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COPI COATED ER-TO-GOLGI TRANSPORT COMPLEXES SEGREGATE FROM COPII IN CLOSE PROXIMITY TO ER EXIT SITES

Stephens, D.J.*, Lin-Marq, N.+, Pagano, A.+, Pepperkok, R.* and Paccaud, J.-P.+

* Department of Cell Biology and Biophysics, EMBL-Heidelberg, D-69117, Germany.
+ Department of Morphology, University of Geneva, 1211 Geneva 4, Switzerland.

Figure 2A:

Time lapse imaging of GFP-SEC24Dp expressed in Vero cells. Overlayed on to this image in red, yellow, blue and cyan, are the trajectories followed by five randomly chosen structures during 10 minutes of imaging. Most structures undergo only limited movement in a confined area while a minority of structures move greater distances (>5 microns). Images were taken every 10 seconds.

Figure 2B:

Time lapse imaging of GFP-SEC24Dp expressed in Vero cells following 30 minutes incubation in the presence of 5 mM nocodazole. Overlayed on to the first frame of the movie in red, yellow, blue and cyan, are the trajectories followed by six randomly chosen structures during 10 minutes of imaging. Images were taken every 10 seconds.

Figure 3A:

Fluorescence recovery after photobleaching of GFP-SEC24Dp structures. The marked (square) area was photobleached for 3 seconds using 100% laser power of the 488 nm line of a Zeiss LSM510 confocal microscope at t=0. Fluorescence recovery was subsequently monitored by acquiring images every 7.3 seconds at 0.2% laser power.

Figure 4A:

Time lapse imaging of YFP-SEC24Dp (pseudocoloured red) and CFP-ER (pseudocoloured green) expressing cells. YFP-SEC24Dp positive structures are associated with the endoplasmic reticulum and their movements are primarily due to movement of the ER network itself. Note that there is considerable movement from these starting points (arrowhead) during the course of the experiment but that the YFP-SEC24Dp structures remain associated with the ER membrane at all times. Images were acquired at 1 second intervals.

Figure 4B:

Enlarged region of the centre of A. As in figure 4A, YFP-SEC24Dp is pseudocoloured red and CFP-ER, pseudocoloured green. Images were acquired at 1 second intervals.

Figure 5B:

Dual visualisation of YFP-SEC24p and CFP-VSV-G in microinjected Vero cells. A. COPII (red) and VSV-G (green) initially colocalise but VSV-G translocates to the Golgi alone. Images were acquired at 1 second intervals.

Figure 5C:

CFP-VSV-G (green) structures are frequently visualised moving towards the Golgi apparatus but do not contain YFP-SEC24p (red). Images were acquired at 1 second intervals.

Figure 6A:

Dual visualisation of COPI and COPII complexes. COPII was labelled by expression of GFP-SEC24p, COPI by microinjection of expressing cells with Cy3-labelled CM1A10 which labels but does not interfere with the function of the COPI complex. A. COPI (red) and COPII (green) are seen to localise very close to one another initially, after which COPI alone is transported to the Golgi apparatus. Images were acquired at 1 second intervals.

Figure 6B:

COPI TCs (red) are often seen trafficking to the Golgi without being associated with COPII (green) at any point during imaging. Images were acquired at 1 second intervals.

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