Lateral Phase Separation Research Articles

Specific Adsorption of Cytochrome c on Cardiolipin−Glycerophospholipid Monolayers and Bilayers

In this study, we examined the adsorption of cytochrome c (cyt c) on monolayers and liposomes formed from (i) pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), or cardiolipin (CL) and on (ii) the more thermodynamically stable binary mixtures of POPE/CL (0.8:0.2 mol/mol) and POPC/CL (0.6:0.4 mol/mol). Constant surface pressure experiments showed that the maximum and minimum interactions occurred in the pure CL (anionic phospholipid) and the pure POPE (zwitterion) monolayers, respectively. Observation by atomic force microscopy (AFM) of the images of Langmuir-Blodgett (LB) films extracted at 30 mN m-1 suggests that the different interactions of cyt c with POPE/CL and the POPC/CL monolayers could be due to lateral phase separation occurring in the POPE/CL mixture. The competition between 8-anilino-1-naphthalene sulfonate (ANS) and cyt c for the same binding sites in liposomes that have identical nominal compositions with respect to those of the monolayers was used to obtain binding parameters. In agreement with the monolayer experiments, the most binding was observed in POPE/CL liposomes. All of our observations strongly support the existence of selective adsorption of cyt c on CL, which is modulated differently by different neutral phospholipids (POPE and POPC).

Phosphatidic Acid Domains in Membranes: Effect of Divalent Counterions

Phosphatidic acid (PA) is emerging as a key phospholipid in a wide range of biological processes such as signal transduction, secretion, or membrane fusion. In most cases, the biological functionality of PA is associated with the presence of micromolar to millimolar calcium concentrations. It has been argued that PA can create defects in the packing of lipids in membranes due to lateral phase separation by divalent ions, which in turn aggregate proteins with high affinity for PA. In this article, we present a detailed investigation of the properties of PA domains in the presence of divalent ions by a combination of molecular dynamics simulations and theoretical methods. Our results show that PA is extremely effective in binding divalent ions through its oxygen atoms, with a broad distribution of binding constants and exhibiting the phenomenon of charge inversion (a total number of bound counterion charges that exceeds the negative PA charge). We predict that a PA-rich domain undergoes a drastic reorganization when divalent cations reach micromolar concentrations (i.e., typical physiological conditions), as PA lipids become doubly charged by releasing their protons. We also present a detailed investigation of the properties of interfacial water, which determine the binding of proteins or other molecules. We conclude with a discussion of the implications of our results in the context of recent experimental studies in model systems and in real cells.

Observation of vertical honeycomb structure in InAlN∕GaN heterostructures due to lateral phase separation

The microstructure of InxAl1−xN∕GaN heterostructures (where x∼0.13–0.19), grown by molecular beam epitaxy, was investigated by transmission electron microscopy. Observations in the cross-section and plan-view geometries show evidence for lateral phase separation originating at the GaN surface that results in a vertical honeycomblike structure within the InAlN layers. The lateral dimensions of the honeycomb cells are ∼5–10nm. The vertical walls are In rich with a width of ∼1–2nm and align roughly perpendicular to ⟨112¯0⟩ and ⟨11¯00⟩ directions. The phase separation is attributed to random compositional fluctuations during the early stages of growth, possibly associated with misfit-strain relaxation.

Molecular dynamics simulations of surface-controlled phase separation of binary fluid mixtures confined in slit pores - Online only

When binary mixtures are confined into nanoscopic slit pores, an intricate interplay between surface enrichment (wetting) of one component and lateral phase separation occurs. After a brief review of the static equilibrium phase diagram of such systems, a discussion of the kinetics of phase separation is given. Considering quenches from an initially homogeneous distribution of the two species in the slit, it is shown by molecular dynamics simulation that typically in the initial stages a stratified structure develops, with enrichment layers of the preferred component at the walls of the slit pore. Then this laterally homogeneous structure breaks up into domains, which coarsen with time according to a power law with a 2/3 exponent. This growth law must be attributed to a hydrodynamic mechanism, since corresponding simulations of a diffusive Ginzburg-Landau model yield an exponent of 1/3 only. The relation to spinodal decomposition in d=2 space dimensions is briefly discussed.

Order and Disorder in a Liquid Crystalline Bilayer: Pulsed Field Gradient NMR Studies of Lateral Phase Separation

The pulsed field gradient NMR method utilized for determination of lipid lateral diffusion in macroscopically aligned lipid bilayers is described. This technique is proposed to have an appreciable potential for biophysical investigations in the field of lipid (lipidomics) and membrane biology. Topics like transport of molecules both across and in the plane of the membrane can be studied successfully, as well as the formation of lipid domains and their intrinsic dynamics can be scrutinized. In this review some recent results on lipid membrane systems are presented. In particular investigations, not easily monitored with other spectroscopic methods, of so‐called “raft membranes” are presented. In these membranes two‐phase coexistence between liquid disordered (l d) and liquid ordered (l o) phases occurs. Methods for obtaining good oriented lipid membranes, necessary for the NMR method to be efficiently used, are also described. Quite unexpectedly, cholesterol (CHOL) seems to partition into both phases to roughly the same extent, indicating that CHOL has no particular preference for any of the l d and l o phases, and that there are no specific interactions between CHOL and saturated lipids. Another interesting observation is that the lateral diffusion is the same for all components, independent of the molecular structure (thus including CHOL), if they reside in the same domain in the membrane. Furthermore, it is found that the domain formation process is sensitive to small changes in the phospholipid chain composition and the structure of the sterol. However, more studies are needed to explain the effect on the raft‐forming properties from changes in the positions and the number of double bonds in the sterol ring system.

Effect of the Nature of the 3β-Substitution in Manoyl Oxides on the Thermotropic Behavior of DPPC Lipid Bilayer and on DPPC Liposomes

Functionalized manoyl oxide derivatives have been proved over the years to evoke several biological responses. Among them, 3β-hydroxy-manoyl oxide (1) and 3β-acetoxy-manoyl oxide (2) have been shown to exhibit in vitro antimicrobial and cytotoxic activity, while N-imidazole-3 β-thiocarbonyl ester of manoyl oxide (3) was found to exhibit potent cytotoxic effect. Their partitioning into phospholipid bilayers may lead to membrane structure modifications that are crucial in liposome development as they may influence their maintenance and integrity. DSC was used to study the modifications induced in DPPC bilayers by incorporating increasing concentrations of the three manoyl oxide derivatives. All derivatives were found to strongly affect the bilayer structural organization in terms of a decrease of the cooperativity, the fluidization and partially destabilization of the gel phase and the induction of a lateral phase separation in clustering domains. Derivatives 1 and 3 were incorporated into DPPC liposomes and their physicochemical stability was monitored at 4°C. The stability of liposomes was strongly influenced by the presence of 1 and 3 at any molar ratio studied. DPPC/1 liposomes were found to retain its stability for 48 h at low concentration of 10% mol, while at higher concentrations up to 30% mol they collapsed into aggregated material. In all cases DPPC/3 liposomes were found unstable and sticky aggregated structures precipitated from the bulk suspension.

Nanowire array fabricated by Al–Ge phase separation

Phase-separated Al–Ge films, composed of Al nanowire array with diameters ranging from less than 10 to 18 nm embedded in an amorphous Ge matrix, have been prepared by a sputtering method. Similar to lateral phase separation of Al–Si system, sputtered Al and Ge form an Al nanowire array surrounded by a Ge matrix during film growth when the preparation conditions are optimized. Removal of the Al nanowires from the phase-separated Al–Ge films by etching in acid solution provides nanoporous films with a pore density on the order of 10 15 pores/m 2. Template-assisted growth of Ni nanowires into the nanopores was carried out to increase the potential applications of the phase-separated Al–Ge films.

Spatial distribution of deep level defects in crack-free AlGaN grown on GaN with a high-temperature AlN interlayer

The deep level luminescence of crack-free Al0.25Ga0.75N layers grown on a GaN template with a high-temperature grown AlN interlayer has been studied using spatially resolved cathodoluminescence (CL) spectroscopy. The CL spectra of Al0.25Ga0.75N grown on a thin AlN interlayer present a deep level aquamarine luminescence (DLAL) band at about 2.6eV and a deep level violet luminescence (DLVL) band at about 3.17eV. Cross-section line scan CL measurements on a cleaved sample edge clearly reveal different distributions of DLAL-related and DLVL-related defects in AlGaN along the growth direction. The DLAL band of AlGaN is attributed to evolve from the yellow luminescence band of GaN, and therefore has an analogous origin of a radiative transition between a shallow donor and a deep acceptor. The DLVL band is correlated with defects distributed near the GaN∕AlN∕AlGaN interfaces. Additionally, the lateral distribution of the intensity of the DLAL band shows a domainlike feature which is accompanied by a lateral phase separation of Al composition. Such a distribution of deep level defects is probably caused by the strain field within the domains.

Lateral Phase Separation Gives Multiple Lamellar Phases in a “Binary” Surfactant/Water System: The Phase Behavior of Sodium Alkyl Benzene Sulfonate/Water Mixtures

We have examined the structure of the lamellar phase (Lalpha) that coexists with a micellar solution (L1) for a commercial sodium alkyl benzene sulfonate (LAS) mixed with water. The surfactant is a mixture containing C10-C13 alkyl chains, having all positional isomers of the benzene sulfonate group present except the 1-isomer. Unusually for ionic surfactants, the difference in compositions between the coexisting L1 and Lalpha phases is large (L1 = approximately 20 wt % LAS; Lalpha = approximately 65 wt %). The main technique employed was X-ray diffraction, supplemented by optical microscopy and differential scanning calorimetry (DSC). At ambient temperatures, the lamellar phase gives a single diffraction pattern with the main reflection (d) at approximately 32.5 A, whatever the composition. However, above 40 degrees C, the diffraction peak becomes broader and moves to higher d values. At higher temperatures still, several distinct and different diffraction peaks are observed, differing in detail according to composition. The largest d values (approximately 42-4 A) are observed for the lowest LAS concentrations, while the largest number of separate reflections (five) occurs for samples with approximately 44-50% LAS, both at the highest temperatures. Although there are some differences in the data between heating and cooling cycles, the d values return to the original value at low temperature. There are no observable transitions in DSC, nor is there any heterogeneity in the lamellar phase observable by microscopy. The data clearly indicate that there is some lateral separation of the different LAS isomers within the bilayers, which results in the formation of local lamellar regions having different surfactant compositions. This lateral phase separation may arise from the presence of an (electrostatic) attractive interaction, which gives rise to an upper consolute loop within the lamellar phase region of a pure LAS isomer. Similar mechanisms may occur in biological membranes and could be responsible for the occurrence of membrane lipid patches.

Calorimetric study on the induction of interdigitated phase in hydrated DPPC bilayers by bioactive labdanes and correlation to their liposome stability: The role of chemical structure

Labd-7,13-dien-15-ol (1), labd-13-ene-8α,15-diol (2), and labd-14-ene-8,13-diol (sclareol) have been found to exhibit cytotoxic and cytostatic effects. Their partitioning into phospholipid bilayers may induce membrane structure modifications, crucial in the development of liposomes. DSC was used to elucidate the profile of modifications induced in DPPC bilayers by incorporating increasing concentrations of the labdanes. Labdanes 1, 2 and sclareol were incorporated into SUV liposomes composed of DPPC their physicochemical stability was monitored (4 °C) and was compared to liposomes incorporating cholesterol. All labdanes strongly affect the bilayer organization in a concentration dependent manner in terms of a decrease of the cooperativity, the fluidization and partially destabilization of the gel phase, the induction of a lateral phase separation and the possible existence of interdigitated domains in the bilayer. The physicochemical stability of liposomes was strongly influenced by the chemical features of the labdanes. The liposomal preparations were found to retain their stability at low labdane concentration (10 mol%), while at higher concentrations up to 30 mol% a profound decrease in intact liposomes occurred, and a possible existence of interdigitated sheets was concluded.

Comparative Calorimetric and Spectroscopic Studies of the Effects of Lanosterol and Cholesterol on the Thermotropic Phase Behavior and Organization of Dipalmitoylphosphatidylcholine Bilayer Membranes

We carried out comparative DSC and Fourier transform infrared spectroscopic studies of the effects of cholesterol and lanosterol on the thermotropic phase behavior and organization of DPPC bilayers. Lanosterol is the biosynthetic precursor of cholesterol and differs in having three rather than two axial methyl groups projecting from the β-face of the planar steroid ring system and one axial methyl group projecting from the α-face, whereas cholesterol has none. Our DSC studies indicate that the incorporation of lanosterol is more effective than cholesterol is in reducing the enthalpy of the pretransition. Lanosterol is also initially more effective than cholesterol in reducing the enthalpies of both the sharp and broad components of the main phase transition. However, at sterol concentrations of 50 mol %, lanosterol does not abolish the cooperative hydrocarbon chain-melting phase transition as does cholesterol. Moreover, at higher lanosterol concentrations (∼30–50 mol %), both sharp and broad low-temperature endotherms appear in the DSC heating scans, suggestive of the formation of lanosterol crystallites, and of the lateral phase separation of lanosterol-enriched phospholipid domains, respectively, at low temperatures, whereas such behavior is not observed with cholesterol at comparable concentrations. Our Fourier transform infrared spectroscopic studies demonstrate that lanosterol incorporation produces a less tightly packed bilayer than does cholesterol, which is characterized by increased hydration in the glycerol backbone region of the DPPC bilayer. These and other results indicate that lanosterol is less miscible in DPPC bilayers than is cholesterol, but perturbs their organization to a greater extent, probably due primarily to the rougher faces and larger cross-sectional area of the lanosterol molecule and perhaps secondarily to its decreased ability to form hydrogen bonds with adjacent DPPC molecules. Nevertheless, lanosterol does appear to produce a lamellar liquid-ordered phase in DPPC bilayers, although this phase is not as tightly packed as comparable cholesterol/DPPC mixtures.

Domain-Formation in DOPC/SM Bilayers Studied by pfg-NMR: Effect of Sterol Structure

Pulsed field gradient (pfg)-NMR spectroscopy was utilized to determine lipid lateral diffusion coefficients in oriented bilayers composed of 25 mol % sterol and equimolar amounts of dioleoylphosphatidylcholine and sphingomyelin. The occurrence of two lipid diffusion coefficients in a bilayer was used as evidence of lateral phase separation into liquid ordered and liquid disordered domains. It was found that cholesterol, ergosterol, sitosterol, and lathosterol induced domains, whereas lanosterol, stigmasterol, and stigmastanol resided in homogeneous membranes in the temperature interval of 24–70°C. Among the domain-forming sterols, differences in the upper miscibility temperature indicated that the stability of the liquid ordered phase could be modified by small changes in the sterol structure. The domain-forming capacity for the different sterols is discussed in terms of the ordering effect of the sterols on the lipids, and it is proposed that the driving force for the lateral phase separation is the reduced solubility of the unsaturated lipid in the highly ordered phase.

Phase Separation in Bilayer Lipid Membranes: Effects on the Inner Leaf Due to Coupling to the Outer Leaf

The combined effects of the tendency of cholesterol to order lipids in the liquid phase and the coupling between lipids in the two leaves of a bilayer are investigated theoretically utilizing a Landau free energy. We show that as a consequence of these combined effects, lateral phase separation in the outer leaf between cholesterol-rich and -poor liquids causes a similar, but weaker, phase separation in the inner leaf. Just as the areal density of lipids in the outer leaf increases in the cholesterol-rich regions, so the areal density of lipids also increases in the inner leaf. Thus, the areal density in the inner leaf varies spatially, reflecting spatial variations of the areal density in the outer leaf. This provides a mechanism for proteins attached to the inner leaf via a hydrocarbon tether to respond to variations in the composition of the outer leaf. We also note that the effect of coupling between the leaves should be observable in artificial bilayers.

A Time-Resolved Fluorescence Diphenylhexatriene (DPH) Anisotropy Characterization of a Series of Model Lipid Constructs for the Sperm Plasma Membrane

We present a steady-state and time-resolved fluorescence anisotropy analysis, using the probe diphenylhexatriene (DPH), of a hierarchical series of lipid membrane constructs that have been designed to resemble sperm plasma membranes. Anisotropy and lipid order parameters determined for the model membranes are compared to those of lipid membranes reconstituted from rabbit sperm plasma membranes. In addition, we study the response of all membrane systems to surfactant attack. The surfactants used in this investigation have current or potential use as spermicidal and antimicrobial agents. We show that our highest-level approximation of the sperm plasma membrane captures the essential characteristics of the sperm extract membranes, in terms of lipid order and response to surfactant attack. Our studies also highlight the importance of sphingomyelin as a regulator of membrane properties and also show indirect evidence of lateral phase separation in the membrane constructs that contain both cholesterol and sphingomyelin, as revealed by a preservation of lipid order under surfactant attack.

Phase behavior of polyelectrolyte-surfactant complexes at planar surfaces

We investigate theoretically the phase diagram of an insoluble charged surfactant monolayer in contact with a semidilute polyelectrolyte solution (of opposite charge). The polyelectrolytes are assumed to have long-range and attractive (electrostatic) interaction with the surfactant molecules. In addition, we introduce a short-range (chemical) interaction which is either attractive or repulsive. The surfactant monolayer can have a lateral phase separation between dilute and condensed phases. Three different regimes of the coupled system are investigated depending on system parameters. A regime where the polyelectrolyte is depleted due to short range repulsion from the surface, and two adsorption regimes, one being dominated by electrostatics, whereas the other by short range chemical attraction (similar to neutral polymers). When the polyelectrolyte is more attracted (or at least less repelled) by the surfactant molecules as compared with the bare water-air interface, it will shift upwards the surfactant critical temperature. For repulsive short-range interactions the effect is opposite. Finally, the addition of salt to the solution is found to increase the critical temperature for attractive surfaces, but does not show any significant effect for repulsive surfaces.

Photovoltaic Performance and Morphology of Polyfluorene Blends: The Influence of Phase Separation Evolution

We report the composition profile in blends of hole-accepting and electron-accepting polyfluorenes. During solvent evaporation to form thin (∼150 nm thickness) solid films, demixing of the polymers produces micrometer scale lateral phase separation. We show here that demixing is more complete in regions closer to the interface between the two phases. We propose that carriers transport is easier in these regions than in the bulk. We show how this affects the photovoltaic efficiency of diodes made with these blends, which show a linear dependence of efficiency on the length of the interface between the phases.

Sphingosine Increases the Permeability of Model and Cell Membranes

Sphingosine, at 5–15 mol % total lipids, remarkably increases the permeability to aqueous solutes of liposomal and erythrocyte ghost membranes. The increased permeability cannot be interpreted in terms of leakage occurring at the early stages of a putative membrane solubilization by sphingosine, nor is it due to a sphingosine-induced generation of nonlamellar structures, or flip-flop lipid movement. Instead, sphingosine stabilizes (rigidifies) gel domains in membranes, raising their melting temperatures and increasing the transition cooperativity. Structural defects originating during the lateral phase separation of the “more rigid” and “less rigid” domains are likely sites for the leakage of aqueous solutes to the extravesicular medium. The presence of coexisting domains in the plasma membrane makes it a target for sphingosine permeabilization. The sphingosine-induced increase in rigidity and breakdown of the plasma membrane permeability barrier could be responsible for some of the physiological effects of sphingosine.

Biological physics in four lectures and three applications

An introduction to Biological Physics is provided by three applications of Statistical Mechanics to current problems of biological interest. They are the possibility of lateral phase separation in the plasma membrane, the design of a vesicle sensitive to its environment which could be used for drug delivery, and the process of fusion of biological membranes.

Lateral Phase Separation in Lipid-Coated Microbubbles

In the design of lipid-coated microbubble ultrasound contrast agents for molecular imaging and targeted drug delivery, the surface distribution of the shell species is important because it dictates such properties as ligand location, brush coverage, and amount of drug loading. We used a combination of spectroscopy and microscopy techniques to test the prevailing notion that the main phosphatidyl choline (PC) and lipopolymer species are completely miscible within the monolayer shell. NMR spectroscopy showed that the shell composition is roughly equivalent to the bulk lipid ratio. FTIR spectroscopy showed a sharp melting peak corresponding to the main phase-transition temperature of the main PC species, with no observed pretransitions while scanning from room temperature, indicating a single PC-rich ordered phase. Electron and fluorescence microscopy showed a heterogeneous microstructure with dark (ordered) domains and bright (disordered) regions. Domain formation was thermotropic and reversible. Fluorescent labeling of the lipopolymer following shell formation showed that it partitions preferentially into the disordered interdomain regions. The ordered domains, therefore, are composed primarily of PC, and the disordered interdomain regions are enriched in lipopolymer. Phase heterogeneity was observed at all lipopolymer concentrations (0.5 to 20 mol %), and the degree of phase separation increased with lipopolymer content. The composition and temperature dependence of the microstructure indicates that phase separation is driven thermodynamically rather than being a kinetically trapped relic of the shell-formation process. The overall high variation in microstructure, including the existence of anomalous three-phase coexistence, highlights the nonequilibrium (history-dependent) nature of the monolayer shell.

Lipid lateral diffusion in bilayers with phosphatidylcholine, sphingomyelin and cholesterol: An NMR study of dynamics and lateral phase separation

Pulsed field gradient (pfg)-NMR measurements of the lipid lateral diffusion coefficients in several macroscopically aligned bilayer systems were summarized from previous and new studies. The aim was to carry out a comparison of the translational dynamics for bilayers with various mixtures of l,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) and chicken egg yolk sphingomyelin (eSM), with or without cholesterol. New useful information was obtained on the dynamics in these lipid bilayers that has not been previously appreciated. Thus, we were able to propose that the driving force behind the phase separation into l d and l o phases evolves from the increasing difficulty to incorpotate DOPC into a highly ordered phase. Our results suggest that DOPC has a preference to be located in a disordered phase, while DPPC and eSM prefer the ordered phase. Quite unexpectedly, CHOL seems to partition into both phases to roughly the same extent, indicating that CHOL has no particular preference for any of the l d or l o phases, and there are no specific interactions between CHOL and saturated lipids.