Figure 7.

Quantitation of fluorescence intensity in post Golgi intermediates. (A) Upper and lower non-Golgi regions (white rectangles) of a cell expressing VSVG–GFP were repetitively photobleached using high energy illumination 40 min after a shift to 32°C. The entire cell was then imaged with low illumination levels for 36 min at 3.4 images/min. See Quicktime movie sequence at http://dir.nichd.nih.gov/cbmb/pb7labob.html. The ROI that was analyzed is shown in the far right panel. (B) The representative ROI at different times after photobleaching. Bottom panel is an overlay of all the images taken during the 36 min after photobleaching. Note the many paths followed by individual PGCs to the plasma membrane. (C) The compartmental model used to determine the fraction of VSVG–GFP conveyed to the PM in PGCs (Materials and Methods). (D) The fluorescent intensity in PGCs (defined as structures larger than 0.2 μm² or five pixels) (solid circles) and the total fluorescence (i.e., the sum of fluorescence intensity in PGCs, PM, and all other fluorescence; open circles) within the ROIs are plotted against time. The optimal model solution generated by the model in C is shown by solid lines (Materials and Methods). The rate constants obtained by least squares fitting were 1.94 ± 0.47 (SD)% per min for the Golgi-to-PGC pathway and 1.22 ± 0.32 (SD)% per min for the Golgi-to-other pathway. Given this model structure, the data also permitted resolution of the VSVG–GFP residence time in measured PGCs (3.38 ± 0.26 [SD] min), but could not resolve the VSVG–GFP residence time in carriers associated with the “other” pathway. Optimization required that the residence time in the “other” pathway was no more than 0.2 min.