JCS019463 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Relationship between protein-sorting dynamics in the ER and fluorescence-intensity variance values. (A) Images of COS7 cells expressing VSVG-GFP at 0 and 30 minutes after shift from non-permissive (40°C) to permissive (34°C) temperature. Scale bar:10 µm. (B) A scheme demonstrating the redistribution of VSVG-GFP shown in A. (C) Two tables representing digital images of a region of interest within the cells in A. Both tables have the same total (360) and average (10) fluorescence intensity values: the differential distribution is detected only in the variance values.

• Supplemental Figure S2 -

  Fig. S2. Analysis of ER-to-ER exit site (ERES) sorting of VSVG-FP at two permissive temperatures. (A) VSVG-GFP-expressing COS7 cells were incubated for 24 hours at non-permissive 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 prior to incubation for an additional 20 minutes at 40°C. Image analysis was initiated upon transfer of the cells to permissive temperatures (18°C top panel, 32°C lower panel). Images were captured at 20 second intervals. (B) Time-dependent increase in relative PFIVar, calculated as described in Materials and Methods. Average PFIVar values for 14 cells at 18°C (black full circles) and 12 cells at 32°C (red full circles) are plotted against time. Error bars are s.e.m. and lines are a fit to the equation y=a exp^kx. Obtained k values were 3.64±0.070% per minute and 0.93±0.010% per minute for 32°C and 18°C, respectively.

• Supplemental Figure S3 -

  Fig. S3. FRAP analysis of VSVG-FP recycling from ARF1Q71L-locked membranes. Cells coexpressing VSVG-YFP and ARFQ71L-CFP were treated with nocodazole as described previously: 25 minutes after shift to the permissive temperature, part of the cell (yellow region of interest) was photobleached using a high-power 514 nm Ar laser line (area circled in yellow). Images were captured at 30 second intervals for an additional 20 minutes. Scale bar: 10 µM.

• Supplemental Figure S4 -

  Fig. S4. FRAP analysis of VSVG sorting into brefeldin A (BFA)- and nocodazole-mediated dilated ERESs. Cells co-expressing VSVG-CFP and VSVG-YFP were treated with nocodazole during a 20-min incubation at 4°C and BFA was added 30 minutes after shift to permissive temperature (34°C) as described in Fig. 7C. At 55 minutes after the shift to the permissive temperature, the rectangular boxed area over the cell was photobleached using a high-power 514 nm Ar laser line. Images were captured at 27 second intervals for 77 minutes. Right-hand side: merged or inverted images of the bleached box. (B) Quantitative analysis of the FRAP in A. Time-dependent changes in average fluorescence intensities of a region of interest over the entire cell (red filled dots), the entire bleach box (black filled dots), and a region of interest including ER outside the bleached box (green filled dots). Lines were generated by fitting to exponential equations. (C) Analysis of two dilated ERESs: one inside the bleached box (black filled dots) and the other outside it (red filled dots). Lines were fitted to exponential equations. The decrease at 1200 seconds is due to blink-out.

• Movie 1 -

  Movie 1. 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 mg/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. See Fig. 3A

• Movie 2 -

  Movie 2. Pseudocolor image of a single ERES during cargo sorting. See Fig. 3C

• Movie 3 -

  Movie 3. Cells expressing VSVG-GFP were treated with nocodazole. 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. See Fig. 4A

• Movie 4 -

  Movie 4. COS7 cells co-expressing VSVG-YFP (green) and Sec31-CFP (red) after transfer to permissive temperature (34°C). Images were acquired at 2-minute intervals. See Fig. 5A

• Movie 5 -

  Movie 5. 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. See Fig. 7A.

• Movie 6 -

  Movie 6. 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. 5 µg/ml BFA was added 15 minutes after the shift to the permissive temperature. Confocal images were captured at 20 second intervals. See Fig. 7C.