Miscibility Phase Diagrams of Giant Vesicles Containing Sphingomyelin

Saturated sphingomyelin (SM) lipids are implicated in lipid rafts in cell plasma membranes. Here we use fluorescence microscopy to observe coexisting liquid domains in vesicles containing SM, an unsaturated phosphatidylcholine lipid (either DOPC or POPC), and cholesterol. We note similar phase behavior in a model membrane mixture without SM ($\mathrm{DOPC}/\mathrm{DPPC}/\mathrm{Chol}$), but find no micron-scale liquid domains in membranes of $\mathrm{POPC}/\mathrm{PSM}/\mathrm{Chol}$. We delineate the onset of solid phases below the miscibility transition temperature, and detail indirect evidence for a three-phase coexistence of one solid and two liquid phases.

Figure 1

Giant vesicles of $\mathrm{DOPC}/\mathrm{PSM}/\mathrm{Chol}$ at $25°\mathrm{C}$. Vesicles are imaged before domains fully coalesce, and compositions are mole fractions. By examining the fraction of bright and dark phases in vesicles, we estimate that the dark liquid phase is rich in PSM and Chol, while the bright liquid phase is rich in DOPC [3]. The same trend is evident in $\mathrm{POPC}/\mathrm{PSM}/\mathrm{Chol}$ (not shown). Scale bars are $20\mu \mathrm{m}$.

Figure 2

Miscibility phase boundaries in ternary lipid mixtures of (a) $\mathrm{DOPC}/\mathrm{PSM}/\mathrm{Chol}$, (b) $\mathrm{DOPC}/\mathrm{DPPC}/\mathrm{Chol}$, (c) $\mathrm{POPC}/\mathrm{PSM}/\mathrm{Chol}$, and (d) $\mathrm{POPC}/\mathrm{DPPC}/\mathrm{Chol}$. Compositions where a miscibility transition is observed are denoted by black circles and the colored surface reports an extrapolated fit of measured values of $T_{\mathrm{mix}}$. Errors in Fig. 3 ($\pm 1$ to $\pm 5°\mathrm{C}$) apply here. Higher temperatures occur at higher fractions of saturated lipid. Open circles indicate that only one liquid phase is present down to $10°\mathrm{C}$. Grey square symbols indicate that solid domains nucleate directly from a uniform liquid phase as temperature is lowered. Coexisting liquid domains are not observed for all points shown in membranes of $\mathrm{POPC}/\mathrm{DPPC}/\mathrm{Chol}$ between $10–60°\mathrm{C}$.

Figure 3

Partial phase diagrams for vesicles of $\mathrm{DOPC}/\mathrm{PSM}/\mathrm{Chol}$ with a constant ratio of saturated to unsaturated lipid. Black circle symbols mark $T_{\mathrm{mix}}$ and gray square symbols mark where solid phases are first observed as temperature is lowered. The three phase states observed are: solid-liquid ($S\mathrm{\text{−}}L$), liquid-liquid ($L\mathrm{\text{−}}L$), and one uniform liquid phase ($L$). Error bars record the range of temperatures where a transition is observed in a single vesicle preparation, and all transitions are fully reversible. Lines are drawn to guide the eye and are not a fit to any theory.

Figure 4

Partial phase diagrams for vesicles of (a)–(c) $\mathrm{DOPC}/\mathrm{PSM}/\mathrm{Chol}$ and (d)–(f) $\mathrm{POPC}/\mathrm{PSM}/\mathrm{Chol}$ at constant temperatures. White symbols denote that membranes are in one uniform phase, either liquid (circles) or solid (squares). Black circles denote coexisting liquid phases, and gray squares denote coexisting solid and liquid phases.

Figure 5

Experimental data in diagrams (a) and (b) are both identical to the fluorescence microscopy data in Fig. 4e for $\mathrm{POPC}/\mathrm{PSM}/\mathrm{Chol}$ at $23°\mathrm{C}$. Underlying phase diagrams contain dark gray three-phase regions and light gray two-phase regions. (a) Our results are fit well by a diagram that includes a three-phase triangle. (b) Our results are poorly fit by a previously published diagram of the same mixture [15]. Our experiments do not exclude more complicated phase diagrams, including diagrams with additional two- and three-phase regions.