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Imaging of procollagen transport reveals COPI-dependent cargo sorting during ER-to-Golgi transport in mammalian cells

Cell Biology and Cell Biophysics Programme, EMBL Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg, Germany

View larger version (123K): [in a new window]   Fig. 1. Expression-level-dependent and ascorbate-dependent localisation of PC-GFP. Cells expressing low levels of PC-GFP, 3 hours after microinjection of plasmid DNA, localise the protein to the ER in the absence of ascorbate (A), but 10 minutes after the addition of ascorbate, PC-FP localises to punctate TCs that traffic to the Golgi (B). Time lapse movies showing the dynamics of procollagen in the absence (Movie 1) and presence (movie 2) of ascorbate are available at jcs.biologists.com/supplemental . In contrast cells expressing high levels of PC-GFP, 6 hours after microinjection of plasmid DNA, localise the protein, in the absence of ascorbate, to aggregates located within the ER (C). The exposure times for (B) and (C) are the same; arrowheads are ndicated TCs in (B) and aggregates in (C). Cells expressing such high levels of PC-FP were not used during any of the experiments described here. PC-GFP is intact when expressed in cells. Bars=5 µm. (D) shows an immunoblot with the anti-procollagen antibody LF-39 of mock-transfected HeLa cells (lane 1) or HeLa cells transfected with PC-GFP (lane 2). The mobility shift in lane 2 is caused by the presence of the GFP moiety and partial glycosylation.

View larger version (81K): [in a new window]   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 .

View larger version (90K): [in a new window]   Fig. 6. Transport of PC-FP through the early secretory pathway is dependent on the function of the COPI and COPII coat complexes. (A,B) Cells expressing PC-FP were microinjected with a control plasmid (pcDNA3.1 (A); arrowheads indicate examples of TCs) or plasmid expressing SAR1a(H79G) (B) and incubated at 37°C for 2 hours followed by addition of ascorbate. Images were taken 30 minutes after ascorbate addition. Efficacy of injected SAR1a(H79G) expression was confirmed by anti-ERGIC-53 labelling (not shown). Control cells (A) were located on the same dish as SAR1a(H79G) expressing cells (B) for these experiments. (C,D) Cells expressing PC-FP were microinjected with a control plasmid (C; arrowheads indicate examples of TCs) or plasmid expressing pARF1(Q71L) (D). Ascorbate was added to the medium 4 hours after microinjection. Cells were imaged 30 minutes after the addition of ascorbate. (E,F) Microinjection of anti-EAGE blocks transport of PC-FP through the early secretory pathway. Cells expressing PC-CFP were injected with anti-EAGE in the absence of ascorbate and subsequently incubated with ascorbate for 1 hour following microinjection of anti-EAGE. (E) shows the cell before injection, (F) shows the same cell 1 hour after antibody injection. Note the differences in the ER labelling pattern showing the viability of the cell after injection. Bars=5 µm.

View larger version (120K): [in a new window]   Fig. 3. PC-GFP transport complexes do not label for ERGIC-53 or COPI during transport to the Golgi. Cells expressing PC-GFP in the presence of ascorbic acid were fixed 10 minutes after the addition of ascorbate and processed for immunofluorescence with antibodies directed against COPI (B) or ERGIC-53 (D). Note that PC-FP structures (A) do not label for COPI (B) and similarly PC-GFP TCs (C) do not label for ERGIC-53 (D, arrows). Bars=5 µm.

View larger version (101K): [in a new window]   Fig. 4. PC-GFP transport complexes do not label for ts-045-G during transport to the Golgi. Those that do label for COPI contain both PC-FP and ts-045-G. Cells expressing PC-FP and ts-045-G-FP were incubated for 16 hours at 39.5°C, after which ascorbate and cycloheximide were added to the culture medium for 30 minutes. Cells were then incubated for 6 minutes at 32°C, fixed with paraformaldehyde and processed for immunofluorescence. PC-CFP TCs (A,D) largely exclude ts-045-G (B,E; arrow) and COPI (C,F). (D-F) An enlargement of the corresponding regions in (A-C), respectively. Those TCs that do label for both PC-CFP (A,D; arrowhead) and ts-045-G-YFP (B,E; arrowhead) also label for COPI (C,F; arrowhead). In contrast, TCs containing ts-045-G-YFP (G; arrowhead) also contain ERGIC-53 (H; arrowhead) and COPI (I; arrowhead). Bars=5 µm.

View larger version (153K): [in a new window]   Fig. 5. Segregation of PC from ERGIC-53 occurs at the level of exit from the ER. Cells expressing PC-FP were incubated, in the presence of cycloheximide, at 15°C for 60 minutes prior to addition of ascorbate and further incubation at 15°C for 90 minutes. Cell were then fixed with paraformaldehyde at 15°C and processed for immunofluorescence with anti-ERGIC-53. The majority of PC-FP containing structures (A; arrows) do not colocalise with ERGIC-53 (B; arrows). Similarly, the majority of PC-FP TCs (C; arrows) do not colocalise with COPI labelled TCs (D; arrows). Occasional examples of COPI-labelled PC-FP containing TCs are seen (C,D; arrowhead). For quantitation of results see Table 1. Bars=5 µm.

View larger version (122K): [in a new window]   Fig. 7. PC-GFP dynamics following injection of anti-EAGE. Cells expressing PC-FP were incubated in the presence of ascorbate for 10 minutes prior to microinjection of anti-EAGE. When imaged immediately after injection, most PC-GFP-labelled structures were immobile (A; arrowhead). A small proportion (1-5 per cell) remained motile and trafficked to the Golgi (A; arrow) and (A1-A4, which shows time lapse images of the cell in A taken 3 seconds apart) (see also Movie 4 at jcs.biologists.org/supplemental ). At later time periods after injection (30 minutes in the continued presence of ascorbate) few punctate structures remain and significant ER labelling is seen in the cells but those that are still visible are immobile (B1-B4, showing images 3s apart, arrowheads mark static structures) (see also Movie 5 at jcs.biologists.org/supplemental ). Bars=5 µm.

View larger version (103K): [in a new window]   Fig. 8. Inhibition of COPI function by microinjection of anti-EAGE results in colocalisation of PC-FP with ERGIC-53. (A) and (B) show control cells expressing PC-FP (A) to which ascorbate was added 30 minutes before microinjection of control IgG. After a further 30 minutes at 37°C, cells were fixed and immunolabelled with anti-ERGIC-53 (B). Note structures containing PC-FP in (A; arrows) that do not label for ERGIC-53 in (B; arrows). (C,D) show cells expressing PC-FP (C) that, 30 minutes after the addition of ascorbate, were microinjected for anti-EAGE, incubated for a further 30 minutes at 37°C, fixed and immunolabelled for anti-ERGIC-53 (D). Note structures containing PC-FP in (C; arrowheads) that also label for ERGIC-53 in (D; arrowheads). The insets in (C) and (D) show zoomed areas from the highlighted regions. Bar=5 µm (applies to all panels).