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First published online 10 February 2009
doi: 10.1242/jcs.028738

Journal of Cell Science 122, 656-666 (2009)
Published by The Company of Biologists 2009
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Dynamic conformational changes in the FERM domain of FAK are involved in focal-adhesion behavior during cell spreading and motility

Ekaterina Papusheva1, Fernanda Mello de Queiroz1, Jeremie Dalous2, Yunyun Han1, Alessandro Esposito3, Elizabeth A. Jares-Erijmanxa4,5, Thomas M. Jovin5,6 and Gertrude Bunt1,2,5,*

1 Molecular Biology of Neuronal Signals, Max-Planck Institute of Experimental Medicine, 37075 Göttingen, Germany
2 Department of Neuro- and Sensory Physiology, University Medicine Göttingen, 37073 Göttingen, Germany
3 Cellular Biophysics, European Neuroscience Institute, 37073 Göttingen, Germany
4 Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
5 DFG Research Center for Molecular Physiology of the Brain (CMPB), 37073 Göttingen, Germany
6 Laboratory of Cellular Dynamics, Max-Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany


Figure 1
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  		Fig. 1. The FERM sensor reports on an intramolecular conformational change in the FERM domain of FAK. (A) Overview of the FAK-FRET probes used. In the FERM sensor, CFP (donor) is inserted at site 391 to measure FRET with YFP (acceptor) at the N-terminus. (B,C) FRET-efficiency (E%) distributions of the FERM sensor compared with co-transfected singly labeled Y(FP)-FAK and FERM-C(FP) FAK constructs in FAK–/– fibroblasts as detected by acceptor photobleaching. (B) Cumulative histograms (as probability density functions, PDF) of the measured FRET-efficiency distributions over the indicated number of cells (n). (C) FRET-efficiency images of representative cells. YFP pre and YFP post indicate YFP fluorescence intensities before and after photobleaching, respectively. YFP was bleached in the boxed area. FRET efficiencies are displayed in pseudo-color scale as indicated. Scale bars: 10 µm.

 

Figure 2
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  		Fig. 2. Functional characterization of the FERM sensor. (A) The FERM sensor rescues FAK–/– cell motility: the number of cells migrating through the fibronectin-coated filter of a Thincert chamber after 6 hours is shown for cells expressing GFP, GFP-wtFAK or the FERM sensor. Results are the average of three separate experiments; n, number of counted fields. (B) Western blot analysis of Tyr397 and Tyr576 phosphorylation for GFP-wtFAK and the FERM sensor in FAK–/– cells. Serum-starved cells were grown on fibronectin (FN) for 24 hours or kept in suspension (S) for 1 hour. (C) FRAP analysis of cells expressing YFP-wtFAK or the FERM sensor shows identical turnover and recovery between the two.

 

Figure 3
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  		Fig. 3. The FERM sensor responds to integrin stimulation. (A) Acceptor-photobleaching FRET-efficiency (E%) images of FAK–/– fibroblasts expressing the FERM sensor, plated on poly-L-ornithine/BSA (PLO) for 30 minutes (upper row) or on fibronectin overnight (lower row). YFP was bleached in the boxed area. FRET efficiencies are shown in pseudo-color. Scale bars: 10 µm. (B) Cumulative FRET distribution histograms (probability density function, PDF) of PLO-plated cells, cytosol (Cyto) and FAs of fibronectin (FN)-adherent cells. Insert shows the corresponding integrated cumulative distributions (cumulative density function, CDF) for statistical analysis by KS testing, in the same color-coding.

 

Figure 4
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  		Fig. 4. Adhesion-induced conformational changes in the FERM domain of FAK during early and late phases of spreading. (A) REF-52 cells expressing the FERM sensor were held in suspension for 1 hour and were re-plated on fibronectin for the indicated time points before fixation. Cells plated on PLO for 30 minutes were taken as control. FRET was determined by ratiometric imaging. FRET ratios (YFP/CFP) are shown in pseudo-color as indicated. Scale bars: 10 µm. (B) Proportion of FAs with FRET ratios exceeding 1.66 (60% of scale maximum), i.e. high-FRET FAs, for the different time points. Values are indicated with s.e.m.; statistical significance was tested by one-way ANOVA with Tukey post-analysis (**P<0.005, ***P<0.001).

 

Figure 5
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  		Fig. 5. The conformational change in the FERM domain involves the polybasic KAKTLR sequence in the F2 lobe, but is independent of intrinsic kinase activity and Tyr397 phosphorylation. (A) Acceptor-photobleaching FRET-efficiency (E%) images of FAK–/– fibroblasts expressing the FERM sensor (WT FERM) or the Arg454 mutant, Phe397 mutant or KAKTLR mutant of the FERM sensor. YFP was bleached in the boxed area. FRET efficiencies are shown in pseudo-color. Scale bars: 10 µm. (B) Cumulative FRET distribution histograms of the cytosol and FAs of cells expressing the Arg454 (upper), Phe397 (middle) or KAKTLR (lower) mutant FERM sensor as compared with the native (WT) FERM sensor. The corresponding integrated cumulative distributions (CDF) for statistical analysis by KS testing are shown in the right column, in the same color-coding. Statistical significance (by KS testing) of differences between mutant and native sensor in the cytosolic (Cyto) and FA compartments is indicated (*P<0.05, **P<0.005).

 

Figure 6
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  		Fig. 6. KAKTLR mutation of the FERM sensor reduces Tyr397 and Tyr576 phosphorylation. Western blot analysis showing Tyr397 and Tyr576 phosphorylation of the FERM-KAKTLR mutant as compared with the native (WT) FERM sensor and GFP-wtFAK in serum-starved FAK–/– cells that are adherent on fibronectin.

 

Figure 7
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  		Fig. 7. The conformation of the FERM domain in FAK correlates with distinct FA behaviors. Randomly migrating FAK–/– fibroblasts expressing the FERM sensor were imaged at 5-minute intervals. (A) YFP fluorescence intensity distributions of a representative cell (upper row). Arrows indicate examples of centripetally sliding FAs. Dynamic FA behavior is visualized by red and green overlaying of the images at the indicated time points (middle row). FRET was determined by ratiometric imaging (lower row). FRET ratios (YFP/CFP) are shown in pseudo-color as indicated. The 5th to 95th percentile FRET-ratio range of cytosol values (c) is indicated. Scale bar: 10 µm. (B) Fates of 222 individual FA sites in the time-lapse images of randomly moving FAK–/– fibroblasts expressing the FERM sensor. FA behavior was classified as growing, shrinking, sliding or stable, and the proportions of FAs with high FRET (ratios exceeding 60% of scale maximum, T, which is indicated in the color bar in A) were determined for each category. Most growing and sliding FAs exhibit high FRET; most shrinking and stable FAs exhibit low FRET.

 

Figure 8
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  		Fig. 8. ROCK-mediated tension signaling affects the conformation of the FERM domain of FAK. (A-C) YFP fluorescence intensity images of FAK–/– fibroblasts expressing the FERM sensor: (A) untreated; (B,C) treated with either 10 µM ML7 for 20 minutes (B) or 10 µM Y27632 for 10 minutes (C). Scale bar: 10 µm. (D,E) Corresponding cumulative FRET distribution histograms (left column) and cumulative distributions for statistical analysis by KS testing (right column) as measured in FAs by acceptor photobleaching upon treatment with either ML7 (D) or Y27632 (E). Statistical significance (**P<0.005) upon stimulation by Y27632 is indicated.

 

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© The Company of Biologists Ltd 2009