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Expression of BRG1, a human SWI/SNF component, affects the organisation of actin filaments through the RhoA signalling pathway

Patrik Asp, Margareta Wihlborg, Mattias Karlén* and Ann-Kristin Östlund Farrants{ddagger}

Department of Zoological Cell Biology, The Wenner-Gren Institute, Stockholm University, SE-106 91 Stockholm, Sweden
* Present address: Laboratory for Developmental Biology, Cell and Molecular Biology, Karolinska Institute, SE-171 77 Stockholm, Sweden



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  		Fig. 1. The formation of stress-fibre-like structures in BRG1-expressing clones. Immunofluorescence images show a BRG1-expressing clone of SW13 cells seeded for 2 days before being fixed and stained with affinity-purified anti-BRG1 antibodies against the N-terminal part of the protein visualised by TRITC-conjugated secondary anti-rabbit antibodies (A) and FITC-conjugated phalloidin (B) to visualise actin filament structures from the same cells. In (C), an immunofluorescence image of a second BRG1-expressing clone grown for 5 days before fixation and staining with phalloidin is shown. Immunofluorescence images of a BRG1-K798R-expressing clone stained with affinity-purified anti-BRG1 antibodies as in A (D), and the same cells stained with phalloidin (E). Immunofluorescence images of SW13 cells fixed and stained with affinity-purified anti-BRG1 antibodies as in A (F), and the same cells stained with phalloidin (G). Bar, 20 µm; the magnification is the same in A-G. (H) Cell lysates were made from untransfected SW13 cells and from clones expressing the BRG1 protein or the BRG1-K798R protein. The proteins (20 µg/sample) were separated on a 7% SDS-PAGE for detection of BRG1 and a 15% SDS-PAGE for the detection of actin, and transferred to a membrane that was subsequently probed with anti-BRG1 antiserum or monoclonal ß-actin antibodies. The lane marked `sw13' is lysate from untransfected cells, `BRG1' is from the BRG1-expressing clone and `K798R' is from the BRG1-K798R-expressing clone.

 


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  		Fig. 2. The effect of a specific antisense BRG1-RNA on the formation of stress-fibre-like actin filament structures. Cells were transfected with a vector expressing a specific BRG1 antisense RNA and the green fluorescent protein (GFP) as a marker. (A) Cell lysates (20 µg/sample) from BRG1-expressing cells grown for 72 hours after transfection and from untransfected BRG1-expressing cells were separated using 7% SDS-PAGE and probed with anti-BRG1 serum (top) and anti-{alpha}-tubulin (bottom). The amounts of antisense vector (µg) used for transfection of 0.6x106 BRG1-expressing cells are indicated above the lanes. (B) An immunofluorescence image shows BRG1-expressing cells transfected with the antisense BRG1-vector stained with TRITC-conjugated phalloidin. (C) The same cells visualised by GFP-fluorescence as a marker for transfected cells. BRG1-expressing cells were also transfected with the GFP-vector without the antisense insert and stained with TRITC-conjugated phalloidin (D). The same cells were visualised by GFP-fluorescence as a marker (E). Bar, 20 µm; the magnification is the same in B-E.

 


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  		Fig. 3. The effect of TSA-treatment on the actin filament organisation. Immunofluorescence images show SW13 cells treated for 24 hours with 125 ng/ml of TSA, fixed and stained with FITC-phalloidin (A), BRG1-expressing cells selected for 10 days and stained with FITC-phalloidin (B), and BRG1-expressing cells selected for 10 days, treated with 125 ng of TSA/ml for 24 hours and stained with FITC-phalloidin (C). Bars, 20 µm (A); 100 µm (B,C).

 


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  		Fig. 4. The relationship in time between BRG1 expression and the appearance of stress-fibre-like actin filaments. (A) Immunoblots of cell lysates (20 µg/sample) of cells transfected with the pBJ5-BRG1 expression vector (indicated above the lanes) or the pBJ5-BRG1-K798R vector (indicated above the lanes) taken at the time points indicated above the lanes. The lane marked `0' is untransfected SW13 cells lysed just prior to transfection. The {alpha}-tubulin levels in the same samples are shown as a control. (B) SW13 cells were transfected with pBJ5-BRG1 or the ATPase-deficient pBJ5-BRG1-K798R on glass coverslips and samples were taken at 24, 48 and 72 hours. The cells were double-stained with FITC-phalloidin and anti-BRG1 antiserum visualised by TRITC-secondary antibodies after fixation. Between 30 and 50 cells expressing the BRG1 protein or the mutated BRG1-K798R protein in the nucleus were counted blindly in each separate experiment for each group (four separate experiments for 24 hours and 48 hours). The percentage of BRG1/mutated BRG1-K798R-expressing cells that had formed thick actin bundles in the cell body was determined for each sample. The standard deviation is given for each group; n is the number of separate experiments in the group. The two values for the 72 hour point were 65 and 72% for BRG1-expressing cells, and 3 and 13% for BRG1-K798R-expressing cells. Immunofluorescence images of cells transfected with the BRG1-vector stained with phalloidin (C) and with the BRG-antiserum against the N-terminal (D) 24 hours after transfection and stained with phalloidin (E) and the anti-BRG1 antiserum as in D (F), 48 hours after transfection are shown. Bar, 20 µm.

 


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  		Fig. 5. Activation of the RhoA pathway by LPA and serum stimulation. Immunofluorescence images show SW13 cells starved (0.25% fetal calf serum) for 6 hours (A) and exposed to 10 µM LPA after 6 hours of starvation (B) before being fixed and stained with FITC-phalloidin. Immunofluorescence images show BRG1-expressing cells starved (0.25% fetal calf serum) for 18 hours (C), exposed to 5 µM LPA for 20 minutes after starvation (D) and given 5% serum for 20 minutes after starvation (E). Immunofluorescence images show BRG1-expressing cells expressing a myc-tagged RhoA(N19) protein stained with FITC-phalloidin (F) and the anti-9E-myc antibody (G) for detection of cells expressing the RhoA(N19) protein. All images are at the same magnification; bar, 20 µm.

 


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  		Fig. 6. The activation state of the RhoA-GTPase. (A) Immunoblots of control pull-downs using the human form of rhotekin-RBD showing that it is specific for the activated form of RhoA. Approximately 104 SW13 cells were seeded in 5 cm dishes and left untreated or transfected with the vector as indicated above the lanes for 24 hours prior to the pull-down. Myc-tagged mutant forms of RhoA were detected using the anti-myc-9E antibody. (B) The activation states of endogenous RhoA in SW13 cells, a BRG1-expressing clone and a BRG1-K798R-expressing clone were measured 24 hours after seeding at ~60% confluence. The protein concentration of each sample was measured in the crude lysates and the protein input adjusted accordingly. The RhoA protein was detected using anti-RhoA antibodies. (C) The activation state of RhoA in SW13 cells, BRG1 and BRG1-K798R-expressing clones seeded at ~60% confluence, starved for 18 hours in serum-free medium and stimulated with 5% serum for 20 minutes as indicated above the lanes is shown. (The experiment presented is representative of three experiments.)

 


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  		Fig. 7. The effect of BRG1 expression on the protein levels of Rho-kinase/ROCK proteins. Immunfluorescence images of BRG1-expressing cells exposed to the Rho-kinase/ROCK inhibitor Y-27632 for 1 hour, fixed and stained with FITC-phalloidin (A) and native SW13 cells exposed to the same treatment as BRG1-expressing cells (B). Bar, 20 µm. (C) Immunoblots of cell lysate (20 µg/sample) of SW13 cells transfected with the pBJ5-BRG1 or the pBJ5-BRG1-K798R expression vector. Samples were taken at the time points indicated above the lanes. The membranes were probed with a C-terminal antiserum against BRG1, ROCK1 antibodies, ROCK2 antibodies, a Dia1 antiserum, ß-actin antibodies and {alpha}-tubulin antibodies as indicated on the left. The BRG1-transfection is shown on the left and the BRG1-K798R on the right as indicated (representative of one experiment of five). (D) Immunoblots of the level of ROCK1, ROCK2, Dia1, RhoA, HDAC2, P65 of NF{kappa}B and {alpha}-tubulin in cell lysate (20 µg/lane) of BRG1-expressing cells (labelled `0'), cells that were transfected with a 5 µg GFP-expressing vector (labelled `V') or a GFP-expressing vector co-expressing a specific antisense BRG1-fragment. The amount of GFP-BRG1-antisense vector used is given above the lanes.

 


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  		Fig. 8. The effect of Rho-kinase/ROCK expression in SW13 cells. (A,B) Immunofluoroscence images show SW13 cells expressing a myc-tagged, constitutively active Rho-kinase/ROCK fragment habouring the kinase domain, which were fixed and stained with FITC-phalloidin (A) and the anti-9E-myc antibody (B) for detection of cells expressing the Rho-kinase/ROCK fragment. Immunofluorescence images of cells expressing a wild-type, myc-tagged ROCK1 protein stained with FITC-phalloidin (C) and anti-9E-myc antibodies (D), and SW13 cells expressing a wild-type, myc-tagged ROCK2 protein stained with FITC-phalloidin (E) and anti-myc-9E-antibodies (F). Immuofluorescence images of BRG1-expressing cells transfected with a dominant negative, myc-tagged ROCK1 (KD-IA), stained with FITC-phalloidin (G) and anti-9E-myc antibodies (H). Bar, 20 µm; the same magnification was used in A-H.

 

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