Asymmetric Lipid Membranes Research Articles

Energy Transfer on Cytoskeleton of Red Blood Cell Ghosts and their Efficiency Control by KCl Concentration-induced Cell Shrinkage.

Lipid bilayer membranes such as liposomes have been utilized as platforms for bioinspired artificial photosynthesis. Embedding functional compounds, including chromophores and catalysts, into two-dimensional lipid membranes allows their high local concentration and proximity, resulting in enhanced reactivity compared to that of homogeneous solutions. The control of photoreactions by the physical and chemical properties of membranes, such as fluidity and phase separation, has also been well studied in recent years. In contrast, it remains difficult to control chemical reactions via dynamic membrane deformation. Here, we report on the control of excitation energy transfer using red blood cell ghosts (RBCGs) as scaffolds, relying on their asymmetric lipid membranes and inherent and unique deformability. RBCGs, in which donor and acceptor molecules were chemically conjugated to a two-dimensional cytoskeleton located beneath the inner membrane, exhibited energy transfer, and their efficiency varied depending on the amount and ratio of donor and acceptor modifications, as confirmed by experimental and theoretical analysis. Furthermore, the KCl concentration-induced RBCG shrinkage enhanced the energy transfer efficiency. Our proposed method is expected to facilitate the construction of photoreaction systems that can be controlled via membrane deformation.

Structure of symmetric and asymmetric lipid membranes from joint SAXS/SANS.

Small-angle X-ray and neutron scattering (SAXS/SANS) techniques excel in unveiling intricate details of the internal structure of lipid membranes under physiologically relevant temperature and buffer conditions, all without the need to resort to bulky labels. By concurrently conducting and analyzing neutron and X-ray data, these methods harness the complete spectrum of contrast and resolution from various components constituting lipid membranes. Despite this, the literature exhibits only a sparse presence of applications compared to other techniques in membrane biophysics. This chapter serves as a primer for conducting joint SAXS/SANS analyses on symmetric and asymmetric large unilamellar vesicles, elucidating fundamental elements of the analysis process. Specifically, we introduce the basics of interactions of X-rays and neutrons with matter that lead to the scattering contrast and a description of membrane structure in terms of scattering length density profiles. These profiles allow fitting of the experimentally observed scattering intensity. We further integrate practical insights, unveiling strategies for successful data acquisition and providing a comprehensive assessment of the technique's advantages and drawbacks. By amalgamating theoretical underpinnings with practical considerations, this chapter aims to dismantle barriers hindering the adoption of joint SAXS/SANS approaches, thereby encouraging an influx of studies in this domain.

The rise of FTIR spectroscopy in the characterization of asymmetric lipid membranes

In contrast to symmetric unilamellar liposomes (sLUVs) prepared from a mixture of different lipids, asymmetric ones (aLUVs) with different lipid composition in the inner and outer membrane leaflets are more suitable model systems of eukaryotic plasma membranes. However, apart from the challenging preparation of asymmetric liposomes and small amounts of obtained asymmetric unilamellar liposomes (aLUVs), a major drawback is the qualitative characterization of asymmetry, as each of the techniques used so far has certain limitations. In this regard, we prepared aLUVs composed dominantly of DPPC(out)/DPPS(in) lipids and, along with 1H NMR and DSC characterization, we showed for the first time how FTIR spectroscopy can be used in the presence of (a)symmetry between DPPC/DPPS lipid bilayers. Using second derivative FTIR spectra we demonstrated not only that the hydration of lipids glycerol backbone and choline moiety of DPPC differs in s/aLUVs, but in addition that the lateral interactions between hydrocarbon chains during the phase change display different trend in s/aLUVs. Molecular dynamics simulations confirmed different chain ordering and packing between s/a bilayers, with a significant influence of temperature, i.e. membrane phase.

Efficient Simulations of Solvent Asymmetry Across Lipid Membranes Using Flat-Bottom Restraints.

The routinely employed periodic boundary conditions complicate molecular simulations of physiologically relevant asymmetric lipid membranes together with their distinct solvent environments. Therefore, separating the extracellular fluid from its cytosolic counterpart has often been performed using a costly double-bilayer setup. Here, we demonstrate that the lipid membrane and solvent asymmetry can be efficiently modeled with a single lipid bilayer by applying an inverted flat-bottom potential to ions and other solute molecules, thereby restraining them to only interact with the relevant leaflet. We carefully optimized the parameters of the suggested method so that the results obtained using the flat-bottom and double-bilayer approaches become mutually indistinguishable. Then, we apply the flat-bottom approach to lipid bilayers with various compositions and solvent environments, covering ions and cationic peptides to validate the approach in a realistic use case. We also discuss the possible limitations of the method as well as its computational efficiency and provide a step-by-step guide on how to set up such simulations in a straightforward manner.

Native Planar Asymmetric Suspended Membrane for Single-Molecule Investigations: Plasma Membrane on a Chip.

Cellular plasma membranes, in their role as gatekeepers to the external environment, host numerous protein assemblies and lipid domains that manage the movement of molecules into and out of cells, regulate electric potential, and direct cell signaling. The ability to investigate these roles on the bilayer at a single-molecule level in a controlled, in vitro environment while preserving lipid and protein architectures will provide deeper insights into how the plasma membrane works. A tunable silicon microarray platform that supports stable, planar, and asymmetric suspended lipid membranes (SLIM) using synthetic and native plasma membrane vesicles for single-molecule fluorescence investigations is developed. Essentially, a "plasma membrane-on-a-chip" system that preserves lipid asymmetry and protein orientation is created. By harnessing the combined potential of this platform with total internal reflection fluorescence (TIRF) microscopy, the authors are able to visualize protein complexes with single-molecule precision. This technology has widespread applications in biological processes that happen at the cellular membranes and will further the knowledge of lipid and protein assemblies.

Asymmetric Lipid Membranes under Shear Flows: A Dissipative Particle Dynamics Study.

We investigate the phase behavior of the asymmetric lipid membranes under shear flows, using the dissipative particle dynamics simulation. Two cases, the weak and strong shear flows, are considered for the asymmetric lipid microstructures. Three typical asymmetric structures, the membranes, tubes, and vesicle, are included in the phase diagrams, where the effect of two different types of lipid chain length on the formation of asymmetric membranes is evaluated. The dynamic processes are demonstrated for the asymmetric membranes by calculating the average radius of gyration and shape factor. The result indicates that different shear flows will affect the shape of the second type of lipid molecules; the shape of the first type of lipid molecules is more stable than that of the second type of lipid molecules. The mechanical properties are investigated for the asymmetric membranes by analyzing the interface tension. The results reveal an absolute pressure at the junctions of different types of particles under the weak shear flow; the other positions are almost in a state of no pressure; there is almost no pressure inside the asymmetric lipid membrane structure under the strong shear flow. The findings will help us to understand the potential applications of asymmetric lipid microstructures in the biological and medical fields.

Stabilizing Leaflet Asymmetry in a Highly Coarse-Grained Lipid Membrane Model

Lipid bilayer asymmetry is an essential aspect of biomembranes that leads to properties such as spontaneous curvature and differential stress, which in turn influence the large-scale membrane geometry. This presents a dilemma in simulations: models at atomistic resolution require potentially prohibitive computational resources, while highly coarse-grained models struggle to maintain asymmetry due to accelerated flip-flop dynamics. To address this issue, we present here a version of the coarse-grained Cooke lipid model, amended to stabilize asymmetric lipid membranes.

Long chain sphingomyelin depletes cholesterol from the cytoplasmic leaflet in asymmetric lipid membranes.

The transbilayer distribution of cholesterol (CHL) in complex asymmetric lipid membranes remains controversial, with contrasting investigations suggesting that there is more CHL either in the exoplasmic, outer leaflet (OL) or the cytoplasmic, inner leaflet (IL) depending on cell type or model, membrane composition, and method of investigation. Here, we launch systematic coarse-grained molecular dynamics simulations to investigate the impact of the sphingomyelin (SM) acyl chain length upon CHL distribution in asymmetric lipid membrane mixtures which account for the variation of the most abundant headgroups and acyl chain unsaturation in the two membrane leaflets. We find that there is always more CHL in the OL, but longer chain SM depletes more CHL from the IL than short chain SM in simple membrane mixtures containing SM and 16 : 0, 18 : 1 phospholipids. The difference between longer and shorter chain SM is neutralised in a more complex asymmetric membrane, where there are more saturated tails in the outer leaflet. We propose that interdigitation of long-chain SM into the opposing IL pushes cytoplasmic CHL towards the OL, but higher chain saturation of the outer leaflet compensates for the effect of SM chain length.

Antimicrobial peptide activity in asymmetric bacterial membrane mimics.

We report on the response of asymmetric lipid membranes composed of palmitoyl oleoyl phosphatidylethanolamine and palmitoyl oleoyl phosphatidylglycerol, to interactions with the frog peptides L18W-PGLa and magainin 2 (MG2a), as well as the lactoferricin derivative LF11-215. In particular we determined the peptide-induced lipid flip-flop, as well as membrane partitioning of L18W-PGLa and LF11-215, and vesicle dye-leakage induced by L18W-PGLa. The ability of L18W-PGLa and MG2a to translocate through the membrane appears to correlate with the observed lipid flip-flop, which occurred at the fastest rate for L18W-PGLa. The higher structural flexibility of LF11-215 in turn allows this peptide to insert into the bilayers without detectable changes of membrane asymmetry. The increased vulnerability of asymmetric membranes to L18W-PGLa in terms of permeability, appears to be a consequence of tension differences between the compositionally distinct leaflets, but not due to increased peptide partitioning.

Stabilizing Leaflet Asymmetry under Differential Stress in a Highly Coarse-Grained Lipid Membrane Model.

We present a version of the coarse-grained Cooke lipid model, modified to simulate asymmetric lipid membranes. It is inspired by a method employed by Wang et al. [ Commun. Comput. Phys. 2013, 13, 1093-1106] for artificially penalizing lipid flip-flop but copes more robustly with differential stress, at the cost of one additional bead per lipid and the concomitant increase in computational overhead. Bilayer asymmetry ultimately breaks down beyond a system size dependent critical differential stress, which can be predicted from a simple analytical model. We remeasure many important material parameters for the new model and find it to be consistent with typical fluid lipid membranes. Maintaining a stable stress asymmetry has many applications, and we give two examples: (i) connecting monolayer stress to lipid number asymmetry in order to directly measure the monolayer area modulus and (ii) finding its strain-dependent higher-order correction by monitoring the equilibrium bilayer area.

Creating Asymmetric Phospholipid Vesicles via Exchange With Lipid-Coated Silica Nanoparticles.

Recently, effort has been placed into fabricating model free-floating asymmetric lipid membranes, such as asymmetric vesicles. Here, we report on the use of lipid-coated silica nanoparticles to exchange lipids with initially symmetric vesicles to generate composition-controlled asymmetric vesicles. Our method relies on the simple and natural exchange of lipids between membranes through an aqueous medium. Using a selected temperature, time, and ratio of lipid-coated silica nanoparticles to vesicles, we produced a desired highly asymmetric leaflet composition. At this point, the silica nanoparticles were removed by centrifugation, leaving the asymmetric vesicles in solution. In the present work, the asymmetric vesicles were composed of isotopically distinct dipalmitoylphosphatidylcholine lipids. Lipid asymmetry was detected by both small-angle neutron scattering (SANS) and proton nuclear magnetic resonance (1H NMR). The rate at which the membrane homogenizes at 75 °C was also assessed.

Development of Artificial Cell Models Using Microfluidic Technology and Synthetic Biology.

Giant lipid vesicles or liposomes are primarily composed of phospholipids and form a lipid bilayer structurally similar to that of the cell membrane. These vesicles, like living cells, are 5–100 μm in diameter and can be easily observed using an optical microscope. As their biophysical and biochemical properties are similar to those of the cell membrane, they serve as model cell membranes for the investigation of the biophysical or biochemical properties of the lipid bilayer, as well as its dynamics and structure. Investigation of membrane protein functions and enzyme reactions has revealed the presence of soluble or membrane proteins integrated in the giant lipid vesicles. Recent developments in microfluidic technologies and synthetic biology have enabled the development of well-defined artificial cell models with complex reactions based on the giant lipid vesicles. In this review, using microfluidics, the formations of giant lipid vesicles with asymmetric lipid membranes or complex structures have been described. Subsequently, the roles of these biomaterials in the creation of artificial cell models including nanopores, ion channels, and other membrane and soluble proteins have been discussed. Finally, the complex biological functions of giant lipid vesicles reconstituted with various types of biomolecules has been communicated. These complex artificial cell models contribute to the production of minimal cells or protocells for generating valuable or rare biomolecules and communicating between living cells and artificial cell models.

Spontaneous Curvature, Differential Stress, and Bending Modulus of Asymmetric Lipid Membranes

On time scales shorter than lipid flip-flop time, lipid bilayers in cells assume asymmetric non-equilibrium steady states in which the physical characteristics of the two leaflets are different. The most notable difference is in lipid composition, but it can also involve different lateral stresses in each leaflet that add up to an overall (usually vanishing) tension. We explore the interplay between these two sources of asymmetry using theoretical modeling and coarse-grained simulation. By minimizing total elastic energy, we find a preferred spontaneous curvature that balances torques between bending moments and differential stress, which can at times lead to unexpected consequences. For example, compositionally asymmetric flat bilayers whose area per lipid in each leaflet is matched with corresponding symmetric tensionless flat bilayers, still exhibit residual differential stress, demonstrating that the conditions of zero area strain and zero bending moment are not identical. Furthermore, we observe that the bending modulus can significantly increase if differential stress exceeds a certain minimum threshold, but that compositional asymmetry alone cannot cause this. We attribute this effect to a stiffening in the compressed leaflet, which seems to be related to its gel transition, but not exactly coinciding with it. Finally, we show that the presence of rapidly flip-flopping lipid species (such as cholesterol) will not necessarily lead to total elimination of differential stress, but may in fact even induce it, depending on whether the partitioning energy prefers one leaflet over the other.

Spontaneous Curvature, Differential Stress, and Bending Modulus of Asymmetric Lipid Membranes

Lipid bilayers can exhibit asymmetric states, in which the physical characteristics of one leaflet differ from those of the other. This most visibly manifests in a different lipid composition, but it can also involve opposing lateral stresses in each leaflet that combine to an overall vanishing membrane tension. Here, we use theoretical modeling and coarse-grained simulation to explore the interplay between a compositional asymmetry and a nonvanishing differential stress. Minimizing the total elastic energy leads to a preferred spontaneous curvature that balances torques due to both bending moments and differential stress, with sometimes unexpected consequences. For instance, asymmetric flat bilayers, whose specific areas in each leaflet are matched to those of corresponding tensionless symmetric flat membranes, still exhibit a residual differential stress because the conditions of vanishing area strain and vanishing bending moment differ. We also measure the curvature rigidity of asymmetric bilayers and find that a sufficiently strong differential stress, but not compositional asymmetry alone, can increase the bending modulus. The likely cause is a stiffening of the compressed leaflet, which appears to be related to its gel transition but not identical with it. We finally show that the impact of cholesterol on differential stress depends on the relative strength of elastic and thermodynamic driving forces: if cholesterol solvates equally well in both leaflets, it will redistribute to cancel both leaflet tensions almost completely, but if its partitioning free energy prefers one leaflet over the other, the resulting distribution bias may even create differential stress. Because cells keep most of their lipid bilayers in an asymmetric nonequilibrium steady state, our findings suggest that biomembranes are elastically more complex than previously thought: besides a spontaneous curvature, they might also exhibit significant differential stress, which could strongly affect their curvature energetics.

Molecular Dynamics Simulations Reveal Leaflet Coupling in Compositionally Asymmetric Phase-Separated Lipid Membranes.

The eukaryotic plasma membrane has an asymmetric distribution of its component lipids. Rafts that result from liquid-liquid phase separation are a feature of its exoplasmic leaflet, but how these exoplasmic leaflet domains are coupled to the cytoplasmic leaflet is not understood. These rafts can be studied in model membranes of three-component mixtures that produce coexisting liquid ordered (Lo) and liquid disordered (Ld) domains. We conducted all-atom molecular dynamics simulations of compositionally asymmetric lipid bilayers that reflect a more realistic model of the plasma membrane. One leaflet contained phase-separated domains with phosphatidylcholine and cholesterol, representing the exoplasmic leaflet, whereas the other contained phosphatidylethanolamine, phosphatidylserine, and cholesterol, which are the predominant components of the cytoplasmic leaflet. Inspired by findings of domain alignment across the two leaflets in compositionally symmetric model membranes, we examined the coupling between the two leaflets to see how the single-phase cytoplasmic leaflet would respond to phase separation in the other leaflet and if information could be communicated across the membrane. We found the region of the single-phase leaflet apposing the Lo domain to be slightly more ordered and thicker than the region apposing the Ld domain. The region across from the Lo domain is somewhat enriched in cholesterol and significantly depleted of polyunsaturated lipids.

Thermal conductivity and rectification in asymmetric archaeal lipid membranes.

Nature employs lipids to construct nanostructured membranes that self-assemble in an aqueous environment to separate the cell interior from the exterior environment. Membrane composition changes among species and according to environmental conditions, which allows organisms to occupy a wide variety of different habitats. Lipid bilayers are phase-change materials that exhibit strong thermotropic and lyotropic phase behavior in an aqueous environment, which may also cause thermal rectification. Among different types of lipids, archaeal lipids are of great interest due to their ability to withstand extreme conditions. In this paper, nonequilibrium molecular dynamics simulations were employed to study the nanostructures and thermal properties of different archaeols and to investigate thermal rectification effects in asymmetric archaeal membranes. In particular, we are interested in understanding the role of bridged phytanyl chains and cyclopentane groups in controlling the phase transition temperature and heat flow across the membrane. Our results indicate that the bridged phytanyl chains decrease the molecular packing of lipids, whereas the existence of cyclopentane rings on the tail groups increases the molecular packing by enhancing the interactions between isoprenoid chains. We found that macrocyclic archaeols have the highest thermal conductivity, whereas macrocyclic archaeols with two cyclopentane rings have the lowest. The effect of the temperature on the variation of thermal conductivity was found to be progressive. Our results further indicate that small thermal rectification effects occur in asymmetric archaeol bilayer membranes at around 25 K temperature gradient. The calculated thermal rectification factor was around 0.09 which is in the range of rectification factor obtained experimentally for nanostructures such as carbon nanotubes (0.07). Such phenomena may be of biological significance and could also be optimized for use in various engineering applications.

Label-free and charge-sensitive dynamic imaging of lipid membrane hydration on millisecond time scales

Biological membranes are highly dynamic and complex lipid bilayers, responsible for the fate of living cells. To achieve this function, the hydrating environment is crucial. However, membrane imaging typically neglects water, focusing on the insertion of probes, resonant responses of lipids, or the hydrophobic core. Owing to a recent improvement of second-harmonic (SH) imaging throughput by three orders of magnitude, we show here that we can use SH microscopy to follow membrane hydration of freestanding lipid bilayers on millisecond time scales. Instead of using the UV/VIS resonant response of specific membrane-inserted fluorophores to record static SH images over time scales of >1,000 s, we SH imaged symmetric and asymmetric lipid membranes, while varying the ionic strength and pH of the adjacent solutions. We show that the nonresonant SH response of water molecules aligned by charge-dipole interactions with charged lipids can be used as a label-free probe of membrane structure and dynamics. Lipid domain diffusion is imaged label-free by means of the hydration of charged domains. The orientational ordering of water is used to construct electrostatic membrane potential maps. The average membrane potential depends quadratically on an applied external bias, which is modeled by nonlinear optical theory. Spatiotemporal fluctuations on the order of 100-mV changes in the membrane potential are seen. These changes imply that membranes are very dynamic, not only in their structure but also in their membrane potential landscape. This may have important consequences for membrane function, mechanical stability, and protein/pore distributions.

Effects of temperature and PEG grafting density on the translocation of PEGylated nanoparticles across asymmetric lipid membrane

Plasma membrane internalization of nanoparticles (NPs) is important for their biomedical applications such as drug-delivery carriers. On one hand, in order to improve their half-life in circulation, PEGylation has been widely used. However, it may hinder the NPs’ membrane internalization ability. On the other hand, higher temperature could enhance the membrane permeability and may affect the NPs’ ability to enter into or exit from cells. To make full use of their advantages, we systematically investigated the effects of temperature and PEG density on the translocation of PEGylated nanoparticles across the plasma asymmetric membrane of eukaryotic cells, using near-atom level coarse-grained molecular dynamics simulations. Our results showed that higher temperature could accelerate the translocation of NPs across membranes by making lipids more disorder and faster diffusion. On the contrary, steric hindrance effects of PEG would inhibit NPs’ translocation process and promote lipids flip-flops. The PEG chains could rearrange themselves to minimize the contacts between PEG and lipid tails during the translocation, which was similar to ‘snorkeling effect’. Moreover, lipid flip-flops were affected by PEGylated density as well as NPs’ translocation direction. Higher PEG grafting density could promote lipid flip-flops, but inhibit lipid extraction from bilayers. The consequence of lipid flip-flop and extraction was that the membranes got more symmetric.

What Can We Learn about Cholesterol's Transmembrane Distribution Based on Cholesterol-Induced Changes in Membrane Dipole Potential?

Cholesterol is abundant in the plasma membranes of animal cells and is known to regulate a variety of membrane properties. Despite decades of research, the transmembrane distribution of cholesterol is still a matter of debate. Here we consider this outstanding issue through atomistic simulations of asymmetric lipid membranes, whose composition is largely consistent with eukaryotic plasma membranes. We show that the membrane dipole potential changes in a cholesterol-dependent manner. Remarkably, moving cholesterol from the extracellular to the cytosolic leaflet increases the dipole potential on the cytosolic side, and vice versa. Biologically this implies that by altering the dipole potential, cholesterol can provide a driving force for cholesterol molecules to favor the cytosolic leaflet, in order to compensate for the intramembrane field that arises from the resting potential.

Asymmetric cationic liposomes designed for heat-activated association with cells

Improved anticancer drugs and drug carriers are needed in combination therapies, such as hyperthermia-assisted chemotherapy. Liposomal drug carriers with advanced functions are attractive candidates for targeted accumulation and drug release in response to heat stimulus. We report on the design of liposomes with a heat-activated surface function. Our design is based on asymmetric lipid membranes with a defined gel to liquid-crystalline phase-transition temperature around 41°C. Asymmetry between the inner and the outer membrane leaflets was generated through selective PEGylation of cationic lipids in the outer membrane leaflet. In a physiological buffer, the PEGylated asymmetric liposomes had a neutral zeta potential and did not bind to planar anionic model membranes. In contrast, following upon heat-activation, binding of liposomes to the model membranes occurred. Release of a hydrophilic dye encapsulated in the asymmetric liposomes occurred at 40°C. Enhanced uptake of the asymmetric liposomes by hypopharyngeal carcinoma cells (FaDu cells) was observed when hyperthermia was applied compared to experiments performed at 37°C. These results show the potential of asymmetric liposomes for localized delivery of drugs into cells in response to (external) temperature stimulus.