Figure 4.
Fluorescence anisotropy reports homo-FRET between cholesterol sensors. (A) Representative confocal images showing membrane-localized eGFP-GRAW–W fluorescence emission in a naïve DRG neuron, acquired in perpendicular and parallel directions. (B) Corresponding anisotropy-based heatmaps derived from the images in A, shown for both pre- and post-WSC conditions, illustrating a reduction in anisotropy after WSC treatment. (C–E) Summary time course of the changes in GRAM-W fluorescence anisotropy and estimated homo-FRET efficiency for tsA cells (C, n = 10, P = 5e−4, 5e−3, and 2e−3 at 5, 10, and 15 min time points), naïve DRG neurons (D, n = 6, P = 0.03 for all time points), and DRG neurons of SNI model animals (E, n = 5, P = 3e−3, 0.01, and 0.03 at 5, 10, and 15 min time points). Since the homo-FRET data are derived from the measured anisotropy, the statistical significance is only shown for the anisotropy and remains the same for the homo-FRET. (F–H) Summary time course of changes in the OlyA fluorescence anisotropy and estimated homo-FRET efficiency for tsA cells (F, n = 4, P = 0.11, 0.08, and 0.04 at 5, 10, and 15 min time points), naïve DRG neurons (G, n = 6, P = 0.12, 0.2, and 0.12 at 5, 10, and 15 min time points), and DRG neurons of SNI model animals (H, n = 4, P = 0.08, 0.032, and 0.049 at 5, 10, and 15 min time points). Data shown are mean ± SEM, *P < 0.05, **P < 0.01 using a one-sided paired t test, justified by the directional hypothesis that increased sensor crowding leads to higher homo-FRET. Refer to the image caption for details. Part A displays a phasor plot where data points cluster on a universal semicircle with a phase lifetime (tau(phi)) of 2.57 ns before W S C treatment, including an inset of a circular fluorescent cell membrane. Part B shows the same phasor plot and inset five minutes after the application of water-soluble cholesterol (W S C), with the phase lifetime remaining constant at 2.57 n s. Part C presents a time-course graph tracking the lifetime of G R A M-W-e G F P in nanoseconds over 15 minutes, with green square data points remaining stable near 2.6 n s. Statistical significance is noted as n.s. (not significant) for all time points, confirming that cholesterol enrichment does not statistically alter the fluorescence lifetime of the G R A M-W-e G F P probe. All values are approximated.

Fluorescence anisotropy reports homo-FRET between cholesterol sensors. (A) Representative confocal images showing membrane-localized eGFP-GRAW–W fluorescence emission in a naïve DRG neuron, acquired in perpendicular and parallel directions. (B) Corresponding anisotropy-based heatmaps derived from the images in A, shown for both pre- and post-WSC conditions, illustrating a reduction in anisotropy after WSC treatment. (C–E) Summary time course of the changes in GRAM-W fluorescence anisotropy and estimated homo-FRET efficiency for tsA cells (C, n = 10, P = 5e−4, 5e−3, and 2e−3 at 5, 10, and 15 min time points), naïve DRG neurons (D, n = 6, P = 0.03 for all time points), and DRG neurons of SNI model animals (E, n = 5, P = 3e−3, 0.01, and 0.03 at 5, 10, and 15 min time points). Since the homo-FRET data are derived from the measured anisotropy, the statistical significance is only shown for the anisotropy and remains the same for the homo-FRET. (F–H) Summary time course of changes in the OlyA fluorescence anisotropy and estimated homo-FRET efficiency for tsA cells (F, n = 4, P = 0.11, 0.08, and 0.04 at 5, 10, and 15 min time points), naïve DRG neurons (G, n = 6, P = 0.12, 0.2, and 0.12 at 5, 10, and 15 min time points), and DRG neurons of SNI model animals (H, n = 4, P = 0.08, 0.032, and 0.049 at 5, 10, and 15 min time points). Data shown are mean ± SEM, *P < 0.05, **P < 0.01 using a one-sided paired t test, justified by the directional hypothesis that increased sensor crowding leads to higher homo-FRET.

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