Modelling lipid rafts formation through chemo-mechanical interplay triggered by receptor-ligand binding.

Cell membranes, mediator of many biological mechanisms from adhesion and metabolism up to mutation and infection, are highly dynamic and heterogeneous environments exhibiting a strong coupling between biochemical events and structural re-organisation. This involves conformational changes induced, at lower scales, by lipid order transitions and by the micro-mechanical interplay of lipids with transmembrane proteins and molecular diffusion. Particular attention is focused on lipid rafts, ordered lipid microdomains rich of signalling proteins, that co-localise to enhance substance trafficking and activate different intracellular biochemical pathways. In this framework, the theoretical modelling of the dynamic clustering of lipid rafts implies a full multiphysics coupling between the kinetics of phase changes and the mechanical work performed by transmembrane proteins on lipids, involving the bilayer elasticity. This mechanism produces complex interspecific dynamics in which membrane stresses and chemical potentials do compete by determining different morphological arrangements, alteration in diffusive walkways and coalescence phenomena, with a consequent influence on both signalling potential and intracellular processes. Therefore, after identifying the leading chemo-mechanical interactions, the present work investigates from a modelling perspective the spatio-temporal evolution of raft domains to theoretically explain co-localisation and synergy between proteins' activation and raft formation, by coupling diffusive and mechanical phenomena to observe different morphological patterns and clustering of ordered lipids. This could help to gain new insights into the remodelling of cell membranes and could potentially suggest mechanically based strategies to control their selectivity, by orienting intracellular functions and mechanotransduction.

Systematic comparison of unilamellar vesicles reveals that archaeal core lipid membranes are more permeable than bacterial membranes.

One of the deepest branches in the tree of life separates the Archaea from the Bacteria. These prokaryotic groups have distinct cellular systems including fundamentally different phospholipid membrane bilayers. This dichotomy has been termed the lipid divide and possibly bestows different biophysical and biochemical characteristics on each cell type. Classic experiments suggest that bacterial membranes (formed from lipids extracted from Escherichia coli, for example) show permeability to key metabolites comparable to archaeal membranes (formed from lipids extracted from Halobacterium salinarum), yet systematic analyses based on direct measurements of membrane permeability are absent. Here, we develop a new approach for assessing the membrane permeability of approximately 10 µm unilamellar vesicles, consisting of an aqueous medium enclosed by a single lipid bilayer. Comparing the permeability of 18 metabolites demonstrates that diether glycerol-1-phosphate lipids with methyl branches, often the most abundant membrane lipids of sampled archaea, are permeable to a wide range of compounds useful for core metabolic networks, including amino acids, sugars, and nucleobases. Permeability is significantly lower in diester glycerol-3-phosphate lipids without methyl branches, the common building block of bacterial membranes. To identify the membrane characteristics that determine permeability, we use this experimental platform to test a variety of lipid forms bearing a diversity of intermediate characteristics. We found that increased membrane permeability is dependent on both the methyl branches on the lipid tails and the ether bond between the tails and the head group, both of which are present on the archaeal phospholipids. These permeability differences must have had profound effects on the cell physiology and proteome evolution of early prokaryotic forms. To explore this further, we compare the abundance and distribution of transmembrane transporter-encoding protein families present on genomes sampled from across the prokaryotic tree of life. These data demonstrate that archaea tend to have a reduced repertoire of transporter gene families, consistent with increased membrane permeation. These results demonstrate that the lipid divide demarcates a clear difference in permeability function with implications for understanding some of the earliest transitions in cell origins and evolution.

A study on attempt for determination of permeation kinetics of coumarin at lipid bilayer of liposomes by using capillary electrophoresis with moment analysis theory.

It was tried to develop a moment analysis method for the determination of lipid membrane permeability. The first absolute and second central moments of elution peaks measured by liposome electrokinetic chromatography (LEKC) are analyzed by using moment equations. As a concrete example, elution peak profiles of coumarin in a LEKC system, in which liposomes consisting of 1-palmitoyl-2-oleoyl-sn­glycero-3-phosphocholine (POPC) and phosphatidylserine (PS) are used as a pseudo-stationary phase, were analyzed. It seems that lipid membrane permeability of coumarin across the lipid bilayer of POPC/PS liposomes was measured by the moment analysis method because previous permeability measurements using parallel artificial membrane permeability assay (PAMPA) and Caco-2 cells indicated that coumarin is permeable across lipid bilayer. However, it was also pointed out that the moment analysis method with LEKC is not effective for the determination of lipid membrane permeability and that it provides information about adsorption/desorption kinetics at lipid bilayer of liposomes. Therefore, different moment equations were also developed for the determination of adsorption/desorption rate constants of coumarin from the LEKC data. It was demonstrated that permeation rate constants at lipid bilayer or adsorption/desorption rate constants can be determined from the LEKC data on the basis of moment analysis theory for the mass transfer phenomena of coumarin at the lipid bilayer of POPC/PS liposomes. Mass transfer kinetics of solutes at lipid bilayer should be determined under the conditions that liposomes originally be because they are self-assembling and dynamic systems formed through weak interactions between phospholipid monomers. The moment analysis method using LEKC is effective for the experimental determination of the mass transfer rate constants at the lipid bilayer of liposomes because neither immobilization nor chemical modification of liposomes is necessary when LEKC data are measured. It is expected that the results of this study contribute to the dissemination of an opportunity for the determination of permeation rate constants or adsorption/desorption rate constants at the lipid bilayer of liposomes to many researchers because capillary electrophoresis is widespread.

Artificial peptides to induce membrane denaturation and disruption and modulate membrane composition and fusion.

Membranes consisting of phospholipid bilayers are an essential constituent of eukaryotic cells and their compartments. The alteration of their composition, structure, and morphology plays an important role in modulating physiological processes, such as transport of molecules, cell migration, or signaling, but it can also lead to lethal effects. The three main classes of membrane-active peptides that are responsible for inducing such alterations are cell-penetrating peptides (CPPs), antimicrobial peptides (AMPs), and fusion peptides (FPs). These peptides are able to interact with lipid bilayers in highly specific and tightly regulated manners. They can either penetrate the membrane, inducing nondestructive, transient alterations, or disrupt, permeabilize, or translocate through it, or induce membrane fusion by generating attractive forces between two bilayers. Because of these properties, membrane-active peptides have attracted the attention of the pharmaceutical industry, and naturally occurring bioactive structures have been used as a platform for synthetic modification and the development of artificial analogs with optimized therapeutic properties to transport biologically active cargos or serve as novel antimicrobial agents. In this review, we focus on synthetic membrane interacting peptides with bioactivity comparable with their natural counterparts and describe their mechanism of action.

Interaction of Graphitic Carbon Nitride with Cell Membranes: Probing Phospholipid Extraction and Lipid Bilayer Destruction.

Understanding how nanomaterials interact with cell membranes has important implications for ecotoxicology and human health. Here, we investigated the interactions between graphitic carbon nitride (g-C3N4, CN) and red blood cells, a plausible contact target for nanoparticles when they enter the bloodstream. Through a hemolysis assay, the cytotoxicity of CN derived from different precursors was quantitatively assessed, which is highly related to the surface area of CN. Reactive oxygen species (ROS) generation and lipid peroxidation detection confirmed that CN causes rapid cell membrane rupture by a physical interaction mechanism rather than ROS-related chemical oxidation. Dye leakage assay and theoretical simulation indicated that the less-layered CN is prone to folding inward to wrap and extract lipid molecules from cell membranes. The electron-rich inherent pores of CN play a dominant role in capturing the headgroups of phospholipids, whereas the hydrophobic interaction is critical for the anchoring of lipid tails. Our further experimental evidence demonstrated that the destructive extraction of phospholipids from cell membranes by CN occurs primarily in the outer leaflet, and phosphatidylcholine is the most easily extracted lipid. Moreover, the formation of protein corona on CN was found to decrease the nonspecific interactions but increase steric repulsion, thus mitigating CN cytotoxicity. Overall, our data provide a molecular basis for CN's cytotoxicity.

CAMLG-CDG: a novel congenital disorder of glycosylation linked to defective membrane trafficking.

The transmembrane domain recognition complex (TRC) pathway is required for the insertion of C-terminal tail-anchored (TA) proteins into the lipid bilayer of specific intracellular organelles such as the endoplasmic reticulum (ER) membrane. In order to facilitate correct insertion, the recognition complex (consisting of BAG6, GET4 and UBL4A) must first bind to TA proteins and then to GET3 (TRC40, ASNA1), which chaperones the protein to the ER membrane. Subsequently, GET1 (WRB) and CAML form a receptor that enables integration of the TA protein within the lipid bilayer. We report an individual with the homozygous c.633 + 4A>G splice variant in CAMLG, encoding CAML. This variant leads to aberrant splicing and lack of functional protein in patient-derived fibroblasts. The patient displays a predominantly neurological phenotype with psychomotor disability, hypotonia, epilepsy and structural brain abnormalities. Biochemically, a combined O-linked and type II N-linked glycosylation defect was found. Mislocalization of syntaxin-5 in patient fibroblasts and in siCAMLG deleted Hela cells confirms this as a consistent cellular marker of TRC dysfunction. Interestingly, the level of the v-SNARE Bet1L is also drastically reduced in both of these models, indicating a fundamental role of the TRC complex in the assembly of Golgi SNARE complexes. It also points towards a possible mechanism behind the hyposialylation of N and O-glycans. This is the first reported patient with pathogenic variants in CAMLG. CAMLG-CDG is the third disorder, after GET4 and GET3 deficiencies, caused by pathogenic variants in a member of the TRC pathway, further expanding this novel group of disorders.

Affinity-Based Enrichment of Extracellular Vesicles with Lipid Nanoprobes.

Extracellular vesicles (EVs) are lipid-bilayer-enclosed vesicles with sub-micrometer size that are released by various cells. EVs contain a tissue-specific signature wherein a variety of proteins and nucleic acids are selectively packaged. Growing evidence has shown important biological roles and clinical relevance of EVs in diseases. For EV-related studies to thrive, rapid efficient isolation of pure EVs is a prerequisite. However, lengthy procedure, low yield, low throughput, and high contaminants stemmed from existing isolation approaches hamper both basic research and large-scale clinical implementation. We have shown that lipid nanoprobes (LNP) enable spontaneous labeling and rapid isolation of EVs by coupling with magnetic enrichment. Recently, we further developed a one-step EV isolation platform that utilizes EV size-matched silica nanostructures and surface-conjugated LNPs with an integrated microfluidic mixer. EVs, derived from up to 2-ml clinical plasma, can be processed with this point-of-care device using optimized flow rate. Subsequently, contents of isolated EVs can be extracted on-chip and eluted from the device for downstream molecular analyses. The LNP-functionalized microfluidic device combined with state-of-the-art analysis platforms could have great potential in promoting EV-centered research and clinical use in the future.

Measuring microtubule binding kinetics of membrane-bound kinesin motors using supported lipid bilayers.

Membrane-bound cargos in cells are generally transported by multiple kinesin motors. Quantifying the bimolecular on-rate of motors for their microtubule track is important for understanding of multi-motor transport but is complicated by diffusion of the motors in the plane of the lipid bilayer. Here, we describe a method to measure the kinesin on-rate that uses a modified microtubule gliding assay performed on a supported lipid bilayer and detects motor binding by a local increase in fluorescence. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2019).

Engineering the bilayer: Emerging genetic tool kits for mechanistic lipid biology.

The structural diversity of lipids underpins the biophysical properties of cellular membranes, which vary across all scales of biological organization. Because lipid composition results from complex metabolic and transport pathways, its experimental control has been a major goal of mechanistic membrane biology. Here, we argue that in the wake of synthetic biology, similar metabolic engineering strategies can be applied to control the composition, physicochemical properties, and function of cell membranes. In one emerging area, titratable expression platforms allow for specific and genome-wide alterations in lipid biosynthetic genes, providing analog control over lipidome stoichiometry in membranes. Simultaneously, heterologous expression of biosynthetic genes and pathways has allowed for gain-of-function experiments with diverse lipids in non-native systems. Finally, we highlight future directions for tool development, including recently discovered lipid transport pathways to intracellular lipid pools. Further tool development providing synthetic control of membrane properties can allow biologists to untangle membrane lipid structure-associated functions.

Out-of-Plane Nanoscale Reorganization of Lipid Molecules and Nanoparticles Revealed by Plasmonic Spectroscopy.

Lipid bilayers assembled on solid substrates have been extensively studied with single-molecule resolution as the constituent molecules diffuse in 2D; however, the out-of-plane motion is typically ignored. Here we present the subnanometer out-of-plane diffusion of nanoparticles attached to hybrid lipid bilayers (HBLs) assembled on metal surfaces. The nanoscale cavity formed between the Au nanoparticle and Au film provides strongly enhanced optical fields capable of locally probing HBLs assembled in the gaps. This allows us to spectroscopically resolve the nanoparticles assembled on bilayers, near edges, and in membrane defects, showing the strong influence of charged lipid rafts. Nanoparticles sitting on the edges of the HBL are observed to flip onto and off of the bilayer, with flip energies of ∼10 meV showing how thermal energies dynamically modify lipid arrangements around a nanoparticle. We further resolve the movement of individual lipid molecules by doping the HBL with low concentrations of Texas Red (TxR) dye-labeled lipids.

Multimodal Multiphoton Imaging of the Lipid Bilayer by Dye-Based Sum-Frequency Generation and Coherent Anti-Stokes Raman Scattering.

Coherent anti-Stokes Raman scattering (CARS) imaging is widely used for imaging molecular vibrations inside cells and tissues. Lipid bilayers are potential analytes for CARS imaging due to their abundant CH2 vibrational bonds. However, identifying the plasma membrane is challenging since it possesses a thin structure and is closely apposed to lipid structures inside the cells. Since the plasma membrane provides the most prominent asymmetric location within cells, orientation sensitive sum-frequency generation (SFG) imaging is a promising technique for selective visualization of the plasma membrane labeled by a nonfluorescent and SFG-specific dye, Ap3, when using a CARS microscope system. In this study, we closely compare the characteristics of lipid bilayer imaging by dye-based SFG and CARS using giant vesicles (GVs) and N27 rat dopaminergic neural cells. As a result, we show that CARS imaging can be exploited for the visualization of whole lipid structures inside GVs and cells but is insufficient for identification of the plasma membrane, which instead can be achieved using dye-based SFG imaging. In addition, we demonstrate that these unique properties can be combined and applied to the live-cell tracking of intracellular lipid structures such as lipid droplets beneath the plasma membrane. Thus, multimodal multiphoton imaging through a combination of dye-based SFG and CARS can serve as a powerful chemical imaging tool to investigate lipid bilayers in GVs and living cells.

Kinetics of lipid raft formation at lipid monolayer-bilayer junction probed by surface plasmon resonance.

A label-free, non-dispruptive, and real-time analytical device to monitor the dynamic features of biomolecules and their interactions with neighboring molecules is an essential prerequisite for biochip- and diagonostic assays. To explore one of the central questions on the lipid-lipid interactions in the course of the liquid-ordered (lo) domain formation, called rafts, we developed a method of reconstituting continuous but spatially heterogeneous lipid membrane platforms with molayer-bilayer juntions (MBJs) that enable to form the lo domains in a spatiotemporally controlled manner. This allows us to detect the time-lapse dynamics of the lipid-lipid interactions during raft formation and resultant membrane phase changes together with the raft-associated receptor-ligand binding through the surface plasmon resonance (SPR). For cross-validation, using epifluorescence microscopy, we demonstrated the underlying mechanisms for raft formations that the infiltration of cholesterols into the sphingolipid-enriched domains plays a crucial roles in the membrane phase-separation. Our membrane platform, being capable of monitoring dynamic interactions among lipids and performing the systematic optical analysis, will unveil physiological roles of cholesterols in a variety of biological events.

Amyloid-ß Peptide-Lipid Bilayer Interaction Investigated by Supercritical Angle Fluorescence.

The understanding of the interaction between the membrane of neurons and amyloid-ß peptides is of crucial importance to shed light on the mechanism of toxicity in Alzheimer's disease. This paper describes how supercritical angle fluorescence spectroscopy was applied to monitor in real-time the interaction between a supported lipid bilayer (SLB) and the peptide. Different forms of amyloid-ß (40 and 42 amino acids composition) were tested, and the interfacial fluorescence was measured to get information about the lipid integrity and mobility. The results show a concentration-dependent damaging process of the lipid bilayer. Prolonged interaction with the peptide up to 48 h lead to an extraction and clustering of lipid molecules from the surface and a potential disruption of the bilayer, correlated with the formation of peptide aggregates. The natural diffusion of the lipid was slightly hindered by the interaction with amyloid-ß(1-42) and closely related to the oligomerization of the peptide. The adsorption and desorption of Amyloid-ß was also characterized in terms of affinity. Amyloid-ß(1-42) exhibited a slightly higher affinity than amyloid-ß(1-40). The former was also more prone to aggregate and to adsorb on the bilayer as oligomer.

Comment on 'Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol'.

The plasma membrane in mammalian cells is rich in cholesterol, but how the cholesterol is partitioned between the two leaflets of the plasma membrane remains a matter of debate. Recently, Liu et al. used domain 4 (D4) of perfringolysin O as a cholesterol sensor to argue that cholesterol is mostly in the exofacial leaflet (Liu et al., 2017). This conclusion was made by interpreting D4 binding in live cells using in vitro calibrations with liposomes. However, liposomes may be unfaithful in mimicking the plasma membrane, as we demonstrate here. Also, D4 binding is highly sensitive to the presence of cytosolic proteins. In addition, we find that a D4 variant, which requires >35 mol% cholesterol to bind to liposomes in vitro, does in fact bind to the cytoplasmic leaflet of the plasma membrane in a cholesterol-dependent manner. Thus, we believe, based on the current evidence, that it is unlikely that there is a significantly higher proportion of cholesterol in the exofacial leaflet of the plasma membrane compared to the cytosolic leaflet.

The architecture of the OmpC-MlaA complex sheds light on the maintenance of outer membrane lipid asymmetry in Escherichia coli.

A distinctive feature of the Gram-negative bacterial cell envelope is the asymmetric outer membrane (OM), where lipopolysaccharides and phospholipids (PLs) reside in the outer and inner leaflets, respectively. This unique lipid asymmetry renders the OM impermeable to external insults, including antibiotics and bile salts. In Escherichia coli, the complex comprising osmoporin OmpC and the OM lipoprotein MlaA is believed to maintain lipid asymmetry by removing mislocalized PLs from the outer leaflet of the OM. How this complex performs this function is unknown. Here, we defined the molecular architecture of the OmpC-MlaA complex to gain insights into its role in PL transport. Using in vivo photo-cross-linking and molecular dynamics simulations, we established that MlaA interacts extensively with OmpC and is located entirely within the lipid bilayer. In addition, MlaA forms a hydrophilic channel, likely enabling PL translocation across the OM. We further showed that flexibility in a hairpin loop adjacent to the channel is critical in modulating MlaA activity. Finally, we demonstrated that OmpC plays a functional role in maintaining OM lipid asymmetry together with MlaA. Our work offers glimpses into how the OmpC-MlaA complex transports PLs across the OM and has important implications for future antibacterial drug development.

Real-Time Quartz Crystal Microbalance Monitoring of Free Docosahexaenoic Acid Interactions with Supported Lipid Bilayers.

Docosahexaenoic acid (DHA) is the most abundant polyunsaturated omega-3 fatty acid found in mammalian neuronal cell membranes. Although DHA is known to be important for neuronal cell survival, little is know about how DHA interacts with phospholipid bilayers. This study presents a detailed quartz crystal microbalance with dissipation monitoring (QCM-D) analysis of free DHA interactions with individual and mixed phospholipid supported lipid bilayers (SLB). DHA incorporation and subsequent changes to the SLBs viscoelastic properties were observed to be concentration-dependent, influenced by the phospholipid species, the headgroup charge, and the presence or absence of calcium ions. It was observed that 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) SLBs incorporated the greatest amount of DHA concentration, whereas the presence of phospholipids, phosphatidylserine (PS), and phosphatidylinositol (PI) in a POPC SLB significantly reduced DHA incorporation and changed the SLBs physicochemical properties. These observations are hypothesized to be due to a substitution event occurring between DHA and phospholipid species. PS domain formation in POPC/PS 8:2 SLBs was observed in the presence of calcium ions, which favored DHA incorporation to a similar level as for a POPC only SLB. The changes in SLB thickness observed with different DHA concentrations are also presented. This work contributes to an understanding of the physical changes induced in a lipid bilayer as a consequence of its exposure to different DHA concentrations (from 50 to 200 µM). The capacity of DHA to influence the physical properties of SLBs indicates the potential for dietary DHA supplementation to cause changes in cellular membranes in vivo, with subsequent physiological consequences for cell function.

Detergent-Free Membrane Protein Purification.

Membrane proteins are localized within a lipid bilayer; in order to purify them for functional and structural studies the first step must involve solubilizing or extracting the protein from these lipids. To date this has been achieved using detergents which disrupt the bilayer and bind to the protein in the transmembrane region. However finding conditions for optimal extraction, without destabilizing protein structure, is time consuming and expensive. Here we present a recently-developed method using a styrene-maleic acid (SMA) co-polymer instead of detergents. The SMA co-polymer extracts membrane proteins in a small disc of lipid bilayer which can be used for affinity chromatography purification, thus enabling the purification of membrane proteins while maintaining their native lipid bilayer environment.

Electrophoretic separation method for membrane pore-forming proteins in multilayer lipid membranes.

In this paper, we report on a novel electrophoretic separation and analysis method for membrane pore-forming proteins in multilayer lipid membranes (MLMs) in order to overcome the problems related to current separation and analysis methods of membrane proteins, and to obtain a high-performance separation method on the basis of specific properties of the lipid membranes. We constructed MLMs, and subsequently characterized membrane pore-forming protein behavior in MLMs. Through the use of these MLMs, we were able to successfully separate and analyze membrane pore-forming proteins in MLMs. To the best of our knowledge, this research is the first example of membrane pore-forming protein separation in lipid membranes. Our method can be expected to be applied for the separation and analysis of other membrane proteins including intrinsic membrane proteins and to result in high-performance by utilizing the specific properties of lipid membranes.

Analytical investigation of bilayer lipid biosensor based on graphene.

Graphene is another allotrope of carbon with two-dimensional monolayer honeycomb. Owing to its special characteristics including electrical, physical and optical properties, graphene is known as a more suitable candidate compared to other materials to be used in the sensor application. It is possible, moreover, to use biosensor by using electrolyte-gated field effect transistor based on graphene (GFET) to identify the alterations in charged lipid membrane properties. The current article aims to show how thickness and charges of a membrane electric can result in a monolayer graphene-based GFET while the emphasis is on the conductance variation. It is proposed that the thickness and electric charge of the lipid bilayer (LLP and QLP) are functions of carrier density, and to find the equation relating these suitable control parameters are introduced. Artificial neural network algorithm as well as support vector regression has also been incorporated to obtain other models for conductance characteristic. The results comparison between analytical models, artificial neural network and support vector regression with the experimental data extracted from previous work show an acceptable agreement.

Quantifying Lateral Inhomogeneity of Cholesterol-Containing Membranes.

Lateral inhomogeneity plays a critical role for many properties of cholesterol-containing membranes, yet the thermodynamic forces involved in inhomogeneity remain poorly understood. Based on coarse-grained simulations of cholesterol in four increasingly unsaturated phospholipids, we computed lateral density fluctuations and free energies of domain formation, and we quantitatively relate those to variations in the chemical potential of cholesterol. Our simulations suggest that the lateral organization is dominated by weak repulsive cholesterol interactions, leading to a significantly more homogeneous distribution as compared to a two-dimensional ideal gas. Hence, phospholipids provide a "good" solvent for cholesterol. Unexpectedly, the degree of unsaturation of the phospholipid has only a minor effect on the lateral inhomogeneity of cholesterol in binary lipid mixtures. These results provide a link between functional properties and thermal fluctuations in lipid membranes.