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Fig. 1. Brefeldin A treatment induces changes in the architecture of vimentin networks. (a) SW13 v+ cells were either treated with methanol (Control) or 10 µg/ml BFA for 30 minutes or 1 hour at 37°C. Cells were fixed and stained with monoclonal antibodies directed against vimentin and visualized by epifluorescence microscopy. BFA treatment induces either retraction of vimentin networks to the perinuclear region or the generation of process-like formations when compared with untreated cells. The severity of the phenotype increases with longer treatment. Bars, 20 µm. (b) High magnification (100x) images of cells treated for 30 minutes with 10 µg/ml BFA and 4x magnification of process marked by asterisk (*) show that vimentin processes are made up of densely clustered vimentin filaments. Scale bars are 20 µm. (c) Quantification of results from a. Number of cells showing either retracted vimentin networks or process formation after either 30 minutes or 1 hour of BFA treatment were counted and expressed as a percent of the total number of cells. (d) SW13 v+ cells stably expressing GFP-vimentin were either treated with methanol (Control, 
) or 10 µg/ml BFA (BFA, 
) for 15 minutes before beginning FRAP. Images were taken every 30 seconds for 
30 minutes. No differences were seen in recovery of the bleached region of the cell between control and BFA-treated cells, indicating that BFA treatment had no effect on subunit-exchange dynamics. (e) Spinning-disc confocal time-lapse imaging of vimentin bundling. SW13 cells stably expressing GFP-vimentin were treated with 10 µg/ml BFA for 1 hour. Both process formation (top panels) and retraction (bottom panels) appeared to arise from preexisting vimentin filaments (see movie 1 in supplementary material).
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