JCS03058 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. MDCK cells overexpressing an ER-restricted variant of b5R generate SER patches upon treatment with PDMP. MDCK cells stably expressing the G2A mutant of b5R (Borgese et al., 1996) were immunostained with anti-b5R (A,B) or with anti-calnexin polyclonals (C). In the b5R-expressing cells, PDMP treatment induced the formation of SER patches with similar appearance to those seen in the GFP-b(5)tail HeLa TetOff cells. Smaller patches were seen only rarely (<5% of the cells) in non-transfected cells (C). Panels show brightest point projections of five optical sections taken at 0.2 μm intervals. Bars, 10 μm.

• Supplemental Figure 2A
• Supplemental Figure 2B -

  Fig. S2. Distribution of marker proteins in PDMP-treated HeLa TetOff cells expressing GFP-b(5)tail. Cells grown without doxycycline for 3 days were treated with 100 μM PDMP for 3 hours (with the exception of the cells in the second row of panel B), then fixed with paraformaldehyde and permeabilized. (A) Control for penetration of antibodies into saponin-permeabilised, RNase-treated cells (top row). Staining of cells with anti-GFP antibodies (central panel) results in a pattern superimposable on the endogenous GFP fluorescence; middle row: illustration of the variability of calreticulin staining in the SER patches. The arrow indicates an obvious SER patch that is poorly stained with the anti-calreticulin antibodies; bottom row: staining with monoclonal antibodies against α-tubulin (clone B-5-1-2 from Sigma) reveals a normal organisation of microtubules, which are seen to course through the SER patches. (B) Analysis of COPII and ERGIC-53. Samples were stained with anti-mammalian Sec23 antibodies (from Affinity Bioreagents, top row) or with anti-ERGIC-53 antiserum (a gift from Dr Stefano Bonatti, University of Naples Federico II, middle and bottom rows). Note that COPII-positive puncta appear to be excluded from SER patches, but are often seen at their periphery. In untreated GFP-b(5)tail-expressing cells, ERGIC-53 shows its typical punctate distribution, partly at peripheral sites, but more concentrated in paranuclear position (Schweizer, 1988). After PDMP treatment (bottom row) ERGIC-53 is present in SER patches, at varying concentration, but still remains concentrated in the paranuclear area. The asterisks indicate cells in which ERGIC-53 is either poorly represented in SER patches (red asterisks) or is more concentrated within them (green asterisks). Bars, 10 μm.

• Movie 1 -

  Movie 1. Live-cell analysis of PDMP-induced SER patch formation. Immediately after addition of PDMP, cells were placed in a thermostated CO₂ incubator and imaged for 3 hours. Frames were taken every 5 minutes. SER patches appeared after 1 hour of treatment in some cells and were present in essentially all the cells of this field after 3 hours. Once formed, the patches underwent shape changes, movements and fusion and fission events.

• Movie 2 -

  Movie 2. FRAP analysis of SER patches shows their continuity with the ER network. 20 frames were acquired every 10 seconds immediately after bleaching the boxed area. See legend to Fig. 3 for further explanation.

• Movie 3 -

  Movie 3. Rapid dispersal of PDMP-induced SER patches after removal of the drug. PDMP-treated cells were placed in drug-free medium and immediately subjected to time-lapse imaging by wide-field microscopy. 14 frames were acquired at 1-minute intervals. See legend to Fig. 5 for further explanation.