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Lumenal targeted GFP, used as a marker of soluble cargo, visualises rapid ERGIC to Golgi traffic by a tubulo-vesicular network

Robert Blum^1,2, David J. Stephens³ and Irene Schulz¹

lumenal GFP: ERGIC-to-Golgi path, rewarming from 15°C to 30°C.

The perinuclear area of the investigated cells was bleached by high energy confocal laser scans. Subsequently, cells were rewarmed to visualise the direction of fluorescence movement. As can been seen, an ERGIC-element flows via a tubular process to the Golgi complex and recovers fluorescence in the Golgi complex. After arrival in the Golgi area, the movement stops. Details in Fig. 3A originate from this data set. The cell expresses low levels of luminal GFP. Resolution: 0.098µm/pixel, interval: 7 sec.

lumenal GFP: ERGIC-to-Golgi path, rewarming from 15°C to 33°C.

The translocation of pre-Golgi carriers from the ERGIC to the Golgi complex occurred on or through pre-existing tubular structures. Before rewarming, the perinuclear Golgi area had been partially bleached. Formation of a pre-Golgi network that serves as "track" for carrier movements is visible. Details in Fig. 3B originate from this data set. Resolution: 0.118µm/pixel, interval 7 sec.

lumenal GFP: ERGIC-to-Golgi path, rewarming from 15°C to 34°C.

De novo formation of a pre-Golgi tubular network. Before rewarming, the perinuclear Golgi area had partially been bleached. An ERGIC element was the starting point for the formation of a pre-Golgi tubular network. Resolution: 0.098µm/pixel, interval 7 sec.

lumenal GFP: ERGIC-to-Golgi path, rewarming from 15°C to 30°C.

To verify that the transport tubules really represent transport of lum-GFP in the anterograde ERGIC-to-Golgi and not in the retrograde direction we performed experiments of fluorescence recovery after photobleaching (FRAP). Before rewarming of the cells, the perinuclear Golgi area was bleached by high energy scans with a confocal microscope and recovery of the fluorescence in the perinuclear Golgi area was visualised over time. Living cells revealed that pre-Golgi carriers and long tubules were stained if lum-GFP travelled over long-distances from the peripheral ERGIC to perinuclear Golgi and recover fluorescence in the previously bleached perinuclear Golgi area. Resolution: 0.118µm/pixel, interval 7 sec.

lumenal GFP: ERGIC to cell surface, rewarming from 15°C to 34°C.

In living cells, the time point at which tubular-vesicular pre-Golgi transport was reduced and mainly typical post-Golgi transport structures were visible could be determined at 12 to 18 minutes after rewarming. In this movie two white squares in the left lower site of the movie mark the time period between 12 to 15 min after rewarming to 33°C. In this time period, the main portion of tubular pre-Golgi elements disappears and after that period mainly post-Golgi transport carriers are visible. Four cells exhibiting different lum-GFP expression rates are visible. Resolution: 0.196µm/pixel, interval 7 sec.

lumenal GFP: Golgi to cell surface, 33°C, 20min after rewarming.

20min after rewarming, luminal GFP stains mainly "small" vesicle-like post-Golgi transport carriers. Some of these carriers move to the cell surface where they disappear, indicating that the fluorescent material has been extruded. Resolution: 0.098µm/pixel, interval 7 sec.

lumenal GFP: ERGIC-to-Golgi, rewarming 15°C to 30°C

Example of typical structures (tubular elements, pre-Golgi carrier-like elements) appearing after rewarming. Simultaneously to pre-Golgi structures, some typical post-Golgi containers are visible on their way to the plasmamembrane (especially in the right lower part of the movie). Resolution: 0.098µm/pixel, interval 10 sec.

GFP-p23: ERGIC-to-Golgi, rewarming 15°C to 33°C.C

GFP-p23 is the putative COPI-receptor and is known to travel from the ERGIC to the Golgi by pre-Golgi carriers (Blum et al, 1999, J. Cell Science, 112, 537-548). The movie gives an example of typical GFP-p23 carrier-like movement. Movement of GFP-p23 from the ERGIC to Golgi also involves small tubular elements that often appear if peripheral elements are in movement. GFP-p23 does not stain tubular-vesicular networks as they appear if luminal-GFP flows anterogradely. Additionally, small vesicle-like carriers are visible that move from the Golgi area to the periphery. In other time-lapse sequences, small vesicle-like carriers also move from the periphery to the Golgi area. Resolution: 0.118µm/pixel, interval 7 sec.

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