Panel A shows a schematic illustration depicting Förster Resonance Energy Transfer (F R E T) between C T x B A F-488 (donor) and C T x B A F-647 (acceptor) occurring in large Outer Membrane Domains (O M D s) but not in small O M D s. Panels B and C: Graphs modeling C T x B occupancy (n equals k) within O M D s using Poisson statistics. Panel B shows probability curves for 0 to 4 C T x B molecules, while Panel C shows the cumulative probability for k greater than or equal to 2 C T x B per O M D as a function of mean O M D diameter (nanometer). Panels D and E: Representative phasor plots (S versus G) of membrane-localized fluorescence in t s A-201 cells. The F R E T efficiency (E) increases from 0.05 before W S C treatment to 0.09 after W S C treatment. Insets provide magnified views of the data clusters. Panel F: A time course graph (0 to 7 minutes) showing normalized fluorescence (green) and F L I M-F R E T (blue) post-W S C application. Both metrics show a sharp increase within the first two minutes, indicating rapid membrane reorganization following cholesterol enrichment. All values are approximate.
Combining the CTxB-based FRET approach and a cholesterol sensor in assessing the modulation of PM properties by cholesterol enrichment. (A) Cartoon showing the FRET between CTxB AF-488 and CTxB AF-647 under conditions of small OMDs and large OMDs. Only one fluorophore is shown for the CTxB pentamer for simplicity. (B and C) CTxB occupancy within OMDs was modeled using Poisson statistics with a nonlinear dependence on OMD diameter (B) to interpret CTxB-based FLIM-FRET responses to OMD expansion. The increased FRET with larger OMDs reflects a higher probability of multiple CTxB molecules occupying the same OMD (C). (D and E) Representative phasor plots of membrane-localized fluorescence of tsA201 cells from donor alone (CTxB AF-488 only) or with the acceptor CTxB AF-647 before (D) and after (E) the WSC treatment. The FRET efficiency was calculated using the FRET trajectory (in red). The insets show amplified views of the phasor plots. A confocal image of membrane-localized fluorescence of a tsA cell labeled with CTxB is also shown. (F) Time course of the change in normalized fluorescence intensity at the PM and the CTxB-based FLIM-FRET efficiency after 0.5 mg/ml WSC application in tsA201 cells. Data shown are mean + SEM; WSC was added to the culture medium at the start of time-lapse imaging, which was performed at intervals of ∼27 s for FRET (n = 4) and ∼20 s for GRAM-W (n = 3). Time zero refers to the steady state prior to WSC addition. The purple bar denotes the duration of WSC pipetting into the recording chamber.