Line tension and stability of domains in cell-adhesion zones mediated by long and short receptor-ligand complexes

Submicron scale domains of membrane-anchored receptors play an important role in cell signaling. Central questions concern the stability of these microdomains, and the mechanisms leading to the domain formation. In immune-cell adhesion zones, microdomains of short receptor-ligand complexes form next to domains of significantly longer receptor-ligand complexes. The length mismatch between the receptor-ligand complexes leads to membrane deformations and has been suggested as a possible cause of the domain formation. The domain formation is a nucleation and growth process that depends on the line tension and free energy of the domains. Using a combination of analytical calculations and Monte Carlo simulations, we derive here general expressions for the line tension between domains of long and short receptor-ligand complexes and for the adhesion free energy of the domains. We argue that the length mismatch of receptor-ligand complexes alone is sufficient to drive the domain formation, and obtain submicron-scale minimum sizes for stable domains that are consistent with the domain sizes observed during immune-cell adhesion.     

Flicker spectroscopy of thermal lipid bilayer domain boundary fluctuations

This contribution describes measurements of lipid bilayer domain line tension based on two-dimensional thermal undulations of membranes with liquid ordered/liquid disordered phase coexistence and near-critical composition at room temperature. Lateral inhomogeneity of lipid and protein composition is currently a subject of avid research aimed at determining both fundamental properties and biological relevance of membrane domains. Line tension at fluid lipid bilayer membrane domain boundaries controls the kinetics of domain growth and therefore regulates the size of compositional heterogeneities. High line tension promotes membrane domain budding and fission. Line tension could therefore be an important control parameter regulating functional aspects of biological membranes. Here the established method of fluid domain flicker spectroscopy is applied to examine thermal domain wall fluctuations of phase-separated bilayer membranes. We find a Gaussian probability distribution for the first few excited mode amplitudes, which permits an analysis by means of appropriately specialized capillary wave theory. Time autocorrelation functions are found to decay exponentially, and relaxation times are fitted by means of a hydrodynamic theory relating line tensions and excited mode relaxation kinetics. Line tensions below 1 pN are obtained, with these two approaches yielding similar results. We examine experimental artifacts that perturb the Fourier spectrum of domain traces and discuss ways to identify the number of modes that yield reliable line tension information.     

Coupling between line tension and domain contact angle in heterogeneous membranes

The compositional differences between domains in phase-separated membranes are associated with differences in bilayer thickness and moduli. The resulting packing deformation at the phase boundary gives rise to a line tension, the one dimensional equivalent of surface tension. In this paper we calculate the line tension between a large membrane domain and a continuous phase as a function of the thickness mismatch and the contact angle between the phases. We find that the packing-induced line tension is sensitive to the contact angle, reaching a minimum at a specific value. The difference in the line tension between a flat domain (that is within the plane of the continuous phase) and a domain at the optimal contact angle may be of order 40%. This could explain why previous calculations of the thickness mismatch based line tension tend to yield values that are higher than those measured experimentally.     

Coupling between line tension and domain contact angle in heterogeneous membranes

The compositional differences between domains in phase-separated membranes are associated with differences in bilayer thickness and moduli. The resulting packing deformation at the phase boundary gives rise to a line tension, the one dimensional equivalent of surface tension. In this paper we calculate the line tension between a large membrane domain and a continuous phase as a function of the thickness mismatch and the contact angle between the phases. We find that the packing-induced line tension is sensitive to the contact angle, reaching a minimum at a specific value. The difference in the line tension between a flat domain (that is within the plane of the continuous phase) and a domain at the optimal contact angle may be of order 40%. This could explain why previous calculations of the thickness mismatch based line tension tend to yield values that are higher than those measured experimentally.     

Lateral redistribution of transmembrane proteins and liquid-ordered domains in lipid membranes with inhomogeneous curvature

A number of processes in living cells are accompanied by significant changes of the geometric curvature of lipid membranes. In turn, heterogeneity of the lateral curvature can lead to spatial redistribution of membrane components, most important of which are transmembrane proteins and liquid-ordered lipid-protein domains. These components have a so-called hydrophobic mismatch: the length of the transmembrane domain of the protein, or the thickness of the bilayer of the domain differ from the thickness of the surrounding membrane. In this work we consider redistribution of membrane components with hydrophobic mismatch in membranes with non-uniform geometric curvature. Dependence of the components’ energy on the curvature is calculated in terms of theory of elasticity of liquid crystals adapted to lipid membranes. According to the calculations, transmembrane proteins prefer regions of the membrane with zero curvature. Liquid-ordered domains having a size of a few nm distribute mainly into regions of the membrane with small negative curvature appearing in the cell plasma membrane in the process of endocytosis. The distribution of domains of a large radius is determined by a decrease of their perimeter upon bending; these domains distribute into membrane regions with relatively large curvature.     

Organization and dynamics of Fas transmembrane domain in raft membranes and modulation by ceramide

To comprehend the molecular processes that lead to the Fas death receptor clustering in lipid rafts, a 21-mer peptide corresponding to its single transmembrane domain (TMD) was reconstituted into mammalian raft model membranes composed of an unsaturated glycerophospholipid, sphingomyelin, and cholesterol. The peptide membrane lateral organization and dynamics, and its influence on membrane properties, were studied by steady-state and time-resolved fluorescence techniques and by attenuated total reflection Fourier transformed infrared spectroscopy. Our results show that Fas TMD is preferentially localized in liquid-disordered membrane regions and undergoes a strong reorganization as the membrane composition is changed toward the liquid-ordered phase. This results from the strong hydrophobic mismatch between the length of the peptide hydrophobic stretch and the hydrophobic thickness of liquid-ordered membranes. The stability of nonclustered Fas TMD in liquid-disordered domains suggests that its sequence may have a protective function against nonligand-induced Fas clustering in lipid rafts. It has been reported that ceramide induces Fas oligomerization in lipid rafts. Here, it is shown that neither Fas TMD membrane organization nor its conformation is affected by ceramide. These results are discussed within the framework of Fas membrane signaling events.     

Domains and Rafts in Membranes – Hidden Dimensions of Selforganization

Both biomembranes and biomimetic membranes such as lipid bilayers withseveral components contain intramembrane domains and rafts.Macromolecules, which are anchored to the membrane but have no tendeney tocluster, induce curved nanodomains. Clustering of membrane componentsleads to larger domains which can grow up to a certain maximal size andthen undergo a budding process. The maximal domain size depends on theinterplay of spontaneous curvature, bending rigidity, and line tension.It is argued that this interplay governs the formation of bothclathrin-coated buds and caveolae. Finally, membrane adhesion often leadsto domain formation within the contact zone.     

Real-time imaging of lipid domains and distinct coexisting membrane protein clusters

A detailed understanding of biomembrane architecture is still a challenging task. Many in vitro studies have shown lipid domains but much less information is known about the lateral organization of membrane proteins because their hydrophobic nature limits the use of many experimental methods. We examined lipid domain formation in biomimetic Escherichia coli membranes composed of phosphatidylethanolamine and phosphatidylglycerol in the absence and presence of 1% and 5% (mol/mol) membrane multidrug resistance protein, EmrE. Monolayer isotherms demonstrated protein insertion into the lipid monolayer. Subsequently, Brewster angle microscopy was applied to image domains in lipid matrices and lipid-protein mixtures. The images showed a concentration dependent impact of the protein on lipid domain size and shape and more interestingly distinct coexisting protein clusters. Whereas lipid domains varied in size (14-47μm), protein clusters exhibited a narrow size distribution (2.6-4.8μm) suggesting a non-random process of cluster formation. A 3-D display clearly indicates that these proteins clusters protrude from the membrane plane. These data demonstrate distinct co-existing lipid domains and membrane protein clusters as the monofilm is being compressed and illustrate the significant mutual impact of lipid-protein interactions on lateral membrane architecture.     

Differential Effect of Plant Lipids on Membrane Organization: SPECIFICITIES OF PHYTOSPHINGOLIPIDS AND PHYTOSTEROLS*

The high diversity of the plant lipid mixture raises the question of their respective involvement in the definition of membrane organization. This is particularly the case for plant plasma membrane, which is enriched in specific lipids, such as free and conjugated forms of phytosterols and typical phytosphingolipids, such as glycosylinositolphosphoceramides. This question was here addressed extensively by characterizing the order level of membrane from vesicles prepared using various plant lipid mixtures and labeled with an environment-sensitive probe. Fluorescence spectroscopy experiments showed that among major phytosterols, campesterol exhibits a stronger ability than β-sitosterol and stigmasterol to order model membranes. Multispectral confocal microscopy, allowing spatial analysis of membrane organization, demonstrated accordingly the strong ability of campesterol to promote ordered domain formation and to organize their spatial distribution at the membrane surface. Conjugated sterol forms, alone and in synergy with free sterols, exhibit a striking ability to order membrane. Plant sphingolipids, particularly glycosylinositolphosphoceramides, enhanced the sterol-induced ordering effect, emphasizing the formation and increasing the size of sterol-dependent ordered domains. Altogether, our results support a differential involvement of free and conjugated phytosterols in the formation of ordered domains and suggest that the diversity of plant lipids, allowing various local combinations of lipid species, could be a major contributor to membrane organization in particular through the formation of sphingolipid-sterol interacting domains.     

Role of fission yeast myosin I in organization of sterol-rich membrane domains

Specialized membrane domains containing lipid rafts are thought to be important for membrane processes such as signaling and trafficking. An unconventional type I myosin has been shown to reside in lipid rafts and function to target a disaccharidase to rafts in brush borders of intestinal mammalian cells. In the fission yeast Schizosaccharomyces pombe, distinct sterol-rich membrane domains are formed at the cell division site and sites of polarized cell growth at cell tips. Here, we show that the sole S. pombe myosin I, myo1p, is required for proper organization of these membrane domains. myo1 mutants lacking the TH1 domain exhibit a uniform distribution of sterol-rich membranes all over the plasma membrane throughout the cell cycle. These effects are independent of endocytosis because myo1 mutants exhibit no endocytic defects. Conversely, overexpression of myo1p induces ectopic sterol-rich membrane domains. Myo1p localizes to nonmotile foci that cluster in sterol-rich plasma membrane domains and fractionates with detergent-resistant membranes. Because the myo1p TH1 domain may bind directly to acidic phospholipids, these findings suggest a model for how type I myosin contributes to the organization of specialized membrane domains.     

Lo/Ld phase coexistence modulation induced by GM1

Lipid rafts are assumed to undergo biologically important size-modulations from nanorafts to microrafts. Due to the complexity of cellular membranes, model systems become important tools, especially for the investigation of the factors affecting “raft-like” L_o domain size and the search for L_o nanodomains as precursors in L_o microdomain formation. Because lipid compositional change is the primary mechanism by which a cell can alter membrane phase behavior, we studied the effect of the ganglioside GM1 concentration on the L_o/L_d lateral phase separation in PC/SM/Chol/GM1 bilayers. GM1 above 1 mol % abolishes the formation of the micrometer-scale L_o domains observed in GUVs. However, the apparently homogeneous phase observed in optical microscopy corresponds in fact, within a certain temperature range, to a L_o/L_d lateral phase separation taking place below the optical resolution. This nanoscale phase separation is revealed by fluorescence spectroscopy, including C₁₂NBD-PC self-quenching and Laurdan GP measurements, and is supported by Gaussian spectral decomposition analysis. The temperature of formation of nanoscale L_o phase domains over an L_d phase is determined, and is shifted to higher values when the GM1 content increases. A “morphological” phase diagram could be made, and it displays three regions corresponding respectively to L_o/L_d micrometric phase separation, L_o/L_d nanometric phase separation, and a homogeneous L_d phase. We therefore show that a lipid only-based mechanism is able to control the existence and the sizes of phase-separated membrane domains. GM1 could act on the line tension, “arresting” domain growth and thereby stabilizing L_o nanodomains.     

Bile Acids Modulate Signaling by Functional Perturbation of Plasma Membrane Domains

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.     

Macroscopic domain formation during cooling in the platelet plasma membrane: An issue of low cholesterol content

There has been ample debate on whether cell membranes can present macroscopic lipid domains as predicted by three-component phase diagrams obtained by fluorescence microscopy. Several groups have argued that membrane proteins and interactions with the cytoskeleton inhibit the formation of large domains. In contrast, some polarizable cells do show large regions with qualitative differences in lipid fluidity. It is important to ask more precisely, based on the current phase diagrams, under what conditions would large domains be expected to form in cells. In this work we study the thermotropic phase behavior of the platelet plasma membrane by FTIR, and compare it to a POPC/Sphingomyelin/Cholesterol model representing the outer leaflet composition. We find that this model closely reflects the platelet phase behavior. Previous work has shown that the platelet plasma membrane presents inhomogeneous distribution of DiI18:0 at 24 °C, but not at 37 °C, which suggests the formation of macroscopic lipid domains at low temperatures. We show by fluorescence microscopy, and by comparison with published phase diagrams, that the outer leaflet model system enters the macroscopic domain region only at the lower temperature. In addition, the low cholesterol content in platelets (～ 15 mol%), appears to be crucial for the formation of large domains during cooling.     

Domain nucleation rates and interfacial line tensions in supported bilayers of ternary mixtures containing galactosylceramide

Domains within the plane of the plasma membrane, referred to as membrane rafts, have been a topic of considerable interest in the field of membrane biophysics. Although model membrane systems have been used extensively to study lipid phase behavior as it relates to the existence of rafts, very little work has focused on either the initial stage of lipid domain nucleation, or the relevant physical parameters such as temperature and interfacial line tension which control nucleation. In this work, we utilize a method in which the kinetic process of lipid domain nucleation is imaged by atomic force microscopy and modeled using classical theory of nucleation to map interfacial line tension in ternary lipid mixtures. These mixtures consist of a fluid phase lipid component (1,2-dilauroyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, or 1,2-dioleoyl-sn-glycero-3-phosphocholine), a solid phase component (galactosylceramide), and cholesterol. Interfacial line tension measurements of galactosylceramide-rich domains track with our previously measured area/perimeter ratios and height mismatches measured here. Line tension also follows known trends in cholesterol interactions and partitioning, as we observed previously with area/perimeter ratios. Our line tension measurements are discussed in combination with recent line tension measurements to address line tension regulation by cholesterol and the dynamic nature of membrane rafts.     

Photo-activated phase separation in giant vesicles made from different lipid mixtures

Using giant unilamellar vesicles (GUVs) made from POPC, DPPC, cholesterol and a small amount of a porphyrin-based photosensitizer that we name PE-porph, we investigated the response of the lipid bilayer under visible light, focusing in the formation of domains during the lipid oxidation induced by singlet oxygen. This reactive species is generated by light excitation of PE-porf in the vicinity of the membrane, and thus promotes formation of hydroperoxides when unsaturated lipids and cholesterol are present. Using optical microscopy we determined the lipid compositions under which GUVs initially in the homogeneous phase displayed Lo-Ld phase separation following irradiation. Such an effect is attributed to the in situ formation of both hydroperoxized POPC and cholesterol. The boundary line separating homogeneous Lo phase and phase coexistence regions in the phase diagram is displaced vertically towards the higher cholesterol content in respect to ternary diagram of POPC:DPPC:cholesterol mixtures in the absence of oxidized species. Phase separated domains emerge from sub-micrometer initial sizes to evolve over hours into large Lo-Ld domains completely separated in the lipid membrane. This study provides not only a new tool to explore the kinetics of domain formation in mixtures of lipid membranes, but may also have implications in biological signaling of redox misbalance.     

A Lipid-Specific Toxin Reveals Heterogeneity of Sphingomyelin-Containing Membranes

Little is known about the heterogenous organization of lipids in biological membranes. Sphingomyelin (SM) is a major plasma membrane lipid that forms lipid domains together with cholesterol and glycolipids. Using SM-specific toxin, lysenin, we showed that in cultured epithelial cells the accessibility of the toxin to SM is different between apical and basolateral membranes. Apical membranes are highly enriched with glycolipids. The inhibitory role of glycolipids in the binding of lysenin to SM was confirmed by comparing the glycolipid-deficient mutant melanoma cell line with its parent cell. Model membrane experiments indicated that glycolipid altered the local density of SM so that the affinity of the lipid for lysenin was decreased. Our results indicate that lysenin recognizes the heterogenous organization of SM in biomembranes and that the organization of SM differs between different cell types and between different membrane domains within the same cell. Isothermal titration calorimetry suggests that lysenin binding to SM is presumably the result of a SM-lysenin complex formation of specific stoichiometry, thus supporting the idea of the existence of small condensed lipid complexes consisting of just a few lipid molecules in living cells.     

Defect formation of lytic peptides in lipid membranes and their influence on the thermodynamic properties of the pore environment

We present an experimental study of the pore formation processes of small amphipathic peptides in model phosphocholine lipid membranes. We used atomic force microscopy to characterize the spatial organization and structure of alamethicin- and melittin-induced defects in lipid bilayer membranes and the influence of the peptide on local membrane properties. Alamethicin induced holes in gel DPPC membranes were directly visualized at different peptide concentrations. We found that the thermodynamic state of lipids in gel membranes can be influenced by the presence of alamethicin such that nanoscopic domains of fluid lipids form close to the peptide pores, and that the elastic constants of the membrane are altered in their vicinity. Melittin-induced holes were visualized in DPPC and DLPC membranes at room temperature in order to study the influence of the membrane state on the peptide induced hole formation. Also differential scanning calorimetry was used to investigate the effect of alamethicin on the lipid membrane phase behaviour.     

Defect formation of lytic peptides in lipid membranes and their influence on the thermodynamic properties of the pore environment

We present an experimental study of the pore formation processes of small amphipathic peptides in model phosphocholine lipid membranes. We used atomic force microscopy to characterize the spatial organization and structure of alamethicin- and melittin-induced defects in lipid bilayer membranes and the influence of the peptide on local membrane properties. Alamethicin induced holes in gel DPPC membranes were directly visualized at different peptide concentrations. We found that the thermodynamic state of lipids in gel membranes can be influenced by the presence of alamethicin such that nanoscopic domains of fluid lipids form close to the peptide pores, and that the elastic constants of the membrane are altered in their vicinity. Melittin-induced holes were visualized in DPPC and DLPC membranes at room temperature in order to study the influence of the membrane state on the peptide induced hole formation. Also differential scanning calorimetry was used to investigate the effect of alamethicin on the lipid membrane phase behaviour.     

Eisosome proteins assemble into a membrane scaffold

Spatial organization of membranes into domains of distinct protein and lipid composition is a fundamental feature of biological systems. The plasma membrane is organized in such domains to efficiently orchestrate the many reactions occurring there simultaneously. Despite the almost universal presence of membrane domains, mechanisms of their formation are often unclear. Yeast cells feature prominent plasma membrane domain organization, which is at least partially mediated by eisosomes. Eisosomes are large protein complexes that are primarily composed of many subunits of two Bin-Amphiphysin-Rvs domain-containing proteins, Pil1 and Lsp1. In this paper, we show that these proteins self-assemble into higher-order structures and bind preferentially to phosphoinositide-containing membranes. Using a combination of electron microscopy approaches, we generate structural models of Pil1 and Lsp1 assemblies, which resemble eisosomes in cells. Our data suggest that the mechanism of membrane organization by eisosomes is mediated by self-assembly of its core components into a membrane-bound protein scaffold with lipid-binding specificity.     

Transbilayer organization of membrane cholesterol at low concentrations: Implications in health and disease

Cholesterol is an essential and representative lipid in higher eukaryotic cellular membranes and is often found distributed nonrandomly in domains in biological membranes. A large body of literature exists on the organization of cholesterol in plasma membranes or membranes with high cholesterol content. However, very little is known about organization of cholesterol in membranes containing low amounts of cholesterol such as the endoplasmic reticulum or inner mitochondrial membranes. In this review, we have traced the discovery and subsequent development of the concept of transbilayer cholesterol dimers (domains) in membranes at low concentrations. We have further discussed the role of membrane curvature and thickness on the transbilayer organization of cholesterol. Interestingly, this type of cholesterol organization could be relevant in cellular sorting and trafficking, and in pathological conditions.