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. 2008 Apr 7;181(1):105–118. doi: 10.1083/jcb.200710093

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Copyright © 2008, The Rockefeller University Press

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Figure 6. — Analysis of FP-22 and VSVG-YFP during export from the same ERES. (a) Imaging of fixed cells. CV1 cells, microinjected with the cDNAs coding for Cerulean-22 and VSVG-Venus, were incubated for 75 min at 39.5°C and then for 3 h at 10°C before fixation. Cells were immunostained for Sec23, using Cy5-conjugated secondary antibodies, and analyzed by confocal microscopy. Single-channel images for the three proteins, and a merged image with the indicated pseudocolors are shown. The arrowheads indicate Sec23-positive structures containing both FP-22 and VSVG. The arrows indicate a structure positive for Sec23 and VSVG but lacking Cerulean-22. Bar, 10 μm. (b) Time-lapse imaging of carriers leaving the ER with VSVG and FP-22 cargo. Cells were microinjected, incubated as described in a, and then imaged while warming to 20°C (see Materials and methods). Each column shows images of the same field at the indicated times after the start of imaging in the Cerulean and YFP channels, as well as the merge of the two channels (bottom) with the indicated pseudocolors. Arrowheads indicate a moving carrier and open circles in the merge indicate the initial position of the structure. Bar, 5 μm. (c) Results of quantification of images like the one in a. The percentage of ERES positive for the indicated protein is shown. Numbers are means ± SD (n = 6). (d) Mean displacement versus time of moving carriers. Initiation of movement at different ERES was not synchronous, and the 0 time point corresponds to the last image before initiation of movement for each carrier. For each carrier at each time point, the displacement was calculated as the sum of displacements occurring during all preceding time intervals. Shown are means ± SD. (e) FI in the Cerulean and YFP channels of moving carriers normalized to the FI at their origin. Carriers in each of seven images were classified according to displacement from the origin within the distance intervals indicated on the abscissa. Means ± SD are shown. The data in d and e were acquired from the analysis of five cells.

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