The dipole potential correlates with lipid raft markers in the plasma membrane of living cells

Abstract

The dipole potential generating an electric field much stronger than any other type of membrane potential influences a wide array of phenomena, ranging from passive permeation to voltage-dependent conformational changes of membrane proteins. It is generated by the ordered orientation of lipid carbonyl and membrane-attached water dipole moments. Theoretical considerations and indirect experimental evidence obtained in model membranes suggest that the dipole potential is larger in liquid-ordered domains believed to correspond to lipid rafts in cell membranes. Using three different dipole potential-sensitive fluorophores and four different labeling approaches of raft and nonraft domains, we showed that the dipole potential is indeed stronger in lipid rafts than in the rest of the membrane. The magnitude of this difference is similar to that observed between the dipole potential in control and sphingolipid-enriched cells characteristic of Gaucher's disease. The results established that the heterogeneity of the dipole potential in living cell membranes is correlated with lipid rafts and imply that alterations in the lipid composition of the cell membrane in human diseases can lead to substantial changes in the dipole potential.

Keywords: Gaucher’s disease; fluorescence and confocal imaging; fluorescence microscopy; lipid rafts; membrane dipole potential; membranes.

Fig. 1.

The effect of an increased dipole potential on the emission ratio of PPZ8 and F66. A–D: A431 (A, C) and SKBR-3 (B, D) cells were treated with the dipole potential-increasing sterol, 6-ketocholestanol, and control cells were treated only with Pluronic F-127. Cells were labeled with the dipole potential-sensitive indicators PPZ8 or F66, followed by determining the emission ratio on a pixel-by-pixel basis. Representative histograms display the distribution of pixelwise ratios calculated from single cells. The mean intensity ratios of control (Pluronic F-127) and 6-ketocholestanol-treated cells calculated from 20 to 30 cells are displayed (E, F). The error bars represent the standard errors of the mean. *P < 0.05, significant difference found between the control and 6-ketocholestanol-treated cells by three-way ANOVA, followed by Tukey’s honest significance test (HSD) test.

Fig. 2.

Determination of the dipole potential inside and outside lipid rafts. SKBR-3 and A431 cells were transfected with GFP-GPI or labeled with AlexaFluor647-CTX-B. Labeling with the dipole potential-sensitive dyes, di-8-ANEPPS (A, B) and F66 (C), was carried out 2 days after transfection with GFP-GPI or concomitantly with CTX-B staining. Images were segmented to raft (CTX-B high, GFP-GPI high) and nonraft (CTX-B low, GFP-GPI low) regions, as is shown in supplemental Fig. S4, and the intensity ratios characteristic of the dipole potential were determined separately for the two masks. The intensity ratios were normalized to the ratio determined for the nonraft region in every cell. The error bars represent the standard errors of the mean. *P < 0.05, significant difference found between the intensity ratios inside and outside lipid rafts by two-way ANOVA, followed by Tukey’s HSD test.

Fig. 3.

Emission spectra of F66 and PPZ8 inside and outside lipid rafts. Cells were labeled with the dipole potential sensitive dyes F66 or PPZ8 and with the lipid raft marker, AlexaFluo647-CTX-B. The emission spectrum of the dipole potential-sensitive dyes was recorded on a pixel-by-pixel basis (a.u., arbitrary units). Lipid rafts correspond to high-intensity areas identified in the CTX-B images. The emission spectra were averaged separately for pixels in the raft and nonraft regions. Nonnormalized spectra are shown in the top panels. To cancel the effect of water quenching, we normalized spectra to the total fluorescence emission, and these normalized curves are shown in the bottom panels.

Fig. 4.

Effect of sphingolipid accumulation in a model of Gaucher’s disease on the dipole potential. THP-1 monocytes were differentiated to macrophages with PMA in the absence (control) and presence (Gaucher) of CBE, followed by labeling them with three different dipole potential-sensitive dyes: di-8-ANEPPS (A, D), PPZ8 (B, E), and F66 (C, F). The intensity ratios were calculated in 20–30 cells, and their means (±SEM) are displayed in the bar graphs (A–C). Representative histograms showing the distribution of pixelwise fluorescence ratios are displayed in (D–F). *P < 0.05, significant difference found between the intensity ratios in control and Gaucher-type cells by two-way ANOVA, followed by Tukey’s HSD test.