Scale bar = 1 µm. (D and E) Budding of a carrier from ERES in Sec13-mCherry (red) CRISPR/CAS12 knock-in HeLa cell clone transfected with VSVG-EGFP (green). White arrows and arrowheads point to carriers. Scale bar = 1 µm.