Fig. 4

a Schematic representation of the experimental setup in both in vitro and in vivo systems. COS-1 cells are transfected with DNA constructs expressing WT and mutant ARF3-mCherry (magenta) and EGFP-GalT (trans-Golgi marker, green) and analyzed by live confocal microscopy between 4 and 6 h post-transfection. Zebrafish embryos are injected at 1 cell stage with WT and mutant ARF3-mCherry and EGFP-GalT mRNA. mKOFP-CAAX mRNA is used as a membrane marker (cyan). Animals are analyzed by live confocal microscopy during gastrulation (~6?7 hpf). b, c Maximum intensity projections of confocal images of a single time-lapse experiment (Supplementary Movie 1) performed in transfected COS-1 cells at 15 min (~4 h post-transfection) and 120 min later (~6 h post-transfection) from the start of the time-lapse experiment. The images show diffused EGFP-GalT signal (trans-Golgi fragmentation) in ARF3^K127E over time (white arrows). Scale bar = 20 ?m. d, e 3D image reconstructions from live confocal acquisitions of the animal pole in developing zebrafish embryos expressing ARF3^WT and ARF3^K127E at the mid-gastrulation stage (~6 hpf). White arrowheads indicate a compact trans-Golgi morphology surrounding the nucleus (?ribbon?) in the EVL cells. Yellow arrowheads indicate cells showing ?punta? morphology of the trans-Golgi dispersed throughout the cytosol. Scale bars = 20 and 50 ?m. The images are representative of embryos from two independent batches. Quantification is shown in Supplementary Fig.  10a. Source data are provided as a Source Data file.