Lipid Phase Separation Research Articles

Domain-selective disruption and compression of phase-separated lipid vesicles by amphiphilic Janus nanoparticles.

Janus nanoparticles (NPs) with anisotropic surface functionalities enable unique biomedical applications, but their interaction with the biomembranes cannot be predicted by models derived from nanoparticles with uniform surface chemistry. Here, we combine experiments with molecular dynamics (MD) simulations to investigate the interaction of amphiphilic Janus NPs, which are cationic and hydrophobic on opposite sides, with lipid vesicles exhibiting phase-separated microdomains. We demonstrate that Janus NPs preferentially bind to and extract lipids from liquid-disordered domains over a broad range of vesicle compositions. This domain-selective membrane disruption and the inter-particle attractions concurrently generate a compression force on the vesicle, causing the remaining liquid-ordered domains to bulge and the entire vesicle to wrinkle. The NP-induced membrane compression and deformation are critically driven by the surface anisotropy of the Janus NPs. The findings highlight the feasibility of using the surface anisotropy of NPs to tailor their interactions with different biological membranes.

Possible Role of Bent Structure of Methylated Lithocholic Acid on Artificial and Plasma Membranes.

Bile acids form micelles that are essential for the absorption of dietary lipids. However, excessive bile acid micelles can disrupt the plasma membrane by removing phospholipids, resulting in cell death. We hypothesized that the bent geometrical structure of the steroid scaffold of bile acids decreases the lipid order (similar to unsaturated phospholipids with cis double bonds), disrupting the plasma membrane. Here, lithocholic acid (LCA), a bile acid, was methylated to prevent micellization. Methylated lithocholic acid (Me-LCA) was mixed with a thin phase-separated lipid bilayer comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol (Chol). Me-LCA was not localized in the DPPC-rich rigid phase but localized in the DOPC-rich fluid phase, and excess Me-LCA did not affect the phase separation. Me-LCA is distributed in the plasma and organelle membranes. However, Me-LCA with bent structure did not affect the membrane properties, membrane fluidity, and hydrophobicity of liposomes composed of DOPC, DPPC, and Chol and also did not affect the proliferation of cells.

Enzyme-Free Liposome Active Motion via Asymmetrical Lipid Efflux

As a class of biocompatible, water-dispersed colloids, liposomes have found widespread applications ranging from food to drug delivery. Adding mobility to these colloids, i.e., liposome micromotors, represents an attractive approach to next-generation liposome carriers with enhanced functionality and effectiveness. Currently, it remains unclear as to the scope of material features useful for building liposome micromotors or how they may differ functionally from their inorganic/polymer counterparts. In this work, we demonstrate liposome active motion taking advantage of mainly a pair of intrinsic material properties associated with these assemblies: lipid phase separation and extraction. We show that global phase separation of ternary lipid systems (such as DPPC/DOPC/cholesterol) within individual liposomes yields stable Janus particles with two distinctive liquid domains. While these anisotropic liposomes undergo pure Brownian diffusion in water, similar to their homogeneous analogues, adding extracting agents, cyclodextrins, to the system triggers asymmetrical cholesterol efflux about the liposomes, setting the latter into active motion. We present detailed analyses of liposome movement and cholesterol extraction kinetics to establish their correlation. We explore various experimental parameters as well as mechanistic details to account for such motion. Our results highlight the rich possibility to hierarchically design lipid-based artificial motors, from individual lipids, to their organization, surface chemistry, and interfacial mechanics.

Control of Line Tension at Phase-Separated Lipid Domain Boundaries: Monounsaturated Fatty Acids with Different Chain Lengths and Osmotic Pressure.

Line tension at phase-separated lipid domain boundaries is an important factor that governs the stability of the phase separation. We studied the control of the line tension in lipid membranes composed of dioleoylphosphocholine (DOPC), dipalmitoylphosphocholine (DPPC), and cholesterol (Chol) by the addition of the following three monounsaturated fatty acids (MUFAs) with different chain lengths: palmitoleic acid (PaA), oleic acid (OA), and eicosenoic acid (EiA). In addition, we attempted to alter the line tension by applying osmotic pressure. The phase behavior of the MUFA-containing lipid membranes in the presence and absence of osmotic stress was observed by fluorescence and confocal laser scanning microscopy. The line tension was quantitatively measured from the domain boundary fluctuation by flicker spectroscopy, and the interactions between the lipids and MUFAs were examined by differential scanning calorimetry. PaA and OA, which are shorter MUFAs, decreased the line tension, whereas EiA changed the liquid domain to a solid domain. The osmotic pressure increased the line tension, even in the presence of MUFAs. It may be possible to control the line tension by combining the chemical approach of MUFA addition and the physical approach of applying osmotic pressure.

Efficient calculation of the free energy for protein partitioning using restraining potentials

An approach for the efficient simulation of phase-separated lipid bilayers, for use in the calculation of equilibrium free energies of partitioning between lipid domains, is proposed. The methodology exploits restraint potentials and rectangular aspect ratios that enforce lipid phase separation, allowing for the simulation of smaller systems that approximately reproduce bulk behavior. The utility of this approach is demonstrated through the calculation of potentials of mean force for the translation of a transmembrane protein between lipid domains. The impact of the imposed restraints on lipid tail ordering and lipid packing are explored, providing insight into how restraints can best be employed to compute accurate free-energy surfaces. This approach should be useful in the accurate calculation of equilibrium partition coefficients for transmembrane protein partitioning in heterogeneous membranes, providing insight into the thermodynamic driving forces that control this fundamental biophysical phenomenon.

Exploring the binding kinetics and behaviors of self-aggregated beta-amyloid oligomers to phase-separated lipid rafts with or without ganglioside-clusters

Lipid binding kinetics and energetics of self-aggregated and disordered beta-amyloid oligomers of various sizes, from solution to lipid raft surfaces, were investigated using MD simulations. Our systems include small (monomers to tetramers) and larger (octamers and dodecamers) oligomers. Our lipid rafts contain saturated and unsaturated phosphatidylcholine (PC), cholesterol, and with or without asymmetrically distributed monosialotetrahexosylganglioside (GM1). All rafts exhibited dynamic and structurally diversified domains including liquid-ordered (Lo), liquid-disordered (Ld), and interfacial Lod domains. For rafts without GM1, all oligomers bound to the Lod domain. For GM1-containing rafts, all small oligomers and most larger oligomers bound specifically to the GM1-clusters embedded in the Lo domain. Lipid-protein binding energies followed an order of GM1 >> unsaturated PC > saturated PC > cholesterol for all rafts. In addition, protein-induced membrane structural disruption increased progressively with the size of the oligomer for the annular lipids surrounding the membrane-bound protein in non-GM1-containing rafts. We propose that the tight binding of beta-amyloid oligomers to the GM1-clusters and the structural perturbation of lipids surrounding the membrane-bound proteins at the Lod domain are early molecular events of the beta-amyloid aggregation process on neuronal membrane surfaces that trigger the onset of Alzheimer's.

Characterization of Phase Separated Planar Lipid Bilayer Membrane by Fluorescence Ratio Imaging and Scanning Probe Microscope

The lipid membrane forms nanodomains (rafts) and shows heterogeneous properties. These nanodomains relate to significant roles in various cell functions, and thus the analysis of the nanodomains in phase-separated lipid membranes is important to clarify the function and role of the nanodomains. However, the lipid membrane possesses small-sized nanodomains and shows a small height difference between the nanodomains and their surroundings at certain lipid compositions. In addition, nanodomain analysis sometimes requires highly sensitive and expensive apparatus, such as a two-photon microscope. These have prevented the analysis by the conventional fluorescence microscope and by the topography of the scanning probe microscope (SPM), even though these are promising methods in macroscale and microscale analysis, respectively. Therefore, this study aimed to overcome these problems in nanodomain analysis. We successfully demonstrated that solvatochromic dye, LipiORDER, could analyze the phase state of the lipid membrane at the macroscale with low magnification lenses. Furthermore, we could prove that the phase mode of SPM was effective in the visualization of specific nanodomains by properties difference as well as topographic images of SPM. Hence, this combination method successfully gave much information on the phase state at the micro/macro scale, and thus this would be applied to the analysis of heterogeneous lipid membranes.

Membrane asymmetry enhances nanotube formation and limits pore resealing after electroporation

Research in cell membranes provides a fascinating intersection of the natural world, bioengineering, and fundamental physics. Model membranes form by the self-assembly of amphiphilic phospholipids into two-molecule-thick fluid sheets spanning up to tens of microns. Increasingly, model membranes include leaflet asymmetry, lipid phase separation, and spontaneous curvature to enable examination of the interplay of diverse physical characteristics that potentially govern complex natural behaviors. For example, lipid phase separation and leaflet asymmetry both encourage the formation of membrane curvature, but the degree to which living cells exploit these relationships is unknown.

Molecular View on the Impact of DHA Molecules on the Physical Properties of a Model Cell Membrane.

Docosahexaenoic acid (DHA) is a ω-3 polyunsaturated fatty acid, which can be uptaken by cells and is essential for proper neuronal and retinal function. However, the detailed physical impact of DHA molecules on the plasma membrane is still unclear. Hence, in this work, we carried out μs-scale coarse-grained molecular dynamics (MD) simulations to reveal the interactions between DHA molecules and a model cell membrane. As is known, the cell membrane can segregate into liquid-ordered (Lo) and liquid-disordered (Ld) membrane domains due to the differential interactions between lipids and proteins. In order to capture this feature, we adopted the three-component phase-separated lipid membranes and considered both anionic and neutral DHA molecules in the current work. Our results showed that DHA molecules can spontaneously self-assemble into nanoclusters, fuse with lipid membranes, and localize preferably in Ld membrane domains. During the membrane fusion process, DHA molecules can change the intrinsic transmembrane potential of the lipid membrane, and the effects of anionic DHA molecules are much more significant. Besides, the presence of DHA molecules mainly in the Ld membrane domains could regulate the differences in the lipid chain order, membrane thickness, cholesterol preference, and cholesterol flip-flop basically in a concentration-dependent manner, which further promote the stability of the intraleaflet dynamics and inhibit the interleaflet dynamics (or promote membrane domain registration) of the membrane domains. In short, the impact of DHA molecules on the physical properties of a model cell membrane on the molecular level revealed in our work will provide useful insights for understanding the biological functions of DHA molecules.

Spontaneous evolution of equilibrium morphology in phospholipid-cholesterol monolayers.

Competition between intradomain electrostatic repulsions and interdomain line tension leads to domain shape transitions in phase-separating lipid monolayers. The question remains if these morphologies are energy minima or are kinetically trapped metastable states. We show the reversible evolution of uniform width stripe domains from polydisperse semicircular domains in monolayers of dipalmitoylphosphatidylcholine (DPPC), hexadecanol (HD) or palmitic acid (PA), and dihydrocholesterol (DChol). The initial semicircular domains grow at a fixed 2:1 DPPC:HD (or PA) stoichiometry, depleting the liquid phase of HD, leaving behind a liquid enriched in DPPC and DChol. At higher surface pressures, the remaining DPPC precipitates onto existing domains, decreasing the ratio of line tension to the square of the dipole density difference, λ/μ2. Theory predicts that, as λ/μ2 decreases, circular domains reversibly transform to uniform width stripes as the minimum energy structure. Measuring the stripe width provides the first estimates of λ/μ2 at liquid condensed–liquid expanded phase coexistence.

Peptide-Folding Triggered Phase Separation and Lipid Membrane Destabilization in Cholesterol-Rich Lipid Vesicles

Liposome-based drug delivery systems are widely used to improve drug pharmacokinetics but can suffer from slow and unspecific release of encapsulated drugs. Membrane-active peptides, based on sequences derived or inspired from antimicrobial peptides (AMPs), could offer means to trigger and control the release. Cholesterol is used in most liposomal drug delivery systems (DDS) to improve the stability of the formulation, but the activity of AMPs on cholesterol-rich membranes tends to be very low, complicating peptide-triggered release strategies. Here, we show a de novo designed AMP-mimetic peptide that efficiently triggers content release from cholesterol-containing lipid vesicles when covalently conjugated to headgroup-functionalized lipids. Binding to vesicles induces peptide folding and triggers a lipid phase separation, which in the presence of cholesterol results in high local peptide concentrations at the lipid bilayer surface and rapid content release. We anticipate that these results will facilitate the development of peptide-based strategies for controlling and triggering drug release from liposomal drug delivery systems.

Lipid Phase Separation in Vesicles Enhances TRAIL-Mediated Cytotoxicity.

Ligand spatial presentation and density play important roles in signaling pathways mediated by cell receptors and are critical parameters when designing protein-conjugated therapeutic nanoparticles. Here, we harness lipid phase separation to spatially control the protein presentation on lipid vesicles. We use this system to improve the cytotoxicity of TNF-related apoptosis inducing ligand (TRAIL), a therapeutic anticancer protein. Vesicles with phase-separated TRAIL presentation induce more cell death in Jurkat cancer cells than vesicles with uniformly presented TRAIL, and cytotoxicity is dependent on TRAIL density. We assess this relationship in other cancer cell lines and demonstrate that phase-separated vesicles with TRAIL only enhance cytotoxicity through one TRAIL receptor, DR5, while another TRAIL receptor, DR4, is less sensitive to TRAIL density. This work demonstrates a rapid and accessible method to control protein conjugation and density on vesicles that can be adopted to other nanoparticle systems to improve receptor signaling by nanoparticles.

Imidazole-Based pH-Sensitive Convertible Liposomes for Anticancer Drug Delivery.

In efforts to enhance the activity of liposomal drugs against solid tumors, three novel lipids that carry imidazole-based headgroups of incremental basicity were prepared and incorporated into the membrane of PEGylated liposomes containing doxorubicin (DOX) to render pH-sensitive convertible liposomes (ICL). The imidazole lipids were designed to protonate and cluster with negatively charged phosphatidylethanolamine-polyethylene glycol when pH drops from 7.4 to 6.0, thereby triggering ICL in acidic tumor interstitium. Upon the drop of pH, ICL gained more positive surface charges, displayed lipid phase separation in TEM and DSC, and aggregated with cell membrane-mimetic model liposomes. The drop of pH also enhanced DOX release from ICL consisting of one of the imidazole lipids, sn-2-((2,3-dihexadecyloxypropyl)thio)-5-methyl-1H-imidazole. ICL demonstrated superior activities against monolayer cells and several 3D MCS than the analogous PEGylated, pH-insensitive liposomes containing DOX, which serves as a control and clinical benchmark. The presence of cholesterol in ICL enhanced their colloidal stability but diminished their pH-sensitivity. ICL with the most basic imidazole lipid showed the highest activity in monolayer Hela cells; ICL with the imidazole lipid of medium basicity showed the highest anticancer activity in 3D MCS. ICL that balances the needs of tissue penetration, cell-binding, and drug release would yield optimal activity against solid tumors.

Lipid-driven CFTR clustering is impaired in cystic fibrosis and restored by corrector drugs.

ABSTRACTMembrane proteins often cluster in nanoscale membrane domains (lipid rafts) that coalesce into ceramide-rich platforms during cell stress, however the clustering mechanisms remain uncertain. The cystic fibrosis transmembrane conductance regulator (CFTR), which is mutated in cystic fibrosis (CF), forms clusters that are cholesterol dependent and become incorporated into long-lived platforms during hormonal stimulation. We report here that clustering does not involve known tethering interactions of CFTR with PDZ domain proteins, filamin A or the actin cytoskeleton. It also does not require CFTR palmitoylation but is critically dependent on membrane lipid order and is induced by detergents that increase the phase separation of membrane lipids. Clustering and integration of CFTR into ceramide-rich platforms are abolished by the disease mutations F508del and S13F and rescued by the CFTR modulators elexacaftor plus tezacaftor. These results indicate CF therapeutics that correct mutant protein folding restore both trafficking and normal lipid interactions in the plasma membrane.This article has an associated First Person interview with the first author of the paper.

Seipin forms a flexible cage at lipid droplet formation sites

Lipid droplets (LDs) form in the endoplasmic reticulum by phase separation of neutral lipids. This process is facilitated by the seipin protein complex, which consists of a ring of seipin monomers, with a yet unclear function. Here, we report a structure of S. cerevisiae seipin based on cryogenic-electron microscopy and structural modeling data. Seipin forms a decameric, cage-like structure with the lumenal domains forming a stable ring at the cage floor and transmembrane segments forming the cage sides and top. The transmembrane segments interact with adjacent monomers in two distinct, alternating conformations. These conformations result from changes in switch regions, located between the lumenal domains and the transmembrane segments, that are required for seipin function. Our data indicate a model for LD formation in which a closed seipin cage enables triacylglycerol phase separation and subsequently switches to an open conformation to allow LD growth and budding.

Fluidity is the way to life: lipid phase separation in bacterial membranes.

A hallmark of biological membranes is the dynamic localization of lipids and proteins. Lipids respond to temperature reduction below a critical point with phase separation, and poikilothermic animals and also bacteria adapt their lipid content to prevent gel phase formation in membranes. In a new study, Gohrbandt etal (2022) show that reduced membrane fluidity in bacterial cells causes reversible phase separation without membrane rupture invivo, highlighting the physical robustness of biological membranes.

Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria

All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane‐adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel‐fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis, we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large‐scale lipid phase separation and protein segregation in intact, protein‐crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase separation process induced by low fluidity is biologically reversible.

Key Factors Governing Initial Stages of Lipid Droplet Formation.

Lipid droplets (LDs) are neutral lipid storage organelles surrounded by a phospholipid (PL) monolayer. LD biogenesis from the endoplasmic reticulum is driven by phase separation of neutral lipids, overcoming surface tension and membrane deformation. However, the core biophysics of the initial steps of LD formation remains relatively poorly understood. Here, we use a tunable, phenomenological coarse-grained model to study triacylglycerol (TG) nucleation in a bilayer membrane. We show that PL rigidity has a strong influence on TG lensing and membrane remodeling: when membrane rigidity increases, TG clusters remain more planar with high anisotropy but a minor degree of phase nucleation. This finding is confirmed by advanced sampling simulations that calculate nucleation free energy as a function of the degree of nucleation and anisotropy. We also show that asymmetric tension, controlled by the number of PL molecules on each membrane leaflet, determines the budding direction. A TG lens buds in the direction of the monolayer containing excess PL molecules to allow for better PL coverage of TG, consistent with the reported experiments. Finally, two governing mechanisms of the LD growth, Ostwald ripening and merging, are observed. Taken together, this study characterizes the interplay between two thermodynamic quantities during the initial LD phases, the TG bulk free energy and membrane remodeling energy.

Domain dynamics of phase-separated lipid membranes under shear flow.

The dynamical behaviour of lateral domains on phase-separated lipid vesicles under external flow is reported. A microfluidic chamber was used for the immobilization of vesicles and the application of shear. Microscopic observation revealed that domains tended to be localized at the vortex center and to exhibit a stripe morphology as the flow speed increased. We clarified the dependency of domain behaviors on the flow speed and lipid mixing fraction. The cholesterol ratio in the membrane affected these domain behaviors. Next, we investigated the growth of domains under flow. We discuss the mechanism of these trends by considering the free energy of phase separation, and reproduce the experimental results by numerical simulations. These findings may lead to a better understanding of the dynamical properties of the membrane under nonequilibrium situations and the biophysical mechanism of cellular mechanotransduction.

Assessing the use of ellipsoidal microparticles for determining lipid membrane viscosity

The viscosity of lipid membranes sets the timescales of membrane-associated motions, whether driven or diffusive, and therefore influences the dynamics of a wide range of cellular processes. Techniques to measure membrane viscosity remain sparse, however, and reported measurements to date, even of similar systems, give viscosity values that span orders of magnitude. To address this, we improve a method based on measuring both the rotational and translational diffusion of membrane-anchored microparticles and apply this approach and one based on tracking the motion of phase-separated lipid domains to the same system of phase-separated giant vesicles. We find good agreement between the two methods, with inferred viscosities within a factor of two of each other. Our single-particle tracking technique uses ellipsoidal microparticles, and we show that the extraction of physically meaningful viscosity values from their motion requires consideration of their anisotropic shape. The validation of our method on phase-separated membranes makes possible its application to other systems, which we demonstrate by measuring the viscosity of bilayers composed of lipids with different chain lengths ranging from 14 to 20 carbon atoms, revealing a very weak dependence of two-dimensional viscosity on lipid size. The experimental and analysis methods described here should be generally applicable to a variety of membrane systems, both reconstituted and cellular.