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First published online 27 July 2004
doi: 10.1242/jcs.01289

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Analysis of Smad nucleocytoplasmic shuttling in living cells

Francisco J. Nicolás^*,, Karolien De Bosscher^,, Bernhard Schmierer and Caroline S. Hill^¶

Laboratory of Developmental Signalling, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK

View larger version (47K): [in a new window]   Fig. 2. TGF-ß-induced nuclear translocation of GFPSmads is dependent upon continuous receptor signalling. (A) HaCaT cell lines stably expressing GFPSmad2, GFPSmad3 or GFPSmad4 were pretreated with cycloheximide and were then incubated with TGF-ß1 for 1 hour, followed by SB-431542 (7.5 µM) for up to 160 minutes. In the case of the GFPSmad4 cells, leptomycin B (LMB) was added 90 minutes after SB-431542 addition (150-minute time-point). Fluorescence images are shown at different time-points after initial TGF-ß treatment. Arrows indicate representative examples of cells demonstrating nucleocytoplasmic shuttling. For GFPSmad3 cells, the boxed region is shown magnified below to demonstrate that GFPSmad3 is partially excluded from the nucleoli upon TGF-ß treatment. Below are graphs showing quantitation of nuclear fluorescence, with fluorescence images collected every 3 minutes. The left-hand graph shows the average of the TGF-ß-induced nuclear fluorescence of the GFPSmad2 and GFPSmad4 cells marked with an arrow. Means and standard deviations are shown. The right-hand graphs show quantifications of the nuclear fluorescence for GFPSmad2 and GFPSmad4 throughout the whole experiment for one of the indicated cells in each case. (B) HeLa TK- cells were transiently transfected with plasmids expressing GFPSmad2 or GFPSmad4 together with FLAG-Smad2 and treated with cycloheximide, TGF-ß1, SB-431542 and LMB as in A. Fluorescence images are shown at different time-points after initial TGF-ß treatment. Arrows indicate representative examples of cells demonstrating nucleocytoplasmic shuttling. The punctate fluorescence observed in the cytoplasm of transiently transfected HeLa TK- cells is not seen in the stable HaCaT cell lines and thus seems to be a consequence of transient transfection. The experiments shown are representatives from at least three independent experiments.

View larger version (44K): [in a new window]   Fig. 1. GFPSmad2, GFPSmad3 and GFPSmad4 fusion proteins form DNA-bound complexes that are transcriptionally active in response to TGF-ß. (A) HeLa TK- cells expressing either GFPSmad3, GFPSmad4, HA-Smad4 or FLAG-Smad3 as indicated were either untreated or treated with TGF-ß1 for 1 hour. Cell extracts were assayed by bandshift analysis using the c-jun SBR as a probe. The positions of migration of complexes containing different combinations of tagged Smad3 and Smad4 are indicated. The same extracts were western blotted with antibodies recognising Smad3, phosphorylated Smad3 (P-Smad3) or Smad4 (below). The right-hand panel shows transcription assays in which MDA-MB468 cells were transfected with the CAGA12-luciferase reporter gene, plasmids expressing the different GFPSmad fusion proteins and pEF-lacZ as an internal control for transfection efficiency. Cells were treated with or without TGF-ß1 for 8 hours. Luciferase was quantitated relative to ß-galactosidase from the pEF-lacZ internal control. The data are means and standard deviations of a representative experiment performed in triplicate. (B) HeLa TK- cells were transfected with plasmids expressing XFoxH1a, GFPSmad2 or GFPSmad4 as indicated. Total cell extracts from cells either left untreated or treated with TGF-ß1 for 1 hour were assayed by bandshift analysis using the activin response element (ARE) as probe. DNA-bound complexes containing either XFoxH1a and endogenous Smad2 and Smad4 or XFoxH1a and GFPSmad2 and GFPSmad4 are indicated. The same extracts were western blotted with antibodies recognising Smad2/3, phosphorylated Smad2 (P-Smad2) or Smad4 (below). The right-hand panel shows transcription assays performed as above, where MDA-MB468 cells were transfected with the ARE3-luciferase reporter gene, plasmids expressing XFoxH1a, the different GFPSmad fusion proteins and pEF-lacZ. The data are means and standard deviations of a representative experiment performed in triplicate.

View larger version (54K): [in a new window]   Fig. 3. Controls for FLIP analysis. (A) HeLa TK- cells were transiently transfected with plasmids expressing GFP, GFPRanBP1NES or GFPPKC. Cells were unstimulated except for those expressing GFPPKC which were stimulated with TPA. Cells were photobleached in the cytoplasm as indicated by the red diamond. The fluorescence images shown prior to photobleaching (time=0 seconds) and then after three consecutive 80-second bleaching periods. In all cases, the plane of focus was a cross-section through the cell, except for the TPA-induced cells expressing GFPPKC where the plane of focus was in the plasma membrane. The right-hand graphs show the FLIP analysis. The fluorescence was quantitated at the bleach point and at a reporting point indicated by the blue square. The intensity of fluorescence is represented in arbitrary units. (B) Stimulation of cells with TPA induces the translocation of GFPPKC to the membrane. HeLa TK- cells expressing GFPPKC were either untreated or treated with TPA for up to 10 minutes. Fluorescence images are shown. Here, the plane of focus is again a cross-section through the cell. The experiments shown are representatives from at least three independent experiments.

View larger version (95K): [in a new window]   Fig. 4. FLIP analysis of GFPSmad2 and GFPSmad4 proves nucleocytoplasmic shuttling. (A) HaCaT cell lines stably expressing GFPSmad2 or GFPSmad4 remained either unstimulated (i,iii) or were treated for 1 hour with TGF-ß1 (ii,iv). The bleach region in the cytoplasm is indicated (red diamond). Each row shows the fluorescence image prior to bleaching (time=0 seconds) and after three consecutive 80-second bleaching periods. The fluorescence was quantitated at the bleach point, at a reporting point in the nucleus of the same cell (blue square) and at a reporting point in the nucleus of an adjacent cell (white triangle). In each case the fluorescence was normalised to the initial fluorescence prior to photobleaching, and the relative fluorescence was plotted. Note that in unstimulated cells expressing GFPSmad4, the cell to the right of the bleached cell also bleaches because the bleach point also contacts the cytoplasm of that cell. The data are representatives from at least three different experiments.

View larger version (57K): [in a new window]   Fig. 5. FLIP analysis reveals that GFPSmad4 is more mobile in the cytoplasm than is GFPSmad2. FLIP analysis was performed on either HeLa TK- cells transiently transfected with plasmids expressing GFPSmad2 or GFPSmad4 and FLAG-Smad2 (A) or HaCaT cell lines stably expressing GFPSmad2 or GFPSmad4 (B). Each row shows the fluorescence image prior to bleaching (time=0 seconds) and then after three consecutive 80-second bleaching periods. The fluorescence was quantitated at the bleach point (red diamond) and at a distant reporting point in the cytoplasm of the same cell (blue square) and at a reporting point in the cytoplasm of an adjacent cell (white triangle). In each case the fluorescence was normalised to the initial fluorescence prior to photobleaching, and the relative fluorescence was plotted. The data are representatives from at least three different experiments.

View larger version (43K): [in a new window]   Fig. 6. FLIP/FRAP analysis reveals a TGF-ß-dependent change in mobility of GFPSmad2 and GFPSmad4 in the nucleus. HeLa TK- cells were transiently transfected with plasmids expressing GFPNLS, GFPß-galactosidaseNLS, GFPPKC, GFPSmad2 or GFPSmad4 with FLAG-Smad2. Cells were either unstimulated or pretreated for 1 hour with LMB or TGF-ß1 as indicated. The cells expressing GFPPKC were pretreated for 10 minutes with TPA. Prior to bleaching, eight measurements of fluorescence were taken at the bleach point in the nucleus (red square) over a period of 2 seconds, then cells were bleached for 11 seconds. The recovery of fluorescence in the bleached area and fluorescence loss at a distant point also in the nucleus (blue square) were measured at 250-millisecond intervals. The levels of fluorescence were normalised to the mean of fluorescence registered before the photobleaching. In all cases, the plane of focus is a cross-section through the cell, except for the TPA-induced cells expressing GFPPKC where the plane of focus was in the plasma membrane. The data are representatives from three different experiments.