Asymmetric Lipid Bilayer Research Articles

Supported lipid bilayers as models for studying membrane domains.

Supported lipid bilayers have been in use for over 30 years. They have been employed to study the structure, composition, and dynamics of lipid bilayer phases, the binding and distribution of soluble, integral, and lipidated proteins in membranes, membrane fusion, and interactions of membranes with elements of the cytoskeleton. This review focuses on the unique ability of supported lipid bilayers to study liquid-ordered and liquid-disordered domains in membranes. We highlight methods to produce asymmetric lipid bilayers with lipid compositions that mimic those of the extracellular and cytoplasmic leaflets of cell membranes and the functional reconstitution of membrane proteins into such systems. Questions related to interleaflet domain coupling and membrane protein activation have been addressed and answered using advanced reconstitution and imaging procedures in symmetric and asymmetric supported membranes with and without coexisting lipid phase domains. Previously controversial topics regarding anomalous and anisotropic diffusion in membranes have been resolved by using supported membrane approaches showing that the propensity of certain lipid compositions to form "rafts" are important but overlaid with "picket-fence" interactions that are imposed by a subtended cytoskeletal network.

Molecular Dynamics Simulation Studies of Interactions of E. coli-K12 with OmpF in Outer Membranes: Effects of LPS Structures on Monoclonal Antibodies Binding

Gram-negative bacterial outer membrane, which is a highly asymmetric lipid bilayer, is composed of an outer membrane protein along with phospholipids (PLs) forming the inner leaflet and lipopolysaccharides (LPSs) forming the outer leaflet. Outer membrane protein F (OmpF) porin is a trimeric integral membrane protein responsible for the passive transport of small hydrophilic molecules such as nutrients and waste products across the outer membrane of Escherichia coli (E. coli). Here, we report the structural properties of a model of the E. coli-K12 outer membrane and its interaction with OmpF using molecular dynamics simulations. Immunochemical and other experiments suggested several cell-surface exposed epitopes (antibody binding sites), which were recognized by various monoclonal antibodies (MAbs). Molecular details of interaction between LPS core sugars and surface of OmpF suggest the importance of LPS core sugars in shielding of these epitopes. Results are compared with experimental evidence, which showed that with shortening of the LPS core sugars (rfa E. coli-K-12 mutants), the number of MAbs, that recognized porin surface epitopes, increased sequentially. To check the stabilizing effect of LPS structure on the extracellular loop conformations, results of interaction between OmpF and asymmetric E. coli-K12 bilayer are compared to those from OmpF simulations in a DMPC bilayer. In addition, overall membrane properties such as, electron density profiles, per-lipid surface area of each lipid type, and chain order parameters are compared with E.coli-K12 outer membrane in absence of OmpF. These results are also compared to our previous molecular dynamics results with E. coli-R1 outer membrane with OmpLA. Overall, membrane structural properties are comparable, however, individual lipid properties showed slight variations.

Impact of PI(3,4,5)P3-Mediated Beta-Arrestin-1 Recruitment on Structure of Asymmetric Lipid Bilayers

The membrane spanning G-protein coupled receptors (GPCRs) facilitate crucial physiological responses to a variety of extracellular ligands, such as hormones, neurotransmitters, ions, photons, and other stimuli [1], thus 50% of drugs available in the market today target GPCRs [2]. The biological mechanisms behind GPCR-signaling involves several other biomolecules, including the key protein group of β-arrestins. These cytosolic adaptor proteins regulate signaling through several different pathways, classically by removing receptors from the plasma membrane via clathrin-mediated endocytosis [3].In membrane binding experiments using both confocal fluorescence microscopy and quartz crystal microbalance, we found that β-arrestin-1 binds specifically to supported lipid bilayers (SLBs) containing phosphatidylinositol trisphosphate lipids, PI(3,4,5)P3. We have consistently shown, using both methods, that such binding is lipid specific and not driven by membrane charge. We have also shown that the membrane-protein interaction depends on protein concentration and membrane composition, and that β-arrestins can induce membrane curvature, which may play a role in the endocytic pathway. This is for the first time quantified with respect to dynamics and respective contributions of specific vs non-specific binding.Having established an assay for PI(3,4,5)P3-mediated recruitment of β-arrestin-1, we conducted a series of neutron reflection experiments to study the protein-lipid interaction on a structural level. Both hydrogenated and deuterated protein was used in reflectometry experiments with diverse contrast matching of the bulk media. We present here the detailed structures of asymmetric lipid bilayers containing PI(3,4,5)P3, and demonstrate that β-arrestin-1 not only binds but also reorganizes the membrane structure.1. Rosenbaum, D. M. et al. Nature. 459(7245), 356-363 (2009).2. Gudermann, T. et al. J Mol Med-Jmm. 73(2), 51-63 (1995).3. DeWire, S. M. et al. Annu Rev Physiol. 69, 483-510 (2007).

Effect of Asymmetry on Bilayer Membrane Systems

Many biological processes, such as membrane fusion and the action of microbial proteins, involve asymmetric uptake of proteins. In addition, it has been demonstrated that large mechaosensitive channels open by the asymmetric inclusion of lyso-lipids in the bilayer in the absence of external pressure. These phenomena can be interpreted in terms of the asymmetric local pressure profile in the bilayer, which lacks the quantitative understanding. By noting that such asymmetric pressure profile can be induced in bilayer membranes with asymmetric number of lipids between leaflets, to provide quantitative understanding, we performed molecular dynamics simulations of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer membranes for a set of systems with different level of asymmetry with and without gramicidin A (gA) or WALP23. We observed that even highly asymmetric lipid bilayers with and without gA or WALP23 were stable during the whole simulation time, which is consistent with the previous study of dipalmitoyl-phosphatidylcholine (DPPC) bilayers [J. Am. Chem. Soc. 131, 15194 (2009)]. As the number of lipids in the lower leaflet becomes smaller, the lipids are more tightly packed in the top leaflet and less ordered in the bottom leaflet, which are quantified in terms the lateral pressure profile, the order parameter, the area per lipid, etc. However, the hydrophobic thickness for pure lipid bilayers does not show meaningful dependence on the asymmetry, which results from the stretched and less ordered lipids in the upper and lower leaflets, respectively. We will also discuss the influence of the asymmetry on the properties of protein-lipid interactions, such as the distance dependence of the lipid properties and the interaction patterns around gA or WALP23.

Bacterial Outer Membranes and Interactions with Membrane Proteins

Because the bacteria's outer membrane (OM) acts as an effective barrier against the permeation of both hydrophobic and hydrophilic compounds, gram-negative cell permeation is one of the biggest challenges to the discovery of novel antibiotics for bacterial infections and antibiotic resistance worldwide. The bacterial OM is a unique and highly asymmetric lipid bilayer composed of phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. An LPS molecule is a complex amphiphatic compound consisting of lipid A, a core oligosaccharide, and an O-antigen polysaccharide. Despite the direct relationship of gram-negative bacteria to the public health and also the fact that there are over 190 identified O-serotypes for Escherichia coli, our molecular-level understanding of how the bacterial OMs behave and work for various types of bacteria, how membrane proteins behave in the OM, and how known drug molecules and potential drugs can enter through the OMs is rudimentary at best. This talk presents our ongoing efforts on all-atom modeling and simulations of these complex bacterial OMs with and without various outer membrane proteins using the CHARMM36 (protein, lipid, carbohydrate) force fields. In addition, various technical aspects and perspectives are also discussed together with future developments of CHARMM-GUI LPS Modeler and OM Builder.

High-throughput formation of lipid bilayer membrane arrays with an asymmetric lipid composition.

We present a micro-device in which more than 10,000 asymmetric lipid bilayer membranes are formed at a time on micro-chamber arrays. The arrayed asymmetric lipid bilayers, where lipid compositions are different between the inner and outer leaflets, are formed with high efficiency of over 97% by injecting several types of liquids into a micro-device that has hydrophilic-in-hydrophobic surfaces. The lipid compositional asymmetry is an intrinsic property of bio-membranes, and therefore, this micro-device extends the versatility of artificial lipid-bilayer systems, which were previously limited to symmetric bilayer formation, and could contribute to the understanding of the role of lipid compositional asymmetry in cell physiology and also to further analytical and pharmacological applications.

Cholesterol behavior in asymmetric lipid bilayers: insights from molecular dynamics simulations.

Asymmetric lipid composition of the cell membranes plays an important role in the multitude of important biological functions. Much less is known, however, about the distribution and dynamics of cholesterol in asymmetric biological membranes. In this work we show how this issue could be addressed computationally by molecular dynamics simulations. The influence of the lipid head group charge, acyl chain saturation, spontaneous membrane curvature and the surface tension of the membrane on cholesterol distribution in asymmetric lipid bilayers is investigated. Four asymmetric bilayers containing DOPC, DOPS, DSPC, or DSPS lipids, were simulated on the time scale extended to tens of microseconds. We show that cholesterol strongly prefers anionic lipids to neutral and saturated lipid tails to unsaturated with distribution ratio ~0.4-0.6. Multiple flip-flop transitions of cholesterol were observed directly and their mean times range from 350 to 2,000 ns. It was shown that the distribution of cholesterol in the asymmetric bilayer depends not only on the type of lipids but also on the local membrane curvature and the surface tension. The geometric shape of spontaneously curved asymmetric bilayer changes dramatically in the presence of cholesterol. The membrane curvature becomes less homogeneous with large patches of flattened regions interleaved by rather sharp bends.

Bilayer registry in a multicomponent asymmetric membrane: dependence on lipid composition and chain length.

A question of considerable interest to cell membrane biology is whether phase segregated domains across an asymmetric bilayer are strongly correlated with each other and whether phase segregation in one leaflet can induce segregation in the other. We answer both these questions in the affirmative, using an atomistic molecular dynamics simulation to study the equilibrium statistical properties of a 3-component asymmetric lipid bilayer comprising an unsaturated palmitoyl-oleoyl-phosphatidyl-choline, a saturated sphingomyelin, and cholesterol with different composition ratios. Our simulations are done by fixing the composition of the upper leaflet to be at the coexistence of the liquid ordered (l(o))-liquid disordered (l(d)) phases, while the composition of the lower leaflet is varied from the phase coexistence regime to the mixed l(d) phase, across a first-order phase boundary. In the regime of phase coexistence in each leaflet, we find strong transbilayer correlations of the l(o) domains across the two leaflets, resulting in bilayer registry. This transbilayer correlation depends sensitively upon the chain length of the participating lipids and possibly other features of lipid chemistry, such as degree of saturation. We find that the l(o) domains in the upper leaflet can induce phase segregation in the lower leaflet, when the latter is nominally in the mixed (l(d)) phase.

Atomistic simulations of anionic Au144(SR)60 nanoparticles interacting with asymmetric model lipid membranes

Experimental observations indicate that the interaction between nanoparticles and lipid membranes varies according to the nanoparticle charge and the chemical nature of their protecting side groups. We report atomistic simulations of an anionic Au nanoparticle (AuNP−) interacting with membranes whose lipid composition and transmembrane distribution are to a large extent consistent with real plasma membranes of eukaryotic cells. To this end, we use a model system which comprises two cellular compartments, extracellular and cytosolic, divided by two asymmetric lipid bilayers. The simulations clearly show that AuNP− attaches to the extracellular membrane surface within a few tens of nanoseconds, while it avoids contact with the membrane on the cytosolic side. This behavior stems from several factors. In essence, when the nanoparticle interacts with lipids in the extracellular compartment, it forms relatively weak contacts with the zwitterionic head groups (in particular choline) of the phosphatidylcholine lipids. Consequently, AuNP− does not immerse deeply in the leaflet, enabling, e.g., lateral diffusion of the nanoparticle along the surface. On the cytosolic side, AuNP− remains in the water phase due to Coulomb repulsion that arises from negatively charged phosphatidylserine lipids interacting with AuNP−. A number of structural and dynamical features resulting from these basic phenomena are discussed. We close the article with a brief discussion of potential implications.

Molecular interactions between amantadine and model cell membranes.

Sum frequency generation (SFG) vibrational spectroscopy was applied to study molecular interactions between amantadine and substrate supported lipid bilayers serving as model cell membranes. Both isotopically asymmetric and symmetric lipid bilayers were used in the research. SFG results elucidated how the water-soluble drug, amantadine, influenced the packing state of each leaflet of a lipid bilayer and how the drugs affected the lipid flip-flop process. It is difficult to achieve such detailed molecular-level information using other analytical techniques. Especially, from the flip-flop rate change of isotopically asymmetric lipid bilayer induced by amantadine, important information on the drug-membrane interaction mechanism can be derived. The results show that amantadine can be associated with zwitterionic PC bilayers but has a negligible influence on the flip-flop behavior of PC molecules unless at high concentrations. Different effects of amantadine on the lipid bilayer were observed for the negatively charged DPPG bilayer; low concentration amantadine (e.g., 0.20 mM) in the subphase could immediately disturb the outer lipid leaflet and then the lipid associated amantadine molecules gradually reorganize to cause the outer leaflet to return to the original orderly packed state. Higher concentration amantadine (e.g., 5.0 mM) immediately disordered the packing state of the outer lipid leaflet. For both the high and low concentration cases, amantadine molecules only bind to the outer PG leaflet and cannot translocate to the inner layer. The presence of amantadine within the negatively charged lipid layers has certain implications for using liposomes as drug delivery carriers for amantadine. Besides, by using PC or PG bilayers with both leaflets deuterated, we were able to examine how amantadine is distributed and/or oriented within the lipid bilayer. The present work demonstrates that SFG results can provide an in-depth understanding of the molecular mechanisms of interactions between water-soluble drugs and model cell membranes.

Determination of mean and Gaussian curvatures of highly curved asymmetric lipid bilayers: the case study of the influence of cholesterol on the membrane shape.

Although molecular dynamics simulations of highly curved lipid bilayers have become increasingly popular in recent years, there is no simple and general method of computing the shape and curvature of the bilayer, which is bent arbitrarily in three dimensions. In this work we propose a method, which allows computing local normal, mean and Gaussian curvatures at any point of an arbitrarily curved lipid membrane using molecular dynamics trajectories. The method is based on the analysis of local membrane patches and is applicable to the membranes of any shape and topology - bilayers, vesicles, micelles, bicelles, etc. The method is applied to a highly curved asymmetric DOPC/DOPS lipid bilayer simulated by means of extended coarse-grained molecular dynamics simulations. It is shown that addition of cholesterol makes the membrane more topologically heterogeneous by increasing the content of highly curved regions with either saddle-like or sphere-like topology. The topology of the DOPS lipid domains is more sensitive to the addition of cholesterol than DOPC domains.

Dissecting Escherichia coli outer membrane biogenesis using differential proteomics.

The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device. While advances in understanding the mechanisms that govern the biogenesis of the cell envelope have been recently made, only few studies are available on how bacterial cells respond to severe envelope biogenesis defects on a global scale. Here we report the use of differential proteomics based on Multidimensional Protein Identification Technology (MudPIT) to investigate how Escherichia coli cells respond to a block of lipopolysaccharide transport to the outer membrane. We analysed the envelope proteome of a lptC conditional mutant grown under permissive and non permissive conditions and identified 123 proteins whose level is modulated upon LptC depletion. Most such proteins belong to pathways implicated in cell envelope biogenesis, peptidoglycan remodelling, cell division and protein folding. Overall these data contribute to our understanding on how E. coli cells respond to LPS transport defects to restore outer membrane functionality.

E. coli Outer Membrane and Interactions with OmpLA

The outer membrane of Gram-negative bacteria is a unique asymmetric lipid bilayer composed of phospholipids (PLs) in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet. Its function as a selective barrier is crucial for the survival of bacteria in many distinct environments, and it also renders Gram-negative bacteria more resistant to antibiotics than their Gram-positive counterparts. Here, we report the structural properties of a model of the Escherichia coli outer membrane and its interaction with outer membrane phospholipase A (OmpLA) utilizing molecular dynamics simulations. Our results reveal that given the lipid composition used here, the hydrophobic thickness of the outer membrane is ∼3 Å thinner than the corresponding PL bilayer, mainly because of the thinner LPS leaflet. Further thinning in the vicinity of OmpLA is observed due to hydrophobic matching. The particular shape of the OmpLA barrel induces various interactions between LPS and PL leaflets, resulting in asymmetric thinning around the protein. The interaction between OmpLA extracellular loops and LPS (headgroups and core oligosaccharides) stabilizes the loop conformation with reduced dynamics, which leads to secondary structure variation and loop displacement compared to that in a DLPC bilayer. In addition, we demonstrate that the LPS/PL ratios in asymmetric bilayers can be reliably estimated by the per-lipid surface area of each lipid type, and there is no statistical difference in the overall membrane structure for the outer membranes with one more or less LPS in the outer leaflet, although individual lipid properties vary slightly.

Cationic Au Nanoparticle Binding with Plasma Membrane-like Lipid Bilayers: Potential Mechanism for Spontaneous Permeation to Cells Revealed by Atomistic Simulations

Despite being chemically inert as a bulk material, nanoscale gold can pose harmful side effects to living organisms. In particular, cationic Au nanoparticles (AuNP+) of 2 nm diameter or less permeate readily through plasma membranes and induce cell death. We report atomistic simulations of cationic Au nanoparticles interacting with realistic membranes and explicit solvent using a model system that comprises two cellular compartments, extracellular and cytosolic, divided by two asymmetric lipid bilayers. The membrane–AuNP+ binding and membrane reorganization processes are discovered to be governed by co-operative effects where AuNP+, counterions, water, and the two membrane leaflets all contribute. On the extracellular side, we find that the nanoparticle has to cross a free energy barrier of about 5 kBT prior forming a stable contact with the membrane. This results in a rearrangement of the zwitterionic lipids and nanoparticle side groups in the contact area, giving rise to the initial stage of pore format...

Determination of the shape and curvature of nonplanar lipid bilayers that are bent in a single plane in molecular dynamics simulations

The curvature of biological membranes is known to be an important influence on important phenomena such as membrane fusion, endocytosis, and the functioning of integral membrane proteins. There is a growing demand for analytical tools that are able accommodate molecular dynamics trajectories of significantly curved lipid bilayers. In this work, an algorithm for determining the shape and curvature of a nonplanar lipid bilayer in molecular dynamics simulations is proposed. The algorithm calculates the coordinates of the midline and the curvature of the bilayer as well as the local normal to it at any point on the membrane, which is bent arbitrarily in a single plane and is topologically equivalent to an infinite bilayer. The algorithm is implemented as a C++ program and tested by exploring the molecular dynamic trajectories of a highly curved meander-like asymmetric lipid bilayer. The algorithm is general enough to allow it to be easily applied to other geometries of nonplanar membrane systems.

Lipid Bilayer Modules as Determinants of K+ Channel Gating

The crystal structure of the sensorless pore module of a voltage-gated K(+) (Kv) channel showed that lipids occupy a crevice between subunits. We asked if individual lipid monolayers of the bilayer embody independent modules linked to channel gating modulation. Functional studies using single channel current recordings of the sensorless pore module reconstituted in symmetric and asymmetric lipid bilayers allowed us to establish the deterministic role of lipid headgroup on gating. We discovered that individual monolayers with headgroups that coat the bilayer-aqueous interface with hydroxyls stabilize the channel open conformation. The hydroxyl need not be at a terminal position and the effect is not dependent on the presence of phosphate or net charge on the lipid headgroup. Asymmetric lipid bilayers allowed us to determine that phosphoglycerides with glycerol or inositol on the extracellular facing monolayer stabilize the open conformation of the channel. This indirect effect is attributed to a change in water structure at the membrane interface. By contrast, inclusion of the positively charged lysyl-dioleoyl-phosphatidylglycerol exclusively on the cytoplasmic facing monolayer of the bilayer increases drastically the probability of finding the channel open. Such modulation is mediated by a π-cation interaction between Phe-19 of the pore module and the lysyl moiety anchored to the phosphatidylglycerol headgroup. The new findings imply that the specific chemistry of the lipid headgroup and its selective location in either monolayer of the bilayer dictate the stability of the open conformation of a Kv pore module in the absence of voltage-sensing modules.

A Molecular Dynamics Simulation Study of Outer Membrane Phospholipase a (OMPLA) Structure and Dynamics in an Asymmetric Lipopolysaccharide Membrane

The outer membrane of Gram-negative bacteria is a unique and highly asymmetric lipid bilayer composed of phospholipids in the inner leaflet and mostly lipopolysaccharide (LPS) in the outer leaflet. Outer membrane phospholipase A (OmpLA) is an integral membrane enzyme in Escherichia coli. The structure of monomeric OmpLA consists of a 12-stranded antiparallel β-strands with a convex and a flat side, six loops at the extracellular side and five turns at the periplasmic side of the membrane. Utilizing the latest C36 CHARMM lipid and carbohydrate force field, we have constructed a model of OmpLA embedded in an asymmetric lipid bilayer with rough LPS molecules (without O-antigen) in one leaflet and phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin in the other leaflet to model the realistic outer membrane environment. The simulation results will be discussed in terms of the key structural properties of the bacterial outer membrane including hydrophobic thickness, area per lipid, and acyl chain order parameter. We will also show the difference of OmpLA structure and dynamics compared to that in a DLPC bilayer. At the same time a comparison between simulations with different numbers of LPS molecules on the outer leaflet will elucidate the potential technical difficulties in building asymmetric bilayer.

Lipid-coated calcium phosphate nanoparticles for nonviral gene therapy.

Though calcium phosphate (CaP) nanoparticles have been utilized as transfection agents in laboratory settings for nearly half a century, their power as a platform for therapeutically focused approaches has only recently been realized. With regard to modern advances in lipid- and polymer-mediated transfection, we present liposome CaP, a novel combination of a stable yet acid-labile CaP core with a surface functional, asymmetric lipid bilayer. Herein, the structural and functional dynamics of such nanoparticles are described in detail, including alteration of hepatocellular tropism through PEGylation and a multifunctional approach to endosomal escape, along with primary therapeutic advances, which these nanoparticles contribute to the field overall. Through these and other multifunctional nanotechnologies for gene therapy, great promise is shown in their ability to tackle truly complex and mechanistically demanding diseases.

YciM is an essential gene required for regulation of lipopolysaccharide synthesis in Escherichia coli

The outer membrane of Gram-negative bacteria is an asymmetric lipid bilayer consisting of an essential glycolipid lipopolysaccharide (LPS) in its outer leaflet and phospholipids in the inner leaflet. Here, we show that yciM, a gene encoding a tetratricopeptide repeat protein of unknown function, modulates LPS levels by negatively regulating the biosynthesis of lipid A, an essential constituent of LPS. Inactivation of yciM resulted in high LPS levels and cell death in Escherichia coli; recessive mutations in lpxA, lpxC or lpxD that lower the synthesis of lipid A, or a gain of function mutation in fabZ that increases the formation of membrane phospholipids, alleviated the yciM mutant phenotypes. A modest increase in YciM led to significant reduction of LPS and increased sensitivity to hydrophobic antibiotics. YciM was shown to regulate LPS by altering LpxC, an enzyme that catalyses the first committed step of lipid A biosynthesis. Regulation of LpxC by YciM was contingent on the presence of FtsH, an essential membrane-anchored protease known to degrade LpxC, suggesting that FtsH and YciM act in concert to regulate synthesis of lipid A. In summary, this study demonstrates an essential role for YciM in regulation of LPS biosynthesis in E. coli.

Model of a Raft in Both Leaves of an Asymmetric Lipid Bilayer

We present a theory of inhomogeneities in the plasma membrane, or rafts, that can exist in both leaves of the plasma membrane. We note that although neither of the major phospholipid components of the outer leaf, sphingomyelin (SM) nor phosphatidylcholine (PC), evinces a tendency to form phases characterized by nonzero curvature, one of the major components of the inner leaf, phosphatidylethanolamine (PE), displays a strong tendency to do so whereas the other, phosphatidylserine (PS), does not. Therefore, we posit that the concentration difference of PS and PE couples to height fluctuations of the plasma membrane bilayer. This brings about a microemulsion in the inner leaf. Coupling of the concentration difference between PS and PE in the inner leaf and SM and PC in the outer leaf propagates the microemulsion to that leaf as well. The characteristic size of the inhomogeneities is equal to the square-root of the ratio of the bending modulus of the bilayer to its surface tension, a size which is ∼100 nm for the plasma membrane. If the coupling between leaves were to be provided by the interchange of cholesterol, then our model raft would consist of SM and cholesterol in the outer leaf and PS and cholesterol in the inner leaf floating in a sea of PC and PE in both leaves.