The Spatiotemporal Pattern of Src Activation at Lipid Rafts Revealed by Diffusion-Corrected FRET Imaging
Figure 11
The assessment of the diffusion model accuracy and the subtraction of biosensor diffusion effects from the apparent FRET images.
(A) The bar graph shows the apparent diffusion coefficients (mean±S.E.M.) of Lyn-Src and KRas-Src biosensors in control cells [0.11±0.01 µm2/sec (n = 43) and 0.18±0.02 µm2/sec (n = 17), respectively], and in cells treated with MβCD [0.17±0.01 µm2/sec (n = 20) and 0.20±0.01 µm2/sec (n = 22), respectively]. (B) The bar graph shows the coefficients of determination (mean±S.E.M.) of the diffusion models for Lyn-Src and KRas-Src biosensors in control cells [0.79±0.033 (n = 50) and 0.56±0.06 (n = 15), respectively], and in cells treated with MβCD [0.68±0.07 (n = 11) and 0.47±0.06 (n = 16), respectively]. Asterisks in (A) and (B) denote significant differences (p<0.05) between different groups as indicated. (C) The FRET signals before and after the subtraction of the effect of Lyn-Src biosensor diffusion from the apparent FRET ratio images. Top panels show the apparent FRET images and lower panels show the corresponding diffusion-subtracted FRET images of the Lyn-Src biosensor, at 0.7, 3.1, 6.1 min after EGF stimulation as indicated. The spatial-temporal dynamics of FRET signals before and after subtracting diffusion is also shown in Movie S2.