LRBA-positive tubules are highly dynamic sites of clathrin-coated vesicle formation. (A) HeLa cells stably expressing 3xFlag-EGFP-LRBA and ARF1-mScarlet were imaged live. Scale bars 10 µm (left merged) or 1 µm (right merged). (B) HeLa cells stably expressing 3xFlag-EGFP-NBEA and ARF1-mScarlet were imaged live. Scale bars 10 µm (left merged) or 1 µm (right merged). (C) HeLa cells stably expressing 3xFlag-EGFP-NBEAL1 and ARF1-mScarlet were imaged live. Scale bars 10 µm (left merged) or 1 µm (right merged). (D) HeLa cells stably expressing 3xFlag-EGFP-NBEAL2 and ARF1-mScarlet were imaged live. Scale bars 10 µm (left merged) or 1 µm (right merged). (E) Quantification of colocalization between structures positive for either LRBA, NBEA, NBEAL1 or NBEAL2, and ARF1. HeLa cells stably expressing ARF1-mScarlet and the indicated BDCP tagged with 3xFlag-EGFP were imaged with confocal microscopy. EGFP or mScarlet positive structures were segmented and each dot represent the percentage of BDCP positive objects in each cell partially or completely overlapping with ARF1 positive object. n ≥ 15 per condition. (F) Schematic summary of the colocalization of LRBA/NBEA, NBEAL1, and NBEAL2-positive tubular compartments with the clathrin/COPI coat recruitment factor ARF1. PM- plasma membrane, EE/SE- early/sorting endosomes, RE- recycling endosomes, GtPM- Golgi to plasma membrane carriers, TGN- trans-Golgi network. (G) HeLa cells stably expressing 3xFlag-EGFP-LRBA and mScarlet-SEC31A (COPII marker, left panel) or mScarlet-COPE (COPI marker, right panel) were imaged live. Scale bars 10 µm (main figure) or 1 µm (inset magnifications). (H) HeLa cells stably expressing 3xFlag-EGFP-LRBA together with clathrin coat adaptors AP1M1-mScarlet (upper left), AP3M1-mScarlet (middle left), GGA1-mScarlet (lower left), GGA3-mScarlet (upper right), mScarlet-EpsinR (middle right), and mScarlet-clathrin heavy chain (lower right) were imaged live. Scale bars 10 µm (left merged) or 1 µm (right merged).