Lipid Domain Formation Research Articles

The cytoplasmic tail of myelin protein zero induces morphological changes in lipid membranes

The major myelin protein expressed by the peripheral nervous system Schwann cells is protein zero (P0), which represents 50% of the total protein content in myelin. This 30-kDa integral membrane protein consists of an immunoglobulin (Ig)-like domain, a transmembrane helix, and a 69-residue C-terminal cytoplasmic tail (P0ct). The basic residues in P0ct contribute to the tight packing of myelin lipid bilayers, and alterations in the tail affect how P0 functions as an adhesion molecule necessary for the stability of compact myelin. Several neurodegenerative neuropathies are related to P0, including the more common Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS) as well as rare cases of motor and sensory polyneuropathy. We found that high P0ct concentrations affected the membrane properties of bicelles and induced a lamellar-to-inverted hexagonal phase transition, which caused bicelles to fuse into long, protein-containing filament-like structures. These structures likely reflect the formation of semicrystalline lipid domains with potential relevance for myelination. Not only is P0ct important for stacking lipid membranes, but time-lapse fluorescence microscopy also shows that it might affect membrane properties during myelination. We further describe recombinant production and low-resolution structural characterization of full-length human P0. Our findings shed light on P0ct effects on membrane properties, and with the successful purification of full-length P0, we have new tools to study the role of P0 in myelin formation and maintenance in vitro.

The mechanistic basis of the membrane-permeabilizing activities of the virulence-associated protein A (VapA) from Rhodococcus equi.

Pathogenic Rhodococcus equi release the virulence-associated protein A (VapA) within macrophage phagosomes. VapA permeabilizes phagosome and lysosome membranes and reduces acidification of both compartments. Using biophysical techniques, we found that VapA interacts with model membranes in four steps: (i) binding, change of mechanical properties, (ii) formation of specific membrane domains, (iii) permeabilization within the domains, and (iv) pH-specific transformation of domains. Biosensor data revealed that VapA binds to membranes in one step at pH 6.5 and in two steps at pH 4.5 and decreases membrane fluidity. The integration of VapA into lipid monolayers was only significant at lateral pressures <20 mN m-1 indicating preferential incorporation into membrane regions with reduced integrity. Atomic force microscopy of lipid mono- and bilayers showed that VapA increased the surface heterogeneity of liquid disordered domains. Furthermore, VapA led to the formation of a new microstructured domain type and, at pH 4.5, to the formation of 5 nm high domains. VapA binding, its integration and lipid domain formation depended on lipid composition, pH, protein concentration and lateral membrane pressure. VapA-mediated permeabilization is clearly distinct from that caused by classical microbial pore formers and is a key contribution to the multiplication of Rhodococcus equi in phagosomes.

The Membrane-Mediated Interaction of Liquid-Ordered Lipid Domains in the Presence of Amphipathic Peptides.

The lipid membranes of living cells are composed of a large number of lipid types and can undergo phase separation with the formation of nanometer-scale liquid-ordered lipid domains, also called rafts. Raft coalescence, i.e., the fusion of lipid domains, is involved in important cell processes, such as signaling and trafficking. In this work, within the framework of the theory of elasticity of lipid membranes, we explore how amphipathic peptides adsorbed on lipid membranes may affect the domain-domain fusion processes. We show that the elastic deformations of lipid membranes drive amphipathic peptides to the boundary of lipid domains, which leads to an increase in the average energy barrier of the domain-domain fusion, even if the surface concentration of amphipathic peptides is low and the domain boundaries are only partially occupied by the peptides. This inhibition of the fusion of lipid domains may lead to negative side effects of using amphipathic peptides as antimicrobial agents.

Unravelling the role of lipid composition on liposome-protein interactions

Upon in vivo administration of nanoparticles, a protein corona forms on their surface and affects their half-life in circulation, biodistribution properties, and stability; in turn, the composition of the protein corona depends on the physico-chemical properties of the nanoparticles. We have previously observed lipid composition-dependent in vitro and in vivo microRNA delivery from lipid nanoparticles. Here, we carried out an extensive physico-chemical characterisation to understand the role of the lipid composition on the in vivo fate of lipid-based nanoparticles. We used a combination of differential scanning calorimetry (DSC), membrane deformability measurements, isothermal titration calorimetry (ITC), and dynamic light scattering (DLS) to probe the interactions between the nanoparticle surface and bovine serum albumin (BSA) as a model protein. The lipid composition influenced membrane deformability, improved lipid intermixing, and affected the formation of lipid domains while BSA binding to the liposome surface was affected by the PEGylated lipid content and the presence of cholesterol. These findings highlight the importance of the lipid composition on the protein-liposome interaction and provide important insights for the design of lipid-based nanoparticles for drug delivery applications.

Phase separation on giant liposomes prepared by gel-assisted swelling.

Giant unilamellar vesicles (GUVs) are often used as models to study lipid membrane structure and function. The most common method for GUV formation is electroformation, though this technique has its own inherent drawbacks such as low yield in ionic solutions or in the presence of charged lipids. In recent years, the method of gel-assisted swelling has been explored as an alternative for electroformation and has been reported to overcome some of the limitations of this technique. This study aims to assess the ability of gel-assisted swelling to form GUVs of ternary lipid compositions that are known to undergo phase separation. To this end, we utilize different ratios of a 3-component mixture of DOPC/DPPC/Cholesterol to form vesicles capable of separating into phases of liquid-ordered (Lo) and liquid-disordered (Ld). This lipid composition was chosen as previous studies have shown that at room temperature, many of its possible compositions exhibit distinct phase separation. We apply confocal fluorescence microscopy to evaluate the resultant GUVs, in terms of yield, size, and phase behavior. We compare the results of this method to that of electroformation with a focus on GUV size and quantity, as well as formation of lipid domains, their number and area fraction on GUVs. Ultimately, the results show that agarose gel-assisted swelling can parallel electroformation in forming phase-separating GUVs over a series of ternary lipid compositions.

Lateral compression of lipids drives transbilayer coupling of protein condensates.

Protein phase separation that is restricted to a two-dimensional lipid membrane has recently gained much attention due to its involvement in various cellular processes. Two popular in vitro model membrane systems, giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs), have been used to study it. However, due to the accessibility to only one side of the membrane in these systems, protein phase separation on both sides of the membrane remains unexplored. Here, we introduce a freestanding planar lipid membrane array as an effective platform to study membrane-associated protein phase separation. Protein condensates on the membrane display liquid-like features, and environmental stimuli, such as salt concentration and temperature, effectively control the observed phase separation results in our system with the application of several intrinsically disordered proteins. Interestingly, we observe transbilayer coupling of protein condensates such that protein-rich domains on one side of the membrane are found to colocalize with those on the other side of the membrane. How do these protein phases communicate across the lipid bilayer? Based on data from lipid probe partitioning and photobleaching experiments, we hypothesize that protein condensation on the membrane exerts lateral compressive stress on the underlying lipids, driving the formation of lipid domains with reduced fluidity. Specifically, lipid domains with reduced fluidity and increased lipid packing, formed by protein condensation on each membrane leaflet, are attracted to one another, leading to transbilayer coupling of protein condensates. These findings suggest a previously unknown mechanism by which cellular signals from one membrane leaflet, triggered by membrane-associated protein phase separation, can be transferred to the opposing leaflet.

Applying machine learning to investigate lipid domain formation behaviors in the presence of amyloidogenic proteins.

Cell membranes are not homogeneous but contain lipid nanodomains distributed on both leaflets of the lipid bilayers. Using microsecond molecular dynamics (MD) simulations, we have simulated phase-separated lipid bilayers (raft membranes) containing lipid nanodomains that mimic the heterogeneous structures of neuronal plasma membranes. Our recent studies indicate that these lipid nanodomains, e.g., the mixed liquid-ordered (Lo) and liquid-disordered (Ld) boundaries or Lod and ganglioside-cluster, represent non-specific and specific membrane damage targets of amyloidogenic protein, e.g., tau, aggregates (oligomers), in raft membranes. However, a robust computational tool to classify lipids into these lipid nanodomains and quantitate the spatial and temporal evolutions of the domain structures in the presence of amyloidogenic oligomers is not available. In this study, we have modified two unsupervised Machine Learning (ML) algorithms, Non-negative Matrix Factorization (NMFk) and K-Mean Clustering, to investigate lipid domain formation behaviors. These algorithms are currently used in mutation analysis of cancer genomes and feature extractions. In this work, NMFk was used to decompose our time-resolved lipid-lipid proximity data matrix into two latent “signature” and “activity” matrices, which contain a set of probability configurations and the time-dependent activity of those configurations, respectively, of the lipids participating in each nanodomain, e.g., Lo and Lod. On the other hand, by implementing additional spatial information to the dataset used in NMFk, the K-Mean Clustering sorts lipids into nanodomains and determines the latent temporal and spatial evolutions of each domain. Hence, by combining both ML algorithms, this study provides new insights into understanding protein-induced membrane damage mechanisms of amyloidogenic oligomers associated with lipid domain disruption behaviors that may contribute to the pathogenesis of Alzheimer's. (Supported by NSF [OAC 153159] and Welch Foundation [W-2057-2021-327].)

Abstract 2471: Dissecting the role of sterol structure to lipid domain formation in living cells

Abstract 2471: Dissecting the role of sterol structure to lipid domain formation in living cells

Domain formation in model lipid–cholesterol liquid-crystalline aggregation

ABSTRACT To understand the mechanism of cell-membrane compartmentalisation, we studied coarse-grained model systems consisting of lipid and cholesterol molecules. Cholesterol plays crucial role in lateral phase segregation in the bilayered lipid membrane to form cholesterol-rich liquid-ordered domains. In this NVT Molecular Dynamics simulation study, we have investigated the role of cholesterol in lipid bilayer destabilisation and domain formation. This study also reveals that the strength of cholesterol interaction is an important factor behind the phase segregation to form cholesterol-rich domains, whereas the relative dipolar strength of lipid and cholesterol molecules along with the concentration of cholesterol controls the formation of multi-bilayered stack.

Staphylococcus aureus Carotenoids Modulate the Thermotropic Phase Behavior of Model Systems That Mimic Its Membrane Composition.

Staphylococcus aureus (S. aureus) is a pathogenic gram-positive bacterium that normally resides in the skin and nose of the human body. It is subject to fluctuations in environmental conditions that may affect the integrity of the membrane. S. aureus produces carotenoids, which act as antioxidants. However, these carotenoids have also been implicated in modulating the biophysical properties of the membrane. Here, we investigate how carotenoids modulate the thermotropic phase behavior of model systems that mimic the phospholipid composition of S. aureus. We found that carotenoids depress the main phase transition of DMPG and CL, indicating that they strongly affect cooperativity of membrane lipids in their gel phase. In addition, carotenoids modulate the phase behavior of mixtures of DMPG and CL, indicating that they may play a role in modulation of lipid domain formation in S. aureus membranes.

Interplay of receptor-ligand binding and lipid domain formation during cell adhesion.

Cell adhesion involved in biological processes such as cell migration, immune responses, and cancer metastasis, is mediated by the specific binding of receptor and ligand proteins. Some of these proteins exhibit affinity for nanoscale lipid clusters in cell membranes. A key question is how these nanoscale lipid clusters influence and react to the receptor-ligand binding during cell adhesion. In this article, we review recent computational studies that shed new light on the interplay of the receptor-ligand binding and the formation of lipid domains in adhering membranes. These studies indicate that the receptor-ligand binding promotes coalescence of lipid clusters into mesoscale domains, which, in turn, enhances both the affinity and cooperativity of the receptor-ligand binding in cell-cell adhesion with mobile ligands. In contrast, in the case of cell-extracellular matrix adhesion with immobile ligands, the receptor-ligand binding and the lipid cluster coalescence can be correlated or anti-correlated, depending strongly on the ligand distribution. These findings deepen our understanding of correlations between cell adhesion and membrane heterogeneities.

Advanced DNA Zipper Probes for Detecting Cell Membrane Lipid Domains.

The cell membrane is a complex mixture of lipids, proteins, and other components. By forming dynamic lipid domains, different membrane molecules can selectively interact with each other to control cell signaling. Herein, we report several new types of lipid-DNA conjugates, termed as "DNA zippers", which can be used to measure cell membrane dynamic interactions and the formation of lipid domains. Dependent on the choice of lipid moieties, cholesterol- and sphingomyelin-conjugated DNA zippers specifically locate in and detect membrane lipid-ordered domains, while in contrast, a tocopherol-DNA zipper can be applied for the selective imaging of lipid-disordered phases. These versatile and programmable probes can be further engineered into membrane competition assays to simultaneously detect multiple types of membrane dynamic interactions. These DNA zipper probes can be broadly used to study the correlation between lipid domains and various cellular processes, such as the epithelial-mesenchymal transition.

Mechanochemical coupling of lipid organization and protein function through membrane thickness deformations

Cell membranes are composed of a great variety of protein and lipid species with distinct unperturbed hydrophobic thicknesses. To achieve hydrophobic matching, the lipid bilayer tends to deform around membrane proteins so as to match the protein hydrophobic thickness at bilayer-protein interfaces. Such protein-induced distortions of the lipid bilayer hydrophobic thickness incur a substantial energy cost that depends critically on the bilayer-protein hydrophobic mismatch, while distinct conformational states of membrane proteins often show distinct hydrophobic thicknesses. As a result, hydrophobic interactions between membrane proteins and lipids can yield a rich interplay of lipid-protein organization and transitions in protein conformational state. We combine here the membrane elasticity theory of protein-induced lipid bilayer thickness deformations with the Landau-Ginzburg theory of lipid domain formation to systematically explore the coupling between local lipid organization, lipid and protein hydrophobic thickness, and protein-induced lipid bilayer thickness deformations in membranes with heterogeneous lipid composition. We allow for a purely mechanical coupling of lipid and protein composition through the energetics of protein-induced lipid bilayer thickness deformations as well as a chemical coupling driven by preferential interactions between particular lipid and protein species. We find that the resulting lipid-protein organization can endow membrane proteins with diverse and controlled mechanical environments that, via protein-induced lipid bilayer thickness deformations, can strongly influence protein function. The theoretical approach employed here provides a general framework for the quantitative prediction of how membrane thickness deformations influence the joint organization and function of lipids and proteins in cell membranes.

Fluorescence Studies of Nicotinic Acetylcholine Receptor and Its Associated Lipid Milieu: The Influence of Erwin London's Methodological Approaches.

Erwin London dedicated considerable effort to understanding lipid interactions with membrane-resident proteins and how these interactions shaped the formation and maintenance of lipid phases and domains. In this endeavor, he developed ad hoc techniques that greatly contributed to advancements in the field. We have employed and/or modified/extended some of his methodological approaches and applied them to investigate lipid interaction with the nicotinic acetylcholine receptor (nAChR) protein, the paradigm member of the superfamily of rapid pentameric ligand-gated ion channels (pLGIC). Our experimental systems ranged from purified receptor protein reconstituted into synthetic lipid membranes having known effects on receptor function, to cellular systems subjected to modification of their lipid content, e.g., varying cholesterol levels. We have often employed fluorescence techniques, including fluorescence quenching of diphenylhexatriene (DPH) extrinsic fluorescence and of nAChR intrinsic fluorescence by nitroxide spin-labeled phospholipids, DPH anisotropy, excimer formation of pyrene-phosphatidylcholine, and Förster resonance energy transfer (FRET) from the protein moiety to the extrinsic probes Laurdan, DPH, or pyrene-phospholipid to characterize various biophysical properties of lipid-receptor interactions. Some of these strategies are revisited in this review. Special attention is devoted to the anionic phospholipid phosphatidic acid (PA), which stabilizes the functional resting form of the nAChR. The receptor protein was shown to organize its PA-containing immediate microenvironment into microdomains with high lateral packing density and rigidity. PA and cholesterol appear to compete for the same binding sites on the nAChR protein.

Influence of ceramide on lipid domain stability studied with small-angle neutron scattering: The role of acyl chain length and unsaturation

Ceramides and diacylglycerols are groups of lipids capable of nucleating and stabilizing ordered lipid domains, structures that have been implicated in a range of biological processes. Previous studies have used fluorescence reporter molecules to explore the influence of ceramide acyl chain structure on sphingolipid-rich ordered phases. Here, we use small-angle neutron scattering (SANS) to examine the ability of ceramides and diacylglycerols to promote lipid domain formation in the well-characterized domain-forming mixture DPPC/DOPC/cholesterol. SANS is a powerful, probe-free technique for interrogating membrane heterogeneity, as it is differentially sensitive to hydrogen’s stable isotopes protium and deuterium. Specifically, neutron contrast is generated through selective deuteration of lipid species, thus enabling the detection of nanoscopic domains enriched in deuterated saturated lipids dispersed in a matrix of protiated unsaturated lipids. Using large unilamellar vesicles, we found that upon replacing 10 mol% DPPC with either C16:0 or C18:0 ceramide, or 16:0 diacylglycerol (dag), lipid domains persisted to higher temperatures. However, when DPPC was replaced with short chain (C6:0 or C12:0) or very long chain (C24:0) ceramides, or ceramides with unsaturated acyl chains of any length (C6:1(3), C6:1(5), C18:1, and C24:1), as well as C18:1-dag, lipid domains were destabilized, melting at lower temperatures than those in the DPPC/DOPC/cholesterol system. These results show how ceramide acyl chain length and unsaturation influence lipid domains and have implications for how cell membranes might modify their function through the generation of different ceramide species.

The effect of sterols on the structure and dynamics of membrane domains and the action of polyenes

We will present the results of the effect that sterols (ergosterol and cholesterol) have on the formation, size and mechanical response of gel-phase lipid domains that form in a bilayer composed of POPC/egg SM/sterol at different mole fractions of sterol studied by atomic force microscopy imaging and force versus distance curves. Bilayers formed by a mix of POPC/egg SM that show phase segregation in the form of a thicker bilayer that is around 2 μm in diameter. Addition of sterol reduces the size of the thicker membrane domains until the bilayer looks homogeneous.

Certain, but Not All, Tetraether Lipids from the Thermoacidophilic Archaeon Sulfolobus acidocaldarius Can Form Black Lipid Membranes with Remarkable Stability and Exhibiting Mthk Channel Activity with Unusually High Ca2+ Sensitivity.

Bipolar tetraether lipids (BTL) have been long thought to play a critical role in allowing thermoacidophiles to thrive under extreme conditions. In the present study, we demonstrated that not all BTLs from the thermoacidophilic archaeon Sulfolobus acidocaldarius exhibit the same membrane behaviors. We found that free-standing planar membranes (i.e., black lipid membranes, BLM) made of the polar lipid fraction E (PLFE) isolated from S. acidocaldarius formed over a pinhole on a cellulose acetate partition in a dual-chamber Teflon device exhibited remarkable stability showing a virtually constant capacitance (~28 pF) for at least 11 days. PLFE contains exclusively tetraethers. The dominating hydrophobic core of PLFE lipids is glycerol dialky calditol tetraether (GDNT, ~90%), whereas glycerol dialkyl glycerol tetraether (GDGT) is a minor component (~10%). In sharp contrast, BLM made of BTL extracted from microvesicles (Sa-MVs) released from the same cells exhibited a capacitance between 36 and 39 pF lasting for only 8 h before membrane dielectric breakdown. Lipids in Sa-MVs are also exclusively tetraethers; however, the dominating lipid species in Sa-MVs is GDGT (>99%), not GDNT. The remarkable stability of BLMPLFE can be attributed to strong PLFE–PLFE and PLFE–substrate interactions. In addition, we compare voltage-dependent channel activity of calcium-gated potassium channels (MthK) in BLMPLFE to values recorded in BLMSa-MV. MthK is an ion channel isolated from a methanogenic that has been extensively characterized in diester lipid membranes and has been used as a model for calcium-gated potassium channels. We found that MthK can insert into BLMPLFE and exhibit channel activity, but not in BLMSa-MV. Additionally, the opening/closing of the MthK in BLMPLFE is detectable at calcium concentrations as low as 0.1 mM; conversely, in diester lipid membranes at such a low calcium concentration, no MthK channel activity is detectable. The differential effect of membrane stability and MthK channel activity between BLMPLFE and BLMSa-MV may be attributed to their lipid structural differences and thus their abilities to interact with the substrate and membrane protein. Since Sa-MVs that bud off from the plasma membrane are exclusively tetraether lipids but do not contain the main tetraether lipid component GDNT of the plasma membrane, domain segregation must occur in S. acidocaldarius. The implication of this study is that lipid domain formation is existent and functionally essential in all kinds of cells, but domain formation may be even more prevalent and pronounced in hyperthermophiles, as strong domain formation with distinct membrane behaviors is necessary to counteract randomization due to high growth temperatures while BTL in general make archaea cell membranes stable in high temperature and low pH environments whereas different BTL domains play different functional roles.

Protein induced lipid demixing in homogeneous membranes

Specific lipid environments are necessary for the establishment of protein signaling platforms in membranes, yet their origin has been highly debated. We present a continuum, exactly solvable model of protein induced local demixing of lipid membranes. The coupling between a local composition and a local thickness of the membrane induces lipid domains around inclusions with hydrophobic mismatch, even for temperatures above the miscibility critical point of the membrane. The model qualitatively explains the experimentally observed formation of lipid domains induced by anchoring of reconstituted actin in flat supported lipid bilayers.

Lateral organization of biomimetic cell membranes in varying pH conditions

Many studies have been devoted to investigation of phase separation and formation of lipid domains, which play crucial role in many biological processes. Here we present a complex study on the formation, dynamics, and stability of the phase-separated supported lipid membranes under varying pH conditions. The size and distribution of liquid-ordered (Lo) phase domains were investigated in a wide range (1.7–9.0) of buffer pH values and a strong correlation was found between the size of the Lo domains and pH of the buffer hydrating the lipid bilayer. Interestingly, the dynamics of lipids composing both Lo and Ld phase are insensitive to the pH of the buffer. Our findings demonstrate that by varying pH of the environment one can induce formation of domains with a specific size and shape without any external modification of the solid support or altering the membrane composition. Finally, we show that the architecture of the lipid membrane is stable even upon replacement of the aqueous medium with the buffer of neutral pH. Consequently, this method of patterning of Lo phase domains in biomimetic membranes is applicable to the studies involving binding of proteins or incorporation of other pH-sensitive molecules.

Increased Levels of Short-Chain Ceramides Modify the Lipid Organization and Reduce the Lipid Barrier of Skin Model Membranes

The skin barrier function is attributed to the stratum corneum (SC) intercellular lipid matrix, which is composed primarily of ceramides (CERs), free fatty acids, and cholesterol. These lipids are organized in two lamellar phases: the short and long periodicity phases (SPP and LPP), respectively. The LPP is considered important for the skin barrier function. High levels of short-chain CERs are observed in various inflammatory skin diseases and have been correlated with barrier dysfunction. In this research, we investigated how the increase in the fraction of the short-chain CER with a nonhydroxy C16 acyl chain linked to a C18 sphingosine base CER NS(C16) at the expense of the physiological chain length CER NS with a C24 acyl chain (CER NS(C24)) impacts the microstructure and barrier function of a lipid model that mimicked certain characteristics of the SC lipid organization. The permeability and lipid organization of the model membranes were compared with that of a control model without CER NS(C16). The permeability increased significantly when ≥50% of CER NS(C24) was substituted with CER NS(C16). Employing biophysical techniques, we showed that the lipid packing density reduced with an increasing proportion of CER NS(C16). Substitution of 75% of CER NS(C24) by CER NS(C16) resulted in the formation of phase-separated lipid domains and alteration of the LPP structure. Using deuterium-labeled lipids enabled simultaneous characterization of the C24 and C16 acyl chains in the lipid models, providing insight into the mechanisms underlying the reduced skin barrier function in diseased skin.