Figure 2.
(A) Fluorescence emission spectra of WCY-eENTH in response to binding to POPC/POPS/PI4,5P2 (80–x/20/x: x = 0–2 mol%) LUVs. The spectra were obtained spectrofluorometrically with the excitation wavelength set at 405 nm. The intensity values were normalized using the maximal intensity of the free sensor as the reference. The black line represents the spectrum of the sensor alone and the red line that with 0% PI4,5P2 (i.e., POPC/POPS (80/20)). (B) The ratiometric calibration curve of WCY-eENTH for PI45P2 quantification. WCY-eENTH was mixed with POPC/POPS/PI4,5P2 (80–x/20/x: x = 0–2 mol%) GUVs and the fluorescence emission intensity in two separate channels were measured by a confocal microscope. Nonlinear least-squares analysis of the plot using the equation FB/FY = (FB/FY)min + (FB/FY)max / (1 + Kd/[PI4,5P2]) yielded Kd, (FB/FY)max, and (FB/FY)min values and the calibration curve was constructed using these parameters. FB/FY, Kd, (FB/FY)max, and (FB/FY)min are the ratio of the fluorescence intensity in the blue channel to that in the yellow channel, equilibrium dissociation constant (in mol%), and the maximal and minimal FB/FY values, respectively. The blue channel depicts the membrane-bound sensor whereas the yellow channel shows membrane-bound plus free sensors. Error bars indicate standard deviations calculated from three independent sets of measurements. (C) Representative blue-channel and yellow channel cross-sectional images of a NIH 3T3 cell microinjected with WCY-eENTH. (D) A spatially resolved PI4,5P2 concentration profile at PM calculated from Figure 2C. The z-axis scale indicates mol% of PI4,5P2. A pseudo-coloring scheme with red and blue representing the highest (i.e., 2 mol%) and the lowest (i.e., 0 mol%) concentration respectively is used to illustrate the spatial PI4,5P2 concentration heterogeneity. Scale bars indicate 5 μm.