Figure 2. Ratiometric imaging reveals higher membrane dipole potential in S. japonicus cells as compared to S. pombe.

(A) A schematic representation of the mechanism for estimating membrane dipole potential using the voltage-sensitive probe di-8-ANEPPS. Membrane bilayer composed of varied glycerophospholipid headgroups (light blue, grey and green) and sterol (yellow) incorporated with the probe. Dipole potential is generated due to the anisotropic arrangement of water (shown in proximity to the outer membrane leaflet for clarity) and lipid dipoles in the interfacial region. Charge transfer leads to a shift in the excitation spectrum of the probe, which is associated with changes in the local electric field strength. The final measurable parameter is the fluorescence intensity ratio (R), defined as the ratio of fluorescence intensities at the blue edge (I_blue) and red edge (I_red) of the excitation spectrum, and is proportional to the value of dipole potential. (B) Representative single-plane fluorescence intensity ratio (R) maps (color-coded in a scale of 0.0-0.4) in S. pombe and S. japonicus of indicated genotypes. R is defined as the ratio of fluorescence intensities at an excitation wavelength of 445 nm to that at 514 nm (emission band pass at 650-710 nm in both cases). Ratiometric images were generated using a custom Fiji macro and are shown as representative visual maps. Quantitative R values shown in (C) were calculated from the corresponding raw fluorescence images acquired at the two excitation wavelengths and not from the color-coded ratiometric maps. (C) A plot representing the individual values of fluorescence intensity ratios (R) calculated at the plasma membrane and at the NE/ER in cells of specified genotypes. Representative regions of interest (ROIs) are shown in Supplemental Fig. S2. The same ROI was applied to the corresponding images acquired at 445 and 514 nm, background fluorescence was subtracted separately in each channel, and R was calculated from the mean fluorescence intensities within the ROI. Data points represent measurements from individual cells from three independent biological replicates, with horizontal lines indicating the mean of each biological replicate. p values were obtained by two-tailed unpaired parametric t-tests, using the means of the three independent biological replicates (n = 3).