Live-cell STED reveals segregated ARF1 and ARF4 subpopulations of tubular-vesicular structures defined by COPI machinery. (A and B) ARF1^EN/ARF4^EN-Halo (green) and βCOP^EN-SNAP (magenta) double KI HeLa cells were labeled with JF571-CA and JFX650-BG and imaged with a STED microscope. ARF1 and ARF4 tubular-vesicular structures are highlighted by white arrows and COPI clusters by yellow arrows. (C) Double KI cell lines ARF1^EN-, ARF3^EN-, ARF4^EN-, and ARF5^EN-Halo (magenta) and βCOP^EN-SNAP (green) were labeled with JFX650-CA and JF585-BG and subsequently treated with nocodazole (33 µM) for 3 h. (D) Scatter dot plot with mean and SD represents the quantification of the distances from the edge of the ARF-labeled cisternae to the center of COPI vesicles measured in nocodazole-treated live cells as described in Materials and methods. ARF1 n = 62; ARF3 n = 49; ARF4 n = 49; ARF5 n = 48. n = COPI vesicles from at least three independent experiments. All images were deconvolved, background subtracted, and smoothed as described in Materials and methods. Scale bars are 5 and 1 µm in the cropped images.