Transbilayer Asymmetry Research Articles

Triacylglycerol-droplet-induced bilayer spontaneous curvature in giant unilamellar vesicles

This study investigated the incorporation of triacylglycerol droplets in the bilayers of giant unilamellar vesicles (GUVs) using four triacylglycerols and four phosphatidylcholines by confocal laser scanning microscopy. The triacylglycerol droplets were incorporated between the monolayer leaflets of the GUVs. Among the spherical droplets protruding on only one side of the bilayers, the droplets bound to the outer leaflets outnumbered those bound to the inner leaflets. The more frequent droplet binding to the outer leaflet caused transbilayer asymmetry in the droplet surface density. A vesicle consisting of a single-bilayer spherical segment and a double-bilayer spherical segment was also observed. The yield of these vesicles was comparable with or higher than that of the droplet-incorporating GUVs for many of the phosphatidylcholine-triacylglycerol combinations. In a vesicle consisting of single-bilayer and double-bilayer segments, most of the triacylglycerol droplets were localized on the outermost membrane surface along the segment boundary and in the double-bilayer segment. To rationalize the formation of these vesicle structures, we propose that the transbilayer asymmetry in the droplet surface density induces spontaneous curvature of the bilayer, with the bilayer spontaneously bending away from the droplets. Energy calculations performed assuming the existence of spontaneous curvature of the bilayer corroborated the experimentally determined membrane shapes for the vesicles consisting of unilamellar and bilamellar regions.

Seeing the Membrane from Both Sides Now: Lipid Asymmetry and Its Strange Consequences.

Almost all biomembranes are constructed as lipid bilayers and, in almost all of these, the two opposing monolayers (leaflets) have distinct lipid compositions. This lipid asymmetry arises through the concerted action of a suite of energy-dependent enzymes that maintain living bilayers in a far-from-equilibrium steady-state. Recent discoveries reveal that lipid compositional asymmetry imparts biophysical asymmetries and that this dualistic organization may have major consequences for cellular physiology. Importantly, while transbilayer asymmetry appears to be an essential, near-ubiquitous characteristic of biological membranes, it has been challenging to reproduce in reconstituted or synthetic systems. Although recent methodological developments have overcome some critical challenges, it remains difficult to extrapolate results from available models to biological systems. Concurrently, there are few experimental approaches for targeted, controlled manipulation of lipid asymmetry in living cells. Thus, the biophysical and functional consequences of membrane asymmetry remain almost wholly unexplored. This perspective summarizes the current state of knowledge and highlights emerging themes that are beginning to make inroads into the fundamental question of why life tends toward asymmetry in its bilayers.

Allosteric modulation of integral protein activity by differential stress in asymmetric membranes.

The activity of integral membrane proteins is tightly coupled to the properties of the surrounding lipid matrix. In particular, transbilayer asymmetry, a hallmark of all plasma membranes, might be exploited to control membrane-protein activity. Here, we hypothesized that the membrane-embedded enzyme outer membrane phospholipase A (OmpLA) is susceptible to the lateral pressure differences that build up between such asymmetric membrane leaflets. Upon reconstituting OmpLA into synthetic, chemically well-defined phospholipid bilayers exhibiting different lateral pressure profiles, we indeed observed a substantial decrease in the enzyme's hydrolytic activity with increasing membrane asymmetry. No such effects were observed in symmetric mixtures of the same lipids. To quantitatively rationalize how the differential stress in asymmetric lipid bilayers inhibits OmpLA, we developed a simple allosteric model within the lateral pressure framework. Thus, we find that membrane asymmetry can serve as the dominant factor in controlling membrane-protein activity, even in the absence of specific, chemical cues or other physical membrane determinants such as hydrophobic mismatch.

Fluorescence Spectroscopic Analysis of Lateral and Transbilayer Fluidity of Exosome Membranes.

Exosomes are small extracellular vesicles (sEVs) involved in distal cell-cell communication and cancer migration by transferring functional cargo molecules. Membrane domains similar to lipid rafts are assumed to occur in exosome membranes and are involved in interactions with target cells. However, the bilayer membrane properties of these small vesicles have not been fully investigated. Therefore, we examined the fluidity, lateral domain separation, and transbilayer asymmetry of exosome membranes using fluorescence spectroscopy. Although there were some differences between the exosomes, TMA-DPH anisotropy showing moderate lipid chain order indicated that ordered phases comprised a significant proportion of exosome membranes. Selective TEMPO quenching of the TMA-DPH fluorescence in the liquid-disordered phase indicated that 40-50% of the exosome membrane area belonged to the ordered phase based on a phase-separated model. Furthermore, NBD-PC in the outer leaflet showed longer fluorescence lifetimes than those in the inner leaflets. Therefore, the exosome membranes maintained transbilayer asymmetry with a topology similar to that of the plasma membranes. In addition, the lateral and transbilayer orders of exosome membranes obtained from different cell lines varied, probably depending on the different membrane lipid components and compositions partially derived from donor cells. As these higher membrane orders and asymmetric topologies are similar to those of cell membranes with lipid rafts, raft-like functional domains are possibly enriched on exosome membranes. These domains likely play key roles in the biological functions and cellular uptake of exosomes by facilitating selective membrane interactions with target organs.

The impact of TiO2 nanoparticle exposure on transmembrane cholesterol transport and enhanced bacterial infectivity in HeLa cells

We have previously shown that exposure to TiO2 nanoparticles (NPs) reduces the resistance of HeLa cells to bacterial infection. Here we demonstrate that the increased infectivity is associated with enhanced asymmetry in the cholesterol distribution. We applied a live cell imaging method which uses tunable orthogonal cholesterol sensors to visualize and quantify in-situ cholesterol distribution between the two leaflets of the plasma membrane (PM). In the control culture, we found marked transbilayer asymmetry of cholesterol, with the concentration in the outer plasma membrane (OPM) being 13 ± 2-fold higher than that in the inner plasma membrane (IPM). Exposure of the culture to 0.1 mg/mL of rutile TiO2 NPs increased the asymmetry such that the concentration in the OPM was 51 ± 10 times higher, while the total cholesterol content increased only 21 ± 2%. This change in cholesterol gradient may explain the increase in bacterial infectivity in HeLa cells exposed to TiO2 NPs since many pathogens, including Staphylococcus aureus used in the present study, require cholesterol for proper membrane attachment and virulence. RT-PCR indicated that exposure to TiO2 was responsible for upregulation of the ABCA1 and ABCG1 mRNAs, which are responsible for the production of the cholesterol transporter proteins that facilitate cholesterol transport across cellular membranes. This was confirmed by the observation of an overall decrease in bacterial infection in ABCA1 knockout or methyl-β-cyclodextrin-treated HeLa cells, as regardless of TiO2 NP exposure. Hence rather than preventing bacterial infection, TiO2 nanoparticles upregulate genes associated with membrane cholesterol production and distribution, hence increasing infectivity. Statement of significanceA great deal of work has been done regarding the toxicology of the particles, especially focusing on detrimental outcomes associated with reactive oxygen species (ROS) production. In this paper we show unambiguously a very surprising result, namely the ability of these particles to enhance bacterial infection even at very small exposure levels, where none of the deleterious effects of ROS products can yet be detected. Using a new imaging technique, we are able to demonstrate, in operando, the effect of the particles on cholesterol generation and distribution in live HeLa cells. This paper also represents the first in a series where we explore other consequences of increased membrane cholesterol, due to particle exposure, which are known to have multiple other consequences on human tissue function and development.

Imaging cholesterol depletion at the plasma membrane by methyl-β-cyclodextrin

The most common method to modify cellular cholesterol (Chol) content in vitro is to incubate cells with cyclodextrin (CD). However, it is not well understood how Chol is removed by CD. Recent development of protein probes that bind Chol allowed to visualize and to follow cellular Chol semi-quantitatively. In this image, non-toxic Chol-binding D4 fragment of bacterial toxin, perfringolysin O was conjugated with mCherry (mCherry-D4) (1Abe M. Makino A. Hullin-Matsuda F. Kamijo K. Ohno-Iwashita Y. Hanada K. Mizuno H. Miyawaki A. Kobayashi T. A role for sphingomyelin-rich lipid domains in the accumulation of phosphatidylinositol 4,5-bisphosphate to the cleavage furrow during cytokinesis.Mol. Cell Biol. 2012; 32: 1396-1407Crossref PubMed Scopus (98) Google Scholar) and the protein was transiently expressed in HeLa cells for 48 h. Cells were synchronized to mitosis and incubated with and without 1 mM methyl-b-CD (MbCD) in the presence of EGFP-D4 in the medium. Selected images are shown. Accumulated images are shown in Supplemental videos 1 and 2. Average fluorescence intensities of EGFP-D4 (green) and mCherry-D4 (magenta) were quantified in the area surrounded by the white rectangle and schematically shown in the kymographs. The quantitated data are shown as graphs in Supplemental Fig. 1. Supplemental Fig. 2 shows that equivalent amount of EGFP-D4 and mCherry-D4 displayed similar Chol concentration dependent binding to phosphatidylcholine/Chol membranes. Under this condition, mCherry-D4 and EGFP-D4 bind accessible Chol (2Das A. Brown M.S. Anderson D.D. Goldstein J.L. Radhakrishnan A. Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis.Elife. 2014; 3e02882Crossref Scopus (171) Google Scholar) in the inner and outer leaflet of the plasma membrane, respectively (1Abe M. Makino A. Hullin-Matsuda F. Kamijo K. Ohno-Iwashita Y. Hanada K. Mizuno H. Miyawaki A. Kobayashi T. A role for sphingomyelin-rich lipid domains in the accumulation of phosphatidylinositol 4,5-bisphosphate to the cleavage furrow during cytokinesis.Mol. Cell Biol. 2012; 32: 1396-1407Crossref PubMed Scopus (98) Google Scholar). Due to high threshold of Chol-detection by D4, intracellular organelles were not labeled with mCherry-D4 and depletion of Chol was accompanied by the intracellular accumulation of the protein. MbCD induced the rapid decrease (5-15 min) of inner leaflet Chol below the D4-binding threshold followed by the slow depletion phase in both outer and inner leaflets. The interaction between D4 or MbCD and cultured cells is not fully elucidated. Although MbCD is endocytosed, endocytosis is inhibited in mitotic cells (3Kobayashi T. Pagano R.E. Lipid transport during mitosis. Alternative pathways for delivery of newly synthesized lipids to the cell surface.J. Biol. Chem. 1989; 264: 5966-5973Abstract Full Text PDF PubMed Google Scholar). Binding of acrylodan-labeled D4 to PC/Chol membrane is inhibited by cytosol (4Courtney K.C. Fung K.Y. Maxfield F.R. Fairn G.D. Zha X. Comment on 'Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol'.Elife. 2018; 7e38493Crossref PubMed Scopus (16) Google Scholar). In addition to Chol, MbCD is reported to interact with other lipids and protein components of the membrane. However, extraction of cytosolic protein by MbCD has not been reported. Our result suggests that the removal of outer leaflet Chol by MbCD is accompanied by the rapid flip of Chol from the inner leaflet to the outer leaflet. However, we cannot exclude the possibility that MbCD preferentially removed Chol from the inner leaflet. EQUIPMENT: FV 1000 confocal microscope with a 60x 1.1 NA Plan Apo objective lens (Olympus) equipped with an environmental chamber maintained with humidity, 37°C, and 5% CO2. ImageQuant LAS 500 (Cytiva). REAGENTS: DMEM supplemented with 10% fetal bovine serum, Lipofectamine 3000 (Thermo Fisher Scientific), pET28/His6-EGFP-D4 (RIKEN BRC, Gene Engineering Division, Catalog # RDB13961), pET28/His6-mCherry-D4 (RIKEN BRC, Catalog # RDB14300), pDEST/mCherry-D4 (1), nocodazole (Sigma-Aldrich), MβCD (Cyclolab), SYPRO Ruby (Thermo Fisher Scientific). This article contains supplemental data. The authors declare that they have no conflicts of interest with the contents of this article. We are grateful to Dr. Françoise Hullin-Matsuda from INSA Lyon for critically reading the manuscript. M. A. conceptualization, methodology, investigation, data curation, writing-original draft preparation, reviewing, and editing. T. K. conceptualization, writing-original draft preparation, reviewing, and editing.

The Impact of TiO <sub>2</sub> Nanoparticle Exposure on Transmembrane Cholesterol Transport and Enhanced Bacterial Infectivity

We have previously shown that exposure to TiO2 nanoparticles (NPs) reduces the resistance of HeLa cells to bacterial infection. Here we demonstrate that the increased infectivity is associated with enhanced asymmetry in the cholesterol distribution. We applied a new live cell imaging method which uses tunable orthogonal cholesterol sensors to visualize and quantify in-situ cholesterol distribution between the two leaflets of the plasma membrane (PM). In the control culture, we found marked transbilayer asymmetry of cholesterol, with the concentration in the outer plasma membrane (OPM) being 12.5(2.2)-fold higher than that in the inner plasma membrane (IPM). Exposure of the culture to 0.1 mg/mL of rutile TiO2 NPs increased the asymmetry such that the concentration in the OPM was 50.8(9.5) times higher, while the total cholesterol content increased only 20.5(2.4)%. This change in cholesterol gradient may explain the increase in bacterial infectivity in HeLa cells exposed to TiO2 NPs since many pathogens, including Staphylococcus aureus used in the present study, require cholesterol for proper membrane attachment and virulence. RT-PCR indicated that exposure to TiO2 was responsible for upregulation of the ABCA1 and ABCG1 mRNAs, which are responsible for the production of the cholesterol transporter proteins that facilitate cholesterol transport across cellular membranes. This was confirmed by the observation of an overall decrease in bacterial infection in ABCA1 knockout or methyl-β-cyclodextrin-treated HeLa cells, as regardless of TiO2 NP exposure. Hence rather than preventing bacterial infection, TiO2 nanoparticles upregulate genes associated with membrane cholesterol production and distribution, hence increasing infectivity.

Asymmetry and Rippling in Mixed Surfactant Bilayers from All-Atom and Coarse-Grained Simulations: Interdigitation and Per Chain Entropy

Understanding lateral organizations of self-assembled bilayers is crucial to gain a control on functionally relevant topologies. We study self-assembled bilayers composed of a surfactant, behenyl trimethyl ammonium chloride (BTMAC), a cosurfactant, stearyl alcohol (SA), at a ratio of 2:1 in the presence of water at 283 K employing subsequent all-atom (AA) and coarse-grained (CG) molecular dynamics simulations. Differences in initial configurations lead to the formation of bilayers at ripple or square phases or interdigitated gel phases of varying trans-leaflet asymmetry. The AA ripple and gel phases are reproduced well at the CG level using bonded potentials from Boltzmann inversion of AA canonical sampling and nonbonded potentials from MARTINI. Inhomogeneous populations of disordered chains with higher per chain configurational entropy and tilt result in rippling stabilized by periodic hydrophobic energy barrier and strong interdigitation. Order parameters of the asymmetric bilayers are sufficiently coupled to the per chain entropies at both levels of resolutions to serve as a reflector of the per chain configurational entropy inaccessible by experiments. Thus, trans-bilayer asymmetry may be a controlling parameter to induce rippling in a bilayer of industrial importance. This work will be useful for future investigation on domain-associated transport and signaling in biomembranes at a low temperature.

Toward a new picture of the living plasma membrane.

Our understanding of the plasma membrane structure has undergone a major change since the proposal of the fluid mosaic model of Singer and Nicholson in the 1970s. In this model, the membrane, composed of over thousand lipid and protein species, is organized as a well-equilibrated two-dimensional fluid. Here, the distribution of lipids is largely expected to reflect a multicomponent system, and proteins are expected to be surrounded by an annulus of specialized lipid species. With the recognition that a multicomponent lipid membrane is capable of phase segregation, the membrane is expected to appear as patchwork quilt pattern of membrane domains. However, the constituents of a living membrane are far from being well equilibrated. The living cell membrane actively maintains a trans-bilayer asymmetry of composition, and its constituents are subject to a number of dynamic processes due to synthesis, lipid transfer as well as membrane traffic and turnover. Moreover, membrane constituents engage with the dynamic cytoskeleton of a living cell, and are both passively as well as actively manipulated by this engagement. The extracellular matrix and associated elements also interact with membrane proteins contributing to another layer of interaction. At the nano- and mesoscale, the organization of lipids and proteins emerge from these encounters, as well as from protein-protein, protein-lipid, and lipid-lipid interactions in the membrane. New methods to study the organization of membrane components at these scales have also been developed, and provide an opportunity to synthesize a new picture of the living cell surface as an active membrane composite.

Peptide-Induced Lipid Flip-Flop in Asymmetric Liposomes Measured by Small Angle Neutron Scattering.

Despite the prevalence of lipid transbilayer asymmetry in natural plasma membranes, most biomimetic model membranes studied are symmetric. Recent advances have helped to overcome the difficulties in preparing asymmetric liposomes in vitro, allowing for the examination of a larger set of relevant biophysical questions. Here, we investigate the stability of asymmetric bilayers by measuring lipid flip-flop with time-resolved small-angle neutron scattering (SANS). Asymmetric large unilamellar vesicles with inner bilayer leaflets containing predominantly 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and outer leaflets composed mainly of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) displayed slow spontaneous flip-flop at 37 ◦C (half-time, t1/2 = 140 h). However, inclusion of peptides, namely, gramicidin, alamethicin, melittin, or pHLIP (i.e., pH-low insertion peptide), accelerated lipid flip-flop. For three of these peptides (i.e., pHLIP, alamethicin, and melittin), each of which was added externally to preformed asymmetric vesicles, we observed a completely scrambled bilayer in less than 2 h. Gramicidin, on the other hand, was preincorporated during the formation of the asymmetric liposomes and showed a time resolvable 8-fold increase in the rate of lipid asymmetry loss. These results point to a membrane surface-related (e.g., adsorption/insertion) event as the primary driver of lipid scrambling in the asymmetric model membranes of this study. We discuss the implications of membrane peptide binding, conformation, and insertion on lipid asymmetry.

Effect of Transmembrane Asymmetric Distribution of Lipids and Peptides on Lipid Bilayers.

The transbilayer asymmetry of the biomembrane is generated due to the differences in lipid and protein compositions between two leaflets, which plays important roles in physiological functions. However, transbilayer asymmetry can also be originated due to a nonequal number of lipids or proteins in each leaflet, which has not been well recognized. Therefore, to shed light on this field, here we generated theoretical models for the effect of transbilayer asymmetry originated from the differences in the number of lipids and peptides in each leaflet on the state of lipid bilayers. The first model described the effect of asymmetric lipid distribution on the state of lipid bilayers. We obtained theoretical equations for the fractional change in area per lipid in both leaflets as a function of the ratio of the number of lipids in each leaflet, which agreed with the molecular dynamics simulation results quantitatively. Results indicated that tensions in both leaflets are opposite in direction, and their magnitude is the same. We also performed experiments on the effect of lipid insertion in the outer leaflet on the fractional change in area per lipid. These results agreed quantitatively with the values predicted by the above model. The second model described the effect of asymmetric distribution of peptides on the state of lipid bilayers. We obtained theoretical equations for the area per lipid in both leaflets as a function of the surface concentration of peptides located only in the outer leaflet, which agreed with the results of the antimicrobial peptide magainin 2-induced area change.

Substrate Selectivity of a P4‐ATPase Flippase

Phospholipids, the primary structural components of cell membranes, are distributed unequally across the membrane. The sphingolipids, including sphingomyelin (SM) and glycosphingolipids, are enriched on the exofacial surface of the plasma membrane or the luminal surfaces of internal organelles, while the cytofacial leaflet is enriched in the aminophospholipids, phophatidylserine (PS) and phosphatidylethanolamine (PE). Contributing to maintenance of this asymmetry is a combination of slow passive transbilayer phospholipid movement, trapping by cytofacial proteins and the activity of a number of energy‐dependent transporters. The substrate specificity and directionality of these pumps defines the transbilayer asymmetry of phospholipids. Two transporter superfamilies, ABC transporters and P‐type ATPases, account for these activities and move lipids in either the exofacial (floppase) or cytofacial (flippase) directions, respectively. In previous studies we have characterized the biochemical requirements and substrate specificity for a PS specific flippase activity in human red blood cells. Transport is ATP‐dependent and sensitive to vanadate and sulfhydryl‐modification. This flippase is selective for the sn‐1,2‐isomer of PS. Transport is diminished by modification of the PS headgroup amine (with the exception of mono‐methylation), carboxyl and phosphate groups, while a variety of fatty acyl groups and the D‐serine analog are accommodated. Red blood cells express two members of the P4‐type ATPase subfamily, ATP8A1 and ATP11C, that are likely responsible for this activity. Here, we report on the heterologous expression, purification, and functional assaying of hexahistidine‐tagged constructs of Atp8a1 with and without its cognate accessory subunit, Cdc50a. Purified Atp8a1 is vanadate‐sensitive and dependent upon Cdc50a co‐expression and PS for robust ATPase activity, although lipid specificity is not dependent on the presence of Cdc50a. In addition to reconstituting the flippase activity of Atp8a1 and ATP11c in vitro, our efforts are focused on rational mutagenesis of Atp8a1 to identify the lipid transport path and the determinants of substrate specificity, directed by homology modeling and the lipid‐binding characteristics of the closest members of the P‐type superfamily. X‐ray crystal structures of the SERCA pump in conformation E2 (PDB 2AGV) display a PE glycerol backbone and head group tightly pinched by helices M2 and M4 with PE's acyl chains extending between helices M2 and M6. This PE‐binding site implies a mechanism for how the P4lipid‐binding site and transport pathway evolved from ancestor P‐types designed to move simple cations. With these clues, we aim to identify the PS binding site and deduce the origin of the sn‐1,2‐stereospecificity of Atp8a1.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

C24 Sphingolipids Govern the Transbilayer Asymmetry of Cholesterol and Lateral Organization of Model and Live-Cell Plasma Membranes

Mammalian sphingolipids, primarily with C24 or C16 acyl chains, reside in the outer leaflet of the plasma membrane. Curiously, little is known how C24 sphingolipids impact cholesterol and membrane microdomains. Here, we present evidence that C24 sphingomyelin, when placed in the outer leaflet, suppresses microdomains in giant unilamellar vesicles and also suppresses submicron domains in the plasma membrane of HeLa cells. Free energy calculations suggested that cholesterol has a preference for the inner leaflet if C24 sphingomyelin is in the outer leaflet. We indeed observe that cholesterol enriches in the inner leaflet (80%) if C24 sphingomyelin is in the outer leaflet. Similarly, cholesterol primarily resides in the cytoplasmic leaflet (80%) in the plasma membrane of human erythrocytes where C24 sphingolipids are naturally abundant in the outer leaflet. We conclude that C24 sphingomyelin uniquely interacts with cholesterol and regulates the lateral organization in asymmetric membranes, potentially by generating cholesterol asymmetry.

Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol.

Controlled distribution of lipids across various cell membranes is crucial for cell homeostasis and regulation. We developed an imaging method that allows simultaneous in situ quantification of cholesterol in two leaflets of the plasma membrane (PM) using tunable orthogonal cholesterol sensors. Our imaging revealed marked transbilayer asymmetry of PM cholesterol (TAPMC) in various mammalian cells, with the concentration in the inner leaflet (IPM) being ∼12-fold lower than that in the outer leaflet (OPM). The asymmetry was maintained by active transport of cholesterol from IPM to OPM and its chemical retention at OPM. Furthermore, the increase in the IPM cholesterol level was triggered in a stimulus-specific manner, allowing cholesterol to serve as a signaling lipid. We found excellent correlation between the IPM cholesterol level and cellular Wnt signaling activity, suggesting that TAPMC and stimulus-induced PM cholesterol redistribution are crucial for tight regulation of cellular processes under physiological conditions.

Membrane mechanical properties of synthetic asymmetric phospholipid vesicles.

Synthetic lipid vesicles have served as important model systems to study cellular membrane biology. Research has shown that the mechanical properties of bilayer membranes significantly affects their biological behavior. The properties of a lipid bilayer are governed by lipid acyl chain length, headgroup type, and the presence of membrane proteins. However, few studies have explored how membrane architecture, in particular trans-bilayer lipid asymmetry, influences membrane mechanical properties. In this study, we investigated the effects of lipid bilayer architecture (i.e. asymmetry) on the mechanical properties of biological membranes. This was achieved using a customized micropipette aspiration system and a novel microfluidic technique previously developed by our team for building asymmetric phospholipid vesicles with tailored bilayer architecture. We found that the bending modulus and area expansion modulus of the synthetic asymmetric bilayers were up to 50% larger than the values acquired for symmetric bilayers. This was caused by the dissimilar lipid distribution in each leaflet of the bilayer for the asymmetric membrane. To the best of our knowledge, this is the first report on the impact of trans-bilayer asymmetry on the area expansion modulus of synthetic bilayer membranes. Since the mechanical properties of bilayer membranes play an important role in numerous cellular processes, these results have significant implications for membrane biology studies.

Transbilayer asymmetry and sphingomyelin composition modulate the preferential membrane partitioning of the nicotinic acetylcholine receptor in Lo domains

We have previously shown that the intact nicotinic acetylcholine receptor (AChR) lacks preference for Lo domains when reconstituted in a sphingomyelin (SM), cholesterol (Chol) and POPC (1:1:1) model system (Bermúdez V, Antollini SS, Fernández-Nievas GA, Aveldaño MI, Barrantes FJ. J. Lipid Res. 2010; 51: 2629–2641). Here, we have furthered our studies by characterizing the influence of different lipid host compositions on the distribution of purified AChR reconstituted in two model systems (POPC:Chol, 1:1 and POPC:Chol:SM, 1:1:1), involving a) different SM species (porcine brain SM (bSM), 16:0-SM, 18:0-SM or 24:1-SM); or b) induced transbilayer asymmetry, resulting from enrichment in bSM in the external hemilayer. AChR distribution was evaluated by fluorescence resonance energy transfer efficiency between the AChR intrinsic fluorescence and Laurdan or dehydroergosterol fluorescence, and by analyzing the distribution of AChR in detergent-resistant and detergent-soluble fractions (1% Triton X-100, 4 °C). bSM-induced transbilayer asymmetry or the presence of 16:0-SM and/or 18:0-SM (unlike bSM or 24:1-SM) resulted in the preferential partitioning of AChR in Lo domains, suggesting that the localization of AChR in ordered domains strongly depends on the characteristics of the host lipid membrane, and in particular on the sphingolipid composition and transbilayer asymmetry.

Revisiting transbilayer distribution of lipids in the plasma membrane

Whereas asymmetric transbilayer lipid distribution in the plasma membrane is well recognized, methods to examine the precise localization of lipids are limited. In this review, we critically evaluate the methods that are applied to study transbilayer asymmetry of lipids, summarizing the factors that influence the measurement. Although none of the present methods is perfect, the current application of immunoelectron microscopy-based technique provides a new picture of lipid asymmetry. Next, we summarize the transbilayer distribution of individual lipid in both erythrocytes and nucleated cells. Finally we discuss the concept of the interbilayer communication of lipids.

Asymmetric lipid membranes: towards more realistic model systems.

Despite the ubiquity of transbilayer asymmetry in natural cell membranes, the vast majority of existing research has utilized chemically well-defined symmetric liposomes, where the inner and outer bilayer leaflets have the same composition. Here, we review various aspects of asymmetry in nature and in model systems in anticipation for the next phase of model membrane studies.

To be or not to be in Membrane Domains: Transbilayer Asymmetry and Sphingomyelin-Dependent Preferential Partitioning of the Acetylcholine Receptor

The preferential partitioning of the nicotinic acetylcholine receptor (AChR) in liquid-ordered (Lo) domains, heterogeneous membrane domains commonly known as rafts, is thought to be a part of its clustering mechanism. Previous studies from our group have shown that AChR lacks preference for Lo domains when reconstituted in sphingomyelin (SM), cholesterol (Chol) and POPC (1:1:1) model systems (Bermudez et al., 2010). Here we study the effect on the possible Lo-preferential partitioning of purified AChR reconstituted in two different model systems (POPC:Chol, 1:1 and POPC:Chol:SM, 1:1:1) under: a) induced transbilayer asymmetry, resulting from addition of brain sphingomyelin (bSM) to the external hemilayer; and b) the presence of different pure SM species in the model membrane (bSM, 16:0-SM, 18:0-SM or 24:1-SM). AChR distribution was evaluated by fluorescence resonance energy transfer efficiency between the AChR intrinsic fluorescence and Laurdan or dehydroergosterol fluorescence, and also by determining the presence of AChR in detergent-resistant and detergent-soluble domains (1% Triton X-100, 4°C). Both studies show that the induction of transbilayer asymmetry or the presence of 16:0-SM or 18:0-SM, as opposed to bSM or 24:1-SM, leads to a preferential partitioning of AChR in Lo domains. Thus, the localization of AChR in Lo domains strongly depends on the characteristics of the host lipid membrane.

Membrane Properties Involved in Calcium-Stimulated Microparticle Release from the Plasma Membranes of S49 Lymphoma Cells

This study answered the question of whether biophysical mechanisms for microparticle shedding discovered in platelets and erythrocytes also apply to nucleated cells: cytoskeletal disruption, potassium efflux, transbilayer phospholipid migration, and membrane disordering. The calcium ionophore, ionomycin, disrupted the actin cytoskeleton of S49 lymphoma cells and produced rapid release of microparticles. This release was significantly inhibited by interventions that impaired calcium-activated potassium current. Microparticle release was also greatly reduced in a lymphocyte cell line deficient in the expression of scramblase, the enzyme responsible for calcium-stimulated dismantling of the normal phospholipid transbilayer asymmetry. Rescue of the scrambling function at high ionophore concentration also resulted in enhanced particle shedding. The effect of membrane physical properties was addressed by varying the experimental temperature (32–42°C). A significant positive trend in the rate of microparticle release as a function of temperature was observed. Fluorescence experiments with trimethylammonium diphenylhexatriene and Patman revealed significant decrease in the level of apparent membrane order along that temperature range. These results demonstrated that biophysical mechanisms involved in microparticle release from platelets and erythrocytes apply also to lymphocytes.