Fig. 2. Ascorbate-dependent trafficking of PC-FP in living cells. (A) Low level expression of PC-FP in Vero cells in the absence of ascorbate. (B) Localisation of PC-FP 10 minutes after the addition of ascorbate. (C) and (D) show colocalisation of PC-CFP (C) and SEC24Dp-YFP (D) in living cells 2 minutes after the addition of ascorbate. (E) and (F) show an enlargement of the area bounded by the white box (width 5 µm) in (C) and (D), respectively. Examples of colocalisation of PC-FP with SEC24Dp-YFP are marked with arrowheads. (G-J) PC-FP exits the ER at or in close proximity to COPII-labelled ER exit sites. Still images from a time lapse series are shown, taken 5 minutes after the addition of ascorbate, showing part of a cell expressing PC-CFP (green) and YFP-SEC24Dp (red). Note the movement of SEC24Dp and PC-CFP together (H versus G) followed by segregation of PC-CFP (arrowhead) from YFP-SEC24Dp (arrow). The PC-CFP-labelled structure passes close by another YFP-SEC24Dp structure (J) before becoming lost amongst the large amount of label in the Golgi region. Images were taken 4 seconds apart. Bars=5 µm. Time lapse movies showing the dynamics of procollagen in the absence (Movie 1) and presence (Movie 2) of ascorbate and of the series of images shown in C-F (Movie 3) are available at jcs.biologists.org/supplemental .

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