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First published online 15 August 2006
doi: 10.1242/jcs.03147

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Architecture of the vimentin cytoskeleton is modified by perturbation of the GTPase ARF1

¹ Graduate Program in Biochemistry, Cell, and Developmental Biology, Emory University, Atlanta, GA 30322, USA
² Department of Cell Biology, Emory University, Atlanta, GA 30322, USA
³ Department of Dermatology, Emory University, Atlanta, GA 30322, USA
⁴ Center for Neurodegenerative Diseases, Emory University, Atlanta, GA 30322, USA

View larger version (81K): [in a new window]   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).

View larger version (69K): [in a new window]   Fig. 2. Brefeldin A induces changes in the microtubule cytoskeleton in a vimentin-dependent manner. (a) SW13 cells stably expressing GFP-vimentin were treated with either methanol (Control) or 10 µg/ml BFA for 30 minutes at 37°C. Following treatment, cells were fixed and processed for immunofluorescence and staining with antibodies against -tubulin. Staining was visualized by confocal microscopy. BFA treatment led to the formation of microtubule processes in close apposition to vimentin. (b) SW13 v- and v+ cells were treated with either methanol (Control) or 10 µg/ml BFA for 30 minutes at 37°C and processed for immunofluorescence. Cells were stained with antibodies to -tubulin and with Rhodamine-phalloidin (to visualize F-actin) and observed by confocal microscopy. Changes in microtubule architecture occurred only in the presence of intermediate filaments in SW13 v+ cells. Process formation or retraction of microtubules was not observed in SW13 v- cells. No changes were observed in cortical actin networks. Bars, 10 µm.

View larger version (78K): [in a new window]   Fig. 3. Depolymerization of actin does not inhibit BFA-induced changes in vimentin networks. SW13 v+ cells were treated in either the absence or presence of 10 µg/ml BFA for 30 minutes, 1 µg/ml cytochalasin D for 45 minutes, or pretreated with 1 µg/ml cytochalasin D for 15 minutes followed by the addition of 10 µg/ml BFA for 30 minutes in the continued presence of cytochalasin D at 37°C. Cells were processed and stained with monoclonal antibodies directed against either vimentin, -tubulin, or ß-actin and visualized by epifluorescence microscopy. Cytochalasin D treatment induced actin depolymerization as shown by ß-actin staining. In addition, it also generated retraction of both microtubule and vimentin networks. However, cells treated with both BFA and cytochalasin D exhibited process-like formations of both vimentin and microtubules, indicating that actin was not required for the effects of BFA on vimentin and microtubule networks. Bar, 20 µm.

View larger version (69K): [in a new window]   Fig. 4. Brefeldin A and ilimaquinone have differential effects on the vimentin cytoskeleton. SW13 v+ cells were treated in either the absence or presence of 10 µg/ml BFA or 40 µM IQ for either 30 minutes or 1 hour (vimentin 1 h panel only) at 37°C. Cells were then processed for immunofluorescence and stained with monoclonal antibodies directed against vimentin, GM130, the subunit of AP-1 or the subunit of AP-3. Vimentin staining was visualized by epifluorescence microscopy, and GM130, AP-1 and AP-3 staining was visualized by confocal microscopy. BFA induced changes in vimentin architecture that became more pronounced at 1 hour. IQ did not induce similar changes, although cells were observed to round up, particularly with longer treatments. Both drugs resulted in fragmentation of the Golgi complex, as visualized by GM130 staining. Only BFA resulted in release of the adaptors AP-1 and AP-3 from the membrane. Bars, 20 µm. Insets are of cells indicated by an asterisk (*) and are magnified 3x.

View larger version (97K): [in a new window]   Fig. 5. The effects of BFA on the vimentin cytoskeleton are reversible. SW13 v+ cells were either untreated (Control) or treated with 10 µg/ml BFA for 30 minutes at 37°C. Cells were then washed and allowed to recover in HEPES-buffered DMEM for 0, 5, 15 or 30 minutes. Following recovery, cells were processed for immunofluorescence and stained with monoclonal antibodies directed against vimentin. Vimentin staining was visualized by epifluorescence microscopy. Changes in the morphology of the vimentin cytoskeleton were fully reversible 30 minutes following drug treatment. Bar, 20 µm.

View larger version (85K): [in a new window]   Fig. 6. Vimentin cytoskeletal architecture is modified by the ARF1 dominant-negative mutant T31N. SW13 v+ cells were transiently transfected with either wild-type ARF1, ARF1-T31N or ARF1-Q71L fused to GFP. Cells were then fixed and processed for immunofluorescence and stained with antibodies directed against either vimentin (a) or the subunit of AP-3 (b). (a) ARF1-T31N induced process formation in vimentin networks similar to BFA-treated cells. Approximately 66% of cells expressing ARF1-T31N showed either process formation or retraction (quantification on right). The wild type and Q71L mutants did not show the same effects as T31N on vimentin networks. (b) ARF1-T31N, but not the wild type or Q71L mutants, induced release of the adaptor AP-3 from the membrane. Bars, 10 µm.

View larger version (45K): [in a new window]   Fig. 7. BFA treatment induces a preferential association of adaptor complexes with both soluble vimentin subunits and insoluble vimentin networks. (a) Immunoprecipitations were performed from rat brain cytosol (lanes 1, 2, 4 and 5) or in the absence of cytosol (lane 3) using either beads coated with no antibody (lane 1), control pre-immune antibodies (lane 2) or polyclonal 3 (AP-3) antibodies (lanes 3-5). Immunoprecipitated proteins were resolved by SDS-PAGE and transferred to membranes that were incubated with (lanes 1-4) or without (lane 5) recombinant vimentin (Overlay: Vimentin). A band of 120 kDa specifically binds vimentin and co-migrates with the ß3 AP-3 subunit, shown by immunoblotting using an antibody directed against ß3B (IB: AP-3). Inputs correspond to 10% of the cytosol used for immunoprecipitation. (b) SW13 v+ cells stably expressing GFP-vimentin were treated with 10 µg/ml BFA for 30 minutes at 37°C and were processed for immunofluorescence. Cells were stained with monoclonal antibodies to the subunit of AP-3. Confocal microscopy of staining revealed that BFA treatment induced bundling of intermediate filament networks and that bundles were decorated with AP-3 immunoreactivity. Bar, 10 µm. (c) SW13 vimentin positive (v+) and negative (v-) cells were either treated or not with 10 µg/ml BFA for 30 minutes at 37°C. Cells were then extracted with 1% Triton X-100 at 4°C. Detergent-insoluble extracts (lanes 1 and 2) and input (lanes 3 and 4) were analyzed by immunoblot with AP-3 antibodies ( and 3, the latter not shown), AP-1 antibodies (against the subunit), vimentin and tubulin antibodies. Association of both AP-3 and AP-1 with insoluble vimentin networks is increased by BFA treatment. (d) Quantification of the results presented in c. Data is presented as mean fold increase (± s.e.m.) over the amount of adaptor present in the detergent insoluble pool of SW13 v- cells (lane 1) normalized to tubulin (n=3). (e) Soluble extracts from SW13 v- and v+ cells treated or not with 10 µg/ml BFA were immunoprecipitated using antibodies to AP-1 () or AP-3 (). Immunoprecipitations were then analyzed by immunoblot for the presence of vimentin. BFA treatment induced an association of soluble vimentin protein with both AP-1 and AP-3 adaptors.

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