Lipid phase separation in phospholipid bilayers and monolayers modeling the plasma membrane.

It is postulated that biological membrane lipids are heterogeneously distributed into lipid microdomains. Recent evidence indicates that docosahexaenoic acid-containing phospholipids may be involved in biologically important lipid phase separations. Here we investigate the elastic and thermal properties of a model plasma membrane composed of egg sphingomyelin (SM), cholesterol and 1-stearoyl-2-docosahexaenoyl-sn-glycerophosphoethanolamine (SDPE). Two techniques are employed, pressure-area isotherms on monolayers to examine condensation and interfacial elasticity behavior, and differential scanning calorimetry (DSC) on bilayers to evaluate phase separations. Significant levels of condensation are observed for mixtures of SM and cholesterol. Surface elasticity measurements indicate that cholesterol decreases and SDPE increases the in-plane elasticity of SM monolayers. At X(SDPE)> or =0.15 in SM, a more horizontal region emerges in the pressure-area isotherms indicating 'squeeze out' of SDPE from the monolayers. Addition of cholesterol to equimolar amounts of SM and SDPE further increases the amount of 'squeeze out', supporting the concept of phase separation into a cholesterol- and SM-rich liquid ordered phase and a SDPE-rich liquid disordered phase. This conclusion is corroborated by DSC studies where as little as X(Chol)=0.0025 induces a phase separation between the two lipids.

Liquid crystalline/gel state phase separation in docosahexaenoic acid-containing bilayers and monolayers.

The phase behavior of lipid mixtures containing 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0, 22:6 PC) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was studied with bilayers using differential scanning calorimetry (DSC), and with monolayers monitoring pressure/area isotherms and surface elasticity, and lipid domain formation followed by epifluorescence microscopy. From DSC studies it is concluded that DPPC/18:0, 22:6 PC phase separates into DPPC-rich and 18:0, 22:6 PC-rich phases. In monolayers, phase separation is indicated by changes in pressure-area isotherms implying phase separation where 18:0, 22:6 PC is 'squeezed out' of the remaining DPPC monolayer. Phase separation into lipid domains in the mixed PC monolayer is quantified by epifluorescence microscopy using the fluorescently labeled phospholipid membrane probe, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl). These results further describe the ability of docosahexaenoic acid to participate in lipid phase separations in membranes.

Detection of lipid domains in docasahexaenoic acid-rich bilayers by acyl chain-specific FRET probes.

A major problem in defining biological membrane structure is deducing the nature and even existence of lipid microdomains. Lipid microdomains have been defined operationally as heterogeneities in the behavior of fluorescent membrane probes, particularly the fluorescence resonance energy transfer (FRET) probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl-diacyl-sn-glycero-3-phosphoethan olamine (N-NBD-PE) and (N-lissamine rhodamine B sulfonyl)-diacyl-snglycero-3-phosphoethanolamine (N-Rh-PE). Here we test a variety of N-NBD-PEs and N-Rh-PEs containing: (a) undefined acyl chains, (b) liquid crystalline- and gel-state acyl chains, and (c) defined acyl chains matching those of phase separated membrane lipids. The phospholipid bilayer systems employed represent a liquid crystalline/gel phase separation and a cholesterol-driven fluid/fluid phase separation; phase separation is confirmed by differential scanning calorimetry. We tested the hypothesis that acyl chain affinities may dictate the phase into which N-NBD-PE and N-Rh-PE FRET probes partition. While these FRET probes were largely successful at tracking liquid crystalline/gel phase separations, they were less useful in following fluid/fluid separations and appeared to preferentially partition into the liquid-disordered phase. Additionally, partition measurements indicate that the rhodamine-containing probes are substantially less hydrophobic than the analogous NBD probes. These experiments indicate that acyl chain affinities may not be sufficient to employ acyl chain-specific N-NBD-PE/N-Rh-PE FRET probes to investigate phase separations into biologically relevant fluid/fluid lipid microdomains.

Cholesterol versus cholesterol sulfate: effects on properties of phospholipid bilayers containing docosahexaenoic acid.

The important omega-3 fatty acid docosahexaenoic acid (DHA) is present at high concentration in some membranes that also contain the unusual sterol cholesterol sulfate (CS). The association between these lipids and their effect on membrane structure is presented here. Differential scanning calorimetry (DSC), MC540 fluorescence, erythritol permeability, pressure/area isotherms on lipid monolayers and molecular modeling are used to compare the effect of CS and cholesterol on model phospholipid membranes. By DSC, CS decreases the main phase transition temperature and broadens the transitions of dipalmitolyphosphatidylcholine (DPPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0,18:1 PC) and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0,22:6 PC) to a much larger extent than does cholesterol. In addition CS produces a three-component transition in 18:0,18:1 PC bilayers that is not seen with cholesterol. In a mixed phospholipid bilayer composed of 18:0,18:1 PC/18:0,22:6 PC (1:1, mol/mol), CS at 2.5 membrane mol% or more induces lateral phase separation while cholesterol does not. CS decreases lipid packing density and increases permeability of 18:0,18:1 PC and 18:0,22:6 PC bilayers to a much larger extent than cholesterol. CS disrupts oleic acid-containing bilayers more than those containing DHA. Molecular modeling confirms that the anionic sulfate moiety on CS renders this sterol more polar than cholesterol with the consequence that CS likely resides higher (extends further into the aqueous environment) in the bilayer. CS can therefore be preferentially accommodated into DHA-enriched bilayers where its tetracyclic ring system may fit into the delta 4 pocket of DHA, a location excluded to cholesterol. It is proposed that CS may in part replace the membrane function of cholesterol in DHA-rich membranes.

Cholesterol versus alpha-tocopherol: effects on properties of bilayers made from heteroacid phosphatidylcholines.

The techniques of differential scanning calorimetry, fluorescence of merocyanine 540, fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, proton permeability, and lipid peroxidation are used to compare the perturbations of cholesterol and alpha-tocopherol on lipid bilayer membranes composed of different phosphatidylcholines containing stearic acid in the sn-1 position and an unsaturated fatty acid (either oleic, alpha-linolenic, gamma-linolenic, or docosahexaenoic acid) in the sn-2 position. It is concluded that the structural roles of cholesterol and alpha-tocopherol may be similar with membranes composed of some phosphatidylcholines but are clearly different with membranes composed of other related phosphatidylcholines. alpha-Tocopherol exerts a much larger effect than cholesterol on membranes rich in polyunsaturated fatty acids that have their initial double bond before the delta 9 position. Cholesterol interacts more favorably with fatty acids that do not have an double bond before the delta 9 position. The membrane structural effects are explained in terms of the larger size of the sterol ring structure of cholesterol compared to the smaller chromanol ring of the alpha-tocopherol.

Cholesterol condensation of alpha-linolenic and gamma-linolenic acid-containing phosphatidylcholine monolayers and bilayers.

Cholesterol is demonstrated to condense phosphatidylcholine (PC) monolayers and bilayers containing stearic acid in the sn-1 position and alpha-linolenic acid in the sn-2 position (18:0, alpha-18:3 PC) but has no effect when gamma-linolenic acid occupies the sn-2 position (18:0,gamma-18:3 PC). Cholesterol-induced condensation is measured by area/molecule determinations made on monolayers using a Langmuir trough, while condensation in bilayers is followed by the fluorescent dyes merocyanine (MC540) and dansyllysine. Permeability to erythritol is also demonstrated to be diminished by cholesterol for the condensable 18:0,alpha-18:3 PC bilayer membranes but not the 18:0,gamma-18:3 PC membranes. alpha- and gamma-linolenic acid are isomers containing 18 carbons and three unsaturations. Both fatty acids have unsaturations at positions 9 and 12 and differ only in the location of the third unsaturation, at either position 6 for gamma-linolenic acid (an omega-6 fatty acid) and at position 15 for alpha-linolenic acid (an omega-3 fatty acid). Here lipid-cholesterol interaction is used to distinguish the effect of position of unsaturation on membrane structure.

Use of merocyanine (MC540) in quantifying lipid domains and packing in phospholipid vesicles and tumor cells.

The fluorescent probe merocyanine (MC540) reports qualitatively on several membrane events. Here we demonstrate that MC540 fluorescence can quantify the degree of coexisting liquid-crystalline and gel states in mixed monotectic phosphatidylcholine (PC) bilayers. The probe exhibits disparate fluorescence wavelength maximas and and intensities when incorporated into liquid-crystalline and gel state membranes. The fluorescence measurements partitioning of the EPR spin probe TEMPO between the aqueous environment and the membrane fluid phase. While both techniques can accurately assess the phase transition of synthetic PCs, only MC540 can distinguish between liquid-crystalline phases of different composition. MC540 fluorescence for single-component PC bilayers correlates quantitatively with estimates of the area/molecule determined from surface area/pressure isotherms of lipid monolayers, whereas partitioning of TEMPO fails to assess the relative degree of lipid packing in various fluid state membranes. Additionally, MC540 fluorescence characterizes the interaction of cholesterol with membranes made from condensable (18:0, 18:1-PC) and non-condensable (18:0, 22:6-PC) lipids. Finally MC540 distinguishes tumor cell membranes differing only in the amount of docosahexaenoic acid (DHA). Thus we conclude that MC540 can be used quantitatively to study phospholipid packing and membrane phases with lipid vesicles and to sense subtle differences in the arrangement of phospholipids in biological membranes.