SNAREs prefer liquid-disordered over "raft" (liquid-ordered) domains when reconstituted into giant unilamellar vesicles

Membrane domains ("rafts") have received great attention as potential platforms for proteins in signaling and trafficking. Because rafts are believed to form by cooperative lipid interactions but are not directly accessible in vivo, artificial phase-separating lipid bilayers are useful model systems. Giant unilamellar vesicles (GUVs) offer large free-standing bilayers, but suitable methods for incorporating proteins are still scarce. Here we report the reconstitution of two water-insoluble SNARE proteins into GUVs without fusogenic additives. Following reconstitution, protein functionality was assayed by confocal imaging and fluorescence auto- and cross-correlation spectroscopy. Incorporation into GUVs containing phase-separating lipids revealed that, in the absence of other cellular factors, both proteins exhibit an intrinsic preference for the liquid-disordered phase. Although the picture from detergent resistance assays on whole cells is ambiguous, reconstitutions of components of the exocytic machinery into GUVs by this new approach should yield insight into the dynamics of protein complex associations with hypothesized liquid-ordered phase microdomains, the correspondence between detergent-resistant membranes and liquid-ordered phase, and the mechanism of SNARE-mediated membrane fusion.     

Interaction of influenza virus haemagglutinin with sphingolipid-cholesterol membrane domains via its transmembrane domain.

Sphingolipid-cholesterol rafts are microdomains in biological membranes with liquid-ordered phase properties which are implicated in membrane traffic and signalling events. We have used influenza virus haemagglutinin (HA) as a model protein to analyse the interaction of transmembrane proteins with these microdomains. Here we demonstrate that raft association is an intrinsic property encoded in the protein. Mutant HA molecules with foreign transmembrane domain (TMD) sequences lose their ability to associate with the lipid microdomains, and mutations in the HA TMD reveal a requirement for hydrophobic residues in contact with the exoplasmic leaflet of the membrane. We also provide experimental evidence that cholesterol is critically required for association of proteins with lipid rafts. Our data suggest that the binding to specific membrane domains can be encoded in transmembrane proteins and that this information will be used for polarized sorting and signal transduction processes.     

Visualizing association of lipidated signaling proteins in heterogeneous membranes−Partitioning into subdomains, lipid sorting, interfacial adsorption, and protein association

In a combined chemical biological and biophysical approach, we studied the partitioning of differently fluorescent-labeled palmitoyl and/or farnesyl lipidated peptides, which represent membrane recognition model systems, as well as the full lipidated N-Ras protein into various model membrane systems including canonical model raft mixtures. To this end, two-photon fluorescence microscopy on giant unilamellar vesicles, complemented by tapping-mode atomic force microscopy (AFM) measurements, was carried out. The measurements were performed over a wide temperature range, ranging from 30 to 80 °C to cover different lipid phase states (solid-ordered (gel), fluid/gel, liquid-ordered/liquid-disordered, all-fluid). The results provide direct evidence that partitioning of the lipidated peptides and N-Ras occurs preferentially into liquid-disordered lipid domains, which is also reflected in a faster kinetics of incorporation. The phase sequence of preferential binding of N-Ras to mixed-domain lipid vesicles is liquid-disordered > liquid-ordered ? solid-ordered. Intriguingly, we detect - using the better spatial resolution of AFM - also a large proportion of the lipidated protein located at the liquid-disordered/liquid-ordered phase boundary, thus leading to a favorable decrease in line tension that is associated with the rim of neighboring domains. In an all-liquid-ordered, cholesterol-rich phase, phase separation can be induced by an effective lipid sorting mechanism owing to the high affinity of the lipidated peptides and proteins to a fluid-like lipid environment. At low temperatures, where the overall acyl chain order parameter of the lipid bilayer has markedly increased, such an efficient lipid sorting mechanism is energetically too costly and self-association of the peptide into small clusters takes place. These data reveal the interesting ability of the lipidated peptides and proteins to induce formation of fluid microdomains at physiologically relevant high cholesterol concentrations. Furthermore, our results reveal self-association of the N-Ras protein at the domain boundaries which may serve as an important vehicle for association processes and nanoclustering, which has also been observed in in vivo studies.     

Quantitative Imaging of Molecular Order in Lipid Membranes Using Two-Photon Fluorescence Polarimetry

We present a polarimetric two-photon microscopy technique to quantitatively image the local static molecular orientational behavior in lipid and cell membranes. This approach, based on a tunable excitation polarization state complemented by a polarized readout, is easily implementable and does not require hypotheses on the molecular angular distribution such as its mean orientation, which is a main limitation in traditional fluorescence anisotropy measurements. The method is applied to the investigation of the molecular angular distribution in giant unilamellar vesicles formed by liquid-ordered and liquid-disordered micro-domains, and in COS-7 cell membranes. The highest order contrast between ordered and disordered domains is obtained for dyes locating within the membrane acyl chains.     

Characterization and application of a new optical probe for membrane lipid domains

In this article, we characterize the fluorescence of an environmentally sensitive probe for lipid membranes, di-4-ANEPPDHQ. In large unilamellar lipid vesicles (LUVs), its emission spectrum shifts up to 30 nm to the blue with increasing cholesterol concentration. Independently, it displays a comparable blue shift in liquid-ordered relative to liquid-disordered phases. The cumulative effect is a 60-nm difference in emission spectra for cholesterol containing LUVs in the liquid-ordered state versus cholesterol-free LUVs in the liquid-disordered phase. Given these optical properties, we use di-4-ANEPPDHQ to image the phase separation in giant unilamellar vesicles with both linear and nonlinear optical microscopy. The dye shows green and red fluorescence in liquid-ordered and -disordered domains, respectively. We propose that this reflects the relative rigidity of the molecular packing around the dye molecules in the two phases. We also observe a sevenfold stronger second harmonic generation signal in the liquid-disordered domains, consistent with a higher concentration of the dye resulting from preferential partitioning into the disordered phase. The efficacy of the dye for reporting lipid domains in cell membranes is demonstrated in polarized migrating neutrophils.     

Evolution and development of model membranes for physicochemical and functional studies of the membrane lateral heterogeneity

One of the main questions in the membrane biology is the functional roles of membrane heterogeneity and molecular localization. Although segregation and local enrichment of protein/lipid components (rafts) have been extensively studied, the presence and functions of such membrane domains still remain elusive. Along with biochemical, cell observation, and simulation studies, model membranes are emerging as an important tool for understanding the biological membrane, providing quantitative information on the physicochemical properties of membrane proteins and lipids. Segregation of fluid lipid bilayer into liquid-ordered (Lo) and liquid-disordered (Ld) phases has been studied as a simplified model of raft in model membranes, including giant unilamellar vesicles (GUVs), giant plasma membrane vesicles (GPMVs), and supported lipid bilayers (SLB). Partition coefficients of membrane proteins between Lo and Ld phases were measured to gauze their affinities to lipid rafts (raftophilicity). One important development in model membrane is patterned SLB based on the microfabrication technology. Patterned Lo/Ld phases have been applied to study the partition and function of membrane-bound molecules. Quantitative information of individual molecular species attained by model membranes is critical for elucidating the molecular functions in the complex web of molecular interactions. The present review gives a short account of the model membranes developed for studying the lateral heterogeneity, especially focusing on patterned model membranes on solid substrates.     

Polysialic acid chains exhibit enhanced affinity for ordered regions of membranes

Polysialic acid (polySia) forms linear chains which are usually attached to the external surface of the plasma membrane mainly through the Neural Cell Adhesion Molecule (NCAM) protein. It is exposed on neural cells, several types of cancer cells, dendritic cells, and egg and sperm cells. There are several lipid raft-related phenomena in which polySia is involved; however the mechanisms of polySia action as well as determinants of its localization in lipid raft microdomains are still unknown, although the majority of NCAM molecules in the liquid-ordered raft membrane fractions of neural cells appear to be polysialylated. Here we investigate the affinity of polySia (both soluble and NCAM-dependent plasma membrane-bound) for liquid-ordered- and liquid-disordered regions of lipid vesicle and neuroblastoma cell membranes. Our studies indicate that polySia chains have a higher affinity for ordered regions of membranes as determined by the dissociation constant values for polySia-lipid bilayer complex, the fluorescence intensity of polySia bound to giant vesicles, the polySia-to-membrane FRET signal at the plasma membrane of live cells, and the decrease of the FRET signals after Endo-N treatment of the cells. These results suggest that polysialylation may be one of the determinants of protein association with liquid-ordered membrane lipid raft domains.     

Lipids as modulators of proteolytic activity of BACE: involvement of cholesterol, glycosphingolipids, and anionic phospholipids in vitro

The beta-secretase, BACE, is a membrane spanning aspartic protease, which cleaves the amyloid precursor protein (APP) in the first step of proteolytic processing leading to the formation of the neurotoxic beta-amyloid peptide (Abeta). Previous results have suggested that the regulation of beta-secretase and BACE access to APP is lipid dependent, and involves lipid rafts. Using the baculovirus expression system, we have expressed recombinant human full-length BACE in insect cells and purified milligram amounts to homogeneity. We have studied partitioning of fluorophor-conjugated BACE between the liquid ordered and disordered phases in giant (10-150 mum) unilamellar vesicles, and found approximately 20% to associate with the raft-like, liquid-ordered phase; the fraction associated with liquid-ordered phase increased upon cross-linking of raft lipids. To examine involvement of individual lipid species in modulating BACE activity, we have reconstituted the purified BACE in large ( approximately 100 nm) unilamellar vesicles, and determined its specific activity in vesicles of various lipid compositions. We have identified 3 groups of lipids that stimulate proteolytic activity of BACE: 1) neutral glycosphingolipids (cerebrosides), 2) anionic glycerophospholipids, and 3) sterols (cholesterol).     

Equinatoxin II permeabilizing activity depends on the presence of sphingomyelin and lipid phase coexistence

Equinatoxin II is a pore-forming protein of the actinoporin family. After membrane binding, it inserts its N-terminal α-helix and forms a protein/lipid pore. Equinatoxin II activity depends on the presence of sphingomyelin in the target membrane; however, the role of this specificity is unknown. On the other hand, sphingomyelin is considered an essential ingredient of lipid rafts and promotes liquid-ordered/liquid-disordered phase separation in model membranes that mimic raft composition. Here, we used giant unilamellar vesicles to simultaneously investigate the effect of sphingomyelin and phase separation on the membrane binding and permeabilizing activity of Equinatoxin II. Our results show that Equinatoxin II binds preferentially to the liquid-ordered phase over the liquid-disordered one and that it tends to concentrate at domain interfaces. In addition, sphingomyelin strongly enhances membrane binding of the toxin but is not sufficient for membrane permeabilization. Under the same experimental conditions, Equinatoxin II formed pores in giant unilamellar vesicles containing sphingomyelin only when liquid-ordered and -disordered phases coexisted. Our observations demonstrate the importance of phase boundaries for Equinatoxin II activity and suggest a double role of sphingomyelin as a specific receptor for the toxin and as a promoter of the membrane organization necessary for Equinatoxin II action.     

Specific RNA binding to ordered phospholipid bilayers

We have studied RNA binding to vesicles bounded by ordered and disordered phospholipid membranes. A positive correlation exists between bilayer order and RNA affinity. In particular, structure-dependent RNA binding appears for rafted (liquid-ordered) domains in sphingomyelin-cholesterol-1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. Binding to more highly ordered gel phase membranes is stronger, but much less RNA structure-dependent. All modes of RNA-membrane association seem to be electrostatic and headgroup directed. Fluorometry on 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes indicates that bound RNA broadens the gel-fluid melting transition, and reduces lipid headgroup order, as detected via fluorometric measurement of intramembrane electric fields. RNA preference for rafted lipid was visualized and confirmed using multiple fluorophores that allow fluorescence and fluorescence resonance energy transfer microscopy on RNA molecules closely associated with ordered lipid patches within giant vesicles. Accordingly, both RNA structure and membrane order could modulate biological RNA–membrane interactions.     

Exposure of phosphatidylinositol transfer proteins to sphingomyelin-cholesterol membranes suggests transient but productive interactions with raft-like, liquid-ordered domains

Both isoforms of rat phosphatidylinositol transfer protein (PITP) mediate the intermembrane transfer of sphingomyelin (CerPCho). In the plasma membrane, CerPCho often segregates with cholesterol into microdomains such as lipid rafts and caveolae. To test the hypothesis that PITP exhibits a preference for CerPCho- and cholesterol-rich membranes, we prepared unilamellar vesicles containing variable amounts of these two lipids. We also used CerPCho species with different acyl composition and treated vesicles with agents known to sequester and remove cholesterol. We observed that the beta isoform of rat PITP was more sensitive to membrane cholesterol than was the alpha isoform, as shown by increases in specific activities of lipid transfer of 2-6-fold. A relatively high membrane content of cholesterol (mole fraction > 0.4) was required to elicit such enhancements. Treatment of cholesterol-rich membranes with a series of beta cyclodextrins demonstrated that, upon depletion of cholesterol from participating membranes, the PITPbeta activity changes were fully reversible. We finally noted that the mechanism by which cholesterol enhances the activity of PITPbeta appeared to involve a decreased affinity of the protein for the membrane surface, in a manner that was independent of vesicle size and membrane microviscosity. We conclude that PITPbeta interacts transiently but productively with the liquid-ordered phase formed by CerPCho and cholesterol and discuss the possibility of PITP interactions in vivo with sphingolipid- and cholesterol-rich membrane microdomains.     

Sorting of Lipids and Proteins in Membrane Curvature Gradients

The sorting of lipids and proteins in cellular trafficking pathways is a process of central importance in maintaining compartmentalization in eukaryotic cells. However, the mechanisms behind these sorting phenomena are currently far from being understood. Among several mechanistic suggestions, membrane curvature has been invoked as a means to segregate lipids and proteins in cellular sorting centers. To assess this hypothesis, we investigate the sorting of lipid analog dye trace components between highly curved tubular membranes and essentially flat membranes of giant unilamellar vesicles. Our experimental findings indicate that intracellular lipid sorting, contrary to frequent assumptions, is unlikely to occur by lipids fitting into membrane regions of appropriate curvature. This observation is explained in the framework of statistical mechanical lattice models that show that entropy, rather than curvature energy, dominates lipid distribution in the absence of strongly preferential lateral intermolecular interactions. Combined with previous findings of curvature induced phase segregation, we conclude that lipid cooperativity is required to enable efficient sorting. In contrast to lipid analog dyes, the peripheral membrane binding protein Cholera toxin subunit B is effectively curvature-sorted. The sorting of Cholera toxin subunit B is rationalized by statistical models. We discuss the implications of our findings for intracellular sorting mechanisms.     

EPR and FTIR studies reveal the importance of highly ordered sterol-enriched membrane domains for ostreolysin activity

Ostreolysin is a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), which recognizes specifically and binds to raft-like sterol-enriched membrane domains that exist in the liquid-ordered phase. Its binding can be abolished by micromolar concentrations of lysophospholipids and fatty acids. The membrane activity of ostreolysin, however, does not completely correlate with the ability of a certain sterol to induce the formation of a liquid-ordered phase, suggesting that the protein requires an additional structural organization of the membrane to exert its activity. The aim of this study was to further characterize the lipid membranes that facilitate ostreolysin binding by analyzing their lipid phase domain structure. Fourier-transformed infrared spectroscopy (FTIR) and electron paramagnetic resonance (EPR) were used to analyze the ordering and dynamics of membrane lipids and the membrane domain structure of a series of unilamellar liposomes prepared by systematically changing the lipid components and their ratios. Our results corroborate the earlier conclusion that the average membrane fluidity of ostreolysin-susceptible liposomes alone cannot account for the membrane activity of the protein. Combined with previous data computer-aided interpretation of EPR spectra strongly suggests that chemical properties of membrane constituents, their specific distribution, and physical characteristics of membrane nanodomains, resulting from the presence of sterol and sphingomyelin (or a highly ordered phospholipid, dipalmitoylphosphatidylcholine), are essential prerequisites for ostreolysin membrane binding and pore-formation.     

Membrane order and molecular dynamics associated with IgE receptor cross-linking in mast cells

Cholesterol-rich microdomains (or "lipid rafts") within the plasma membrane have been hypothesized to exist in a liquid-ordered phase and play functionally important roles in cell signaling; however, these microdomains defy detection using conventional imaging. To visualize domains and relate their nanostructure and dynamics to mast cell signaling, we use two-photon (760 nm and 960 nm) fluorescence lifetime imaging microscopy and fluorescence polarization anisotropy imaging, with comparative one-photon anisotropy imaging and single-point lifetime and anisotropy decay measurements. The inherent sensitivity of ultrafast excited-state dynamics and rotational diffusion to the immediate surroundings of a fluorophore allows for real-time monitoring of membrane structure and organization. When the high affinity receptor for IgE (FcepsilonRI) is extensively cross-linked with anti-IgE, molecules associated with cholesterol-rich microdomains (e.g., saturated lipids (the lipid analog diI-C(18) or glycosphingolipids)) and lipid-anchored proteins coredistribute with cross-linked IgE-FcepsilonRI. We find an enhancement in fluorescence lifetime and anisotropy of diI-C(18) and Alexa 488-labeled IgE-FcepsilonRI in the domains where these molecules colocalize. Our results suggest that fluorescence lifetime and, particularly, anisotropy permit us to correlate the recruitment of lipid molecules into more ordered domains that serve as platforms for IgE-mediated signaling.     

Liquid domains in vesicles investigated by NMR and fluorescence microscopy

We use (2)H-NMR, (1)H-MAS NMR, and fluorescence microscopy to detect immiscibility in three particular phospholipid ratios mixed with 30% cholesterol: 2:1 DOPC/DPPC, 1:1 DOPC/DPPC, and 1:2 DOPC/DPPC. Large-scale (>160 nm) phase separation into liquid-ordered (L(o)) and liquid-crystalline (L(alpha)) phases is observed by both NMR and fluorescence microscopy. By fitting superimposed (2)H-NMR spectra, we quantitatively determine that the L(o) phase is strongly enriched in DPPC and moderately enriched in cholesterol. Tie-lines estimated at different temperatures and membrane compositions are based on both (2)H-NMR observations and a previously published ternary phase diagram. (2)H- and (1)H-MAS NMR techniques probe significantly smaller length scales than microscopy experiments (submicron versus micron-scalp), and complex behavior is observed near the miscibility transition. Fluorescence microscopy of giant unilamellar vesicles shows micrometer-scale domains below the miscibility transition. In contrast, NMR of multilamellar vesicles gives evidence for smaller ( approximately 80 nm) domains just below the miscibility transition, whereas large-scale demixing occurs at a lower temperature, T(low). A transition at T(low) is also evident in fluorescence microscopy measurements of the surface area fraction of ordered phase in giant unilamellar vesicles. Our results reemphasize the complex phase behavior of cholesterol-containing membranes and provide a framework for interpreting (2)H-NMR experiments in similar membranes.     

Direct Visualization of the Action of Triton X-100 on Giant Vesicles of Erythrocyte Membrane Lipids

The raft hypothesis proposes that microdomains enriched in sphingolipids, cholesterol, and specific proteins are transiently formed to accomplish important cellular tasks. Equivocally, detergent-resistant membranes were initially assumed to be identical to membrane rafts, because of similarities between their compositions. In fact, the impact of detergents in membrane organization is still controversial. Here, we use phase contrast and fluorescence microscopy to observe giant unilamellar vesicles (GUVs) made of erythrocyte membrane lipids (erythro-GUVs) when exposed to the detergent Triton X-100 (TX-100). We clearly show that TX-100 has a restructuring action on biomembranes. Contact with TX-100 readily induces domain formation on the previously homogeneous membrane of erythro-GUVs at physiological and room temperatures. The shape and dynamics of the formed domains point to liquid-ordered/liquid-disordered (Lo/Ld) phase separation, typically found in raft-like ternary lipid mixtures. The Ld domains are then separated from the original vesicle and completely solubilized by TX-100. The insoluble vesicle left, in the Lo phase, represents around 2/3 of the original vesicle surface at room temperature and decreases to almost 1/2 at physiological temperature. This chain of events could be entirely reproduced with biomimetic GUVs of a simple ternary lipid mixture, 2:1:2 POPC/SM/chol (phosphatidylcholine/sphyngomyelin/cholesterol), showing that this behavior will arise because of fundamental physicochemical properties of simple lipid mixtures. This work provides direct visualization of TX-100-induced domain formation followed by selective (Ld phase) solubilization in a model system with a complex biological lipid composition.     

Direct Visualization of the Action of Triton X-100 on Giant Vesicles of Erythrocyte Membrane Lipids

The raft hypothesis proposes that microdomains enriched in sphingolipids, cholesterol, and specific proteins are transiently formed to accomplish important cellular tasks. Equivocally, detergent-resistant membranes were initially assumed to be identical to membrane rafts, because of similarities between their compositions. In fact, the impact of detergents in membrane organization is still controversial. Here, we use phase contrast and fluorescence microscopy to observe giant unilamellar vesicles (GUVs) made of erythrocyte membrane lipids (erythro-GUVs) when exposed to the detergent Triton X-100 (TX-100). We clearly show that TX-100 has a restructuring action on biomembranes. Contact with TX-100 readily induces domain formation on the previously homogeneous membrane of erythro-GUVs at physiological and room temperatures. The shape and dynamics of the formed domains point to liquid-ordered/liquid-disordered (Lo/Ld) phase separation, typically found in raft-like ternary lipid mixtures. The Ld domains are then separated from the original vesicle and completely solubilized by TX-100. The insoluble vesicle left, in the Lo phase, represents around 2/3 of the original vesicle surface at room temperature and decreases to almost 1/2 at physiological temperature. This chain of events could be entirely reproduced with biomimetic GUVs of a simple ternary lipid mixture, 2:1:2 POPC/SM/chol (phosphatidylcholine/sphyngomyelin/cholesterol), showing that this behavior will arise because of fundamental physicochemical properties of simple lipid mixtures. This work provides direct visualization of TX-100-induced domain formation followed by selective (Ld phase) solubilization in a model system with a complex biological lipid composition.     

Membrane microdomains: Role of ceramides in the maintenance of their structure and functions

Free-standing giant unilamellar vesicles were used to visualize the complex lateral heterogeneity, induced by ceramide in the membrane bilayer at micron scale using C₁₂-NBD-PC probe partitioning under the fluorescence microscope. Ceramide gel domains exist as leaf-like structures in glycerophospholipid/ceramide mixtures. Cholesterol readily increases ceramide miscibility with glycerophospholipids but cholesterol-ceramide interactions are not involved in the organization of the liquid-ordered phase as exemplified by sphingomyelin/cholesterol mixtures. Sphingomyelin stabilizes the gel phase and thus decreases ceramide miscibility in the presence of cholesterol. Gel/liquid-ordered/liquid-disordered phase coexistence was visualized in quaternary phosphatidylcholine/sphingomyelin/ceramide/cholesterol mixtures as occurrence of dark leaf-like and circular domains within a bright liquid phase. Sphingomyelin initiates specific ceramide-sphingomyelin interactions to form a highly ordered gel phase appearing at temperatures higher than pure ceramide gel phase in phosphatidylcholine/ceramide mixtures. Less sphingomyelin is engaged in formation of liquid-ordered phase leading to a shift in its formation to lower temperatures. Sphingomyelinase activity on substrate vesicles destroys micron L_o domains but induces the formation of a gel-like phase. The activation of phospholipase A₂ by ceramide on heterogeneous membranes was visualized. Changes in the phase state of the membrane bilayer initiates such morphological processes as membrane fragmentation, budding in and budding out was demonstrated.     

Targeting membrane proteins to liquid-ordered phases: molecular self-organization explored by fluorescence correlation spectroscopy

The complex and dynamic architecture of biological membranes comprises of various heterogeneities, some of which may include lipid-based and/or protein-based microdomains called "rafts". Due to interactions among membrane components, several types of domains can form with different characteristics and mechanisms of formation. Model membranes, such as giant unilamellar vesicles (GUVs), provide a key system to study lipid-lipid and lipid-protein interactions, which are potentially relevant to raft formation, by (single-molecule) optical microscopy. Here, we review studies of combined confocal imaging and fluorescence correlation spectroscopy (FCS) on lipid dynamics and organization in domains assembled in GUVs, prepared from various lipid mixtures, which are relevant to the problem of raft formation. Finally, we summarize the results on lipid-protein interactions, which govern the targeting of several putative raft- and non-raft-associated membrane proteins to domain-exhibiting GUVs.     

Brij detergents reveal new aspects of membrane microdomain in erythrocytes

Membrane microdomains enriched in cholesterol, sphingolipids (rafts), and specific proteins are involved in important physiological functions. However their structure, size and stability are still controversial. Given that detergent-resistant membranes (DRMs) are in the liquid-ordered state and are rich in raft-like components, they might correspond to rafts at least to some extent. Here we monitor the lateral order of biological membranes by characterizing DRMs from erythrocytes obtained with Brij-98, Brij-58, and TX-100 at 4?°C and 37?°C. All DRMs were enriched in cholesterol and contained the raft markers flotillin-2 and stomatin. However, sphingomyelin (SM) was only found to be enriched in TX-100-DRMs – a detergent that preferentially solubilizes the membrane inner leaflet – while Band 3 was present solely in Brij-DRMs. Electron paramagnetic resonance spectra showed that the acyl chain packing of Brij-DRMs was lower than TX-100-DRMs, providing evidence of their diverse lipid composition. Fatty acid analysis revealed that the SM fraction of the DRMs was enriched in lignoceric acid, which should specifically contribute to the resistance of SM to detergents. These results indicate that lipids from the outer leaflet, particularly SM, are essential for the formation of the liquid-ordered phase of DRMs. At last, the differential solubilization process induced by Brij-98 and TX-100 was monitored using giant unilamellar vesicles. This study suggests that Brij and TX-100-DRMs reflect different degrees of lateral order of the membrane microdomains. Additionally, Brij DRMs are composed by both inner and outer leaflet components, making them more physiologically relevant than TX-100-DRMs to the studies of membrane rafts.