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First published online August 20, 2008
doi: 10.1242/10.1242/jcs.022509

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Rac regulates the interaction of fascin with protein kinase C in cell migration

¹ Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London SE1 1UL, UK
² Department of Cell Biology, Lerner Research Institute, The Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA
³ Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, The Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA

View larger version (40K): [in this window] [in a new window]   Fig. 1. Colocalisation of fascin and PKC is regulated by PKC and Rac activity. (A,B) SW480-Pa cells plated on 15 nM LN for 4 hours in the absence or presence of 1 µM BIM or 100 µM NSC23766 were co-stained with antibodies as stated, and imaged using phase-contrast or confocal microscopy. Scale bars: 10 µm. Insets show higher magnification views of the edges of representative control cells; scale bars: 4 µm. In B, arrows and arrowhead indicate cell edges positive or negative for Rac, respectively. (C,D) PKC activation is Rac dependent. SW480-Pa cells were pre-treated with BIM, TPA, NSC23766 or solvent (Con) as described in the Materials and Methods, lysed and analysed by SDS-PAGE and immunoblotting for total PKC or PKC–T655PO4 as a reporter of PKC activation (C). D shows quantification of results from three independent experiments. Columns represent the mean and bars indicate s.e.m. (E) PKC was immunoprecipitated from LN-adherent cells and treated in vitro with 100 µM NSC23766 or 1 µM BIM before activity analysis by immunoblotting. Only BIM treatment decreased PKC–T655PO4. Representative of three independent experiments.

View larger version (28K): [in this window] [in a new window]   Fig. 2. PKC activity drives the interaction of fascin with PKC. (A) SW480-Pa cells transiently expressing GFP-fascin, alone or plus mRFP-PKC, were treated with solvent or PDBu for the indicated times, and then fixed and imaged using FLIM to measure FRET. PDBu and TPA activate conventional PKC equivalently at early timepoints. (B) Quantification of data from nine cells from three independent experiments as in A. (C) SW480-Pa cells transiently co-expressing either GFP-fascin-S39A or GFP-fascin-S39D with PKC-mRFP were treated with PDBu for 20 minutes, then fixed and imaged using FLIM to measure FRET. Only GFP-fascin-S39D interacted with active PKC. (A,C) Intensity multiphoton GFP images (donor) and, where relevant, epifluorescence images for RFP (acceptor). Lifetime images are depicted using a pseudocolour scale where red is a low lifetime (1.8 nseconds) and blue is high (2.3 nseconds).

View larger version (30K): [in this window] [in a new window]   Fig. 3. Fascin and PKC interact in living carcinoma cells migrating on LN. (A) Level of expression of GFP-Xtfascin in SW480 IKD-F11 Fas–cells compared with the endogenous fascin of SW480 cells. Cells were stained with antibody reactive with human and Xenopus fascin 1 and the mean fluorescence intensity calculated from 10 cells in each population and from three independent experiments. (B) IKD-F11 Fas–cells transiently expressing GFP-Xtfascin alone (top panels) or GFP-Xtfascin and PKC-mRFP (bottom panels) were plated on 15 nM LN for 2 hours. Transfected migrating cells were imaged over time, using FLIM to measure FRET. Panels show intensity multiphoton GFP images from each cell (donor) and, where relevant, an epifluorescence image for mRFP (acceptor) at selected timepoints. Lifetime images are depicted by a pseudocolour scale with red as low lifetime (1.7 nseconds) and blue as high (2.3 nseconds). Arrows indicate examples of strong interaction in filopodia. Representative of a total of nine experiments for control and test cells.

View larger version (21K): [in this window] [in a new window]   Fig. 4. PKC and Rac activities regulate the interaction of fascin and PKC in migrating cells. (A,B) SW480 IKD-F11 Fas–cells transiently transfected with GFP-Xtfascin alone (bottom line in A) or GFP-Xtfascin and mRFP-PKC (all other panels and in B) were plated on 15 nM LN for 2 hours, with or without pretreatment with 1 µM BIM for 30 minutes or 100 µM NSC23766 for 4 hours, then fixed, mounted and imaged using FLIM to measure FRET. (A) Intensity multiphoton GFP images from each cell (donor) and, where relevant, an epifluorescence image for mRFP (acceptor). Lifetime images are depicted by a pseudocolour scale with red as low lifetime (1.7 nseconds) and blue as high (2.3 nseconds). (B) Histogram demonstrating mean cumulative FRET efficiency data from 16 cells per condition and three independent experiments. The graph represents the range of FRET efficiencies seen per cell in each condition, normalised to pixel intensity, and demonstrates a clear leftward shift in BIM- and NSC-treated samples where FRET is inhibited. (C) A kinase-dead form of PKC, PKCK380A, does not interact with fascin in cells migrating on laminin. The cumulative FRET efficiency graph shows the mean FRET efficiency for wild-type and kinase-dead PKC. Each column represents the mean from seven to nine cells per condition and three independent experiments. Insets show the GFP and RFP intensities, and lifetime plot from a representative cell expressing PKC–K380A. Pseudocolour scale as in A.

View larger version (25K): [in this window] [in a new window]   Fig. 5. The fascin–PKC interaction depends specifically on Rac1 activity in migrating cells. (A,B) IKD-F11 Fas–cells cultured in doxycycline were transiently transfected with GFP-Xtfascin and PKC-mRFP in conjunction with either N17Rac1-myc or N17Cdc42-myc, and allowed to migrate on 15 nM laminin for 6 hours. Cells were fixed and antibody stained to detect the GTPase and imaged using FLIM to measure FRET. Only N17Rac1 inhibited the FRET interaction. Lifetime images are depicted with a pseudocolour scale where red is a low lifetime (1.75 nseconds) and blue is high (2.4 nseconds). Representative of at least eight cells in each sample. (C) Cumulative FRET efficiency graph for cells migrating on laminin, under control conditions or in cells expressing N17Rac1-myc or N17Cdc42-myc. The mean FRET efficiency for each condition is from seven to nine cells per condition and three independent experiments.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Constitutively active Rac1 or PKC, but not Cdc42, activates the interaction of fascin and PKC in quiescent cells. (A) Lifetime of cells expressing GFP-Xtfascin alone. (B) IKD-F11 Fas–cells were transiently transfected with GFP-Xtfascin and the indicated mRFP acceptor constructs in the absence or presence of constitutively active Rac1 or Cdc42. Quiescent, serum-starved cells were fixed and antibody-stained to detect the GTPase and imaged using FLIM to detect FRET. Lifetime images are depicted with a pseudocolour scale where red is a low lifetime (1.85 nseconds) and blue is high (2.4 nseconds). FRET was increased in the presence of V12Rac1-HA (arrow indicates examples of strong interaction in filopodia) or PKCA25E-mRFP, but not in the presence of V12Cdc42-HA. (C) Cumulative FRET efficiency graph for control cells or cells expressing the indicated constructs. The mean FRET efficiency for each condition is from seven to nine cells per condition and three independent experiments.

View larger version (27K): [in this window] [in a new window]   Fig. 7. Role of Pak1 in regulating the fascin–PKC interaction in migrating cells. (A) IKD-F11 Fas–cells cultured in doxycycline were transiently transfected with GFP-Xtfascin, PKC-mRFP and either wild-type Pak1-myc or Pak1AID-myc and allowed to migrate on 15 nM laminin for 6 hours. Cells were fixed and antibody-stained to detect Pak1 and imaged using FLIM to measure FRET. Pak1-AID inhibited the FRET interaction. Lifetime images are depicted with a pseudocolour scale where red is a low lifetime (1.85 nseconds) and blue is high (2.4 nseconds). Representative of at least eight cells in each sample. (B) Cumulative FRET efficiency graph for control cells or cells co-expressing the indicated Pak1 constructs. The mean FRET efficiency for each condition is from seven to nine cells per condition and three independent experiments. (C) Pak1 activity is Rac dependent. SW480-Pa cells were pretreated with BIM, NSC23766 or solvent (Con), as described in the Materials and Methods, plated on 15 nM laminin for 2 hours in the continued presence of inhibitors, then lysed and analysed by SDS-PAGE and immunoblotting for total Pak1 or Pak1–T423PO4 as a reporter of Pak1 activation. Representative of three independent experiments.

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