Analysis of the life history of ER-to-Golgi carriers: budding translocation, fusion, and colocalization of cargo with the small GTPase Rab1b. (A) Photobleaching of the Golgi to expose ER-to-Golgi carrier life history. The Golgi region of interest was photobleached in the VSVG-GFP (green) channel after transfer to permissive temperature (32°C), following overnight at 39.5°C. Cells used were a HeLa cell clone expressing an endogenous (endo) Sec13-mCherry (center, and red in merged image) transfected with VSVG-GFP (bottom, and green in merged image). Scale bars = 5 µm. (B) Representative images from a time-lapse series captured after the FRAP in A. Arrowheads point to carrier translocating and fusing with the Golgi. Right: Projection of the images in B. See Video 9. Scale bar = 5 µm. (C) Rab1b localizes with cargo throughout the life history of ER-to-Golgi carriers. Huh7 cells were cotransfected with VSVG-YFP (center, and green in merged image) and Rab1-mCherry (red and inverted, bottom), were shifted to the permissive temperature of 32°C after an overnight in nonpermissive temperature (39.5°C). Representative images captured by confocal microscopy from a time-lapse series are shown with designated times. Arrowheads point to a single carrier budding from an ERES translocating and fusing with the Golgi apparatus. Right: A projection showing the path of the same carrier. See Video 10. Scale bar = 1 µm.