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.     

The impact of line tension on the contact angle of nanodroplets

The line tension for a Lennard–Jones (LJ) fluid on a (9, 3) solid of varying strength was calculated using Monte Carlo simulations. A new perturbation method was used to determine the interfacial tension between liquid–vapour, solid–liquid and solid–vapour phases for this system to determine the Young's equation contact angle. Cylindrical and spherical nanodroplets were simulated for comparison. The contact angles from the cylindrical drops and Young's equation agree very well over the range of surface strengths and cylindrical drop sizes, except on a very weak surface. Tolman length effects were not observable for cylindrical drops. This shows that quite small systems can reproduce macroscopic contact angles. For spherical droplets, a deviation between the contact angle of spherical droplets and Young's equation was evident, but decreased with increasing interaction strengths to be negligible for contact angles less than 90°. Linear fitting of the contact angle data for varying droplet sizes showed no clear effect by line tension on contact angle. All calculated line tension values have a magnitude less than 4 × 10^? 12 J/m with both negative and positive signs. The best estimate of line tension for this system of LJ droplets was 1 × 10^? 13 J/m, which is smaller than the reported estimations in the literature, and is too small to be conclusively positive or negative in value.     

Ganglioside GM1 increases line tension at raft boundary in model membranes

Gangliosides are significant participants in suppression of immune system during tumor processes. It was shown that they can induce apoptosis of T-lymphocytes in a raft-dependent manner. Fluorescence confocal microscopy was used to study distribution and influence of ganglioside GM1 on raft properties in giant unilamellar vesicles. Both raft and non-raft phase markers were utilized. No visible phase separation was observed without GM1 unless lateral tension was applied to the membrane. At 2 mol % of GM1 large domains appeared indicating macroscopic phase separation. Increase of GM1 content to 5 mol % resulted in shape transformation of the domains consistent with growth of line tension at the domain boundary. At 10 mol % of GM1 almost all domains were pinched out from vesicles, forming their own homogeneous liposomes. Estimations showed that the change of the GM1 content from 2 to 5–10 mol % resulted in a several-fold increase of line tension. This finding provides a possible mechanism of apoptosis induction by GM1. Incorporation of GM1 into a membrane leads to an increase of the line tension. This results in a growth of the average size of rafts due to coalescence or merger of small domains. Thus, necessary proteins can find themselves in one common raft and start the corresponding cascade of reactions. The article is published in the original.     

The relationship between compositional phase separation and vesicle morphology: Implications for the regulation of phospholipase A2 by membrane structure

The action of phospholipase A₂ (PLA₂) on bilayer substrates causes the accumulation of reaction products, lyso-phospholipid and fatty acid. These reaction products and the phospholipid substrate generate compositional heterogeneities and then apparently phase separate when a critical mole fraction of reaction product accumulates in the membrane. This putative phase separation drives an abrupt morphologic rearrangement of the vesicle, which may be in turn responsible for modulating the activity of PLA₂. Here we examine the thermotropic properties of the phase-separated lipid system formed upon hydrating colyophilized reaction products (1:1 palmitic acid:1-palmitoyl-2-lyso-phosphatidylcholine) and substrate, dipalmitoylphosphatidylcholine. The mixture forms structures which are not canonical spherical vesicles and appear to be disks in the gel-state. The main gel-liquid transition of these structures is hysteretic. This hysteresis is apparent using several techniques, each selected for its sensitivity to different aspects of a lipid aggregate's structure. The thermotropic hysteresis reflects the coupling between phase separation and changes in vesicle morphology.     

Lowering line tension with high cholesterol content induces a transition from macroscopic to nanoscopic phase domains in model biomembranes

Chemically simplified lipid mixtures are used here as models of the cell plasma membrane exoplasmic leaflet. In such models, phase separation and morphology transitions controlled by line tension in the liquid-disordered (Ld)?+?liquid-ordered (Lo) coexistence regime have been described [1]. Here, we study two four-component lipid mixtures at different cholesterol fractions: brain sphingomyelin (BSM) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol (Chol). On giant unilamellar vesicles (GUVs) display a nanoscopic-to-macroscopic transition of Ld?+?Lo phase domains as POPC is replaced by DOPC, and this transition also depends on the cholesterol fraction. Line tension decreases with increasing cholesterol mole fractions in both lipid mixtures. For the ternary BSM/DOPC/Chol mixture, the published phase diagram [19] requires a modification to show that when cholesterol mole fraction is >~0.33, coexisting phase domains become nanoscopic.     

Disappearance of calcium-induced phase separation in phosphatidylserine-phosphatidylcholine membranes caused by protonation and by electric current

Disappearance of Ca²⁺-induced phase separation in phosphatidylserine-phosphatidylcholine membranes has been studied under several conditions by monitoring electron spin resonance spectrum of spin-labeled phosphatidylcholine. The membranes were prepared in Millipore filters. Electron micrographs of the preparations showed formation of multilayered structures lined on the pore surface. The phase separation was disappeared when the membrane was soaked in non-buffered salt solution (100 ml KCl, pH 5.5). It was markedly contrasting that when the bathing salt solution was buffered no disappearance was observed. Disappearance of the phase separation was also observed when the Ca²⁺-treated membrane was transferred to acidic salt solutions ($\text{?}\text{pH 2.5}$) or to low ionic strength media ($\text{? 10}\text{mM}$) buffered at pH 5.5, and then to the buffered salt solution (100 mM KCl, pH 5.5). These are due to replacement of Ca²⁺ by proton, proton-induced separation, followed by disappearance of the phase separation inthe buffered salt solution. Biological significance of the competition between Ca²⁺ and proton for the phase separation or domain formation in the membranes was emphasized.     

Surface tension induced by sphingomyelin to ceramide conversion in lipid membranes

We have investigated the effect of sphingomyelin (SM) to ceramide enzymatic conversion on lipid bilayers using Giant Unilamellar Vesicles (GUVs). Sphingomyelinase was added externally to GUVs containing various proportions of SM. In situ asymmetrical SM conversion to ceramide reduced the area of one leaflet. In the absence of equilibration of all the lipids between the two leaflets, a mismatch between the two monolayers was generated. The tension generated by this mismatch was sufficient to trigger the formation of membrane defects and total vesicle collapse at relatively low percentage of SM (≈ 5% mol). The formation of nanometric size defects was visualised by AFM in supported bilayers. Vesicle rupture was prevented in two circumstances: (a) in GUVs containing a mixture of l_d and l_o domains and (b) in GUVs containing 5% lyso-phosphatidylcholine. In both cases, the accumulation of enough ceramide (at initial SM concentration of 10%) allowed the formation of ceramide-rich domains. The coupling between the two asymmetrical monolayers and the condensing effect produced by the newly formed ceramide generated a tension that could underlie the mechanism through which ceramide formation induces membrane modifications observed during the late stages of apoptosis.     

Phase transition and lateral phase separation in planar lipid membranes as sensed by the lipophilic ion dipicrylamine

The permeation of the lipophilic ion dipicrylamine through planar lipid membranes formed from dipalmitoylphosphatidylcholine in n-decane shows an anomaly near the main phase transition of this system. Both the rate constant, k_i, of ion translocation across the membrane interior and the interfacial concentration, N, of this ion have a maximum at about 36°C. Analogous experiments were performed with tetraphenylborate. A considerably lesser effect of the phase transition was found. The addition of cholesterol leads to a broadening of the maxima for k_i and N. The time course of the current following a voltage jump shows a characteristic change below a temperature of about 45°C, if the molar ratio cholesterol/ phosphatidylcholine in the membrane forming solution exceeds 1. While the current transient decays exponentially above 45°C, a sum of two exponential terms yields an adequate fit below that temperature. This is regarded as evidence for a lateral phase separation below 45°C into structurally different domains, which provide two different pathways for dipicrylamine.     

Divalent cation-induced surface tension increase in acidic phospholipid membranes: Ion binding and membrane fusion

Chelation binding of divalent cations to phospholipid membranes may cause deformation in the headgroup regions of these lipid molecules. This deformation may be responsible for the observed large increase in surface tension of acidic phospholipid membranes induced by divalent cations. On the other hand, simple binding of monovalent cations without being followed by such a deformation of membrane molecules, does not result in a large surface tension increase in the membrane. A theoretical explanation for the above situation is given and the divalent cation-induced acidic phospholipid membrane fusion as well as other lipid membrane fusions are discussed in terms of the increased surface energy of membranes.     

Interaction between bending and tension forces in bilayer membranes.

A theoretical analysis is presented of the bending mechanics of a membrane consisting of two tightly-coupled leaflets, each of which shears and bends readily but strongly resists area changes. Structures of this type have been proposed to model biological membranes such as red blood cell membrane. It is shown that when such a membrane is bent, anisotropic components of resultant membrane tension (shear stresses) are induced, even when the tension in each leaflet is isotropic. The induced shear stresses increase as the square of the membrane curvature, and become significant for moderate curvatures (when the radius of curvature is much larger than the distance between the leaflets). This effect has implications for the analysis of shape and deformation of freely suspended and flowing red blood cells.     

FRET analysis of domain formation and properties in complex membrane systems

The application of Förster Resonance Energy Transfer (FRET) to the detection and characterization of phase separation in lipid bilayers (both in model systems and in cell membranes) is reviewed. Models describing the rate and efficiency of FRET for both uniform probe distribution and phase separation, and recently reported methods for detection of membrane heterogeneity and determination of phase boundaries, probe partition coefficients and domain size, are presented and critically discussed. Selected recent applications of FRET to one-phase lipid systems, gel/fluid phase separation, liquid ordered/liquid disordered phase separation (lipid rafts), complex systems containing ceramide and cell membranes are presented to illustrate the wealth of information that can be inferred from carefully designed FRET studies of membrane domains.     

Lipid-mediated preferential localization of hypericin in lipid membranes

Subcellular localization of a photosensitizer is critical to its therapeutic outcome during photodynamic therapy (PDT). We delineated the distribution of hypericin, a new generation photosensitizer, in model membrane systems to identify the operating principles of its subcellular accumulation. Results from fluorescence microscopy indicated preferential incorporation of hypericin in lipid of giant unilamellar vesicles. Monolayer fluorescence measurements further identified cholesterol as the key determinant for the observed selectivity of hypericin. The emission spectra of hypericin in lipid monolayers varied in a lipid-dependent manner and Stoke's shift behavior suggests that hypericin may form closely packed structure with cholesterol. Overall, our data lead to the conclusion that cholesterol is the major origin of the selectivity for hypericin in membrane systems. A hypothetical model depicting the intracellular and intravascular co-transport of hypericin and cholesterol because of their high affinity is presented.     

Fluorescence probe partitioning between Lo/Ld phases in lipid membranes

Fluorescence microscopy imaging is an important technique for studying lipid membranes and is increasingly being used for examining lipid bilayer membranes, especially those showing macroscopic coexisting domains. Lipid phase coexistence is a phenomenon of potential biological significance. The identification of lipid membrane heterogeneity by fluorescence microscopy relies on membrane markers with well-defined partitioning behavior. While the partitioning of fluorophores between gel and liquid-disordered phases has been extensively characterized, the same is not true for coexisting liquid phases. We have used fluorescence microscopy imaging to examine a large variety of lipid membrane markers for their liquid phase partitioning in membranes with various lipid compositions. Most fluorescent lipid analogs are found to partition strongly into the liquid-disordered (L_d) phase. In contrast, some fluorescent polycyclic aromatic hydrocarbons with a flat ring system were found to partition equally, but others partition preferentially into liquid-ordered (L_o) phases. We have found these fluorescent markers effective for identification of coexisting macroscopic membrane phases in ternary lipid systems composed of phospholipids and cholesterol.     

Calculation of line tension in various models of lipid bilayer pore edge

The line tension of the edge of the lipid bilayer pore is calculated on the basis of the elastic theory of continuous liquid-crystal medium. Three types of deformations of the membrane were taken into account: bending, lateral stretching/compression, and tilt of the lipidic tails. Various models of structure of the pore edge are considered: models of the cylindrical shape with given radius and optimum radius, “extrapolational” model, “two-coordinate” model, and model with a hydrophobic cavity (“void”). Models can be conventionally divided into two classes. The first class includes models in which membrane monolayers are in contact with each other everywhere. Models of the second class admit appearance of a hydrophobic cavity between monolayers. Models of the first class yield value of the line tension γ, strongly differing from that known from the literature (～10 pN). For example, the value of the line tension γ obtained in the cylindrical model equals to 21 pN; in the two-coordinate model, 19 pN, and in the extrapolational model, 62 pN. At the same time, the model with cavity gives the value of γ eqal ～10 pN, provided that surface tension at the boundary of the lipid tails is close to zero. This value is in a good agreement with the literature data.     

Phase separations in membranes of Anacystis nidulans grown at different temperatures

Freeze fracture electron microscopy studies were performed on samples of Anacystis nidulans quenched from different temperatures. Membrane lipid phase separations were observed to take place over the ranges 15–30°C, 5–25°C and –5–15°C for cultures grown at 38, 28 and 18°C, respectively. Differential scanning calorimetry heating curves showed endotherms which coincided with these temperature ranges. Variations of phase separation temperatures with growth temperature, and hysteresis effects in the calorimetric measurements, were related to changes in the fatty acid composition of membrane lipids.     

Lateral segregation of anionic phospholipids in model membranes induced by cytochrome P450 2B1: bi-directional coupling between CYP2B1 and anionic phospholipid

The lateral segregation of anionic phospholipids phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylserine (PS) was detected after addition of cytochrome P450 2B1 (CYP2B1). The tendency of lipid clustering was highly dependent on the type of anionic phospholipids examined. PA was the most highly clustered while PI and PS clustered to a lesser degree. Moreover, liposomes containing anionic phospholipids form anionic phospholipid-rich microdomains in the presence of CYP2B1. Anionic phospholipids (mostly notably PA) also increased the ability of CYP2B1 to bind to lipid monolayers. In addition to the ability of CYP2B1 to modulate the physical properties of the membrane, the membrane itself can have reciprocal effects on the activity and conformation of CYP2B1. The catalytic activity of CYP2B1 increased as a function of anionic phospholipid concentration and in the presence of 10 mol% PA, the activity increased by 85%. These results suggest a bi-directional coupling between the CYP2B1 and anionic phospholipids.     

Studies on the interaction between nanodiamond and human hemoglobin by surface tension measurement and spectroscopy methods

In this study, a novel method to probe molecular interactions and binding of human hemoglobin (Hb) with nanodiamond (ND) was introduced based on the surface tension measurement. This method complements conventional techniques, which are basically done by zeta potential and dynamic light scattering (DLS) measurements, near and far circular dichroism (CD) spectroscopy, intrinsic and extrinsic fluorescence spectroscopy. Addition of ND to Hb solution increased the surface tension value of Hb–ND complex relative to those of Hb and ND molecules. The zeta potential values reveled that Hb and ND provide identical charge distribution at pH 7.5. DLS measurements demonstrated that Hb, ND, and ND–Hb complex have hydrodynamic radiuses of 98.37 ± 4.57, 122.07 ± 7.88 nm and 62.27 ± 3.70 at pH of 7.5 respectively. Far and near UV-CD results indicated the loss of α-helix structure and conformational changes of Hb, respectively. Intrinsic fluorescence data demonstrated that the fluorescence quenching of Hb by ND was the result of the static quenching. The hydrophobic interaction plays a pivotal role in the interaction of ND with Hb. Fluorescence intensity changes over time revealed conformational change of Hb continues after the mixing of the components (Hb–ND) till 15 min, which is indicative of the denaturation of the Hb relative to the protein control. Extrinsic fluorescence data showed a considerable enhancement of the ANS fluorescence intensity of Hb–ND system relative to the Hb till 60 nM of ND, likely persuaded by greater exposure of nonpolar residues of Hb hydrophobic pocket. The remarkable decrease in T_m value of Hb in Hb–ND complex exhibits interaction of Hb with ND conducts to conformational changes of Hb. This study offers consequential discrimination into the interaction of ND with proteins, which may be of significance for further appeal of these nanoparticles in biotechnology prosecution.     

Identification of a GTP-binding protein in the contact sites between inner and outer mitochondrial membranes

Whilst investigating whether GTP hydrolysis may be required for the import of preproteins into mitochondria we have found that a GTP-binding protein is located at the contact sites between mitochondrial inner and outer membranes. When mitochondrial outer membranes purified from rat liver were UV-irradiated in the presence of [alpha-32P]GTP, a 52 kDa protein was radiolabelled, whereas [alpha-32P]ATP did not label this protein. GTP-binding proteins were also labelled in the cytosolic and microsomal fractions, but the 52 kDa protein was concentrated in mitochondrial membranes and was the only protein specifically labelled by GTP in these membranes. Fractionation of mitochondrial membrane vesicles into outer membranes, inner membranes and contact sites between outer and inner membranes showed that the GTP-binding activity was highly enriched in contact sites, the location at which preprotein import is believed to occur. A protein of almost identical size was also found to be labelled in mitochondria from yeast.     

Properties of bilayer membranes in the phase transition or phase separation region

The increase in passive permeability of bilayer membranes near the phase transition temperature is usually explained as caused by either the increase in the amount of ‘boundary lipid’ present in the membrane, or by the increase in lateral compressibility of the membrane. Since both the amount of ‘boundary lipid’ and the lateral compressibility show a similar anomaly near the transition temperature, it is difficult to distinguish experimentally between the two proposed mechanisms.We have examined some details of both of the proposed pictures. The fluid-solid boundary energy, neglected in previous work, has been computed as a function of the domain size. For a single component uncharged lipid bilayer, the results rule out the existence of even loosely defined solid domains in a fluid phase, or vice versa. Thermodynamic fluctuations, which are responsible for anomalous behaviour near the phase transition temperature, are not intense enough to approximate the formation of a domain of the opposite phase.Turning next to lateral compressibility of bilayer membranes we have considered two-component mixtures in the phase separation region. We present the first calculation of lateral compressibility for such systems. The behaviour shows interesting anomalies, which should correlate with existing and future data on transport across membranes.     

Scratching,dominance, tension,and displacement in male baboons

This paper reports a study designed to test the hypothesis that a relationship exists among dominance rank, tension, and scratch behaviors in anubis baboons (Papio cynocephalus anubis). Our study was conducted on two groups of male baboons, each containing eight unrelated individuals who were approximately 6 years of age and who shared common rearing histories. Focal animal sampling was used to collect behavioral data. Hand scratching, foot scratching, avoid, tension, allogrooming, autogrooming, and manipulation were measured as acts performed per hour of sampling. Dominance matrices were constructed based on net difference of avoid behavior performed and received (adjusted for time sampled). Individual status ranks were grouped into two status classes, high and low. Analysis of variance models demonstrated significant differences in the performance rate of scratching behaviors by dominance rank class, as well as differences in scratching performance by tension class. Individuals in the high status class had significantly higher rates of total scratching, hand scratching, foot scratching, and tension behavior performance than their counterparts in the low status class. No significant difference was found between status classes or tension classes for performance rate of allogrooming, autogrooming, or manipulation behavior. The frequency of scratching and general level of activity were not significantly correlated. The results are interpreted to indicate the possibility that scratching may function as a displacement behavior, which subjectively appears to communicate heightened frustration, anxiety, or arousal.