Miscibility phase diagrams of giant vesicles containing sphingomyelin

Saturated sphingomyelin (SM) lipids are implicated in lipid rafts in cell plasma membranes. Here we use fluorescence microscopy to observe coexisting liquid domains in vesicles containing SM, an unsaturated phosphatidylcholine lipid (either DOPC or POPC), and cholesterol. We note similar phase behavior in a model membrane mixture without SM (DOPC/DPPC/Chol), but find no micron-scale liquid domains in membranes of POPC/PSM/Chol. We delineate the onset of solid phases below the miscibility transition temperature, and detail indirect evidence for a three-phase coexistence of one solid and two liquid phases.     

Combined effects of headgroup charge and tail unsaturation of lipids on lateral organization and diffusion of lipids in model biomembranes

Lateral organization and dynamics of lipids in plasma membranes are crucial for several cellular processes such as signal transduction across the membrane and still remain elusive.In this paper,using coarse-grained molecular dynamics simulation,we theoretically study the combined effects of headgroup charge and tail unsaturation of lipids on the lateral organization and diffusion of lipids in ternary lipid bilayers.In neutral ternary lipid bilayers composed of saturated lipids,unsaturated lipids,and cholesterols,under the conditions of given temperature and components,the main factor for the phase separation is the unsaturation of unsaturated lipids and the bilayers can be separated into liquid-ordered domains enriched in saturated lipids and cholesterols and liquid-disordered domains enriched in unsaturated lipids.Once the headgroup charge is introduced,the electrostatic repulsion between the negatively charged lipid headgroups will increase the distance between the charged lipids.We find that the lateral organization and diffusion of the lipids in the(partially) charged ternary lipid bilayers are determined by the competition between the headgroup charge and the unsaturation of the unsaturated lipids.In the bilayers containing unsaturated lipids with lower unsaturation,the headgroup charge plays a crucial role in the lateral organization and diffusion of lipids.The headgroup charge may make the lipid domains unstable and even can suppress phase separation of the lipids in some systems.However,in the bilayers containing highly unsaturated lipids,the lateral organization and diffusion of lipids are mainly dominated by the unsaturation of the unsaturated lipids.This work may provide some theoretical insights into understanding the formation of nanosized domains and lateral diffusion of lipids in plasma membranes.     

多组分磷脂巨囊泡的相结构和胆固醇对微畴成长的影响

巨囊泡作为细胞的简化模型,其相分离与出芽动力学规律已引起许多领域科学家的关注.本实验采用DPPC/DOPC/Chol的三组分形成的巨囊泡作为模型,借助荧光显微镜观察该三组分体系侧向分离的相结构图,并对微畴的成长过程作了系统的观察研究和理论分析.实验发现:从高温的均相区域淬灭到低温的分相区域,膜表面发生侧向分离形成微畴.体系内胆固醇的掺入量的多少会影响磷脂膜的相结构和膜内微畴的成长,固定DOPC/DPPC为1:1的前提下,微畴尺寸随着胆固醇掺入量的增加而变大.     

多组分磷脂巨囊泡的相结构和胆固醇对微畴成长的影响

摘要：巨囊泡作为细胞的简化模型,其分相与出芽机理及动力学规律已引起许多领域科学家的关注。在富含胆固醇的典型生物膜体系如二棕榈酰磷脂酰胆碱DPPC(2-dihexadecanoyl-rac-glycero-3phosphocholine)/二油酰磷脂酰胆碱DOPC(dioleoyl-phosphatidylcholine)/胆固醇(Chol)的三组分形成的巨囊泡作为模型,从高温退火至低温会发生相分离,形成微畴。实验中借助荧光显微镜观察生物膜体系侧向分离的相结构图。实验发现,体系各组分的不同会影响磷脂膜的相结构和膜内微畴的成长,固定 DOPC/DPPC为1:1的前提下,微畴尺寸随着胆固醇参入量的增加而变大。最后运用理论进一步分析了微畴的成长机理。     

Influence of membrane lipid composition on the activity of functionally reconstituted LmrA under high hydrostatic pressure

In cellular membranes, proteins and lipids are in sensitive macromolecular interaction influencing each other. To evaluate this interaction, the multi-drug transporter LmrA from Lactococcus lactis was functionally reconstituted in vesicles consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), DMPC+10 mol% cholesterol and the model raft mixture DOPC/1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol (1:2:1) and in natural membrane lipids at 30 °C. The lateral structure and organization of these proteoliposomes were modulated using high hydrostatic pressure. A sharp pressure-induced fluid-to-gel phase transition is observed without an extended two-phase region. The possibility for lipid sorting, such as for DMPC/cholesterol bilayers, has an inhibitory effect on the LmrA activity. A fluid-like membrane phase over the whole pressure range with suitable hydrophobic matching, such as for DOPC, prevents the membrane protein from high-pressure inactivation up to 200 MPa. Under high-pressure conditions, highest LmrA activities, exceeding those at ambient pressure, are achieved for heterogeneous lipid matrices with a small hydrophobic mismatch and the ability of lipid sorting.     

Investigating lipid interactions and the process of raft formation in cellular membranes using ToF-SIMS

There is an increased interest in how lipids interact with each other, especially in the lateral separation of lipids into coexisting liquid phases as this is believed to be an attribute of raft formation in cell membranes. ToF-SIMS has shown itself to be an excellent tool for investigating cellular and model membrane systems and will be perhaps the most powerful one for investigating raft formation. Results from our laboratory show the capability of ToF-SIMS at identifying unequivocally the content of coexisting liquid lipid phases. Using supported lipid monolayers we find that the inclusion of dipalmitoylphosphatidylethanolamine (DPPE) to a homogeneous dipalmitoyl-phosphatidylcholine (DPPC)/cholesterol phase results in the formation of cholesterol-rich domains [A.G. Sostarecz, C.M. McQuaw, A.G. Ewing, N. Winograd, J. Am. Chem. Soc. 126 (2004) 13882]. Also, for DPPE/cholesterol systems a single homogeneous DPPE/cholesterol phase is formed at ∼50 mol% cholesterol, whereas DPPC/cholesterol systems form a single phase at 30 mol% cholesterol [C.M. McQuaw, A. Sostarecz, L. Zheng, A.G. Ewing, N. Winograd, Langmuir 21 (2005) 807]. Currently we are exploring the incorporation of sphingomyelin into phospholipid-cholesterol mixtures in an effort to gain a better understanding of its role in raft formation.     

Organization in lipid membranes containing cholesterol

A fundamental attribute of raft formation in cell membranes is lateral separation of lipids into coexisting liquid phases. Using fluorescence microscopy, we observe spontaneous lateral separation in free-floating giant unilamellar vesicles. We record coexisting liquid domains over a range of composition and temperature significantly wider than previously reported. Furthermore, we establish correlations between miscibility in bilayers and in monolayers. For example, the same lipid mixtures that produce liquid domains in bilayer membranes produce two upper miscibility critical points in the phase diagrams of monolayers.     

Fluorescence Lifetime Tuning—A Novel Approach to Study Flip-Flop Kinetics in Supported Phospholipid Bilayers

In the present work we introduce a straightforward fluorescent assay that can be applied in studies of the transbilayer movement (flip-flop) of fluorescent lipid analogues across supported phospholipid bilayers (SPBs). The assay is based on the distance dependent fluorescence quenching by light absorbing surfaces. Applied to SPBs this effect leads to strong differences in fluorescence lifetimes when the dye moves from the outer lipid leaflet to the leaflet in contact with the support. Herein, we present the basic principles of this novel approach, and comment on its advantages over the commonly used methods for investigating flip-flop dynamics across lipid bilayers. We test the assay on the fluorescent lipid analog Atto633-DOPE and the 3-hydroxyflavone F2N12S probe in SPBs composed of DOPC/ DOPS lipids. Moreover, we compare and discuss the flip-flop rates of the probes with respect to their lateral diffusion coefficients.     

Pore formation phase diagrams for lipid membranes

Critical lateral pressure for a pore formation and phase diagram of porous membrane are derived analytically as functions of the microscopic parameters of the lipid chains. The derivation exploits path-integral calculation of the free energy of the ensembles of semi-flexible strings and rigid rods that mimic the hydrophobic tails of lipids in the lipid bilayers and bolalipid membranes respectively. Analytical expressions for the area stretch/compressibility moduli of the membranes are derived in both models.     

Temperature effect on thin lipid film elasticity and phase separation: insights from Langmuir monolayer and fluorescence microscopy techniques

Langmuir monolayer pressure isotherms and compressibility modulus measurements of phospholipid mixtures in several Langmuir monolayer systems at the air/water interface were investigated in this study. The ultimate aim was to carry out a comparison of the elasticity modulus for monolayers with different mixtures of l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and chicken egg yolk sphingomyelin (eSM), in the presence/absence of cholesterol (Chol). In particular, we were able to propose that the leading force beyond the phase separation into liquid expanded (LE-) and liquid condensed (LC-) phases emerges from the increasing barrier to incorporate DOPC molecules into a highly ordered LC-phase. In addition, our findings suggest that DOPC lipid molecules have a priority to incorporate in a disordered LE-phase, while DPPC and eSM prefer the ordered one. Also, Chol seems to split almost equally into both phases, indicating that Chol has no priority for either phase and there are no particular interactions between Chol and saturated lipid molecules.     

拉曼光谱研究人参皂苷Rb1与DPPC双层膜的作用

为深入了解人参皂苷的分子药理学特性,阐明人参皂苷与细胞膜的作用机制,利用拉曼光谱从分子水平研究了不同浓度人参皂苷Rb1与DPPC(二棕榈酰磷脂酸胆碱)双层膜的作用.结果表明,人参皂苷Rb1没有改变DPPC的极性头部O-C-C-N+的稳定构象,极性头仍然平行于膜表面.并且,拉曼峰值比I1096/I1126/1096/I1062和I2848/I288/0随着药物浓度的增加而相应的变大,说明Rbl增加了烃链的无序度,增强了双层膜的流动性.由此推测该药物与DPPC的作用可能由于皂苷分子内及分子间的氢键与磷脂双层膜的极性头部相作用而停留在膜的表面.     

Thermodynamic approach to phase coexistence in ternary phospholipid-cholesterol mixtures

We introduce a simple and predictive model for determining the phase stability of ternary phospholipid-cholesterol mixtures. Assuming that competition between the liquid and gel order of the phospholipids is the main driving force behind lipid segregation, we derive a Gibbs free energy of mixing, based on the thermodynamic properties of the lipids main transition. A numerical approach was devised that enables the fast and efficient determination of the ternary diagrams associated with our Gibbs free energy. The computed phase coexistence diagram of DOPC/DPPC/cholesterol reproduces well-known features for this system at 10 °C, as well as its evolution with temperature.     

Patterning silver nanocubes in monolayers using phase separated lipids as templates

Strategies for assembling silver nanocubes (NCs) into distinct 2D patterns on Langmuir–Blodgett (LB) films are demonstrated using two different lipid mixtures as vehicles: (1) raft mixtures containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SPM), and cholesterol in different mole ratios (2:2:1 and 1:1:1) and (2) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) at a 1:3 mol ratio. Atomic force microscopy was employed to unveil the mechanisms of such pattern formation in the LB film. The results demonstrate that aggregation of NCs into round-like pattern is governed by preferential localization of NCs within the liquid condensed (LC) domains of DOPC/SPM/Cholesterol mixture. Cholesterol was found to govern the size and shape of the rounded islands. On the other hand, incorporation of NCs within the liquid expanded (LE) phase of DPPC/DLPC mixture produced linear-branched chains, oriented normal to the Langmuir film transfer direction. The as engineered patterns of silver NCs exhibited characteristic plasmonic signatures. Our results reveal the potential in assembling plasmonic metal nanoparticles into diverse patterns on solid substrates by exploiting their preferential localization either in LC or LE phase of appropriate lipid mixture in Langmuir film.     

Detection of submicron-sized raft-like domains in membranes by small-angle neutron scattering

Using coarse grained models of heterogeneous vesicles we demonstrate the potential for small-angle neutron scattering (SANS) to detect and distinguish between two different categories of lateral segregation: 1) unilamellar vesicles (ULV) containing a single domain and 2) the formation of several small domains or “clusters” (~10 nm in radius) on a ULV. Exploiting the unique sensitivity of neutron scattering to differences between hydrogen and deuterium, we show that the liquid ordered (lo) DPPC-rich phase can be selectively labeled using chain deuterated dipalymitoyl phosphatidylcholine (dDPPC), which greatly facilitates the use of SANS to detect membrane domains. SANS experiments are then performed in order to detect and characterize, on nanometer length scales, lateral heterogeneities, or so-called “rafts”, in ~30 nm radius low polydispersity ULV made up of ternary mixtures of phospholipids and cholesterol. For 1:1:1 DOPC:DPPC:cholesterol (DDC) ULV we find evidence for the formation of lateral heterogeneities on cooling below 30 °C. These heterogeneities do not appear when DOPC is replaced by SOPC. Fits to the experimental data using coarse grained models show that, at room temperature, DDC ULV each exhibit approximately 30 domains with average radii of ~10 nm.     

Effect of Polyacrylic Acid on Phase State of Lipids and Diffusion in Lipid-Water System

Lipid vesicles interacting with polyanions are promising for controlled drug delivery. However, different aspects of the interaction of these polymers with lipids are far from complete understanding. In this work we studied the influence of polyacrylic acid (PAA) with small concentrations (1–4 mol%) on the change of the phase state, lateral diffusion of these lipids in lamellar phase and transmembrane water diffusion in macroscopically oriented bilayers of lipid-water systems formed by dimiristoylphosphatidylcholine (DMPC) and dioleoylphosphatidylcholine. Measurements were performed by ³¹P nuclear magnetic resonance (NMR) spectroscopy and the ¹H NMR technique with a pulsed field gradient. It was found that the presence of PAA does not change the lamellar structure of the system. However, a part of bilayers changes their originally flat geometry and forms vesicles with a higher surface curvature. Macroscopic orientation of bilayers disappeared. For DMPC the presence of PAA leads to a shift of the gel-to-liquid crystalline phase-transition temperature to higher temperatures. An increase of PAA concentration leads to a monotonous decrease in the lateral diffusion coefficient of lipids that is caused, probably, by the ordering of lipids in bilayers. The transbilayer diffusion coefficient of water increases in the presence of PAA, but it depends slightly on the PAA concentration. An increase of pH leads to a change of the lipid lateral and transbilayer diffusion coefficients to the values typical for a pure bilayer. Authors' address: Andrey Filippov, Kazan State University, Kremlevskaya ulitsa 18, Kazan 420008, Russian Federation     

Water diffusivity in model biological membranes

Water self-diffusion was studied in model biological membranes (lipid bilayers of dioleoylphosphatidylcholine (DOPC) and dimyristoylphosphatidylcholine) by nuclear magnetic resonance with pulsed field gradient. The results for DOPC-water bilayers for three different orientations of the angle ? between the direction of the field gradient and the normal to the bilayer (?=57.4, 90, 0°) were presented. The differences in the diffusion decay shapes and values of the diffusion coefficients, obtained at different bilayer orientations and demonstrating highly anisotropic diffusion of the interbilayer water was discussed. This study also has shown some features of water diffusion in model lipid bilayers at the concentrations corresponding to the presence of bound and quasi-free water. The dependence of the water and lipid diffusion on the water content was considered from the point of view of the bilayers hydration and types of interbilayer water.     

Diffraction studies on natural and model lipid bilayers

In this study we have used neutron diffraction to examine the swelling behaviour and bilayer parameters of membranes reconstituted from polar lipids extracted from B. subtilis and model systems composed of synthetic phospholipids. Evidence for phase separation in the model system (lacking in Lysyl-PG, L-PG) is discussed in relation to its possible contribution to membrane domain formation through lipid-lipid interactions. Comparing these results with those obtained from the bilayers composed of lipids extracted from bacterial cells gives us some indication of the role of L-PG in the B. subtilis plasma membrane.     

Influence of (phospho)lipases on properties of mica supported phospholipid layers

The effect of enzymes: lipase from Candida cylindracea (L_Cc), phospholipase A₂ from hog pancreas (PLA₂) and phospholipase C from Bacillus cereus (PLC) to modulate wetting properties of solid supported phospholipid bilayers was studied via advancing and receding contact angle measurements of water, formamide and diiodomethane, and calculation of the surface free energy and its components from van Oss et al. (LWAB) and contact angle hysteresis (CAH) approaches. Simultaneously, topography of the studied layers was determined by Atomic Force Microscopy (AFM). The investigated lipid bilayers were transferred on mica plates from subphase of pure water by means of Langmuir-Blodgett and Langmuir-Schaefer techniques. The investigated phospolipid layers were: saturated DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), unsaturated DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and their mixture DPPC/DOPC. The obtained results revealed that the lipid membrane degradation by the enzymes caused increase in its surface free energy due to the amphiphilic hydrolysis products, which may accumulate in the lipid bilayer. In result activity of the enzymes may increase and then break down the bilayer structure takes place. It is likely that after dissolution of the hydrolysis reaction products in the bulk phase, patches of bare mica surface are accessible, which contribute to the apparent surface free energy changes. Comparison of AFM images and the free energy changes of the layers gives better insight into changes of their properties. The observed gradual increase in the layer surface free energy allows controlling of the hydrolysis process to obtain the surfaces of defined properties.     

变温拉曼光谱和DSC探讨人参皂苷Rb1分子与DPPC双层膜的作用

研究人参皂苷分子与生物膜的作用对于深入了解中药人参的药理活性及其生物学功效至关重要。DPPC作为具有双分子层结构的脂质分子,常被许多国内外学者作为模拟膜的模型来研究药物分子与细胞膜的作用;Rb1作为中药人参中的重要皂苷成分,具有显著的药理学功效和生物性能。拉曼光谱是探讨分子间作用的有力工具,差示扫描量热技术（differential scanning calorimetry, DSC）是研究脂双层分子单体及其与药物分子作用的常用技术,而将两者结合研究药物分子对细胞膜作用的研究的报道较少。本文采用变温拉曼光谱和DSC探讨了在温度变化条件下人参皂苷Rb1单体分子与DPPC双层膜的作用。通过拉曼光谱测试,在Rb1作用前后,DPPC分子极性头部O—C—C—N+和C—C伸缩振动区域以及烷基链部分C—H键的伸缩振动区域的变化表明,随着温度的增加,含有一定浓度Rb1的DPPC磷脂极性头部旁氏构象没有发生变化,脂酰链的无序性构象增多,侧向排列的无序性增强,DPPC脂双层的流动性增加。由DSC实验得到的几个热力学常数[相变温度（T_m）、半峰宽（ΔT_1/2）及相转变焓值（ΔH）]的变化表明,DSC进一步验证了变温拉曼实验结果,随着Rb1浓度的增大,DPPC双层膜的相变温度显著下降,流动性增强,说明Rb1对DPPC双层膜的影响较大。