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. 2009 Jul 1;284(36):24133–24143. doi: 10.1074/jbc.M109.015271

FIGURE 1.

FIGURE 1.

The Pakabi FRET-based biosensor for PAK1 activation. A, Pakabi design. The Pakabi biosensor is a fusion protein comprising YFP, aa 65 to the C terminus of PAK1, and CFP. In the inactive dimeric state, the proximity of the YFP and CFP fluorophores allows FRET to occur. Upon activation, the conformation of PAK1 changes dramatically, including dissociation of PAK1 dimers, thereby moving away the YFP acceptor from the CFP donor. Because the stoichiometry of YFP to CFP remains 1:1, the level of FRET is easily calculated as the ratio between YFP fluorescence (excitation 430 nm, emission 530 nm) and CFP fluorescence (excitation 430 nm, emission 480 nm). Circles represent phosphorylatable residues. B, biochemical validation of Pakabi. COS-1 cells were transfected with the indicated vectors expressing wild-type Pakabi alone, wild-type Pakabi together with Cdc42V12 or a mutated Pakabi carrying the PAK1 activating mutation L107F. Total cell lysates were analyzed by Western blotting with anti-PAK, anti-phospho-PAK1-S199/204, and anti-phospho-PAK1-T423 antibodies. In parallel, spectrographs of isolated living cells were acquired, and the peak intensities of YFP and CFP were used to calculate FRET, which is normalized to wild-type Pakabi alone taken as 100%. Bars represent standard error of the means (±S.E.). The number of cells (n) analyzed for each condition was ≥14. C, dose-response decrease of FRET for Pakabi upon activation by Cdc42. COS-7 cells were transfected with vectors expressing Pakabi and mRFP-Cdc42V12. For individual living cells, YFP, CFP, and RFP images were acquired and whole cell mean intensities were measured. The cells (n = 110) were divided into categories according to RFP intensity. Single cell FRET values were calculated as YFP/CFP ratio and averaged for each category. D, control with Pakabi unable to bind GTPases. COS-7 cells were transfected with a vector encoding CRIB-mutated Pakabi alone or together with the plasmid expressing mRFP-Cdc42V12. FRET values were measured as above; n ≥ 14 for each condition. E, control with Cdc42V12 not-fused to mRFP. COS-7 cells were transfected with vectors expressing Pakabi alone or together with mRFP-Cdc42V12 or FLAG-Cdc42V12. n ≥ 30 for each condition.