Supplementary Figure 11: MCS⁺ revealed the dynamics of the electrostatic potential of the inner plasma membrane in response to osmolality shock.

(a) Ratiometric images (R/G ratio, pseudo-colored as indicated) of MCS⁺ in live COS-7 cells before and after exchange to hypotonic (150 mM to 40 mM) and hypertonic (150 mM to 450 mM) buffers. Scale bar = 10 μm. (b) Change in in donor (green) and acceptor (red) intensity and FRET efficiency (R/G ratio) for the cells shown in (a). Arrows indicate when solutions were added. Images and data in a-d are representative of n = 5 independent experiments per condition. It is noteworthy that hypotonic treatments resulted solely in an increase in FRET efficiency while hypertonic treatments caused first a rapid loss in FRET efficiency followed by a small increase. Thus quantitative live cell measurements of the effective electrostatic potential with MCS⁺ may be able to identify compensatory mechanisms of membrane channels and pumps to hypertonic treatments because of the reversible response of the sensor.