(A) Design of the FAK conformation biosensor (CYFAK413). In the inactive conformation, the CFP and citrine are in proximity and FRET occurs. In the open conformation, the CFP and citrine are further apart and can rotate more freely, which will result in a reduced FRET signal. (B) Schematic structure of the FAK biosensor and control biosensors. CFP is fused to the N terminus. The numbers in the construct name refer to the citrine insertion site. (C) Normalized spectra of the FAK wild-type biosensor and control biosensors were measured by fluorometry. (D) FRET in the FAK conformation biosensor was verified by acceptor photobleaching in living cells. HeLa cells expressing the biosensor were imaged in both CFP and citrine channels following excitation of CFP at 11-s intervals. The acceptor (citrine) was photobleached by pulse illumination for 6 s at each 11-s interval, after the zero time point. The mean intensity from whole cells was measured at each time point and normalized to the zero time point. Shown are the average results from three experiments ± standard error. (E) Representation of the FERM/kinase domain interface was created using Pymol and illustrates the key interaction between F596 in the kinase domain and a hydrophobic pocket in the F2 subdomain of the FERM domain (17). (F) Normalized spectra of the FAK wild-type biosensor and a FAK biosensor with mutations designed to disrupt the FERM/kinase domain interaction are shown. (G) Normalized FRET/CFP emission ratios of FAK biosensors containing different mutations are shown. The constructs were analyzed as in panel C, and the FRET/CFP ratios of each were normalized to the FRET/CFP ratio of the wild-type biosensor. Shown are the average results from at least three experiments ± standard error. The results were analyzed by one-way analysis of variance (P < 0.0001) and Tukey's multiple comparison post test (CYFAK413 versus each construct; P < 0.001).

(A) HeLa cells expressing the FAK autophosphorylation biosensor or the 397F control were trypsinized and plated on fibronectin-coated coverslips. CFP, FRET, and citrine images were sequentially captured by fluorescence microscopy. CFP and FRET^C/CFP ratio images of the cells are shown. The ratio images are pseudocolored so that hotter colors reflect an increase in FAK autophosphorylation. Shown are representative images (n > 5 cells). The boxed area in the “CFAK+Citrine-dSH2 FRET^c/CFP” image is shown at higher magnification to the right. (B) HeLa cells expressing the FAK biosensor or the constitutively active mutant were trypsinized and plated on fibronectin-coated coverslips. CFP, FRET, and citrine images were sequentially captured by fluorescence microscopy. CFP and CFP/FRET ratio images of the cells are shown. Note that the ratio images are pseudocolored so that hotter colors reflect an increase in the open, active conformation. Representative images are shown (n > 15 cells).

Acidic phospholipids bind the basic patch within the FAK FERM domain. (A) Normalized FRET/CFP emission ratios of the wild-type FAK biosensor and the basic patch mutant are shown. The mutant FRET/CFP ratio was normalized to FRET/CFP ratio of the wild-type biosensor. Shown is a representative experiment (n = 3). (B) The purified recombinant FERM domain was incubated with BODIPY-labeled phospholipids, and binding was measured by fluorescence polarization. Anisotropy (mP, millipolarization units) is plotted against FERM domain concentration. The average of three experiments ± standard deviation is shown. (C) PE/PC vesicles containing increasing amounts of PIP2 were incubated with a GST-FERM fusion protein. The vesicles were sedimented by centrifugation, and the amount of fusion protein in the vesicle containing pellet (P) and the supernatant (S) was determined by SDS-PAGE and Coomassie blue staining. (D) PE/PC vesicles containing 10% (mass ratio) of the indicated lipids were incubated with the GST-FERM fusion protein and analyzed as in panel A. Buf, buffer. (E) PE/PC vesicles containing PIP2 or phosphatidylinositol (PI) were incubated the wild-type GST-FERM domain or a basic patch mutant (KAKTLRK) and analyzed as in panel A. (F and G) HeLa cells expressing a YFP-FERM domain fusion protein or the basic patch mutant (KAKTLRK) were fixed, permeabilized, and then stained with rhodamine-phalloidin. Fixed cells were observed by laser-scanning confocal microscopy. (F) The percentages of cells containing the YFP-FERM constructs in ruffles or at the membrane at the edge of the cell were scored. wt, wild type. Results represent the average from three experiments ± standard deviation with >100 cells counted per experiment. **, P < 0.005; *, P < 0.05. (G) Representative images of cells expressing the YFP-FERM domain (two left panels) or the YFP-FERM KAKTLRK mutant (right panel) are shown. Arrows indicate YFP-FERM localization in ruffles (left panel) and at the membrane at the periphery of the cell (middle panel).

(A) Design of the FAK autophosphorylation biosensor. When tyrosine 397 is unphosphorylated, CFAK does not interact with citrine-dSH2 and FRET does not occur. When tyrosine 397 is phosphorylated, CFAK interacts with citrine-dSH2 and FRET occurs. (C, CFP; Y, citrine). (B) The FAK wild-type autophosphorylation biosensor and the CFAK-Y397F control biosensor were expressed in HEK293 cells and analyzed by fluorometry. CFP was selectively excited at 425 nm, and the resulting emission spectra were normalized to the CFP emission peak. (C) FRET in the FAK biosensor was verified by acceptor photobleaching in living cells. HEK293 cells were photobleached using a 488-nm laser. The emission spectra produced by CFP excitation were recorded before and after photobleaching. Shown are the average results from three experiments ± standard error.

Biochemical characterization of the FAK conformational biosensor. (A) HEK293 cells expressing empty vector (Mock) or the indicated FAK constructs were lysed and immunoprecipitated using a FAK antibody. The immune complexes were incubated in an in vitro kinase assay utilizing recombinant GST-paxillin-N-C3 as an exogenous substrate. Phosphorylation of paxillin was detected using the 4G10 phosphotyrosine (P-Tyr) antibody. Equal amounts of substrate were verified by blotting for paxillin using a polyclonal antiserum. FAK in the immune complexes was verified by blotting for FAK. (B) The wild-type and mutant biosensors were transiently expressed in HeLa cells, and adherent cells (Ad) or cells incubated in suspension (Sus) at 37°C for 1 h were lysed. The biosensors were immunoprecipitated (IP) using a GFP antibody and the immune complexes were analyzed by Western blotting for phosphotyrosine (P-Tyr) using 4G10. Equal amounts of FAK in the immune complexes were verified by blotting for FAK. (C) HeLa cells expressing the FAK biosensor were serum starved and stimulated with LPA (200 ng/ml) for 5 min. The biosensor was immunoprecipitated from cell lysates and blotted with phosphotyrosine or a FAK antibody.

(A) Average CFP/FRET ratio of the cytoplasm or focal adhesions in HeLa cells expressing the wild-type biosensor or the constitutively active variant (average of n = 4 cells). The focal adhesion and cytoplasmic values were analyzed using an unpaired t test (*, P < 0.0005). (B and C) The relationship between biosensor activity and focal adhesion location is shown. The distance of each focal adhesion from the cell margin is plotted versus the average CFP/FRET or FRET^C/CFP ratio, where the highest ratio has been normalized to 100%. Representative data from single cells are shown (n = 4). r, correlation coefficient. (D and E) HeLa cells expressing the FAK biosensor were serum starved and simulated with LPA (200 ng/ml), EGF (50 ng/ml), or PDGF (50 ng/ml). CFP and FRET images were sequentially captured at 1-min intervals. The mean CFP/FRET ratio of cells was calculated and normalized to the ratio at the time of stimulation. The change in the emission ratio of the FAK biosensor following EGF, PDGF, or LPA stimulation is shown in panel A. The change in the emission ratio of the wild-type FAK biosensor is compared with that of the constitutively active FAK biosensor mutant following LPA stimulation in panel B. Shown is the average response ± standard error (n = 3 cells). (F) HeLa cells expressing the biosensor were stimulated with LPA and lysed at the indicated times. The biosensor was immunoprecipitated (IP) and blotted with a phosphotyrosine (P-Tyr) antibody (top) or FAK antibody as a loading control (bottom).

(A) A recombinant fragment of FAK containing the FERM and catalytic domains was incubated with Src (SH3 plus SH2 plus kinase) in the presence of the indicated liposomes in kinase reaction buffer or in buffer alone (buf) for 30 min. As a control substrate, a GST fusion protein containing a peptide mimicking the activation loop of FAK was used. The reaction was terminated by the addition of sample buffer, and phosphorylation of the substrates was examined by Western blotting with a phosphotyrosine (P-Tyr) antibody. (B) The wild-type (WT) biosensor and the F2 basic patch mutant biosensor (KAKTLRK) were coexpressed with PIP5KIα or a catalytically defective mutant of PIP5KIα (KD). The CFP/FRET ratio in each case was determined by fluorometry. The average of three experiments ± standard error is shown. The emission ratios observed in the presence of wild-type and catalytically inactive PIP5KIα were analyzed using an unpaired t test (*, P < 0.05). (C) The average CFP/FRET ratio of the cytoplasm or focal adhesions in HeLa cells coexpressing the wild-type biosensor and wild-type SopB or the catalytically inactive mutant SopBcs is shown (average of n = 6 cells ± standard error). The focal adhesion values in SopB- and SopBcs-expressing cells were analyzed using an unpaired t test (*, P < 0.0025).