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Files in this Data Supplement:
Fig. S1. GFP-ERGIC-53 localizes and oligomerizes like endogenous ERGIC-53. (A) HeLa cells stably expressing GFP-ERGIC-53 were treated with NaBu overnight and recovered from the induction for 24 hours. Together with parental HeLa cells, they were permeabilized with TX-100 and stained for ERGIC-53. Images were taken with a confocal fluorescence microscope using the laser excitations 488 for EGFP and 568 for the antibody staining. Bar, 5 mm. (B) GFP-ERGIC-53 cells treated with NaBu, or non-transfected cells were pulsed with 35S-methionine and chased for the indicated times. Proteins were immunoprecipitated either with anti-ERGIC-53 or with anti-GFP as indicated. Immunoprecipitates were analyzed by non-reducing SDS-PAGE and visualized by fluorography. During the time course, dimers (lower bands) disappear as hexamers (higher bands) form until a steady state is reached when each species is represented to 50%. The arrows indicate endogenous ERGIC-53 dimers (lower arrow) and hexamers (upper arrow). GFP-ERGIC-53 dimers appear as two bands corresponding to homodimers (lower arrowheads) and heterodimers with endogenous ERGIC-53 (lower brackets). These dimers are converted to hetero-hexamers (upper brackets) and homo-hexamers (upper arrowheads).
Movie 1. Fluorescence recovery after photobleaching of GFP-ERGIC-53. HeLa cells expressing GFP-ERGIC-53 were treated with NaBu overnight. They were bleached for 30 seconds (square) and imaged every 10 seconds for 9 minutes during recovery in the presence of 50 mg/ml cycloheximide. The recovery of completely bleached spots and two partially bleached moving structures (arrows) is seen. Movie is ×3 accelerated relative to real time.
Movie 2. Live imaging of GFP-ERGIC-53 (slow imaging). Supplement to Fig. 2A. HeLa cells expressing GFP-ERGIC-53 were treated with NaBu overnight and imaged every 10 seconds for 15 minutes. Several moving structures undergoing fusion, splitting, disappearance and re-emergence are seen. Movie is ×100 accelerated relative to real time.
Movie 3. Live imaging of GFP-ERGIC-53 (4D imaging). Supplement to Fig. 2B. Cells treated as in movie 1 were imaged in the xyz directions every 10 seconds for 33 minutes. Several moving structures undergoing fusion, splitting, disappearance and re-emergence are seen, many of which are long-lived. Movie is ×100 accelerated relative to real time.
Movie 4. Live imaging of GFP-ERGIC-53 (fast imaging). Supplement to Fig. 2C. Cells treated as in movie 1 were imaged every 0.2 second for 1 minute. Several stationary and fast moving structures are seen. Movie is ×4 accelerated relative to real time.
Movie 5. H89 blocks GFP-ERGIC-53 in the ER. Supplement to Fig. 3A. HeLa cells expressing GFP-ERGIC-53 were imaged during H89 block every 10 seconds for 18 minutes. Note that the ERGIC structures in the periphery and in the Golgi area disappear simultaneously while fluorescence of the ER increases. Movie is ×100 accelerated relative to real time.
Movie 6. Recovery from H89 block. Supplement to Fig. 3B. The same cell as movie 4 was imaged during recovery from the H89 block every 10 seconds for 26 minutes. Note that the ERGIC structures in the periphery and in the Golgi area emerge simultaneously while the fluorescence of the ER decreases. Movie is ×100 accelerated relative to real time.
Movie 7. Recovery from H89 block. HeLa cells expressing GFP-ERGIC-53 were incubated for 20 minutes with H89. To exclude possible photo-damage effects, they were imaged only during recovery from the H89 block every 10 seconds for 15 minutes. Note that, as in movie 6, the ERGIC structures in the periphery and in the Golgi area emerge simultaneously while the fluorescence of the ER decreases.100x accelerated.
Movie 8. GFP-ERGIC-53 tubule formation during rewarming from 16°C. Supplement to Fig. 4. HeLa cells stably expressing GFP-ERGIC-53 were imaged every 0.6 second for 1 minute 54 seconds. Several elongated structures are seen to move randomly. Movie is ×6 accelerated relative to real time.
Movie 9. Sorting of GFP-ERGIC-53 and ssDsRed in the ERGIC. Supplement to Fig. 6A. HeLa cells stably expressing GFP-ERGIC-53 and transiently transfected with ssDsRed were imaged during rewarming from a 16°C block every 10 seconds for 20 minutes. Several co-labeled structures are seen to segregate in the periphery (arrows). ER fluorescence of ssDsRed diminishes with time due to transport of ssDsRed to the Golgi. From time 14.10 large ssDsRed spots start to exit the Golgi. These are post-Golgi carriers. Green: GFP-ERGIC-53; red: ssDsred. Movie is ×50 accelerated relative to real time.
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