Condensed complexes, rafts, and the chemical activity of cholesterol in membranes

Abstract

Epifluorescence microscopy studies of mixtures of phospholipids and cholesterol at the air-water interface often exhibit coexisting liquid phases. The properties of these liquids point to the formation of "condensed complexes" between cholesterol and certain phospholipids, such as sphingomyelin. It is found that monolayers that form complexes can incorporate a low concentration of a ganglioside G(M1). This glycolipid is visualized by using a fluorescently labeled B subunit of cholera toxin. Three coexisting liquid phases are found by using this probe together with a fluorescent phospholipid probe. The three liquid phases are identified as a phospholipid-rich phase, a cholesterol-rich phase, and a condensed complex-rich phase. The cholera toxin B labeled ganglioside G(M1) is found exclusively in the condensed complex-rich phase. Condensed complexes are likely present in animal cell membranes, where they should facilitate the formation of specialized domains such as rafts. Condensed complexes also have a major effect in determining the chemical activity of cholesterol. It is suggested that this chemical activity plays an essential role in the regulation of cholesterol biosynthesis. Gradients in the chemical activity of cholesterol should likewise govern the rates and direction of intracellular intermembrane cholesterol transport.

Figure 1

(A–C) Calculated composition-pressure phase diagrams for mixtures containing cholesterol and one or more other lipids. (A) Binary mixture of cholesterol, C, with a non-complex-forming lipid U. (B) Binary mixture of C with a complex-forming lipid S. (C) Ternary mixture of C with U and S present in a 1:1 ratio. Regions of phase coexistence are identified as α, β, and γ. α and β are two-phase regions, and γ is a three-phase region. Compositions of coexisting phases were found by the method of double-tangent construction, using the free energy in Eq. 2 (28), with the parameters p = 2; q = 1; n = 1; K_eq⁰ = 1,000; ΔA = −40 Ų; a_SU = 0; a′_CS = a′_CU = a′_CX = a′_SX = a′_UX = 1/6; π_CS = 12 mN/m; π_CU = 10 mN/m; π_CX = 18 mN/m; π_SX = 2 mN/m; π_UX = 9 mN/m. “X” represents the complex C_nqS_np. (D) Experimental phase diagram showing liquid–liquid miscibility critical points for a mixture of DChol and phospholipids. The phospholipid fraction is a 1:1 molar mixture of egg-SM and DMPC along with 10% GM1. Plotted data points indicate transition pressures where phase coexistence disappears during monolayer compression. Above these pressures, there is a single uniform phase. Stripe superstructure phases, which are characteristic of proximity to a critical point, were observed at the transitions marked by ●. The sketched dotted lines illustrate the possibility of a three-phase region as in C. The “+” symbol represents pressure-composition conditions used in Fig. 2. The error bars represent deviations in three independent phase boundary measurements.

Figure 2

Epifluorescence micrographs of a lipid monolayer containing 31% egg-SM, 31% DMPC, 7% GM1, 30% DChol, and 1% F-DHPE at a surface pressure of 6 mN/m. (The phase diagram of this mixture is shown in Fig. 1D, where the “+” symbol denotes the pressure-composition conditions used here.) The subphase contains Alexa-labeled CT-B. (A) View of the monolayer when observed with a F-DHPE-specific filter that excludes Alexa emission. (B) View of the same monolayer region when observed with an Alexa specific filter that excludes F-DHPE emission. When this monolayer is pressurized, all of the domains go through a critical phase transition at roughly the same pressure, suggestive of proximity to a tricritical point. The probe distribution demonstrates three coexisting phases.

Figure 3

(A) Calculated composition phase diagram for a ternary mixture of cholesterol C, with noncomplex-forming lipid U and complex-forming lipid S, using the same parameters as Fig. 1 A–C, for a surface pressure of π = 4 mN/m. Single-phase regions and regions of two- and three-phase coexistence are indicated by 1φ, 2φ, and 3φ, respectively. ○ indicates a critical point. The black dot denotes a mixture of 18% C and 41% each of S and U. Arrow 1 represents the addition of C to this mixture; arrow 2 represents an increase in the proportion of U in the nonsterol fraction. (B) Chemical activity of cholesterol as a function of cholesterol composition for a 1:1 mixture of S and U. (C) Chemical activity of cholesterol as a function of the proportion of U in the nonsterol fraction of a mixture containing 18% cholesterol. In B and C, the black dot indicates the composition of the mixture described in A; arrows 1 and 2 represent the composition changes described in A.