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Model asymmetric bilayers are useful for studying the coupling between lateral and transverse lipid organization. Here, we used calcium-induced hemifusion to create asymmetric giant unilamellar vesicles (aGUVs) for exploring the phase behavior of 16:0-PC/16:1-PC/Cholesterol, a simplified model for the mammalian plasma membrane. Symmetric GUVs (sGUVs) were first prepared using a composition that produced coexisting liquid-disordered and liquid-ordered phases visible by confocal fluorescence microscopy. The sGUVs were then hemifused to a supported lipid bilayer (SLB) composed of uniformly mixed 16:1-PC/Cholesterol. The extent of outer leaflet exchange was quantified in aGUVs in two ways: (1) from the reduction in fluorescence intensity of a lipid probe initially in the sGUV ("probe exit"); or (2) from the gain in intensity of a probe initially in the SLB ("probe entry"). These measurements revealed a large variability in the extent of outer leaflet exchange in aGUVs within a given preparation, and two populations with respect to their phase behavior: a subset of vesicles that remained phase separated, and a second subset that appeared uniformly mixed. Moreover, a correlation between phase behavior and extent of asymmetry was observed, with more strongly asymmetric vesicles having a greater probability of being uniformly mixed. We also observed substantial overlap between these populations, an indication that the uncertainty in measured exchange fraction is high. We developed models to determine the position of the phase boundary (i.e., the fraction of outer leaflet exchange above which domain formation is suppressed) and found that the phase boundaries determined separately from probe-entry and probe-exit data are in good agreement. Our models also provide improved estimates of the compositional uncertainty of individual aGUVs. We discuss several potential sources of uncertainty in the determination of lipid exchange from fluorescence measurements., Competing Interests: Declaration of interests The authors declare no competing interests.

We performed coarse-grained simulations of the antimicrobial peptides Magainin-2, BP100, MSI-103, and MSI-78 on a phase-separated membrane to study their preference for the different domains. All the peptides displayed a clear preference for the liquid-disordered (Ld) phase over the liquid-ordered (Lo) one. For BP100, MSI-103, and MSI-78 there was a further preference of the peptides for the domain interface. The peptides’ preference toward the disordered phase was shown to reflect a penalization of lipid–lipid interaction enthalpy in the Lo phase, when in the vicinity of peptides. Similar results were observed at the two studied concentrations, although Ld phase saturation at the higher concentration drove some of the peptide excess to the Lo phase. Magainin-2 and MSI-103 were found to dimerize, in agreement with available experimental data. Interestingly, at high concentrations of Magainin-2 toroidal pores spontaneously formed in the Ld phase. We performed additional simulations to characterize this phenomenon, which is likely related to Magainin-2’s membranolytic action.

The nanoscale organization of biological membranes into structurally and compositionally distinct lateral domains is believed to be central to membrane function. The nature of this organization has remained elusive due to a lack of methods to directly probe nanoscopic membrane features. We show here that cryogenic electron micros-copy (cryo-EM) can be used to directly image coexisting nanoscopic domains in synthetic and bioderived membranes without extrinsic probes. Analyzing a series of single-component liposomes composed of synthetic lipids of varying chain lengths, we demonstrate that cryo-EM can distinguish bilayer thickness differences as small as 0.5 Å, comparable to the resolution of small-angle scattering methods. Simulated images from computational models reveal that features in cryo-EM images result from a complex interplay between the atomic distribution normal to the plane of the bilayer and imaging parameters. Simulations of phase-separated bilayers were used to predict two sources of contrast between coexisting ordered and disordered phases within a single liposome, namely differences in membrane thickness and molecular density. We observe both sources of contrast in biomimetic membranes composed of saturated lipids, unsaturated lipids, and cholesterol. When extended to isolatedmammalian plasma membranes, cryo-EM reveals similar nanoscale lateral heterogeneities. The methods reported here for direct, probe-free imaging of nanodomains in unperturbed membranes open new avenues for investigation of nanoscopic membrane organization. [ABSTRACT FROM AUTHOR]

We describe a method for investigating lateral membrane heterogeneity using cryogenic electron microscopy (cryo-EM) images of liposomes. The method takes advantage of differences in the thickness and molecular density of ordered and disordered phases that are resolvable in phase contrast cryo-EM. Compared to biophysical techniques like FRET or neutron scattering that yield ensemble-averaged information, cryo-EM provides direct visualization of individual vesicles and can therefore reveal variability that would otherwise be obscured by averaging. Moreover, because the contrast mechanism involves inherent properties of the lipid phases themselves, no extrinsic probes are required. We explain and discuss various complementary analyses of spatially resolved thickness and intensity measurements that enable an assessment of the membrane's phase state. The method opens a window to nanodomain structure in synthetic and biological membranes that should lead to an improved understanding of lipid raft phenomena., (Copyright © 2024. Published by Elsevier Inc.)

The development of electron cryomicroscopy (cryo-EM) has evolved immensely in the last several decades and is now well-established in the analysis of protein structure both in isolation and in their cellular context. This review focuses on the history and application of cryo-EM to the analysis of membrane architecture. Parallels between the levels of organization of protein structure are useful in organizing the discussion of the unique parameters that influence membrane structure and function. Importantly, the timescales of lipid motion in bilayers with respect to the timescales of sample vitrification is discussed and reveals what types of membrane structure can be reliably extracted in cryo-EM images of vitrified samples. Appreciating these limitations, a review of the application of cryo-EM to examine the lateral organization of ordered and disordered domains in reconstituted and biologically derived membranes is provided. Finally, a brief outlook for further development and application of cryo-EM to the analysis of membrane architecture is provided., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology.)

The impacts of pH, salt concentration (expressed as Debye length), and composition on the phase behavior of hybrid block copolymer-lipid-cholesterol bilayers incorporating carboxyl-terminated poly(butadiene)- block -poly(ethylene oxide) copolymer (PBdPEO1800(−)) or/and non-carboxyl-terminated PBdPEO (PBdPEO1800 or/and PBdPEO950), egg sphingomyelin (egg SM), and cholesterol were examined using fluorescence spectroscopy of laurdan. Laurdan emission spectra were decomposed into three lognormal curves as functions of energy. The ratio of the area of the mid-energy peak to the sum of the areas of all three peaks was evaluated as vesicles were cooled, yielding temperature breakpoint values (T break) expected to be within the range of the phase transition temperature. T break values displayed dependence on pH, Debye length, and vesicle composition consistent with an electrostatic repulsion contribution to vesicle phase behavior. Increased pH and Debye length, for which a greater dissociated fraction of PBdPEO1800(−) and a greater energy of electrostatic repulsion would be expected, resulted in T break values as much as 10 °C less than at low pH or short Debye lengths. Additionally, at Debye lengths comparable to those at physiologically relevant ionic strength, T break at pH 5.9 was observed to be slightly higher than at pH 7.0 for vesicles containing 50 mol% PBdPEO1800(−). Electrostatic effects observed for hybrid vesicles incorporating significant amounts of carboxyl-terminated polymer may have the ability to drive phase separation in response to pH drops—such as those observed after endocytosis—in physiologically relevant conditions, suggesting the utility of such materials for drug delivery. [Display omitted] • Phase behavior of charged hybrid vesicles examined by laurdan multipeak analysis. • At low salt, one-phase to two-phase transition temperature depended strongly on pH. • At physiological salt, dependence on pH was weaker but still measurable. • Applications in the design of pH-sensitive polymer/lipid vesicles for drug delivery [ABSTRACT FROM AUTHOR]

A Finsler geometric surface model is studied as a coarse-grained model for membranes of three components, such as zwitterionic phospholipid (DOPC), lipid (DPPC) and an organic molecule (cholesterol). To understand the phase separation of liquid-ordered (DPPC rich) Lo and liquid-disordered (DOPC rich) Ld, we introduce a binary variable σ(= ±1) into the triangulated surface model. We numerically determine that two circular and stripe domains appear on the surface. The dependence of the morphological change on the area fraction of Lo is consistent with existing experimental results. This provides us with a clear understanding of the origin of the line tension energy, which has been used to understand these morphological changes in three-component membranes. In addition to these two circular and stripe domains, a raft-like domain and budding domain are also observed, and the several corresponding phase diagrams are obtained. [ABSTRACT FROM AUTHOR]

Membrane proteins and polycyclic lipids like cholesterol and hopanoids coordinate phospholipid bilayer ordering. This phenomenon manifests as partitioning of the liquid crystalline phase into liquid-ordered (L o) and liquid-disordered (L d) regions. In Eukaryotes, microdomains are rich in cholesterol and sphingolipids and serve as signal transduction scaffolds. In Prokaryotes, L o microdomains increase pathogenicity and antimicrobial resistance. Previously, we identified spectroscopically distinct chemical shift signatures for all-trans (AT) and trans-gauche (TG) acyl chain conformations, cyclopropyl ring lipids (CPR), and hopanoids in prokaryotic lipid extracts and used Polarization Transfer (PT) SSNMR to investigate bilayer ordering. To investigate how these findings relate to native bilayer organization, we interrogate whole cell and whole membrane extract samples of Burkholderia thailendensis to investigate bilayer ordering in situ. In 13C-13C 2D SSNMR spectra, we assigned chemical shifts for lipid species in both samples, showing conservation of lipids of interest in our native membrane sample. A one-dimensional temperature series of PT SSNMR and transverse relaxation measurements of AT versus TG acyl conformations in the membrane sample confirm bilayer ordering and a broadened phase transition centered at a lower-than-expected temperature. Bulk protein backbone Cα dynamics and correlations consistent with lipid-protein contacts within are further indicative of microdomain formation and lipid ordering. In aggregate, these findings provide evidence for microdomain formation in vivo and provide insight into phase separation and transition mechanics in biological membranes. [Display omitted] • Bilayer ordering is observed in native biological membranes of B. thailendensis. • DP, CP, and rINEPT 1D experiments reveal a low, broad phase transition range. • AT acyl conformation above the phase transition indicates microdomain formation. • Hopanoid-lipid-protein contacts consistent with domain formation are identified. [ABSTRACT FROM AUTHOR]

Phase separation phenomena in hybrid lipid/block copolymer/cholesterol bilayers combining polybutadiene- block -polyethylene oxide (PBdPEO), egg sphingomyelin (egg SM), and cholesterol were studied with fluorescence spectroscopy and microscopy for comparison to lipid bilayers composed of palmitoyl oleoyl phosphatidylcholine (POPC), egg SM, and cholesterol. Laurdan emission spectra were decomposed into three lognormal curves. The temperature dependence of the ratios of the areas of the middle and lowest energy peaks revealed temperature break-point (T break) values that were in better agreement, compared to generalized polarization inflection temperatures, with phase transition temperatures in giant unilamellar vesicles (GUVs). Agreement between GUV and spectroscopy results was further improved for hybrid vesicles by using the ratio of the area of the middle peak to the sum of the areas all three peaks to find the T break values. For the hybrid vesicles, trends at T break are hypothesized to be correlated with the mechanisms by which the phase transition takes place, supported by the compositional range as well as the morphologies of domains observed in GUVs. Low miscibility of PBdPEO and egg SM is suggested by the finding of relatively high T break values at cholesterol contents greater than 30 mol%. Further, GUV phase behavior suggests stronger partitioning of cholesterol into PBdPEO than into POPC, and less miscibility of PBdPEO than POPC with egg SM. These results, summarized using a heat-map, contribute to the limited body of knowledge regarding the effect of cholesterol on hybrid membranes, with potential application toward the development of such materials for drug delivery or membrane protein reconstitution. [Display omitted] • Multipeak analysis provides greater insight into phase behavior than laurdan GP. • Multipeak analysis of laurdan spectra agrees with phase behavior of giant vesicles. • Hybrid lipid/polymer vesicles display phase separation at high cholesterol levels. • Multipeak analysis may reveal phase separation mechanisms in hybrid vesicles. [ABSTRACT FROM AUTHOR]

We present the coarsening dynamics of a liquid-ordered (Lo) domain in a supported lipid membrane under an electric field. In a relatively small nanosmooth region surrounded by geometrical walls of nanocorrugated regions, full coarsening of the Lo domain was produced while in a large nanosmooth region, an intermediate, less-ordered Lo domain was developed so that the diffusion of charged phospholipids in the liquid-disordered (Ld) domain was allowed across the geometrical walls in the presence of an electric field. No appreciable diffusion of the charged lipids into a fully coarsened Lo domain by the electric field implies that the structural ordering of the membrane components plays a significant role on the formation of lipid rafts for biological processes. [ABSTRACT FROM AUTHOR]

Abstract: Liquid-ordered (Lo) and liquid-disordered (Ld) phase coexistence has been suggested to partition the plasma membrane of biological cells into lateral compartments, allowing for enrichment or depletion of functionally relevant molecules. This dynamic partitioning might be involved in fine-tuning cellular signaling fidelity through coupling to the plasma membrane protein and lipid composition. In earlier work, giant plasma membrane vesicles, obtained by chemically induced blebbing from cultured cells, were observed to reversibly phase segregate at temperatures significantly below 37°C. In this contribution, we compare the temperature dependence of fluid phase segregation in HeLa and rat basophilic leukemia (RBL) cells. We find an essentially monotonic temperature dependence of the number of phase-separated vesicles in both cell types. We also observe a strikingly broad distribution of phase transition temperatures in both cell types. The binding of peripheral proteins, such as cholera toxin subunit B (CTB), as well as Annexin V, is observed to modulate phase transition temperatures, indicating that peripheral protein binding may be a regulator for lateral heterogeneity in vivo. The partitioning of numerous signal protein anchors and full length proteins is investigated. We find Lo phase partitioning for several proteins assumed in the literature to be membrane raft associated, but observe deviations from this expectation for other proteins, including caveolin-1. [Copyright &y& Elsevier]

Abstract: The objective of this paper is to review phase behavior and shape characterization of cerebroside-rich domains in binary and ternary lipid bilayers, as obtained by microscopy techniques. These lipid mixtures provide a format to examine molecular (e.g. headgroup, tail unsaturation, and tail hydroxylation) and thermodynamic (e.g. temperature and mole percentages) factors that determine phase behavior, molecular partitioning, crystal/atomic scale structure, and microstructure/shape (particularly of phase-separated domains). Microscopy can provide excellent spatial (often with high resolution) characterization of cerebroside-rich domains (and their surroundings) to identify, describe or infer with high certainty these characteristics. In the introduction to this review we review briefly the molecular structure, phase behavior, and intermolecular interactions of cerebrosides, in comparison to ceramides and sphingomyelins and in some binary and biological systems. The bulk of the review is then devoted to microscopy investigations of cerebroside-rich domain microstructure and shape dynamics in binary and ternary (one component is cholesterol) systems. Quantitative and/or high-resolution microscopy techniques have been used to interrogate cerebroside-rich domains such as freeze-fracture electron microscopy, atomic force microscopy, imaging elipsometry, two-photon fluorescence microscopy, and LAURDAN generalized polarization in addition to the laboratory workhorse technique of epifluorescence microscopy that allows a quick often qualitative assessment of microstructure and dynamics. We particularly focus on the information these microscopy investigations have revealed with respect to phase behavior, cholesterol partitioning, domain shape, and determinants of domain shape. [Copyright &y& Elsevier]

Cell membranes contain a large number of different lipid species. Such a multicomponent mixture exhibits a complex phase behavior with regions of structural and compositional heterogeneity. Especially domains formed in ternary mixtures, composed of saturated and unsaturated lipids together with cholesterol, have received a lot of attention as they may resemble raft formation in real cells. Here we apply a simulation model to assess the molecular nature of these domains at the nanoscale, information that has thus far eluded experimental determination. We are able to show the spontaneous separation of a saturated phosphatidylcholine (PC)/ unsaturated PC/cholesterol mixture into a liquid-ordered and a liquid-disordered phase with structural and dynamic properties closely matching experimental data. The near-atomic resolution of the simulations reveals remarkable features of both domains and the boundary domain interface. Furthermore, we predict the existence of a small surface tension between the monolayer leaflets that drives registration of the domains. At the level of molecular detail, raft-like lipid mixtures show a surprising face with possible implications for many cell membrane processes. [ABSTRACT FROM AUTHOR]

Abstract: Ceramide-induced alterations in the lateral organization of membrane proteins can be involved in several biological contexts, ranging from apoptosis to viral infections. In order to investigate such alterations in a simple model, we used a combined approach of atomic force microscopy, scanning fluorescence correlation spectroscopy and confocal fluorescence imaging to study the partitioning of different membrane components in sphingomyelin/dioleoyl-phosphatidylcholine/cholesterol/ceramide supported bilayers. Such model membranes exhibit coexistence of liquid-disordered, liquid-ordered (raft-like) and ceramide-rich lipid phases. Our results show that components with poor affinity toward the liquid-ordered phase, such as several fluorescent lipid analogues or the synaptic protein Synaptobrevin 2, are excluded from ceramide-rich domains. Conversely, we show for the first time that the raft-associated protein placental alkaline phosphatase (GPI-PLAP) and the ganglioside GM1 are enriched in such domains, while exhibiting a strong decrease in lateral diffusion. Analogue modulation of the local concentration and dynamics of membrane proteins/receptors by ceramide can be of crucial importance for the biological functions of cell membranes. [Copyright &y& Elsevier]

Abstract: Membrane rafts are regions of increased lipid acyl chain order that differ in their lipid and protein composition from the surrounding membrane. By providing an additional level of compartmentalization they have been proposed to serve many functions in cellular signal transduction and trafficking. We will review their potential involvement in different forms of membrane traffic, explicitly excluding signalling, and discuss select aspects of the raft hypothesis in its current form. [Copyright &y& Elsevier]

Abstract: The phase behavior of bilayers of binary mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol has been studied using Raman spectroscopy. It is observed that the shape of the cholesterol vibrational spectrum in lipid–cholesterol binary mixtures does not vary significantly with either the cholesterol concentration or the temperature. This permits determination of the lipid vibrational signatures of the liquid-disordered (ld), solid-ordered (so) and liquid-ordered (lo) phases. Within the phase coexistence region, the measured spectra are described very well by a linear combination of the different spectral components, which permits a quantitative analysis of the phase diagram. In contrast to earlier findings, our experiments provide no indication of a phase boundary at low cholesterol concentration. The upper boundary of the phase coexistence region is found at ∼27 and ∼22mol% for ld–lo and so–lo coexistence region, respectively. Within these phase coexistence regions, the partitioning of cholesterol between the cholesterol-poor and the cholesterol-rich phases is in close agreement with the lever rule. [Copyright &y& Elsevier]

The membrane raft hypothesis postulates the existence of lipid bilayer membrane heterogeneities, or domains, supposed to be important for cellular function, including lateral sorting, signaling, and trafficking. Characterization of membrane lipid heterogeneities in live cells has been challenging in part because inhomogeneity has not usually been definable by optical microscopy. Model membrane systems, including giant unilamellar vesicles, allow optical fluorescence discrimination of coexisting lipid phase types, but thus far have focused on coexisting optically resolvable fluid phases in simple lipid mixtures. Here we demonstrate that giant plasma membrane vesicles (GPMVs) or blebs formed from the plasma membranes of cultured mammalian cells can also segregate into micrometer-scale fluid phase domains. Phase segregation temperatures are widely spread, with the vast majority of GPMVs found to form optically resolvable domains only at temperatures below ≈25°C. At 37°C. these GPMV membranes are almost exclusively optically homogenous. At room temperature, we find diagnostic lipid phase fluorophore partitioning preferences in GPMVs analogous to the partitioning behavior now established in model membrane systems with liquid-ordered and liquid-disordered fluid phase coexistence. We image these GPMVs for direct visual characterization of protein partitioning between coexisting liquid-ordered-like and liquid-disordered-like membrane phases in the absence of detergent perturbation. For example, we find that the transmembrane IgE receptor FcεRI preferentially segregates into liquid-disordered-like phases, and we report the partitioning of additional well known membrane associated proteins. Thus, GPMVs now provide an effective approach to characterize biological membrane heterogeneities. [ABSTRACT FROM AUTHOR]

Although it is well described in model membranes, little is known about phase separation in biological membranes. Here, we provide evidence for a coexistence of at least two different lipid bilayer phases in the apical plasma membrane of epithelial cells. Phase connectivity was assessed by measuring long-range diffusion of several membrane proteins by fluorescence recovery after photo-bleaching in two polarized epithelial cell lines and one fibroblast cell line. In contrast to the fibroblast plasma membrane, in which all of the proteins diffused with similar characteristics, in the apical membrane of epithelial cells the proteins could be divided into two groups according to their diffusion characteristics. At room temperature (≈25°C), one group showed fast diffusion and complete recovery. The other diffused three to four times slower and, more importantly, displayed only partial recovery. Only the first group comprises proteins that are believed to be associated with lipid rafts. The partial recovery is not caused by topological constraints (microvilli, etc.), cytoskeletal constraints, or protein-protein interactions, because all proteins show 100% recovery in fluorescence recovery after photobleaching experiments at 37°C. In addition, the raft-associated proteins cannot be coclustered by antibodies on the apical membrane at 12°C. The interpretation that best fits these data is that the apical membrane of epithelial cells is a phase-separated system with a continuous (percolating) raft phase <25°C in which isolated domains of the nonraft phase are dispersed, whereas at 37°C the nonraft phase becomes the continuous phase with isolated domains of the raft phase dispersed in it. [ABSTRACT FROM AUTHOR]

Abstract: Liquid domains in model lipid bilayers are frequently studied as models of raft domains in cell plasma membranes. Micron-scale liquid domains are easily produced in vesicles composed of ternary mixtures of a high melting temperature lipid, a low melting temperature lipid, and cholesterol. Here, we describe the rich phase behavior observed in binary and ternary systems. We then discuss experimental challenges inherent in mapping phase diagrams of even simple lipid systems. For example, miscibility behavior varies with lipid type, lipid ratio, lipid oxidation, and level of impurity. Liquid domains are often circular, but can become noncircular when membranes are near critical points. Finally, we reflect on applications of phase diagrams in model systems to rafts in cell membranes. [Copyright &y& Elsevier]

Abstract: The concept of lipid rafts and the intense work toward their characterization in biological membranes has spurred a renewed interest in the understanding of domain formation, particularly in the case of cholesterol-containing membranes. The thermodynamic principles underlying formation of domains, rafts, or cholesterol/phospholipid complexes are reviewed here, along with recent work in model and biological membranes. A major motivation for this review was to present those concepts in a way appropriate for the broad readership that has been drawn to the field. Evidence from a number of different techniques points to the conclusion that lipid–lipid interactions are generally weak; therefore, in most cases, massive phase separations are not to be expected in membranes. On the contrary, small, dynamic lipid domains, possibly stabilized by proteins are the most likely outcome. The results on mixed lipid bilayers are used to discuss recent experiments in biological membranes. The clear indication is that proteins partition preferentially into fluid, disordered lipid domains, which is contrary to their localization in ordered, cholesterol/sphingomyelin rafts inferred from detergent extraction experiments on cell membranes. Globally, the evidence appears most consistent with a membrane model in which the majority of the lipid is in a liquid-ordered phase, with dispersed, small, liquid-disordered domains, where most proteins reside. Co-clustering of proteins and their concentration in some membrane areas may occur because of similar preferences for a particular domain but also because of simultaneous exclusion from other lipid phases. Specialized structures, such as caveolae, which contain high concentrations of cholesterol and caveolin are not necessarily similar to bulk liquid-ordered phase. [Copyright &y& Elsevier]

We present evidence that laterally phase separated cholesterol domains constitute a new, equilibrium phase in biological membranes. The domains are characterized in multi-lamellar vesicles (MLV) made of cholesterol and dimyristoylphosphatidylcholine (DMPC) but are also shown to exist in biologically relevant, egg lecithin systems containing a mixture of phospholipids. This work utilizes the fluorescent membrane probes 1-acyl-2-[12-[(5-dimethylamino-1-naphthalenesufonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (DANSYL), and ergosta-5,7,9(11),22-tetraen-3β-ol (ERGO), which have been shown to be minimally invasive mimics of native membrane lipids. The highlight of the work is a heating-induced alleviation of a DANSYL blue shift at relatively high (but undersaturated) cholesterol loadings, which is reversible through at least three heating and cooling cycles. Comparison of the DANSYL spectral shifts with published DMPC–cholesterol phase diagrams shows unequivocally that the spectral results cannot be explained in terms of previously understood phase behavior. Rather, a lateral phase separation occurs within the vesicle bilayer, giving rise to cholesterol micro-domains. The cholesterol domains appear to coexist with, and should not be confused with, the well-known liquid-order phase that arises because of the cholesterol condensation effect. Additional studies involving ERGO–DANSYL energy transfer show a sequestration of probes within the bilayer, confirming the DANSYL spectral data, and a model that includes domains provides the best description of measured energy transfer efficiencies. Best fits of the energy transfer data, using a mathematical model developed to account for the presence of domains, indicates the domain size to be in the range 10–20 nm. [Copyright &y& Elsevier]

Förster resonance energy transfer (FRET) is a powerful tool for investigating heterogeneity in lipid bilayers. In model membrane studies, samples are frequently unilamellar vesicles with diameters of 20−200 nm. It is well-known that FRET efficiency is insensitive to vesicle curvature in uniformly mixed lipid bilayers, and consequently theoretical models for FRET typically assume a planar geometry. Here, we use a spherical harmonic expansion of the acceptor surface density to derive an analytical solution for FRET between donor and acceptor molecules distributed on the surface of a sphere. We find excellent agreement between FRET predicted from the model and FRET calculated from corresponding Monte Carlo simulations, thus validating the model. An extension of the model to the case of a non-uniform acceptor surface density (i.e., a phase-separated vesicle) reveals that FRET efficiency depends on vesicle size when acceptors partition between the coexisting phases, and approaches the efficiency of a uniformly mixed bilayer as the vesicle size decreases. We show that this is an indirect effect of constrained domain size, rather than an intrinsic effect of vesicle curvature. Surprisingly, the theoretical predictions were not borne out in experiments: we did not observe a statistically significant change in FRET efficiency in phase-separated vesicles as a function of vesicle size. We discuss factors that likely mask the vesicle size effect in extruded samples. [ABSTRACT FROM AUTHOR]

We performed coarse-grained simulations of the antimicrobial peptides Magainin-2, BP100, MSI-103, and MSI-78 on a phase-separated membrane to study their preference for the different domains. All the peptides displayed a clear preference for the liquid-disordered (Ld) phase over the liquid-ordered (Lo) one. For BP100, MSI-103, and MSI-78 there was a further preference of the peptides for the domain interface. The peptides' preference toward the disordered phase was shown to reflect a penalization of lipid-lipid interaction enthalpy in the Lo phase, when in the vicinity of peptides. Similar results were observed at the two studied concentrations, although Ld phase saturation at the higher concentration drove some of the peptide excess to the Lo phase. Magainin-2 and MSI-103 were found to dimerize, in agreement with available experimental data. Interestingly, at high concentrations of Magainin-2 toroidal pores spontaneously formed in the Ld phase. We performed additional simulations to characterize this phenomenon, which is likely related to Magainin-2's membranolytic action., (Copyright © 2020 Su, Marrink and Melo.)

Giant plasma membrane vesicles (GPMVs) are isolated directly from living cells and provide an alternative to vesicles constructed of synthetic or purified lipids as an experimental model system for use in a wide range of assays. GPMVs capture much of the compositional protein and lipid complexity of intact cell plasma membranes, are filled with cytoplasm, and are free from contamination with membranes from internal organelles. GPMVs often exhibit a miscibility transition below the growth temperature of their parent cells. GPMVs labeled with a fluorescent protein or lipid analog appear uniform on the micron-scale when imaged above the miscibility transition temperature, and separate into coexisting liquid domains with differing membrane compositions and physical properties below this temperature. The presence of this miscibility transition in isolated GPMVs suggests that a similar phase-like heterogeneity occurs in intact plasma membranes under growth conditions, albeit on smaller length scales. In this context, GPMVs provide a simple and controlled experimental system to explore how drugs and other environmental conditions alter the composition and stability of phase-like domains in intact cell membranes. This chapter describes methods to generate and isolate GPMVs from adherent mammalian cells and to interrogate their miscibility transition temperatures using fluorescence microscopy., (© 2018 Elsevier Inc. All rights reserved.)

A Finsler geometric surface model is studied as a coarse-grained model for membranes of three components, such as zwitterionic phospholipid (DOPC), lipid (DPPC) and an organic molecule (cholesterol). To understand the phase separation of liquid-ordered (DPPC rich) L o and liquid-disordered (DOPC rich) L d , we introduce a binary variable σ ( = ± 1 ) into the triangulated surface model. We numerically determine that two circular and stripe domains appear on the surface. The dependence of the morphological change on the area fraction of L o is consistent with existing experimental results. This provides us with a clear understanding of the origin of the line tension energy, which has been used to understand these morphological changes in three-component membranes. In addition to these two circular and stripe domains, a raft-like domain and budding domain are also observed, and the several corresponding phase diagrams are obtained.

Supported lipid bilayers have been in use for over 30 years. They have been employed to study the structure, composition, and dynamics of lipid bilayer phases, the binding and distribution of soluble, integral, and lipidated proteins in membranes, membrane fusion, and interactions of membranes with elements of the cytoskeleton. This review focuses on the unique ability of supported lipid bilayers to study liquid-ordered and liquid-disordered domains in membranes. We highlight methods to produce asymmetric lipid bilayers with lipid compositions that mimic those of the extracellular and cytoplasmic leaflets of cell membranes and the functional reconstitution of membrane proteins into such systems. Questions related to interleaflet domain coupling and membrane protein activation have been addressed and answered using advanced reconstitution and imaging procedures in symmetric and asymmetric supported membranes with and without coexisting lipid phase domains. Previously controversial topics regarding anomalous and anisotropic diffusion in membranes have been resolved by using supported membrane approaches showing that the propensity of certain lipid compositions to form "rafts" are important but overlaid with "picket-fence" interactions that are imposed by a subtended cytoskeletal network., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Electroporation is the basis for the transfection of genetic material and for drug delivery to cells, including electrochemotherapy for cancer. By means of molecular dynamics many aspects of membrane electroporation have been unveiled at the molecular detail in simple, homogeneous, lipid bilayers. However, the correspondence of these findings \with the process happening in cell membranes requires, at least, the consideration of laterally structured membranes. Here, I present a systematic molecular dynamics study of bilayers composed of different liquid-ordered and liquid-disordered lipid phases subjected to a transversal electric field. The simulations reveal two significant results. First, the electric field mainly affects the properties of the disordered phases, so that electroporation takes place in these membrane regions. Second, the smaller the disordered domains are, the faster they become electroporated. These findings may have a relevant significance in the experimental application of cell electroporation in vivo since it implies that electro-induced and pore-mediated transport processes occur in particularly small disordered domains of the plasma membrane, thus locally affecting only specific regions of the cell., (© 2013.)

Cellular functions are usually associated with the activity of proteins and nucleic acids. Recent studies have shown that lipids modulate the localization and activity of key membrane-associated signal transduction proteins, thus regulating the cell's physiology. Membrane Lipid Therapy aims to reverse cell dysfunctions (i.e., diseases) by modulating the activity of membrane signaling proteins through regulation of the lipid bilayer structure. The present work shows the ability of a series of 2-hydroxyfatty acid (2OHFA) derivatives, varying in the acyl chain length and degree of unsaturation, to regulate the membrane lipid structure. These molecules have shown greater therapeutic potential than their natural non-hydroxylated counterparts. We demonstrated that both 2OHFA and natural FAs induced reorganization of lipid domains in model membranes of POPC:SM:PE:Cho, modulating the liquid-ordered/liquid-disordered structures ratio and the microdomain lipid composition. Fluorescence spectroscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential detergent solubilization experiments showed a destabilization of the membranes upon addition of the 2OHFAs and FAs which correlated with the observed disordering effect. The changes produced by these synthetic fatty acids on the lipid structure may constitute part of their mechanism of action, leading to changes in the localization/activity of membrane proteins involved in signaling cascades, and therefore modulating cell responses., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: The potential use of nucleic acids as therapeutic drugs has triggered the quest for oligonucleotide conjugates with enhanced cellular permeability. To this end, the biophysical aspects of previously reported potential lipid oligodeoxyribonucleotide conjugates were studied including its membrane-binding properties and cellular uptake., Methods: These conjugates were fully characterized by MALDI-TOF mass spectrometry and HPLC chromatography. Their ability to insert into lipid model membrane systems was evaluated by Langmuir balance and confocal microscopy followed by the study of the internalization of a lipid oligodeoxyribonucleotide conjugate bearing a double-tail lipid modification (C28) into different cell lines by confocal microscopy and flow cytometry. This compound was also compared with other lipid containing conjugates and with the classical lipoplex formulation using Transfectin as transfection reagent., Results: This double-tail lipid modification showed better incorporation into both lipid model membranes and cell systems. Indeed, this lipid conjugation was capable of inserting the oligodeoxyribonucleotide into both liquid-disordered and liquid-ordered domains of model lipid bilayer systems and produced an enhancement of oligodeoxyribonucleotide uptake in cells, even better than the effect caused by lipoplexes. In addition, in β2 integrin (CR3) expressing cells this receptor was directly involved in the enhanced internalization of this compound., Conclusions: All these features confirm that the dual lipid modification (C28) is an excellent modification for enhancing nucleic acid delivery without altering their binding properties., General Significance: Compared to the commercial lipoplex approach, oligodeoxyribonucleotide conjugation with C28 dual lipid modification seems to be promising to improve oligonucleotide delivery in mammalian cells., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The lipopeptide surfactin exhibits promising antimicrobial activities which are hampered by haemolytic toxicity. Rational design of new surfactin molecules, based on a better understanding of membrane:surfactin interaction, is thus crucial. We here performed bioimaging of lateral membrane lipid heterogeneity in adherent living human red blood cells (RBCs), as a new relevant bioassay, and explored its potential to better understand membrane:surfactin interactions. RBCs show (sub)micrometric membrane domains upon insertion of BODIPY analogs of glucosylceramide (GlcCer), sphingomyelin (SM) and phosphatidylcholine (PC). These domains exhibit increasing sensitivity to cholesterol depletion by methyl-β-cyclodextrin. At concentrations well below critical micellar concentration, natural cyclic surfactin increased the formation of PC and SM, but not GlcCer, domains, suggesting preferential interaction with lipid assemblies with the highest vulnerability to methyl-β-cyclodextrin. Surfactin not only reversed disappearance of SM domains upon cholesterol depletion but further increased PC domain abundance over control RBCs, indicating that surfactin can substitute cholesterol to promote micrometric domains. Surfactin sensitized excimer formation from PC and SM domains, suggesting increased lipid recruitment and/or diffusion within domains. Comparison of surfactin congeners differing by geometry, charge and acyl chain length indicated a strong dependence on acyl chain length. Thus, bioimaging of micrometric lipid domains is a visual powerful tool, revealing that intrinsic lipid domain organization, cholesterol abundance and drug acyl chain length are key parameters for membrane:surfactin interaction. Implications for surfactin preferential location in domains or at their boundaries are discussed and may be useful for rational design of better surfactin molecules., (Copyright © 2013 Elsevier B.V. All rights reserved.)