Transmembrane Asymmetry Research Articles

Poly(Sitosterol)-Based Hydrophobic Blocks in Amphiphilic Block Copolymers for the Assembly of Hybrid Vesicles.

Amphiphilic block copolymer and lipids can be assembled into hybrid vesicles (HVs), which are an alternative to liposomes and polymersomes. Block copolymers that have either poly(sitostryl methacrylate) or statistical copolymers of sitosteryl methacrylate and butyl methacrylate as the hydrophobic part and a poly(carboxyethyl acrylate) hydrophilic segment are synthesized and characterized. These block copolymers assemble into small HVs with soybean L-α-phosphatidylcholine (soyPC), confirmed by electron microscopy and small-angle X-ray scattering. The membrane's hybrid nature is illustrated by fluorescence resonance energy transfer between labeled building blocks. The membrane packing, derived from spectra when using Laurdan as an environmentally sensitive fluorescent probe, is comparable between small HVs and the corresponding liposomes with molecular sitosterol, although the former show indications of transmembrane asymmetry. Giant HVs with homogenous distribution of the block copolymers and soyPC in their membranes are assembled using the electroformation method. The lateral diffusion of both building blocks is slowed down in giant HVs with higher block copolymer content, but their permeability toward (6)-carboxy-X-rhodamine is higher compared to giant vesicles made of soyPC and molecular sitosterol. This fundamental effort contributes to the rapidly expanding understanding of the integration of natural membrane constituents with designed synthetic compounds to form hybrid membranes.

Unsaturation effects on lipid transmembrane asymmetry.

Within cell plasma membranes, unsaturated lipids are asymmetrically distributed over the inner and outer leaflets, offering an attractive local structural feature. However, the mechanism to keep lipid transmembrane asymmetry and the closely related transmembrane movement (flip-flop) for unsaturated lipids remain poorly understood. Here, we applied sum frequency generation vibrational spectroscopy to investigate this lipid transmembrane asymmetry upon mimicking the cell membrane homeostatic processes. On the one hand, unsaturated lipids were found to hinder the flip-flop process and preserve lipid transmembrane asymmetry in model cell membranes, owing to the steric hindrance caused by their bent tails. On the other hand, local unsaturated lipids in the mixed unsaturated/saturated lipid bilayer were conducive to the formation of the local asymmetry. Therefore, lipid unsaturation can be recognized as an intrinsic key factor to form and maintain lipid transmembrane asymmetry in cell membranes.

Bilayer Charge Asymmetry and Oil Residues Destabilize Membranes upon Poration.

Transmembrane asymmetry is ubiquitous in cells, particularly with respect to lipids, where charged lipids are mainly restricted to one monolayer. We investigate the influence of anionic lipid asymmetry on the stability of giant unilamellar vesicles (GUVs), minimal plasma membrane models. To quantify asymmetry, we apply the fluorescence quenching assay, which is often difficult to reproduce, and caution in handling the quencher is generally underestimated. We first optimize this assay and then apply it to GUVs prepared with the inverted emulsion transfer protocol by using increasing fractions of anionic lipids restricted to one leaflet. This protocol is found to produce highly asymmetric bilayers but with ∼20% interleaflet mixing. To probe the stability of asymmetric versus symmetric membranes, we expose the GUVs to porating electric pulses and monitor the fraction of destabilized vesicles. The pulses open macropores, and the GUVs either completely recover or exhibit leakage or bursting/collapse. Residual oil destabilizes porated membranes, and destabilization is even more pronounced in asymmetrically charged membranes. This is corroborated by the measured pore edge tension, which is also found to decrease with increasing charge asymmetry. Using GUVs with imposed transmembrane pH asymmetry, we confirm that poration-triggered destabilization does not depend on the approach used to generate membrane asymmetry.

Type-B Energetic Processes and Their Associated Scientific Implications

Recently, our work has identified two thermodynamically distinct types (A and B) of energetic processes naturally occurring on Earth: Type-A energy processes such as the classical heat engines, ATP hydrolysis, and many of the known chemical, electrical, and mechanical processes apparently well follow the second law of thermodynamics; and Type-B energy processes, such as the newly discovered thermotrophic function that isothermally utilizes environmental heat energy to do useful work in driving ATP synthesis, which follows the first law of thermodynamics (conservation of mass and energy), but does not necessarily have to be constrained by the second law, owing to their special asymmetric functions. In mitochondria, their special asymmetric functions associated with Type-B processes comprise: 1) The transmembrane asymmetry of inner mitochondrial membrane structure with the protonic outlets of redox-driven proton-pumping protein complexes protruded away from the membrane surface by about 1-3 nm into the bulk liquid p-phase while the protonic inlet of the F0F1-ATP synthase located rightly at the transmembrane electrostatically localized proton (TELP) layer; and 2) The lateral asymmetry of mitochondrial cristae with an ellipsoidal shape that enhances the density of TELP at the cristae tips where the F0F1-ATP synthase enzymes are located in supporting the TELP-associated thermotrophic function. The identification of Type-B energy processes indicates that there is an entirely new world of physical and energy sciences yet to be fully uncovered. Innovative efforts on Type-B processes to enable isothermally utilizing endless environmental heat energy could help to liberate all peoples from their dependence of fossil fuel energy, thus helping to reduce greenhouse gas CO2 emissions and control climate change toward a sustainable future for the humanity on Earth.

Membrane Curvature, Trans-Membrane Area Asymmetry, Budding, Fission and Organelle Geometry.

In biology, the modern scientific fashion is to mostly study proteins. Much less attention is paid to lipids. However, lipids themselves are extremely important for the formation and functioning of cellular membrane organelles. Here, the role of the geometry of the lipid bilayer in regulation of organelle shape is analyzed. It is proposed that during rapid shape transition, the number of lipid heads and their size (i.e., due to the change in lipid head charge) inside lipid leaflets modulates the geometrical properties of organelles, in particular their membrane curvature. Insertion of proteins into a lipid bilayer and the shape of protein trans-membrane domains also affect the trans-membrane asymmetry between surface areas of luminal and cytosol leaflets of the membrane. In the cases where lipid molecules with a specific shape are not predominant, the shape of lipids (cylindrical, conical, or wedge-like) is less important for the regulation of membrane curvature, due to the flexibility of their acyl chains and their high ability to diffuse.

Regulation of the Asymmetric Distribution of Lipids in Human Erythrocyte Membrane in the Presence of Glycerol and Polyethylene Glycol

Cryoprotective agents (CPAs) used to protect erythrocytes during freezing can affect the structural and functional parameters of the membrane, which have an impact on cell viability. The purpose of the study was to examine the effect of glycerol and PEG on the externalization of phosphatidylserine (PS) on the surface of erythrocytes, as well as to determine the role of Ca2+ ions and ATP-dependent processes in the regulation of transmembrane lipid asymmetry in the presence of these CPAs. It was found that the glycerol effect on erythrocytes does not lead to PS externalization. Maintaining the activity of scramblases and flipases in erythrocytes in the presence of glycerol at the level of control parameters allows them to respond similar to native cells to an increase in [Ca2+]in caused by the ionophore A23187 and blocking of ATPase reactions caused by vanadate. The PEG effect contributes to the disruption of the PS distribution in the membrane due to changes in the activity of lipid translocases. However, activation of scramblases and/or inhibition of flipases under the PEG effect does not reach maximum values, as evidenced by an increase in the amount of erythrocytes with externalized PS under cell loading with Ca2+ by ionophore and inhibition of ATP-dependent reactions by vanadate. The effect of PEG on the lipid asymmetry of erythrocyte membranes is apparently mediated by its impact on the Ca2+ level in the cells. Disturbance of the structural parameters of the membrane due to the PS redistribution in the PEG presence, that distinguish it from the glycerol effect, can cause instability of cryopreserved erythrocytes under physiological conditions in vitro.

Disturbance of the transmembrane phosphatidylserine asymmetry in hepatocytes as an apoptosis marker under the action of xenobiotics on rats

Disturbance of the transmembrane phosphatidylserine asymmetry in hepatocytes as an apoptosis marker under the action of xenobiotics on rats

Apoptosis and eryptosis: Striking differences on biomembrane level

The cell plasma membrane plays an essential role in programmed cell death of nucleated cells (apoptosis) and erythrocytes (eryptosis), and its changes due to loss of transmembrane asymmetry are quite similar. However, nucleated cells possess the network of intracellular membranes, which are missing in erythrocytes. Providing comparative studies with series of molecular probes, we observe dramatic differences in membrane lipid order in the course of apoptosis and eryptosis. In contrast to nucleated cells, in which a significant drop of the lipid order in the plasma membrane is observed, the erythrocyte membrane retains the relatively high level of the lipid order. Observation in nucleated cells of significant differences between inner and plasma membranes and detection of apoptotic bodies with different organization suggest that the decrease in the lipid order of their plasma membrane could be at least partially explained by the phospholipid and/or cholesterol exchange between membranes. Such features are absent in erythrocytes.

Intermolecular Headgroup Interaction and Hydration as Driving Forces for Lipid Transmembrane Asymmetry.

Variations between the inner and outer leaflets of cell membranes are crucial for cell functioning and signaling, drug-membrane interactions, and the formation of lipid domains. Transmembrane asymmetry can in principle be comprised of an asymmetric charge distribution, differences in hydration, specific headgroup/H-bonding interactions, or a difference in the number of lipids per leaflet. Here, we characterize the transmembrane asymmetry of small unilamellar liposomes consisting of zwitterionic and charged lipids in aqueous solution using vibrational sum frequency scattering and second harmonic scattering, label-free methods, specifically sensitive to lipid and water asymmetries. For single component liposomes, transmembrane asymmetry is present for the charge distribution and lipid hydration, but the leaflets are not detectably asymmetric in terms of the number of lipids per leaflet, even though geometrical packing arguments would predict so. Such a lipid transmembrane asymmetry can, however, be induced in binary lipid mixtures under conditions that enable H-bonding interactions between phosphate and amine groups. In this case, the measured asymmetry consists of a different number of lipids in the outer and inner leaflet, a difference in transmembrane headgroup hydration, and a different headgroup orientation for the interacting phosphate groups.

Membrane bending by protein crowding is affected by protein lateral confinement

Crowding of asymmetrically-distributed membrane proteins has been recently recognized as an important factor in remodeling of biological membranes, for example during transport vesicle formation. In this paper, we theoretically analyze the effect of protein crowding on membrane bending and examine its dependence on protein size, shape, transmembrane asymmetry and lateral confinement. We consider three scenarios of protein lateral organization, which are highly relevant for cellular membranes in general: freely diffusing membrane proteins without lateral confinement, the presence of a diffusion barrier and interactions with a vesicular coat. We show that protein crowding affects vesicle formation even if the proteins are distributed symmetrically across the membrane and that this effect depends significantly on lateral confinement. The largest crowding effect is predicted for the proteins that are confined to the forming vesicle by a diffusion barrier. We calculate the bending properties of a crowded membrane and find that its spontaneous curvature depends primarily on the degree of transmembrane asymmetry, and its effective bending modulus on the type of lateral confinement. Using the example of COPII vesicle formation from the endoplasmic reticulum, we analyze the energetic cost of vesicle formation. The results provide a novel insight into the effects of lateral and transmembrane organization of membrane proteins, and can guide data interpretation and future experimental approaches.

Влияние криоконсервирования в присутствии криопротектора ПЭГ-1500 на поверхностные характеристики эритроцитов

The studies of changes in surface characteristics of erythrocyte membranes caused by freeze-thawing can be important for assessment of structural and functional integrity of cells and understanding the mechanisms responsible for their damage under extreme conditions. In the research we studied the effect of cryopreservation in presence of exocellular cryoprotectant PEG-1500 on asymmetric distribution of phosphatidylserine in membrane and characteristics of the surface marker CD44 in erythrocyte suspension (the amount of CD44-positive cells and the expression level). It was found that cell amount with the impairments of transmembrane asymmetry of phosphatidylserine increased up to 30% after a short-term exposure of frozen-thawed erythrocytes at 37°C. The decrease in CD44-positive cell amount and lowering of the marker expression were also observed in cryopreserved erythrocyte suspension. Washing of erythrocytes from the cryoprotectant and hemolysis accompanying this process led to restoration of CD44 parameters in suspension of erythrocytes which preserved their integrity. The revealed impairments of surface characteristics of erythrocytes indicate a presence of sublethal membrane injuries in the cells cryopreserved under the protection of PEG-1500. This was true, at least in terms of the changes in the surface marker CD44 indices, since after lysis of cells compromised with sublethal injuries occured during removal of cryoprotectant, the marker indices in erythrocyte suspension did not differ from the control values. Probl Cryobiol Cryomed 2015; 25(2): 104-113

Cardiolipin asymmetry, oxidation and signaling

Cardiolipins (CLs) are ancient and unusual dimeric phospholipids localized in the plasma membrane of bacteria and in the inner mitochondrial membrane of eukaryotes. In mitochondria, two types of asymmetries – trans-membrane and molecular – are essential determinants of CL functions. In this review, we describe CL-based signaling mitochondrial pathways realized via modulation of trans-membrane asymmetry and leading to externalization and peroxidation of CLs in mitophagy and apoptosis, respectively. We discuss possible mechanisms of CL translocations from the inner leaflet of the inner to the outer leaflet of the outer mitochondrial membranes. We present redox reaction mechanisms of cytochrome c-catalyzed CL peroxidation as a required stage in the execution of apoptosis. We also emphasize the significance of CL-related metabolic pathways as new targets for drug discovery. Finally, a remarkable diversity of polyunsaturated CL species and their oxidation products have evolved in eukaryotes vs. prokaryotes. This diversity – associated with CL molecular asymmetry – is presented as the basis for mitochondrial communications language.

Phosphatidylserine Membrane Translocation in Human Spermatozoa: Topography in Membrane Domains and Relation to Cell Vitality

The complex structure of the human spermatozoa membrane comprises five topographic domains. Transmembrane asymmetry of the distribution of phospholipids including phosphatidylserine (PS) is considered a marker of cell activity. The objective of the study was to determine which cytomembrane domains of human spermatozoa are involved in PS membrane translocation and to identify the possible relationship of PS translocation with spermatozoa morphology and vitality. In normozoospermic semen of 35 donors, annexin-V labeling with fluorescein determined PS translocation. Propidium iodide staining distinguished between vital and dead spermatozoa. Three types of PS membrane translocation have been distinguished: (1) in the midpiece, (2) in the acrosomal part and (3) simultaneously in the midpiece and acrosomal part. In morphologically normal vital spermatozoa, PS translocation occurred in the midpiece but never in the equatorial region. In dead spermatozoa, simultaneous PS translocation in the midpiece and acrosomal part was most often observed. The difference between proportions of, respectively, vital and dead spermatozoa presenting PS translocation located in different domains was significant (P < 0.0001). In vital cells, there was no difference in PS translocation prevalence between morphologically normal and abnormal spermatozoa (P > 0.05). The strict relation of PS translocation to specific membrane domains indicates functional specificity. It seems doubtful to include this phenomenon in physiological mechanisms of elimination of abnormal spermatozoa.

Unusual Stability of Archaeal Tetraether Liposomes Against Surfactants

Bipolar tetraether liposomes (BTL) hold great promise for technological applications and serve as an excellent model for studying the plasma membrane of the crenarchaeota, which can thrive in a variety of extreme environments. Physical properties of BTL have thus evolved as an important research subject. In this study, using dynamic light scattering, we have examined the effect of surfactant n-tetradecyl-β-D-maltoside (TDM) on unilamellar vesicles composed of POPC (a monopolar diester hybrid lipid) and the polar lipid fraction E (PLFE, one kind of archaeal bipolar tetraether lipids) isolated from the thermoacidophile Sulfolobus acidocaldarius (growth temperature 72oC; growth pH 2.5) at 25oC. POPC and PLFE differ considerably in their structure; therefore, they were expected to behave differently when exposed to TDM. We found that TDM disrupts the POPC/cholesterol vesicles effectively; however, higher concentrations of TDM were required to disrupt PLFE/POPC vesicles. As the TDM concentration was increased, the size of liposomes reduced. For a given liposome system, the particle size drops abruptly at a specific TDM concentration. The TDM concentration where the vesicle size is reduced to half its original value was designated as C1/2. For PLPF/POPC mixtures with [total lipid] = 0.2 mM, C1/2 changes little with the amount of PLFE until 70 mol%, above which C1/2 undergoes a dramatic increase. According to the C1/2 values, PLFE unilamellar vesicles are ∼14 times more stable against the surfactant TDM than POPC unilamellar vesicles. A surprising result is that, for 100 mol% PLFE, C1/2 is increased as the vesicle diameter is reduced, probably due to a change in transmembrane asymmetry. Tests on the stability of PLFE-based liposomes against other surfactants such as bile salts are being carried out. (supported by NSF DMR 0706410).

The fine structure of the influenza virus envelope and the concept of transmembrane asymmetry of lateral domains in biomembranes

The molecular architectures of enveloped viruses provide a demonstrative example of perfectly arranged macromolecular complexes, which are formed via highly specific interactions of all structural components. Virus morphogenesis is a multistep process that depends on the concerted actions of many viral and cell components, as well as a fitted organization of main viral constituents. The virus envelope is composed of a mixture of lipid raft and nonraft domains. The domains are recruited from the host cell membrane as discrete well-ordered lipid-protein units during virus assembly. The raft-like nature of the influenza virus A envelope was visualized using a novel approach of cold solubilization of detergent-resistant membranes from intact influenza virus A virions with a mixture of NP40 and octyl glucopyranoside, two nonionic detergents drastically differing in their raft-solubilizing activities. The virus envelope is apparently an ensemble of flexibly joint platforms, which are composed of surface glycoproteins (hemagglutinin and neuraminidase), the matrix M1 protein, and lipids. The modern concept of the transmembrane asymmetry of lateral domains in biological membranes was used to explain the solubilization mechanism revealed. Based on the principles of this concept, the M1 protein shell was assumed to provide a structure-forming framework to support asymmetrical rafts in the virus envelope.

Intrinsic Potential of Cell Membranes: Opposite Effects of Lipid Transmembrane Asymmetry and Asymmetric Salt Ion Distribution

Using atomic-scale molecular dynamics simulations, we consider the intrinsic cell membrane potential that is found to originate from a subtle interplay between lipid transmembrane asymmetry and the asymmetric distribution of monovalent salt ions on the two sides of the cell membrane. It turns out that both the asymmetric distribution of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipids across a membrane and the asymmetric distribution of NaCl and KCl induce nonzero drops in the transmembrane potential. However, these potential drops are opposite in sign. As the PC leaflet faces a NaCl saline solution and the PE leaflet is exposed to KCl, the outcome is that the effects of asymmetric lipid and salt ion distributions essentially cancel one another almost completely. Overall, our study highlights the complex nature of the intrinsic potential of cell membranes under physiological conditions.

Lipid Asymmetry in DLPC/DSPC-Supported Lipid Bilayers: A Combined AFM and Fluorescence Microscopy Study

A fundamental attribute of cell membranes is transmembrane asymmetry, specifically the formation of ordered phase domains in one leaflet that are compositionally different from the opposing leaflet of the bilayer. Using model membrane systems, many previous studies have demonstrated the formation of ordered phase domains that display complete transmembrane symmetry; but there have been few reports on the more biologically relevant asymmetric membrane structures. Here we report on a combined atomic force microscopy and fluorescence microscopy study whereby we observe three different states of transmembrane symmetry in phase-separated supported lipid bilayers formed by vesicle fusion. We find that if the leaflets differ in gel-phase area fraction, then the smaller domains in one leaflet are in registry with the larger domains in the other leaflet and the system is dynamic. In a presumed lipid flip-flop process similar to Ostwald ripening, the smaller domains in one leaflet erode away whereas the large domains in the other leaflet grow until complete compositional asymmetry is reached and remains stable. We have quantified this evolution and determined that the lipid flip-flop event happens most frequently at the interface between symmetric and asymmetric DSPC domains. If both leaflets have identical area fraction of gel-phase, gel-phase domains are in registry and are static in comparison to the first state. The stability of these three DSPC domain distributions, the degree of registry observed, and the domain immobility have biological significance with regards to maintenance of lipid asymmetry in living cell membranes, communication between inner leaflet and outer leaflet, membrane adhesion, and raft mobility.

The 3-Hydroxy Group and 4,5- trans Double Bond of Sphingomyelin Are Essential for Modulation of Galactosylceramide Transmembrane Asymmetry

The structural features of SPM that control the transbilayer distribution of β-GalCer in POPC vesicles were investigated by 13C- and 31P-NMR spectroscopy using lipid analogs that share physical similarities with GalCer or SPM. The SPM analogs included N-palmitoyl-4,5-dihydro-SPM, 3-deoxy-SPM, 1-alkyl-2-amidophosphatidylcholine, and dipalmitoylphosphatidylcholine, a popular model “raft lipid”. The transbilayer distributions of the SPM analogs and SPM in POPC vesicles were similar by 31P-NMR. To observe the dramatic change in GalCer transbilayer distribution that occurs when SPM is included in POPC vesicles, the 3-OH group, 4,5- trans double bond, and amide linkage all were required in SPM. However, inclusion of 2 and 10 mol % dihydroSPM in SPM/POPC (1:1) vesicles mitigated and completely abrogated the effect of SPM on the transbilayer distribution of GalCer. Despite sharing some structural features with GalCer and localizing preferentially to the inner leaflet of POPC vesicles, dimyristoylphosphatidylethanolamine did not undergo a change in transbilayer distribution when SPM was incorporated into the vesicles. The results support the hypothesis that specific interactions may be favored among select sphingolipids in curvature-stressed membranes and emphasize the potential importance of the SPM-dihydroSPM ratio in membrane fission and fusion processes associated with vesicle biogenesis and trafficking.

Transmembrane asymmetry and lateral domains in biological membranes.

It is generally assumed that rafts exist in both the external and internal leaflets of the membrane, and that they overlap so that they are coupled functionally and structurally. However, the two monolayers of the plasma membrane of eukaryotic cells have different chemical compositions. This out-of-equilibrium situation is maintained by the activity of lipid translocases, which compensate for the slow spontaneous transverse diffusion of lipids. Thus rafts in the outer leaflet, corresponding to domains enriched in sphingomyelin and cholesterol, cannot be mirrored in the inner cytoplasmic leaflet. The extent to which lipids contribute to raft properties can be conveniently studied in giant unilamellar vesicles. In these, cholesterol can be seen to condense with saturated sphingolipids or phosphatidylcholine to form microm scale domains. However, such rafts fail to model biological rafts because they are symmetric, and because their membranes lack the mechanism that establishes this asymmetry, namely proteins. Biological rafts are in general of nm scale, and almost certainly differ in size and stability in inner and outer monolayers. Any coupling between rafts in the two leaflets, should it occur, is probably transient and dependent not upon the properties of lipids, but on transmembrane proteins within the rafts.

Plasminogen activator inhibitor-1 promotes formation of endothelial microparticles with procoagulant potential.

Endothelial dysfunction is emerging as a common denominator for diverse and highly prevalent cardiovascular diseases. Increased level of plasminogen activator inhibitor-1 (PAI-1) and procoagulant activity have been recognized as hallmarks of endothelial dysfunction. This study was aimed at investigating cellular actions of PAI-1 and a potential link between PAI-1 and procoagulant state. Human umbilical vein endothelial cells treated with PAI-1 were subjected to laser confocal fluorescence microscopy, immunoprecipitation and Western blotting, and FACS analysis for isolation and identification of endothelial microparticles. PAI-1 treatment resulted in a reduced expression of uPAR, its colocalization with caveolin, and the concomitant increase of uPAR abundance in the culture medium. FACS analysis revealed that PAI-1 rapidly and dose-dependently increased the number of endothelial microparticles expressing uPAR and alpha(V)beta3 integrin. This process was attenuated by pretreatment with neutralizing anti-uPAR antibodies. PAI-1 knockout mice showed a significantly decreased number of circulating endothelial microparticles than wild-type mice; however, PAI-1-deficient animals responded to infusion of PAI-1 with a more pronounced rise in the number of microparticles. PAI-1 treatment increased the number of microparticles stained with Annexin V, evidence for the expression of anionic phospholipids. This was accompanied by the accelerated generation of thrombin. The data disclose a novel effect of PAI-1 to dose-dependently promote formation of endothelial microparticles with the reduced transmembrane asymmetry of phospholipids. This phenomenon may be responsible for the observed increase in in vitro thrombin generation. These findings could potentially link these hallmarks of endothelial dysfunction-elevated levels of PAI-1 and propensity toward thrombosis.