Figure 5. Rab31 and OCRL-1 colocalize in carriers that bud from the TGN.

Stably-transfected HeLa cells expressing OCRL-1-ECFP were transiently-transfected with a plasmid encoding Rab31-EYFP. Cells were transferred to recording medium. Formation and the transport of post-TGN carriers were monitored at 32° C by time-lapse fluorescence microscopy, images were collected every 1 s. A) Fluorescence pattern distribution. OCRL-1-ECFP (red), Rab31-EYFP (green) and overlapping of OCRL-1-ECFP and Rab31-EYFP (yellow). Rab31-EYFP and OCRL-1-ECFP co-localize in the TGN (arrow), and in endosomes throughout the cytoplasm (arrowheads). B) Individual frames showing a carrier containing both OCRL-1-ECFP and Rab31-EYFP breaking up from the TGN (white arrowheads). Time in seconds relative to the first image is shown in each frame. C) Maximum pixel projection. OCRL-1-ECFP and Rab31-EYFP co-localized at the TGN and in small vesicles present throughout the cytoplasm. D) Area where the events in B (carrier formation and movement to cell periphery) occurred is indicated by a white lane. E) Kymograph analysis of the area defined in (D). The TGN compartments, vertical yellow trace, is indicated with a white arrow. The carrier formation and its movement, diagonal yellow trace is indicated by the arrowhead. White arrowhead identifies the trace that corresponds to carrier budding shown in (B). Black arrows at the right indicate the time of budding shown in (B). X, distance in μm; t, time in seconds. Bar, 10 μm.