Phase Separation In Membranes Research Articles

Antimicrobial Peptides Increase Line Tension in Raft-Forming Lipid Membranes.

The formation of phase separated membrane domains is believed to be essential for the function of the cell. The precise composition and physical properties of lipid bilayer domains play crucial roles in regulating protein activity and governing cellular processes. Perturbation of the domain structure in human cells can be related to neurodegenerative diseases and cancer. Lipid rafts are also believed to be essential in bacteria, potentially serving as targets for antibiotics. An important question is how the membrane domain structure is affected by bioactive and therapeutic molecules, such as surface-active peptides, which target cellular membranes. Here we focus on antimicrobial peptides (AMPs), crucial components of the innate immune system, to gain insights into their interaction with model lipid membranes containing domains. Using small-angle neutron/X-ray scattering (SANS/SAXS), we show that the addition of several natural AMPs (indolicidin, LL-37, magainin II, and aurein 2.2) causes substantial growth and restructuring of the domains, which corresponds to increased line tension. Contrast variation SANS and SAXS results demonstrate that the peptide inserts evenly in both phases, and the increased line tension can be related to preferential and concentration dependent thinning of the unsaturated membrane phase. We speculate that the lateral restructuring caused by the AMPs may have important consequences in affecting physiological functions of real cells. This work thus shines important light onto the complex interactions and lateral (re)organization in lipid membranes, which is relevant for a molecular understanding of diseases and the action of antibiotics.

Unlocking the potential of zwitterionic vapor-induced phase separation membranes synthesized from maleic anhydride in polyvinylidene fluoride-based systems for antifouling applications

This work presents an approach to form zwitterionic porous poly(vinylidene fluoride) (PVDF) membranes fabricated by vapor-induced phase separation (VIPS). The reaction between maleic anhydride groups, incorporated initially in the casting solution by blending styrene maleic anhydride (SMA), with 3-((3-aminopropyl)dimethylammonio)propane-1-sulfonate led to the desired zwitterionic moieties. XPS and FTIR analyses confirm the functionalization of the membrane. SEM analyses revealed that it did not have any major impact on the membrane morphology deemed to be close to bicontinuous. While the mean pore size of the virgin membrane was 0.57 μm, it increased to 0.85 μm suggesting the pore-forming effect of SMA during the phase-inversion process. Hydration was significantly enhanced, attributed to the zwitterionic groups on the one hand, but also to the formation of carboxylic acid and amide groups following the ring-opening reaction. It led to significant resistance to bacterial attachment as tested with 4 strains of bacteria including Escherichia coli, Stenotrophomonas maltophilia, Staphylococcus aureus and Streptococcus mutans (>99 % reduction for each). Applied in cyclic water/bacterial solution filtration tests, the modified membrane showed clear improvement in overall performances. Hence, for the virgin membrane, the initial water permeability, flux recovery ratio after the first cycle and flux recovery ratio after the second cycle were found to be 3000 L m−2 h−1.bar−1, 12 % and 53 %, respectively. For the optimized zwitterionic membrane, they were measured to be 7000 L m−2 h−1.bar−1, 26 % and 100 %, respectively. The efficacy of styrene maleic anhydride as a precursor for achieving stable and efficient zwitterionization of PVDF microfiltration membranes is evidenced, rendering it valuable for the filtration of microorganism-containing wastewater. Moreover, its potential application extends to the biomedical realm.

Sterol-lipids enable large-scale, liquid-liquid phase separation in bilayer membranes of only 2 components.

A wide diversity of bilayer membranes, from those with hundreds of lipids (e.g., vacuoles of living yeast cells) to those with very few (e.g., artificial vesicles) phase separate into micron-scale liquid domains. The number of components required for liquid-liquid phase separation has been perplexing: only two should be necessary, but more are required except in extraordinary circumstances. What minimal set of molecular characteristics leads to liquid-liquid phase separation in bilayer membranes? This question inspired us to search for single, joined "sterol-lipid" molecules to replace both a sterol and a phospholipid in membranes undergoing liquid-liquid phase separation. By producing phase-separating membranes with only two components, we mitigate experimental challenges in determining tie-lines and in maintaining constant chemical potentials of lipids.

2-Methylpyrazine: A Greener Solvent for Nonsolvent Induced Phase Separation (NIPS) Membrane Fabrication

2-Methylpyrazine: A Greener Solvent for Nonsolvent Induced Phase Separation (NIPS) Membrane Fabrication

Triplet-Triplet Annihilation Upconverting Liposomes: Mechanistic Insights into the Role of Membranes in Two-Dimensional TTA-UC.

Triplet-triplet annihilation upconversion (TTA-UC) implemented in nanoparticle assemblies is of emerging interest in biomedical applications, including in drug delivery and imaging. As it is a bimolecular process, ensuring sufficient mobility of the sensitizer and annihilator to facilitate effective collision in the nanoparticle is key. Liposomes can provide the benefits of two-dimensional confinement and condensed concentration of the sensitizer and annihilator along with superior fluidity compared to other nanoparticle assemblies. They are also biocompatible and widely applied across drug delivery modalities. However, there are relatively few liposomal TTA-UC systems reported to date, so systematic studies of the influence of the liposomal environment on TTA-UC are currently lacking. Here, we report the first example of a BODIPY-based sensitizer TTA-UC system within liposomes and use this system to study TTA-UC generation and compare the relative intensity of the anti-Stokes signal for this system as a function of liposome composition and membrane fluidity. We report for the first time on time-resolved spectroscopic studies of TTA-UC in membranes. Nanosecond transient absorption data reveal the BODIPY-perylene dyad sensitizer has a long triplet lifetime in liposome with contributions from three triplet excited states, whose lifetimes are reduced upon coinclusion of the annihilator due to triplet-triplet energy transfer, to a greater extent than in solution. This indicates triplet energy transfer between the sensitizer and the annihilator is enhanced in the membrane system. Molecular dynamics simulations of the sensitizer and annihilator TTA collision complex are modeled in the membrane and confirm the co-orientation of the pair within the membrane structure and that the persistence time of the bound complex exceeds the TTA kinetics. Modeling also reliably predicted the diffusion coefficient for the sensitizer which matches closely with the experimental values from fluorescence correlation spectroscopy. The relative intensity of the TTA-UC output across nine liposomal systems of different lipid compositions was explored to examine the influence of membrane viscosity on upconversion (UC). UC showed the highest relative intensity for the most fluidic membranes and the weakest intensity for highly viscous membrane compositions, including a phase separation membrane. Overall, our study reveals that the co-orientation of the UC pair within the membrane is crucial for effective TTA-UC within a biomembrane and that the intensity of the TTA-UC output can be tuned in liposomal nanoparticles by modifying the phase and fluidity of the liposome. These new insights will aid in the design of liposomal TTA-UC systems for biomedical applications.

Amantadine interactions with phase separated lipid membranes

Amantadine, a small amphilphic organic compound that consists of an adamantane backbone and an amino group, was first recognized as an antiviral in 1963 and received approval for prophylaxis against the type A influenza virus in 1976. Since then, it has also been used to treat Parkinson’s disease-related dyskinesia and is being considered as a treatment for corona viruses. Since amantadine usually targets membrane-bound proteins, its interactions with the membrane are also thought to be important. Biological membranes are now widely understood to be laterally heterogeneous and certain proteins are known to preferentially co-localize within specific lipid domains. Does amantadine, therefore, preferentially localize in certain lipid composition domains? To address this question, we studied amantadine’s interactions with phase separating membranes composed of cholesterol, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), as well as single-phase DPhPC (1,2-diphytanoyl-sn-glycero-3-phos-phocholine) membranes. From Langmuir trough and differential scanning calorimetry (DSC) measurements, we determined, respectively, that amantadine preferentially binds to disordered lipids, such as POPC, and lowers the phase transition temperature of POPC/DSPC/cholesterol mixtures, implying that amantadine increases membrane disorder. Further, using droplet interface bilayers (DIBs), we observed that amantadine disrupts DPhPC membranes, consistent with its disordering properties. Finally, we carried out molecular dynamics (MD) simulations on POPC/DSPC/cholesterol membranes with varying amounts of amantadine. Consistent with experiment, MD simulations showed that amantadine prefers to associate with disordered POPC-rich domains, domain boundaries, and lipid glycerol backbones. Since different proteins co-localize with different lipid domains, our results have possible implications as to which classes of proteins may be better targets for amantadine.

Shear-Induced Nonequilibrium Patterns in Lipid Bilayer Membranes Exhibiting Phase Separation.

The nonequilibrium dynamics of a fluid lipid membrane under external stimuli is an important issue that spans disciplines such as soft matter, biophysical chemistry, and interface science. This study investigated the dynamic response of lipid vesicles with order-disorder phase separation, which mimics a plasma membrane heterogeneity, to shear flow. Lipid vesicles were immobilized in a microfluidic chamber, and shear-induced nonequilibrium patterns on the membrane surface were observed by an optical microscope. We found that phase-separated membranes exhibit a dissipative structure of stripe patterns along the vortex flow on the membrane surface, and the number of stripes increased with the flow rate. At a high flow rate, the membrane exhibited a stripe-to-wave transition, where striped domains often migrated and the replacement of two different phases happened at vortex centers with time. We obtained a dynamic phase diagram of the shear-induced wave pattern by changing the flow rate, membrane components, and temperature. These findings could provide insight into the dissipative structures of lipid membranes out of equilibrium and flow-mediated mechanotransduction of biological membranes.

Phase separation and water channels in graft-type polymer electrolyte membranes for hydrogen fuel cell

Microstructural complexity in graft-type polymer electrolyte membranes upon operating conditions is still challenging for examination. Here, the phase separation and water channels of poly(styrene sulfonic acid) (PSSA)-grafted poly(ethylene-co-tetrafluoroethylene) (ETFE) polymer electrolyte membranes (ETFE-PEMs) are characterized by a newly applying method based on electron density fluctuation, F, in the small angle X-ray scattering (SAXS) profiles. The ETFE-PEMs exhibit an increase in phase separation in the ion exchange capacity of 0–0.7 mmol/g as qualitatively determined by the increase in the SAXS invariant. The increase in the SAXS invariant is attributed to the evolution of the newly separated phase between the surfaces of crystalline ETFE backbones and the side chains of PSSA grafts. This phase separation allows the formation of water channels upon low relative humidity (RH). The ionic-domain spacing is around 1.52 nm at dry condition and then increases to 1.69 nm at low RH of 10%, reflecting the ion-water aggregation to make larger aggregate size. At 30% relative humidity, the highly ordered lamellar proton-conducting nanochannels (water channels) are formed in the membrane, which correspond to the dramatic increase in proton conductivity. It is interesting to note that these conducting nanochannels remain unchanged upon 30–70% RH. Also, in this range of RH the similar signatures in the pair distribution function suggests that the molecular packing and local structures are highly similar. Above features of phase separation and water channels can elucidate important characteristics of the ETFE-PEMs, especially the high proton conductivity at low RH.

Sterol-lipids enable large-scale, liquid-liquid phase separation in membranes of only two components

Sterol-lipids enable large-scale, liquid-liquid phase separation in membranes of only two components

Unleashing the single-particle power of cryo-EM to study liquid-ordered/liquid-disordered phase separation in model membranes

Unleashing the single-particle power of cryo-EM to study liquid-ordered/liquid-disordered phase separation in model membranes

The effect of electric field-assisted non-solvent induced phase separation method on PVDF membrane morphologies and performances

ABSTRACT Achieving specific functions in polyvinylidene fluoride (PVDF) membranes requires precise control over their crystalline and morphological structure. In this study, we introduce an innovative method involving the application of an electric field as a preliminary treatment step before immersing PVDF in water during the fabrication of non-solvent induced phase separation (NIPS) membranes. Our study reveals that even a brief exposure to an electric field result in a more organized and aligned molecular structure and significantly altered the crystallinity of polyvinylidene fluoride (PVDF). This structural transformation leads to notable improvements in membrane morphology, characterized by reduced macrovoids and enhanced uniformity. Additionally, membranes produced through this method exhibit markedly enhanced ionic conductivity, a pivotal attribute for optimal performance in electrochemical devices. This innovative approach presents an effective alternative for enhancing PVDF membrane properties, with promising implications for high-performance electrochemical devices and separation membranes. Future research endeavors are necessary to elucidate the underlying mechanisms responsible for these enhancements and to assess the long-term stability and practical applicability of the pretreated membranes.

Structural characterization of lateral phase separation in polymer-lipid hybrid membranes.

Hierarchic self-assembly is the main mechanism used to create diverse structures using soft materials. This is a case for both synthetic materials and biomolecular systems, as exemplified by the non-covalent organization of lipids into membranes. In nature, lipids often assemble into single bilayers, but other nanostructures are encountered, such as bilayer stacks and tubular and vesicular aggregates. Synthetic block copolymers can be engineered to recapitulate many of the structures, forms, and functions of lipid systems. When block copolymers are amphiphilic, they can be inserted or co-assembled into hybrid membranes that exhibit synergistic structural, permeability, and mechanical properties. One example is the emergence of lateral phase separation akin to the raft formation in biomembranes. When higher-order structures, such as hybrid membranes, are formed, this lateral phase separation can be correlated across membranes in the stack. This chapter outlines a set of important methods, such as X-ray Scattering, Atomic Force Microscopy, and Cryo-Electron Microscopy, that are relevant to characterizing and evaluating lateral and correlated phase separation in hybrid membranes at the nano and mesoscales. Understanding the phase behavior of polymer-lipid hybrid materials could lead to innovative advancements in biomimetic membrane separation systems.

Phase separation in model lipid membranes investigated with cryogenic electron microscopy.

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.

Lattice-based mesoscale simulations and mean-field theory of cell membrane adhesion.

Adhesion of cell membranes involves multi-scale phenomena, ranging from specific molecular binding at Angstrom scale all the way up to membrane deformations and phase separation at micrometer scale. Consequently, theory and simulations of cell membrane adhesion require multi-scale modeling and suitable approximations that capture the essential physics of these phenomena. Here, we present a mesoscale model for membrane adhesion which we have employed in a series of our recent studies. This model quantifies, in particular, how nanoscale lipid clusters physically affect and respond to the intercellular receptor-ligand binding that mediates membrane adhesion. The goal of this Chapter is to present all details and subtleties of the mean-field theory and Monte Carlo simulations of this mesoscale model, which can be used to further explore physical phenomena related to cell membrane adhesion.

Using the yeast vacuole as a system to test the lipidic drivers of membrane heterogeneity in living cells.

The biophysical drivers of membrane lateral heterogeneity, often termed lipid rafts, have been largely explored using synthetic liposomes or mammalian plasma membrane-derived giant vesicles. Yeast vacuoles, an organelle comparable to mammalian lysosomes, is the only in vivo system that shows stable micrometer scale phase separation in unperturbed cells. The ease of manipulating lipid metabolism in yeast makes this a powerful system for identifying lipids involved in the onset of vacuole membrane heterogeneity. Vacuole domains are induced by stationary stage growth and nutritional starvation, during which they serve as a docking and internalization site for lipid droplet energy stores. Here we describe methods for characterizing vacuole phase separation, its physiological function, and its lipidic drivers. First, we detail methodologies for robustly inducing vacuole domain formation and quantitatively characterizing during live cell imaging experiments. Second, we detail a new protocol for biochemical isolation of stationary stage vacuoles, which allows for lipidomic dissection of membrane phase separation. Third, we describe biochemical techniques for analyzing lipid droplet internalization in vacuole domains. When combined with genetic or chemical perturbations to lipid metabolism, these methods allow for systematic dissection of lipid composition in the structure and function of ordered membrane domains in living cells.

Separation of acetonitrile/water with the combination of superwetting membrane and salt-induced phase separation

Efficient recovery of acetonitrile from its aqueous solution is quite important for both cost reduction and avoiding environmental pollution. The current main strategies including pervaporation, absorptive separation, and extractive distillation are limited by their low separation-flux, low separation-capacity, or high energy-consumption. Herein, we combine the design of superwetting membrane and salt-induced phase separation to propose a new approach for purification of acetonitrile from its aqueous solution, which possesses the advantages of high throughput, continuity, and energy conservation.

Surface nanoroughness impacts the formation and stability of supported lipid bilayers

Solid supported lipid bilayers (SLBs) are excellent platforms for studying the biophysical properties of cell membranes, as well as versatile biomimetic films for biotechnology applications. Among the existing approaches used to form SLBs, vesicle fusion and rupture onto solid supports is the most commonly employed one owing to its straightforward procedure. SLBs are typically formed on atomically flat and very hydrophilic surfaces, overlooking the influence of roughness and topography on membrane formation and organization. As a matter of fact, lipid bilayers in vivo are corrugated at the nanoscale level, as a result of interactions with proteins, fibrils, and other components within the intracellular and extracellular environment. Fundamental studies of the effect of surface roughness on SLBs are scarce and restricted to few contributions, where nanoroughness has shown to affect lipid mobility by a 5-fold decrease and inhibit domain growth in phase-separated membranes. In this work, the impact of nanoroughness on the formation and stability of SLBs onto SiO2 surfaces with different degrees of vertical and lateral surface roughness is studied. Combining quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy with force spectroscopy (AFM-FS), it is shown that nanoroughness affects the formation of SLBs by increasing the activation energy of vesicle fusion, rupture and spreading, and weakens the stability and lateral organization of the formed SLBs.

Intracellular sphingolipid sorting drives membrane phase separation in the yeast vacuole

The yeast vacuole membrane can phase separate into ordered and disordered domains, a phenomenon that is required for micro-lipophagy under nutrient limitation. Despite its importance as a biophysical model and physiological significance, it is not yet resolved if specific lipidome changes drive vacuole phase separation. Here we report that the metabolism of sphingolipids (SLs) and their sorting into the vacuole membrane can control this process. We first developed a vacuole isolation method to identify lipidome changes during the onset of phase separation in early stationary stage cells. We found that early stationary stage vacuoles are defined by increased abundance of putative raft components, including 40% higher ergosterol content and a nearly 3-fold enrichment in complex SLs. These changes were not found in the corresponding whole cell lipidomes, indicating that lipid sorting is associated with domain formation. Several facets of SL composition – headgroup stoichiometry, chain lengths, and increased hydroxylations – were also markers of phase-separated vacuole lipidomes. To test SL function in vacuole phase separation, we carried out a systematic genetic dissection of their biosynthetic pathway. The abundance of CSLs controlled the extent of domain formation and associated micro-lipophagy processes, while their headgroup composition altered domain morphology. These results suggest that lipid trafficking can drive membrane phase separation in vivo and identify SLs as key mediators of this process in yeast.

On Fusogenicity of Positively Charged Phased-Separated Lipid Vesicles: Experiments and Computational Simulations.

This paper studies the fusogenicity of cationic liposomes in relation to their surface distribution of cationic lipids and utilizes membrane phase separation to control this surface distribution. It is found that concentrating the cationic lipids into small surface patches on liposomes, through phase-separation, can enhance liposome's fusogenicity. Further concentrating these lipids into smaller patches on the surface of liposomes led to an increased level of fusogenicity. These experimental findings are supported by numerical simulations using a mathematical model for phase-separated charged liposomes. Findings of this study may be used for design and development of highly fusogenic liposomes with minimal level of toxicity.

Physicochemical properties of the vacuolar membrane and cellular factors determine formation of vacuolar invaginations

Vacuoles change their morphology in response to stress. In yeast exposed to chronically high temperatures, vacuolar membranes get deformed and invaginations are formed. We show that phase-separation of vacuolar membrane occurred after heat stress leading to the formation of the invagination. In addition, Hfl1, a vacuolar membrane-localized Atg8-binding protein, was found to suppress the excess vacuolar invaginations after heat stress. At that time, Hfl1 formed foci at the neck of the invaginations in wild-type cells, whereas it was efficiently degraded in the vacuole in the atg8Δ mutant. Genetic analysis showed that the endosomal sorting complex required for transport machinery was necessary to form the invaginations irrespective of Atg8 or Hfl1. In contrast, a combined mutation with the vacuole BAR domain protein Ivy1 led to vacuoles in hfl1Δivy1Δ and atg8Δivy1Δ mutants having constitutively invaginated structures; moreover, these mutants showed stress-sensitive phenotypes. Our findings suggest that vacuolar invaginations result from the combination of changes in the physiochemical properties of the vacuolar membrane and other cellular factors.