Figure 8. OCRL-1 and newly synthesized VSVG are sorted to different carriers budding from the TGN.

Stably-transfected HeLa cells expressing OCRL-1-ECFP were transfected with plasmid encoding VSVG-Venus, and then incubated at 40° C for 10-12 h to accumulate VSVG-Venus in the ER. Transfected cells were then incubated at 20° C for 3 h, with cycloheximide (100 μg/ml) to trap VSVG-Venus at the TGN. The cells were transferred to recording media at 32 ° C to induce transport of VSVG from Golgi to PM. Time-lapse photographs visualized the formation of carriers containing OCRL-1-ECFP and of carriers containing VSVG-Venus. Images were collected every 1.70 s. A) Fluorescence distribution pattern. Green, VSVG-Venus; red, OCRL-1-ECFP and yellow, overlapping of VSVG-Venus and OCRL-1-ECFP. OCRL-1-ECFP and VSVG-Venus co-localized at the TGN (yellow arrow). Small vesicles present in the cytoplasm only contain VSVG-Venus (green arrowheads) or OCRL-1-ECFP (red arrowheads). B) Top panels, individual frames of the area marked with red box in (A). A TGN tubule containing OCRL-1-ECFP extends (arrow), and breaks up to form a carrier (arrowheads). Bottom panels, individual frames of the area marked with green box in (A). A TGN tubule containing VSVG-Venus extends (arrow), and breaks up to form a carrier (arrowheads). C) Area where the events in B occurred (top panels) is indicated by a white line. D) Kymograph analysis of the area defined in (C). TGN compartments, vertical traces (white arrow). The red trace that corresponds to the budding of carrier containing OCRL-1-ECFP seen in (B) is indicated by a white arrowhead. Arrows at the right indicate the time of budding in (B). E) Area where the events in B occurred (bottom panels) is indicated by a white line. F) Kymograph analysis of the area defined in (E). TGN compartments, vertical traces (white arrow). The green trace that corresponds to the budding of carrier containing VSVG-Venus seen in (B) is indicated by a white arrowhead. Arrows on the right indicate the time of budding in (B). X, distance in μm; t, time in seconds. G) Maximum pixel projection. OCRL-1-ECFP and VSVG-Venus colocalized in the TGN but not in small vesicles present throughout the cytoplasm. Bar, 10 μm.