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First published online 26 February 2008
doi: 10.1242/jcs.019463

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Quantitative live-cell analysis of microtubule-uncoupled cargo-protein sorting in the ER

Department of Pathology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel

View larger version (74K): [in this window] [in a new window]   Fig. 1. ER export of VSVG-FP and Golgi redistribution upon microtubule depolymerization. (A) Confocal images of living COS7 cells coexpressing GalT-YFP (green) and VSVG-CFP (red). For complete microtubule depolymerization, cells were transferred to ice for 20 minutes following a 24 hour incubation at the nonpermissive temperature (40°C) to accumulate VSVG in the ER. Nocodazole (1 µg/ml) was added and images of the cells at the permissive temperature (34°C) were captured for the indicated times. Inserts are enlarged twofold to show the appearance of GalT-YFP and VSVG-CFP in the ER exit sites. Scale bars: 10 µm. (B) Quantitative analysis of the time-dependent decrease in fluorescence intensity (FI) in a region of interest over the Golgi complex of the top cell in A. The line is a simple exponential fit with a rate of 1.23% per minute (R2=0.97). (C) Analysis of the ER export of VSVG-CFP and GalT-YFP in nocodazole-treated cells. Graph shows the time-dependent relative change in variance of fluorescence intensity in a region of interest over the cell in an area that excludes the pre-redistributed Golgi complex.

View larger version (88K): [in this window] [in a new window]   Fig. 2. Secretory traffic of VSVG-FP in the absence of microtubules. Confocal images of fixed COS7 cells coexpressing VSVG-YFP (green, A-B) and either the ER marker Srβ-CFP (A, red) or the ER exit site marker Sec31-CFP (B, red). For complete microtubule depolymerization, cells were transferred to ice for 20 minutes following a 24 hour incubation at the nonpermissive temperature (40°C) to accumulate VSVG in the ER. Nocodazole (1 µg/ml) was added and the cells were incubated at the permissive temperature (34°C) for the indicated times before addition of formaldehyde. Arrowheads indicate reticular and flat ER membranes. Scale bars: 10 µm.

View larger version (40K): [in this window] [in a new window]   Fig. 3. Analysis of ER-to-ER exit site (ERES) sorting of VSVG-FP. (A) VSVG-GFP-expressing COS7 cells were incubated for 24 hours at the nonpermissive temperature (40°C) to accumulate VSVG in the ER. Cells were then transferred to ice for 20 minutes and 1 µg/ml nocodazole was added before an additional 20-minute incubation at 40°C. Image analysis was initiated upon transfer of the cells to the permissive temperature (34°C). Images were captured at 20-second intervals. Lower panels, 4x enlarged inserts to show the accumulation in ERESs (see also supplementary material Movie 1). (B) Time-dependent increase in relative PFIVar, calculated as described in the Materials and Methods (black and gray circles) for two representative cells. Data were fitted to an exponential equation (y = aekx) (lines). R2 values were 0.99 for both data sets, k values were 0.0494 second–1 and 0.0409 second–1. (C) Pseudocolor images (upper panel) and surface-plot (lower panel) of a single ERES during cargo sorting (see also corresponding supplementary material Movie 2). (D) Time-dependent increase in average fluorescence intensity of a region of interest within a single ERES, calculated as described in Materials and Methods (black full circles) for a representative ERES. Data were fitted to the sigmoid equation y=864+(1796/(1+e4.25–0.26x) (lines). R2 value is 0.98. Insert is a plot of normalized and averaged data from 10 cells. Error bars indicate s.e.m.

View larger version (47K): [in this window] [in a new window]   Fig. 4. FRAP analysis of VSVG diffusion and reverse ER exit site-to-ER flux. (A) Cells expressing VSVG-GFP were treated with nocodazole as described previously. 25 minutes after shift to the permissive temperature, most of the cell was photobleached, apart from the area circled in yellow. Images were captured at 20-second intervals for an additional 20 minutes. Lower panel, inverted and magnified images of the boxed areas in the upper panel (see also corresponding supplementary material Movie 3). Scale bar: 10 µm. (B) Quantitative analysis of the average and total (insert) fluorescence intensities of the bleached (open circles) and unbleached (closed circles) regions of interest. Lines in the insert were generated by simulation using SAAM II software. The simple model used is illustrated below the graph. The rate constant obtained from the fit is 4% per minute. Insert was fitted to a linear equation (continuous line) indicating an increase in total fluorescence intensity of 0.23% per minute. (C) FRAP analysis of VSVG-GFP in the ER of nocodazole-treated COS7 cells. The rectangle photobleaching, image acquisition and extraction of the apparent diffusion coefficient are described in the Materials and Methods. Insert is a full-log-scale graph to show the quality of fit at all time points.

View larger version (52K): [in this window] [in a new window]   Fig. 5. Colocalization of VSVG-YFP and Sec31-CFP accumulation in ER exit site (ERES) membranes. (A) COS7 cells coexpressing VSVG-YFP (green) and Sec31-CFP (red) were treated as described in previous figures. Cells were transferred to the permissive temperature (34°C) and images were acquired at 2-minute intervals (see also supplementary material Movie 4). Scale bar: 10 µm. (B) Time-dependent increase in relative PFIVar of VSVG-YFP (green circles) and Sec31-CFP (red circles), calculated as described in the Materials and Methods, for the cell in A. Data were fitted to an exponential equation (y = aekx) (green and red lines for VSVG-YFP and Sec31-CFP, respectively). R2 values were 0.98 and 0.97, and k values were 6.4 and 1.65% per minute, for VSVG and Sec31, respectively.

View larger version (86K): [in this window] [in a new window]   Fig. 6. Relationship between ARF1 and cargo-protein sorting to ER exit sites (ERESs). Confocal images of fixed COS7 cells coexpressing VSVG-YFP (green, A-B) and either the ARF1-CFP (A, red), or ARF1Q71L-CFP (B, red). Cells were transferred to ice for 20 minutes following a 24 hour incubation at the nonpermissive temperature (40°C). Nocodazole (1 µg/ml) was added, and the cells were returned for an additional 20 minutes to the nonpermissive temperature (40°C). After incubation at the permissive temperature (34°C) for the indicated times, cells were fixed by addition of 2% (v/v) formaldehyde and images were captured as described. Scale bars: 10 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 7. Characterization of cargo concentration in brefeldin A (BFA) and nocodazole-induced dilated ER exit site (ERES) membranes. (A) Concentration of VSVG-FP in dilated membranes. COS7 cells, transfected with VSVG-FP, were shifted to the permissive temperature after overnight incubation at 39.5°C and 20 minutes at 4°C when 1 µg/ml nocodazole and 5 µg/ml BFA were added. Confocal images were captured at 20-second intervals (see supplementary material Movie 5). (B) Time-dependent increase in relative PFIVar of VSVG-YFP in the absence (average of 12 cells with s.e.m., filled circles) or presence (empty circles) of 5 µg/ml BFA, calculated as described in the Materials and Methods, for the cell in A. Data were fitted to an exponential equation (y = aekx) (green and red lines for VSVG-YFP and Sec31-CFP, respectively). R2 values were 0.93 and 0.98, and k values were 3.64 and 0.74% per minute, for control and BFA-treated cells, respectively. (C) BFA was added 15 minutes after the shift to the permissive temperature (see supplementary material Movie 6). Scale bars: 20 µm. (D) Three-dimensional reconstruction of a confocal z-section series of a single dilated ERES. (E) Analysis of time-dependent average fluorescence intensity of a single dilated ERES during two cycles of cargo concentration and blink-out. The time zones including an increase in VSVG fluorescence were fitted separately to exponential equations (continuous lines). (F) Pseudocolor images (upper panel) and surface plot (lower panel) of the dilated ERES in E during the time labeled by the blue arrow in E.

View larger version (86K): [in this window] [in a new window]   Fig. 8. Colocalization of secretory organelle markers with dilated ER exit site (ERES) membranes. (A) COS7 cells, cotransfected with CFP- or YFP-labeled VSVG (red) and Srβ-YFP, Sec13-YFP, Sec31-CFP, p58/ERGIC53-YFP and GRASP65-DiHcRED (green) were shifted to the permissive temperature (34°C) after overnight incubation at 39.5°C, followed by 15 minutes at 4°C when nocodazole and brefeldin A (BFA) were added. Confocal images were captured 30 minutes after the temperature shift. (B) COS7 cells were cotransfected with CFP-VSVG (red) and YFP-YFPAIA (green). (C) Scheme depicting the effects of BFA, nocodazole, and both, on ER export. BFA blocks ER export and causes COPI release from ERES membranes. Nocodazole causes the formation of Golgi mini-stacks adjacent to the ERESs. In the presence of nocodazole and BFA, cargo accumulates in the ERESs, which become dilated.

View larger version (40K): [in this window] [in a new window]   Fig. 9. Effect of cholesterol depletion on ER export in the presence of brefeldin A (BFA) and nocodazole. (A) COS7 cells, transfected with VSVG-FP, were shifted to the permissive temperature after overnight incubation at 39.5°C in medium containing LDL-deficient serum and 20 minutes at 4°C when 1 µg/ml nocodazole, 5 µg/ml BFA and 10 mM methyl-β-cyclodextrin were added. Confocal images were captured at 20-second intervals. (B) Analysis of time-dependent average fluorescence intensity (FI) of a single dilated ERES during several cycles of cargo concentration and blink-out in cholesterol-depleted cells. The time zones including an increase in VSVG fluorescence were fitted separately to exponential equations (continuous lines).

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