The ternary system palmitoylsphingomyelin (PSM)/palmitoyloleoylphosphatidylcholine (POPC)/cholesterol is used to model lipid rafts. The phase behavior of the three binary systems PSM/POPC, PSM/cholesterol, and POPC/cholesterol is first experimentally determined. Phase coexistence boundaries are then determined for ternary mixtures at room temperature (23 degrees C) and the ternary phase diagram at that temperature is obtained. From the diagram at 23 degrees C and the binary phase diagrams, a reasonable expectation is drawn for the ternary phase diagram at 37 degrees C. Several photophysical methodologies are employed that do not involve detergent extraction, in addition to literature data (e.g., differential scanning calorimetry) and thermodynamic rules. For the ternary phase diagrams, some tie-lines are calculated, including the one that contains the PSM/POPC/ cholesterol 1:1:1 mixture, which is often used in model raft studies. The diagrams here described are used to rationalize literature results, some of them apparently discrepant, and to discuss lipid rafts within the framework of liquid-ordered/liquid-disordered phase coexistence.

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The effect of physiologically relevant ceramide concentrations (< or = 4 mol %) in raft model membranes with a lipid composition resembling that of cell membranes, i.e., composed of different molar ratios of an unsaturated glycerophospholipid, sphingomyelin, and cholesterol (Chol) along a liquid-disordered-liquid-ordered tie line was explored. The application of a fluorescence multiprobe and multiparameter approach, together with multiple fluorescence resonance energy transfer (FRET) pairs, in the well-characterized palmitoyl-oleoyl-phosphocholine (POPC)/palmitoyl-sphingomyelin (PSM)/Chol ternary mixture, revealed that low palmitoyl-ceramide (PCer) concentrations strongly changed both the biophysical properties and lipid lateral organization of the ternary mixtures in the low-to-intermediate Chol/PSM-, small raft size range (<25 mol % Chol). For these mixtures, PCer recruited up to three PSM molecules for the formation of very small ( approximately 4 nm) and highly ordered gel domains, which became surrounded by rafts (liquid-ordered phase) when Chol/PSM content increased. However, the size of these rafts did not change, showing that PCer did not induce the formation of large platforms or the coalescence of small rafts. In the high Chol/PSM-, large raft domains range (>33 mol % Chol), Chol completely abolished the effect of PCer by competing for PSM association. Lipid rafts govern the biophysical properties and lateral organization in these last mixtures.

To understand the formation and properties of ceramide-enriched domains in cell membranes, the biophysical properties of the binary system palmitoyloleoylphosphatidylcholine (POPC)/palmitoylceramide were thoroughly characterized. Diverse fluorescent probes and parameters were necessary to unravel the complexity of this apparently simple system. For the first time, a complete phase diagram is reported, characterizing the lamellar phases of these mixtures, and providing a quantitative framework integrating biophysical and biological studies. The diagram suggests that in resting cells no ceramide domains exist, but upon apoptotic stimuli, platforms may form. Moreover, our data show that 2 mol% of Cer strongly affects the POPC fluid matrix, suggesting that a small increase in Cer levels can significantly affect cell membrane properties. In this work, we also show that Cer domains, formed in conditions similar to physiological, are extremely ordered and rigid. The domains composition is estimated from the phase diagram and their large size was concluded from fluorescence resonance energy transfer. Dynamic light scattering and electron microscopy were used to characterize the system morphology, which is highly dependent on ceramide content and includes vesiculation and tubular structure formation.

To better understand how ceramide modulates the biophysical properties of the membrane, the interactions between palmitoyl-ceramide (PCer) and palmitoyl-sphingomyelin (PSM) were studied in the presence of the fluid phospholipid palmitoyl-oleoyl-phosphatidylcholine (POPC) in membrane model systems. The use of two fluorescent membrane probes distinctly sensitive to lipid phases allowed a thorough biophysical characterization of the ternary system. In these mixtures, PCer recruits POPC and PSM in the fluid phase to form extremely ordered and compact gel domains. Gel domain formation by low PCer mol fraction (up to 12 mol %) is enhanced by physiological PSM levels (approximately 20-30 mol % total lipid). For higher PSM content, a three-phase situation, consisting of fluid (POPC-rich)/gel (PSM-rich)/gel (PCer-rich) coexistence, is clearly shown. To determine the fraction of each phase a quantitative method was developed. This allowed establishing the complete ternary phase diagram, which helps to predict PCer-rich gel domain formation and explains its enhancement through PSM/PCer interactions.

Large unilamellar vesicles of dimyristoylphosphatidylcholine/cholesterol mixtures were studied using fluorescence techniques (steady-state fluorescence intensity and anisotropy, fluorescence lifetime, and fluorescence resonance energy transfer (FRET)). Three compositions (cholesterol mole fraction 0.15, 0.20, and 0.25) and two temperatures (30 and 40°C) inside the coexistence range of liquid-ordered (l o ) and liquid-disordered (l d ) phases were investigated. Two common membrane probes, N-, which form a FRET pair, were used. The l o /l d partition coefficients of the probes were determined by individual photophysical measurements and global analysis of timeresolved FRET decays. Although the acceptor, Rh-DMPE, prefers the l d phase, the opposite is observed for the donor, NBD-DMPE. Accordingly, FRET efficiency decreases as a consequence of phase separation. Comparing the independent measurements of partition coefficient, it was possible to detect very small domains (Ͻ20 nm) of l o in the cholesterol-poor end of the phase coexistence range. In contrast, domains of l d in the cholesterol-rich end of the coexistence range have comparatively large size. These observations are probably related to different processes of phase separation, nucleation being preferred in formation of l o phase from initially pure l d , and domain growth being faster in formation of l d phase from initially pure l o .

The application of fluorescence lifetime imaging microscopy to study gel/fluid and raftlike lipid domains in giant unilamellar vesicles (GUVs) is demonstrated here. Different regions of the ternary dipalmitoylphosphatidylcholine/dioleoylphosphatidylcholine/cholesterol phase diagram were studied. The head-labeled phospholipid Rhodamine-dioleoylphosphatidylethanolamine (Rhod-DOPE) was used as a fluorescent probe. Gel/fluid and liquid-ordered (l(o))/liquid-disordered (l(d)) phase separation were clearly visualized upon two-photon excitation. Fluorescence intensity decays in different regions of a GUV were also obtained with the microscope in fixed laser-beam configuration. The ensemble behavior of the system was studied by obtaining fluorescence intensity decays of Rhod-DOPE in nongiant vesicle suspensions. The fingerprints for gel/fluid coexistence and for the presence of l(o) raftlike phase, based on fluorescence lifetime imaging microscopy histograms and images, and on the fluorescence intensity decay parameters of Rhod-DOPE, are presented. The presence of three lipid phases in one single GUV is detected unequivocally. From the comparison of lifetime parameters, it can be concluded that the l(o) phase is formed in the binary dipalmitoylphosphatidylcholine/cholesterol but not in the dioleoylphosphatidylcholine/cholesterol mixture. The domains apparent in fluorescence intensity images have a more complex substructure revealed by analysis of the lifetime data. The potential applications of this combined imaging/microscopic/macroscopic methodology are discussed.

A group of proteins with cell membrane remodeling properties is also able to change dramatically the morphology of liposomes in vitro, frequently inducing tubulation. For a number of these proteins, the mechanism by which this effect is exerted has been proposed to be the embedding of amphipathic helices into the lipid bilayer. For proteins presenting BAR domains, removal of an N-terminal amphipathic alpha-helix (H0-NBAR) results in much lower membrane tubulation efficiency, pointing to a fundamental role of this protein segment. Here, we studied the interaction of a peptide corresponding to H0-NBAR with model lipid membranes. H0-NBAR bound avidly to anionic liposomes but partitioned weakly to zwitterionic bilayers, suggesting an essentially electrostatic interaction with the lipid bilayer. Interestingly, it is shown that after membrane incorporation, the peptide oligomerizes as an antiparallel dimer, suggesting a potential role of H0-NBAR in the mediation of BAR domain oligomerization. Through monitoring the effect of H0-NBAR on liposome shape by cryoelectron microscopy, it is clear that membrane morphology is not radically changed. We conclude that H0-NBAR alone is not able to induce vesicle curvature, and its function must be related to the promotion of the scaffold effect provided by the concave surface of the BAR domain.