VAMP4 is packaged into clathrin-coated iISGs near TGNs and VAMP4-positive vesicles fuse with lysosomes. (A and B) INS-1 cells were transfected with EGFP-tagged VAMP4 (green) and mCherry-tagged (pro)insulin (red; A) or EGFP-tagged VAMP4 (green) and Halo-tagged (pro)insulin (red; B) plasmids for 48 h, and live-cell imaging was performed using the Delta Vision OMX V3 system with 100× (NA = 1.40). Scale bars, 5 μm. The enlarged diagrams indicate the Golgi (G) region and peripheral (P) region. Scale bars, 1 μm. (C) INS-1 cells were transfected with EGFP-tagged VAMP4 (green), Halo-tagged (pro)insulin (red), and mCherry-tagged clathrin (magenta) for 48 h, and live-cell imaging was performed using the Delta Vision OMX V3 system with 100× (NA = 1.40). The enlarged images show that VAMP4-positive iISGs colocalize with clathrin (indicated by arrows). Scale bars, 3 μm (left) and 0.5 μm (right). (D) INS-1 cells were transfected with EGFP-tagged VAMP4 (green), mCherry-tagged (pro)insulin (blue), and Halo-tagged LAMP1 (red) for 48 h, and live-cell imaging was performed using a Delta Vision OMX V3 system with 100× (NA = 1.40). The diagram and a snapshot of a consecutive event showed that VAMP4-positive vesicles fused with lysosomes (indicated by arrows). Scale bars, 0.5 μm.