Comparison of three ternary lipid bilayer mixtures: FRET and ESR reveal nanodomains

Abstract

Phase diagrams of ternary lipid mixtures containing cholesterol have provided valuable insight into cell membrane behaviors, especially by describing regions of coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases. Fluorescence microscopy imaging of giant unilamellar vesicles has greatly assisted the determination of phase behavior in these systems. However, the requirement for optically resolved Ld + Lo domains can lead to the incorrect inference that in lipid-only mixtures, Ld + Lo domain coexistence generally shows macroscopic domains. Here we show this inference is incorrect for the low melting temperature phosphatidylcholines abundant in mammalian plasma membranes. By use of high compositional resolution Förster resonance energy transfer measurements, together with electron spin resonance data and spectral simulation, we find that ternary mixtures of DSPC and cholesterol together with either POPC or SOPC, do indeed have regions of Ld + Lo coexistence. However, phase domains are much smaller than the optical resolution limit, likely on the order of the Förster distance for energy transfer (R(0), ∼2-8 nm).

Figure 1

SAE (stimulated acceptor emission) surfaces in DSPC/DOPC/chol show regions of enhanced or reduced FRET efficiency corresponding to phase coexistence regions. Contour plots A and B from 1116 data points, corresponding to 2 mol % sampling of the ternary composition space. Data were smoothed by averaging nearest-neighbor values. The relatively lowest values are blue, and the relatively highest values are red as shown by the scale bar. (A) BoDIPY-PC to Fast-DiI FRET: donor and acceptor colocalization in Ld phase domains results in enhanced FRET, most pronounced near the ordered phase boundary (arrow 1). (B) DHE to BoDIPY-PC FRET: donor and acceptor segregation between ordered and disordered phases results in reduced FRET. Symbols and arrows refer to surface features mentioned in the text. (C and D) Predicted surfaces for the Ld + Lo region corresponding to a best-fit of data in panels A and B (respectively) to Eqs. 1–3. Critical point (star) and tieline field used to model the data are shown.

Figure 2

Lipid and probe K^P in the Ld + Lo tieline field of DSPC/DOPC/chol. Each value of u represents a different tieline, beginning at the critical point (u = 0) and ending at the Ld + Lo segment of the three-phase triangle (u = 1). DHE (dotted), BoDIPY-PC (dashed), and Fast-DiI (dot-dash) K^P are calculated from Eq. 2 and the respective best-fit values of κ₀ and κ₁ listed in Table 1. Lipid K^P (shaded lines) are calculated from tieline endpoints.

Figure 3

SAE surfaces in DSPC/POPC/chol and DSPC/SOPC chol show RRE and REE. Contour plots A and B each from 1116 data points, corresponding to 2% sampling of the ternary composition space. Data were smoothed by averaging nearest-neighbor values. BoDIPY-PC to Fast-DiI FRET in DSPC/POPC/chol (A) and DSPC/SOPC/chol (B). As in Fig. 1, colocalization of these probes in Ld phase domains results in enhanced FRET efficiency at phase-separated compositions. DHE to BoDIPY-PC FRET in DSPC/POPC/chol (C) and DSPC/SOPC/chol (D). Separation of these probes between ordered and disordered phases results in reduced FRET efficiency. Symbols and arrows refer to surface features mentioned in the text.

Figure 4

ESR reveals similarities in phase properties of mixtures forming macroscopic and nanoscopic phases. Compositional trajectories run in the approximate direction of Ld + Lo tielines (see Fig. 1A, dashed line) and differ only in the identity of the low-T_M lipid. (A) Composition-dependent order parameters obtained from ESR spectral simulations in DSPC/DOPC/chol (diamonds), DSPC/POPC/chol (triangles), and DSPC/SOPC/chol (circles). (B) Fraction of 16-PC spin probe in the Lo phase determined by spectral subtraction using Eq. 4 (symbols as in panel A). Predicted fractions from Eq. 5 shown as lines for DSPC/DOPC/chol (solid), DSPC/POPC/chol (dashed), and DSPC/SOPC/chol (dotted), with best-fit parameters listed in Table 2.

Figure 5

Phase diagrams for systems in this study: DSPC/DOPC/chol (solid lines), DSPC/POPC/chol (dashed), and DSPC/SOPC/chol (dotted). Solidus boundary extensions are not well determined in the POPC- and SOPC-containing mixtures.