Raft-like lipid mixtures in the highly coarse-grained Cooke membrane model.

Lipid rafts are nanoscopic assemblies of sphingolipids, cholesterol, and specific membrane proteins. They are believed to underlie the experimentally observed lateral heterogeneity of eukaryotic plasma membranes and implicated in many cellular processes, such as signaling and trafficking. Ternary model membranes consisting of saturated lipids, unsaturated lipids, and cholesterol are common proxies because they exhibit phase coexistence between a liquid-ordered (lo) and liquid-disordered (ld) phase and an associated critical point. However, plasma membranes are also asymmetric in terms of lipid type, lipid abundance, leaflet tension, and corresponding cholesterol distribution, suggesting that rafts cannot be examined separately from questions about elasticity, curvature torques, and internal mechanical stresses. Unfortunately, it is challenging to capture this wide range of physical phenomenology in a single model that can access sufficiently long length- and time scales. Here we extend the highly coarse-grained Cooke model for lipids, which has been extensively characterized on the curvature-elastic front, to also represent raft-like lo/ld mixing thermodynamics. In particular, we capture the shape and tie lines of a coexistence region that narrows upon cholesterol addition, terminates at a critical point, and has coexisting phases that reflect key differences in membrane order and lipid packing. We furthermore examine elasticity and lipid diffusion for both phase separated and pure systems and how they change upon the addition of cholesterol. We anticipate that this model will enable significant insight into lo/ld phase separation and the associated question of lipid rafts for membranes that have compositionally distinct leaflets that are likely under differential stress-like the plasma membrane.

Biomimetic Lipid Raft: Domain Stability and Interaction with Physiologically Active Molecules.

The cell membrane, also called the plasma membrane, is the membrane on the cytoplasmic surface that separates the extracellular from the intracellular. It is thin, about 10 nm thick when viewed with an electron microscope, and is composed of two monolayers of phospholipid membranes (lipid bilayers) containing many types of proteins. It is now known that this cell membrane not only separates the extracellular from the intracellular, but is also involved in sensory stimuli such as pain, itching, sedation, and excitement. Since the "Fluid mosaic model" was proposed for cell membranes, molecules have been thought to be homogeneously distributed on the membrane surface. Later, at the end of the twentieth century, the existence of "Phase-separated microdomain structures" consisting of ordered phases rich in saturated lipids and cholesterol was suggested, and these were termed "Lipid rafts." A model in which lipid rafts regulate cell signaling has been proposed and is the subject of active research.This chapter first outlines the physicochemical properties and thermodynamic models of membrane phase separation (lipid rafts), which play an important role in cell signaling. Next, how physiologically active molecules such as local anesthetics, cooling agents (menthol), and warming agents (capsaicin) interact with artificial cell membranes will be presented.It is undeniable that the plasma membrane contains many channels and receptors that are involved in the propagation of sensory stimuli. At the same time, however, it is important to understand that the membrane exerts a significant influence on the intensity and propagation of these stimuli.

Structure and dynamics of cholesterol-mediated aquaporin-0 arrays and implications for lipid rafts.

Aquaporin-0 (AQP0) tetramers form square arrays in lens membranes through a yet unknown mechanism, but lens membranes are enriched in sphingomyelin and cholesterol. Here, we determined electron crystallographic structures of AQP0 in sphingomyelin/cholesterol membranes and performed molecular dynamics (MD) simulations to establish that the observed cholesterol positions represent those seen around an isolated AQP0 tetramer and that the AQP0 tetramer largely defines the location and orientation of most of its associated cholesterol molecules. At a high concentration, cholesterol increases the hydrophobic thickness of the annular lipid shell around AQP0 tetramers, which may thus cluster to mitigate the resulting hydrophobic mismatch. Moreover, neighboring AQP0 tetramers sandwich a cholesterol deep in the center of the membrane. MD simulations show that the association of two AQP0 tetramers is necessary to maintain the deep cholesterol in its position and that the deep cholesterol increases the force required to laterally detach two AQP0 tetramers, not only due to protein-protein contacts but also due to increased lipid-protein complementarity. Since each tetramer interacts with four such 'glue' cholesterols, avidity effects may stabilize larger arrays. The principles proposed to drive AQP0 array formation could also underlie protein clustering in lipid rafts.

Tensing Flipper: Photosensitized Manipulation of Membrane Tension, Lipid Phase Separation, and Raft Protein Sorting in Biological Membranes.

The lateral organization of proteins and lipids in the plasma membrane is fundamental to regulating a wide range of cellular processes. Compartmentalized ordered membrane domains enriched with specific lipids, often termed lipid rafts, have been shown to modulate the physicochemical and mechanical properties of membranes and to drive protein sorting. Novel methods and tools enabling the visualization, characterization, and/or manipulation of membrane compartmentalization are crucial to link the properties of the membrane with cell functions. Flipper, a commercially available fluorescent membrane tension probe, has become a reference tool for quantitative membrane tension studies in living cells. Here, we report on a so far unidentified property of Flipper, namely, its ability to photosensitize singlet oxygen (1O2) under blue light when embedded into lipid membranes. This in turn results in the production of lipid hydroperoxides that increase membrane tension and trigger phase separation. In biological membranes, the photoinduced segregated domains retain the sorting ability of intact phase-separated membranes, directing raft and nonraft proteins into ordered and disordered regions, respectively, in contrast to radical-based photo-oxidation reactions that disrupt raft protein partitioning. The dual tension reporting and photosensitizing abilities of Flipper enable simultaneous visualization and manipulation of the mechanical properties and lateral organization of membranes, providing a powerful tool to optically control lipid raft formation and to explore the interplay between membrane biophysics and cell function.

Polar Glycerolipids and Membrane Lipid Rafts.

Current understanding of the structure and functioning of biomembranes is impossible without determining the mechanism of formation of membrane lipid rafts. The formation of liquid-ordered and disordered phases (Lo and Ld) and lipid rafts in membranes and their simplified models is discussed. A new consideration of the processes of formation of lipid phases Lo and Ld and lipid rafts is proposed, taking into account the division of each of the glycerophospholipids into several groups. Generally accepted three-component schemes for modeling the membrane structure are critically considered. A four-component scheme is proposed, which is designed to more accurately assume the composition of lipids in the resulting Lo and Ld phases. The role of the polar head groups of phospholipids and, in particular, phosphatidylethanolamine is considered. The structure of membrane rafts and the possible absence of a clear boundary between the Lo and Ld phases are discussed.

Characterization of domain formation in complex membranes.

A widely known property of lipid membranes is their tendency to undergo a separation into disordered (Ld) and ordered (Lo) domains. This impacts the local structure of the membrane relevant for the physical (e.g., enhanced electroporation) and biological (e.g., protein sorting) significance of these regions. The increase in computing power, advancements in simulation software, and more detailed information about the composition of biological membranes shifts the study of these domains into the focus of classical molecular dynamics simulations. In this chapter, we present a versatile yet robust analysis pipeline that can be easily implemented and adapted for a wide range of lipid compositions. It employs Gaussian-based Hidden Markov Models to predict the hidden order states of individual lipids by describing their structure through the area per lipid and the average SCC order parameters per acyl chain. Regions of the membrane with a high correlation between ordered lipids are identified by employing the Getis-Ord local spatial autocorrelation statistic on a Voronoi tessellation of the lipids. As an example, the approach is applied to two distinct systems at a coarse-grained resolution, demonstrating either a strong tendency towards phase separation (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DIPC), cholesterol) or a weak tendency toward phase separation (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PUPC), cholesterol). Explanations of the steps are complemented by coding examples written in Python, providing both a comprehensive understanding and practical guidance for a seamless integration of the workflow into individual projects.

Using lipid binding proteins and advanced microscopy to study lipid domains.

Sphingomyelin is postulated to form clusters with glycosphingolipids, cholesterol and other sphingomyelin molecules in biomembranes through hydrophobic interaction and hydrogen bonds. These clusters form submicron size lipid domains. Proteins that selectively binds sphingomyelin and/or cholesterol are useful to visualize the lipid domains. Due to their small size, visualization of lipid domains requires advanced microscopy techniques in addition to lipid binding proteins. This Chapter describes the method to characterize plasma membrane sphingomyelin-rich and cholesterol-rich lipid domains by quantitative microscopy. This Chapter also compares different permeabilization methods to visualize intracellular lipid domains.

Applications of phase-separating multi-bilayers in protein-membrane domain interactions.

Synthetic model membranes are important tools to elucidate lipid domain and protein interactions due to predefined lipid compositions and characterizable biophysical properties. Here, we introduce a model membrane with multiple lipid bilayers (multi-bilayers) stacked on a mica substrate that is prepared through a spin-coating technique. The spin-coated multi-bilayers are useful in the study of phase separated membranes with a high cholesterol content, mobile lipids, microscopic and reversible phase separation, and easy conjugation with proteins, which make them a good model to study interactions between proteins and membrane domains.

Pore-spanning membranes as a tool to investigate lateral lipid membrane heterogeneity.

Over the years, it has become more and more obvious that lipid membranes show a very complex behavior. This behavior arises in part from the large number of different kinds of lipids and proteins and how they dynamically interact with each other. In vitro studies using artificial membrane systems have shed light on the heterogeneity based on lipid-lipid interactions in multicomponent bilayer mixtures. Inspired by the raft hypothesis, the coexistence of liquid-disordered (ld) and liquid-ordered (lo) phases has drawn much attention. It was shown that ternary lipid mixtures containing low- and high-melting temperature lipids and cholesterol can phase separate into a lo phase enriched in the high-melting lipids and cholesterol and a ld phase enriched in the low-melting lipids. Depending on the model membrane system under investigation, different domain sizes, shapes, and mobilities have been found. Here, we describe how to generate phase-separated lo/ld phases in model membrane systems termed pore-spanning membranes (PSMs). These PSMs are prepared on porous silicon substrates with pore sizes in the micrometer regime. A proper functionalization of the top surface of the substrates is required to achieve the spreading of giant unilamellar vesicles (GUVs) to obtain PSMs. Starting with lo/ld phase-separated GUVs lead to membrane heterogeneities in the PSMs. Depending on the functionalization strategy of the top surface of the silicon substrate, different membrane heterogeneities are observed in the PSMs employing fluorescence microscopy. A quantitative analysis of the heterogeneity as well as the dynamics of the lipid domains is described.

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.

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.

Cryo-EM architecture of a near-native stretch-sensitive membrane microdomain.

Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins1-3. The composition and organization of membrane microdomains remain controversial because few techniques are available that allow the visualization of lipids in situ without disrupting their native behaviour3,4. The yeast eisosome, composed of the BAR-domain proteins Pil1 and Lsp1 (hereafter, Pil1/Lsp1), scaffolds a membrane compartment that senses and responds to mechanical stress by flattening and releasing sequestered factors5-9. Here we isolated near-native eisosomes as helical tubules made up of a lattice of Pil1/Lsp1 bound to plasma membrane lipids, and solved their structures by helical reconstruction. Our structures reveal a striking organization of membrane lipids, and, using in vitro reconstitutions and molecular dynamics simulations, we confirmed the positioning of individual PI(4,5)P2, phosphatidylserine and sterol molecules sequestered beneath the Pil1/Lsp1 coat. Three-dimensional variability analysis of the native-source eisosomes revealed a dynamic stretching of the Pil1/Lsp1 lattice that affects the sequestration of these lipids. Collectively, our results support a mechanism in which stretching of the Pil1/Lsp1 lattice liberates lipids that would otherwise be anchored by the Pil1/Lsp1 coat, and thus provide mechanistic insight into how eisosome BAR-domain proteins create a mechanosensitive membrane microdomain.

Coarse-grained modeling of annexin A2-induced microdomain formation on a vesicle.

Annexin A2 (A2)-induced microdomain formation is a key step in biological processes such as Ca2+-mediated exocytosis in neuroendocrine cells. In this work, a total of 15 coarse-grained molecular dynamics simulations were performed on vesicle models having a diameter of approximately 250 Å for 15 µs each using the Martini2 force field. Five simulations were performed in the presence of 10 A2, 5 in the presence of A2 but absence of PIP2, and 5 simulations in the absence of A2 but presence of PIP2. Consistent results were generated among the simulations. A2-induced PIP2 microdomain formation was observed and shown to occur in three phases: A2-vesicle association, localized A2-induced PIP2 clustering, and A2 aggregation driving PIP2 microdomain formation. The relationship between A2 aggregation and PIP2 microdomain formation was quantitatively described using a novel method which calculated the variance among protein and lipid positions via the Fréchet mean. A large reduction in PIP2 variance was observed in the presence of A2 but not in its absence. This reduction in PIP2 variance was proportional to the reduction observed in A2 variance and demonstrates that the observed PIP2 microdomain formation is dependent upon A2 aggregation. The three-phase model of A2-induced microdomain formation generated in this work will serve as a valuable guide for further experimental studies and the development of novel A2 inhibitors. No microdomain formation was observed in the absence of A2 and minimal A2-membrane interaction was observed in the absence of PIP2.

Measuring the Transmembrane Registration of Lipid Domains in Droplet Interface Bilayers through Tensiometry.

Diverse collections of lipids self-assemble into domains within biological membranes, and these domains are typically organized in both the transverse and lateral directions of the membrane. The ability of the membrane to link these domains across the membrane's interior grants cells control over features on the external cellular surface. Numerous hypothesized factors drive the cross-membrane (or transverse) coupling of lipid domains. In this work we seek to isolate these transverse lipid-lipid influences in a simple model system using droplet interface bilayers (DIBs) to better understand the associated mechanics. DIBs enable symmetric and asymmetric combinations of domain-forming lipid mixtures within a model bilayer, and the evolving energetics of the membrane may be tracked using drop-shape analysis. We find that symmetric distributions of domain-forming lipids produce long-lasting, gradual shifts in the DIB membrane energetics that are not observed in asymmetric distributions of the lipids where the domain-forming lipids are only within one leaflet. The approach selected for this work provides experimental measurement of the mismatch penalty associated with antiregistered lipid domains as well as measurements of the influence of rafts on DIB behaviors with suggestions for their future use as a model platform.

Asymptotic analysis of particle cluster formation in the presence of anchoring sites.

The aggregation or clustering of proteins and other macromolecules plays an important role in the formation of large-scale molecular assemblies within cell membranes. Examples of such assemblies include lipid rafts, and postsynaptic domains (PSDs) at excitatory and inhibitory synapses in neurons. PSDs are rich in scaffolding proteins that can transiently trap transmembrane neurotransmitter receptors, thus localizing them at specific spatial positions. Hence, PSDs play a key role in determining the strength of synaptic connections and their regulation during learning and memory. Recently, a two-dimensional (2D) diffusion-mediated aggregation model of PSD formation has been developed in which the spatial locations of the clusters are determined by a set of fixed anchoring sites. The system is kept out of equilibrium by the recycling of particles between the cell membrane and interior. This results in a stationary distribution consisting of multiple clusters, whose average size can be determined using an effective mean-field description of the particle concentration around each anchored cluster. In this paper, we derive corrections to the mean-field approximation by applying the theory of diffusion in singularly perturbed domains. The latter is a powerful analytical method for solving two-dimensional (2D) and three-dimensional (3D) diffusion problems in domains where small holes or perforations have been removed from the interior. Applications range from modeling intracellular diffusion, where interior holes could represent subcellular structures such as organelles or biological condensates, to tracking the spread of chemical pollutants or heat from localized sources. In this paper, we take the bounded domain to be the cell membrane and the holes to represent anchored clusters. The analysis proceeds by partitioning the membrane into a set of inner regions around each cluster, and an outer region where mean-field interactions occur. Asymptotically matching the inner and outer stationary solutions generates an asymptotic expansion of the particle concentration, which includes higher-order corrections to mean-field theory that depend on the positions of the clusters and the boundary of the domain. Motivated by a recent study of light-activated protein oligomerization in cells, we also develop the analogous theory for cluster formation in a three-dimensional (3D) domain. The details of the asymptotic analysis differ from the 2D case due to the contrasting singularity structure of 2D and 3D Green's functions.

Water Hydrogen-Bond Mediated Layer by Layer Alignment of Lipid Rafts as a Precursor of Intermembrane Processes.

Lipid rafts, which are dynamic nanodomains in the plasma membrane, play a crucial role in intermembrane processes by clustering together and growing in size within the plane of the membrane while also aligning with each other across different membranes. However, the physical origin of layer by layer alignment of lipid rafts remains to be elucidated. Here, by using fluorescence imaging and synchrotron X-ray reflectivity in a phase-separated multilayer system, we find that the alignment of raft-mimicking Lo domains is regulated by the distance between bilayers. Molecular dynamics simulations reveal that the aligned state is energetically preferred when the intermembrane distance is small due to its ability to minimize the volume of surface water, which has fewer water hydrogen bonds (HBs) compared to bulk water. Our results suggest that water HB-driven alignment of lipid rafts plays a role as a precursor of intermembrane processes such as cell-cell fusion, virus entry, and signaling.

Selection of bifunctional RNAs with specificity for arginine and lipid membranes.

The molecular mechanisms of selective RNA loading into exosomes and other extracellular vesicles are not yet completely understood. In order to show that a pool of RNA sequences binds both the amino acid arginine and lipid membranes, we constructed a bifunctional RNA 10Arg aptamer specific for arginine and lipid vesicles. The preference of RNA 10Arg for lipid rafts was visualized and confirmed using FRET microscopy in neuroblastoma cells. The selection-amplification (SELEX) method using a doped (with the other three nucleotides) pool of RNA 10Arg sequences yielded several RNA 10Arg(D) sequences, and the affinities of these RNAs both to arginine and liposomes are improved in comparison to pre-doped RNA. Generation of these bispecific aptamers supports the hypothesis that an RNA molecule can bind both to RNA-binding proteins (RBPs) through arginine within the RBP-binding site and to membrane lipid rafts, thus facilitating RNA loading into exosomes and other extracellular vesicles.

Dynamic Simulations of Interaction of the PEG-DPPE Micelle-Encapsulated Short-Chain Ceramides with the Raft-Included Membrane.

The lipid raft subdomains in cancer cell membranes play a key role in signal transduction, biomolecule recruitment, and drug transmembrane transport. Augmented membrane rigidity due to the formation of a lipid raft is unfavorable for the entry of drugs, a limiting factor in clinical oncology. The short-chain ceramide (CER) has been reported to promote drug entry into membranes and disrupt lipid raft formation, but the underlying mechanism is not well understood. We recently explored the carrier-membrane fusion dynamics of PEG-DPPE micelles in delivering doxorubicin (DOX). Based on the phase-segregated membrane model composed of DPPC/DIPC/CHOL/GM1/PIP2, we aim to explore the dynamic mechanism of the PEG-DPPE micelle-encapsulating DOXs in association with the raft-included cell membrane modulated by C8 acyl tail CERs. The results show that the lipid raft remains integrated and DOX-resistant subjected to free DOXs and the micelle-encapsulating ones. Addition of CERs disorganizes the lipid raft by pushing CHOL aside from DPPC. It subsequently allows for a good permeability for PEG-DPPE micelle-encapsulated DOXs, which penetrate deeper as CER concentration increases. GM1 is significant in guiding drugs' redistributing between bilayer phases, and the anionic PIP2 further helps DOXs attain the inner bilayer surface. These results elaborate on the perturbing effect of CERs on lipid raft stability, which provides a new comprehensive approach for further design of drug delivery systems.

Dehydration of Lipid Membranes Drives Redistribution of Cholesterol Between Lateral Domains.

Cholesterol-rich lipid rafts are found to facilitate membrane fusion, central to processes like viral entry, fertilization, and neurotransmitter release. While the fusion process involves local, transient membrane dehydration, the impact of reduced hydration on cholesterol's structural organization in biological membranes remains unclear. Here, we employ confocal fluorescence microscopy and atomistic molecular dynamics simulations to investigate cholesterol behavior in phase-separated lipid bilayers under controlled hydration. We unveiled that dehydration prompts cholesterol release from raft-like domains into the surrounding fluid phase. Unsaturated phospholipids undergo more significant dehydration-induced structural changes and lose more hydrogen bonds with water than sphingomyelin. The results suggest that cholesterol redistribution is driven by the equalization of biophysical properties between phases and the need to satisfy lipid hydrogen bonds. This underscores the role of cholesterol-phospholipid-water interplay in governing cholesterol affinity for a specific lipid type, providing a new perspective on the regulatory role of cell membrane heterogeneity during membrane fusion.

Novel class of peptides disintegrating biological membranes to aid in the characterization of membrane proteins.

Styrene-maleic acid (SMA) and similar amphiphilic copolymers are known to cut biological membranes into lipid nanoparticles/nanodiscs containing membrane proteins apparently in their relatively native membrane lipid environment. Our previous work demonstrated that membrane raft microdomains resist such disintegration by SMA. The use of SMA in studying membrane proteins is limited by its heterogeneity and the inability to prepare defined derivatives. In the present paper, we demonstrate that some amphiphilic peptides structurally mimicking SMA also similarly disintegrate cell membranes. In contrast to the previously used copolymers, the simple peptides are structurally homogeneous. We found that their membrane-disintegrating activity increases with their length (reaching optimum at 24 amino acids) and requires a basic primary structure, that is, (XXD)n, where X represents a hydrophobic amino acid (optimally phenylalanine), D aspartic acid, and n is the number of repeats of these triplets. These peptides may provide opportunities for various well-defined potentially useful modifications in the study of membrane protein biochemistry. Our present results confirm a specific character of membrane raft microdomains.