Figure 4. FynSensor allows direct visualization of spatially localized active Fyn in cells.

Acceptor photo-bleaching (APB) experiment for visualizing active Fyn in U2OS cells co-expressing FRET donor (Fyn WT) and acceptor (myr-mVenus-F29) FynSensor constructs. (A) APB shows cellular FRET and localized Fyn activity. Representative figure shows donor and acceptor fluorescence images, prior to and after laser-based bleaching of acceptor with 514 nm laser pulse. Also shown is the corresponding ∆Donor image (see Materials and methods) (Scale bar = 10 μm). APB experiments reveal areas showing greater recovery of donor fluorescence after APB indicating spatially localized Fyn activity (B) APB-induced recovery of donor fluorescence is greater at the cell periphery and sensitive to bleaching light dosage. Percentage recovery of donor fluorescence in FynSensor-expressing cells, after acceptor photobleaching at different light dosage (using Equation 2, see Materials and methods), plotted as a function of distance from the cell edge. Extent of recovery also shown at different light dosage. Bleaching is carried out for 10 s using the 514 nm laser line at 2X, 5X and 10X doses (X = 0.069 mW). Lines represent mean of n = 14 cells for 10X, n = 9 cells for 5X, n = 7 cells for 2X, respectively. Values are mean ± s.e.m. (C) FRET signal measured through the APB method is specific (binding dependent) as well as sensitive to APB light doses. Cumulative APB-induced percentage recovery of donor fluorescence observed at the cell periphery in FynSensor-expressing cells (as in B), plotted as a function of light dosage (solid bars). Also, shown extent of recovery when donor-labeled Fyn is used with acceptor-labeled non-binding P41A mutant of F29 (dashed bars). Extent of recovery with non-binding control is minimal and significantly lower than observed for F29 FynSensor. Bars represent mean of n = 14 cells for 10X, n = 9 cells for 5X and n = 7 cells for 2X when binder/F29 is used and n = 14 cells for 10X, n = 10 cells for 5X and n = 8 cells for 2X when non-binder/F29P41A is used, respectively. Student’s unpaired one-tailed t-test was used to determine the p-value. Graph was made using GraphPad Prism. Values are mean ± s.e.m.

Figure 4—figure supplement 1. FynSensor FRET readout is sensitive to kinase activity of Fyn.

FynSensor FRET signal is significantly attenuated when FynSensor expressing cells are treated with an inhibitor of Fyn (SFK) activity. Plotted here is the recovery of donor (mCer-Fyn) fluorescence (425–500 nm) when FynSensor expressing cells are subject to photobleaching at 514 nm, corresponding to absorption wavelength of the acceptor (mVenus-F29 binder). The extent of this recovery is measured as a function of distance from cell periphery. Acceptor-photobleaching (APB) of FynSensor expressing U2OS cells causes a recovery in donor fluorescence showing clear FRET in live cells. This FRET signal (Fyn activity) is observed to be higher at the cell periphery. However, cells treated with 2 µM of SU6656 inhibitor (n = 3 cells) fail to show a similar pattern of recovery of donor fluorescence after APB. Therefore, the FynSensor cellular FRET signal is significantly attenuated on inhibitor treatment. On the contrary, cells treated with DMSO (vehicle control) (n = 4 cells) show an APB-induced recovery of donor fluorescence similar to untreated cells (n = 3 cells). Values are mean ± s.e.m.