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Membrane solubilization induced by Triton X-100 (TX-100) was investigated. Different membrane compositions and phase states were studied along the detergent titration. Expected solubilization profiles were obtained but new information is provided. The fluorescence of nitrobenzoxadiazole (NBD)-labeled lipids indicates that the liquid-ordered (Lo)/liquid-disordered (Ld) phase coexistence is barely unaffected at sub-solubilizing detergent concentrations and highlights the vesicle-to-micelle transition. Moreover, the location of the NBD group in the bilayer emphasizes a detergent-membrane interaction in the case of the insoluble Lo phase membrane. It has also been shown that the molecular packing of the membrane loosens in the presence of TX-100, regardless of the solubilization profile. Motivated by studies on GPMVs, the solubilization of less ordered Lo phase membranes was considered in order to improve the effect of molecular packing on the extent of solubilization. Membranes composed of SM and Chol in an equimolar ratio doped with different amounts of PC were studied. The more ordered the Lo phase membrane is in the absence of detergent, the less likely it is to be solubilized. Furthermore, and in contrast to what is observed for membranes exhibiting an Lo/Ld phase coexistence, a very small decrease in the molecular packing of the Lo phase membrane radically modifies the extent of solubilization. These results have implications for the reliability of TX-100 insolubility as a method to detect ordered domains.

Mitochondria are known as the powerhouse of eukaryotic cells. Energy production occurs in specific dynamic membrane invaginations in the inner mitochondrial membrane called cristae. Although the integrity of these structures is recognized as a key point for proper mitochondrial function, less is known about the mechanisms at the origin of their plasticity and organization, and how they can influence mitochondria function. Here, we review the studies which question the role of lipid membrane composition based mainly on minimal model systems.

Biomimetic lipid bilayer systems are a useful tool for modeling specific properties of cellular membranes in order to answer key questions about their structure and functions. This approach has prompted scientists from all over the world to create more and more sophisticated model systems in order to decipher the complex lateral and transverse organization of cellular plasma membranes. Among a variety of existing biomembrane domains, lipid rafts are defined as small, dynamic, and ordered assemblies of lipids and proteins, enriched in cholesterol and sphingolipids. Lipid rafts appear to be involved in the development of Alzheimer's disease (AD) by affecting the aggregation of the amyloid-β (Aβ) peptide at neuronal membranes thereby forming toxic oligomeric species. In this review, we summarize the laboratory methods which allow to study the interaction of Aβ with lipid rafts. We describe step by step protocols to form giant (GUVs) and large unilamellar vesicles (LUVs) containing raft-mimicking domains surrounded by membrane nonraft regions. Using fluorescence microscopy GUV imaging protocols, one can design experiments to visualize micron-scale raft-like domains, to determine the micron-scale demixing temperature of a given lipid mixture, construct phase diagram, and photogenerate domains in order to assess the dynamics of raft formation and raft size distribution. LUV fluorescence spectroscopy protocols with proper data analysis can be used to measure molecular packing of raft/nonraft regions of the membrane, to report on nanoscale raft formation and determine nanoscale demixing temperature. Because handling of the Aβ requires dedicated laboratory experience, we present illustrated protocols for Aβ-stock aliquoting, Aβ aqueous solubilization, oligomer preparation, determination of the Aβ concentration before and after filtration. Thioflavin binding, dynamic light scattering, and transmission electron microscopy protocols are described as complementary methods to detect Aβ aggregation kinetics, aggregate sizes, and morphologies of observed aggregates.

The study of biomimetic model membrane systems undergoing liquid-ordered (Lo)-liquid-disordered (Ld) phase separation using spectroscopic methods has played an important role in understanding the properties of lipid rafts in plasma membranes. In particular, the membrane-associated fluorescence probe Laurdan has proved to be a very efficient reporter of Lo-Ld phase separation in lipid bilayers using the general polarization (GP) parameter. A limitation of the GP approach is that it monitors only global average packing so that the contribution of each phase remains undetermined. The decomposition of Laurdan emission spectra has been proposed as an additional approach to overcoming this limitation. Here, further developments of this method for the study of Lo-Ld phase separation are described here for Laurdan in sphingomyelin-phosphatidylcholine-cholesterol large unilamellar vesicles. Lipid compositions corresponding to homogeneous Lo or Ld phases as well as undergoing thermally induced Lo-Ld phase separation were investigated. In addition, the occurrence of phase separation was checked by the fluorescence imaging of giant unilamellar vesicles. Decomposition into three log-normal components is used to show that an intermediate energy component is specifically associated with the occurrence of the Lo phase, with a small contribution from this component occurring above the phase-separation temperature being attributable to phase fluctuations. The ratio R X of the relative area of this intermediate-energy peak to that of the low-energy peak is shown to provide a straightforward index of Lo-Ld phase separation as a function of temperature, which is occasionally more sensitive than GP. It is also shown that R X can be used in conjunction with GP to gain further insight into Lo-Ld, the phase-separation processes. This latter feature is illustrated by the influence of the alcohol butanol on the Lo-Ld phase separation in sphingomyelin-phosphatidylcholine-cholesterol bilayers by showing that the effect of the alcohol occurs specifically at the onset of the phase separation, indicating a line tension mechanism. It is proposed that the three components of log-normal decomposition approaching Laurdan emission spectra provide a useful improvement for characterizing Lo-Ld phase-separation phenomena.

Unsaturated lipid oxidation is a fundamental process involved in different aspects of cellular bioenergetics; dysregulation of lipid oxidation is often associated with cell aging and death. To study how lipid oxidation affects membrane biophysics, we used a chlorin photosensitizer to oxidize vesicles of various lipid compositions and degrees of unsaturation in a controlled manner. We observed different shape transitions that can be interpreted as an increase in the area of the targeted membrane followed by a decrease. These area modifications induced by the chemical modification of the membrane upon oxidation were followed in situ by Raman tweezers microspectroscopy. We found that the membrane area increase corresponds to the lipids' peroxidation and is initiated by the delocalization of the targeted double bonds in the tails of the lipids. The subsequent decrease of membrane area can be explained by the formation of cleaved secondary products. As a result of these area changes, we observe vesicle permeabilization after a time lag that is characterized in relation with the level of unsaturation. The evolution of photosensitized vesicle radius was measured and yields an estimation of the mechanical changes of the membrane over oxidation time. The membrane is both weakened and permeabilized by the oxidation. Interestingly, the effect of unsaturation level on the dynamics of vesicles undergoing photooxidation is not trivial and thus carefully discussed. Our findings shed light on the fundamental dynamic mechanisms underlying the oxidation of lipid membranes and highlight the role of unsaturations on their physical and chemical properties., (Copyright © 2019 Biophysical Society. All rights reserved.)

Alzheimer's disease (AD) is characterized by the overproduction of the amyloid-β peptide (Aβ) which forms fibrils under the influence of raft microdomains containing the ganglioside GM1. Raft-mimicking artificial liquid ordered (Lo) domains containing GM1 enhance amyloid-β polymerization. Other experiments suggest that Aβ binds preferably to the non-raft liquid disordered (Ld) phase rather than to the Lo phase in the presence of GM1. Here, the interaction of Aβ(1-42) with GM1-containing biphasic Lo-Ld giant vesicles was investigated. Fluorescence colocalisation experiments confirm that Aβ(1-42) binds preferentially to the Ld phase. The effect of Aβ(1-42) on the Lo-Ld size dynamics was studied using photoinduced spinodal decomposition which mimics the nanodomain-microdomain raft coalescence. Aβ affects the kinetics of the coarsening phase and the size of the resulting microdomains. The effect depends on which phase is in a majority: when the Lo microdomains are formed inside an Ld phase, their growth rate becomes slower and their final size smaller in the presence of Aβ(1-42), whereas when the Ld microdomains are formed inside an Lo phase, the growth rate becomes faster and the final size larger. Fluorimetric measurements on large vesicles using the probe Laurdan indicate that Aβ(1-42) binding respectively increases or decreases the packing of the Ld phase in the presence or absence of GM1. The differential effects of Aβ on spinodal decomposition are accordingly interpreted as resulting from distinct effects of the peptide on the Lo-Ld line tension modulated by GM1. Such modulating effect of Aβ on domain dynamics could be important for lipid rafts in signaling disorders in AD as well as in Aβ fibrillation.

Most biological molecules contain acido-basic groups that modulate their structure and interactions. A consequence is that pH gradients, local heterogeneities and dynamic variations are used by cells and organisms to drive or regulate specific biological functions including energetic metabolism, vesicular traffic, migration and spatial patterning of tissues in development. While the direct or regulatory role of pH in protein function is well documented, the role of hydrogen and hydroxyl ions in modulating the properties of lipid assemblies such as bilayer membranes is only beginning to be understood. Here, we review approaches using artificial lipid vesicles that have been instrumental in providing an understanding of the influence of pH gradients and local variations on membrane vectorial motional processes: migration, membrane curvature effects promoting global or local deformations, crowding generation by segregative polarization processes. In the case of pH induced local deformations, an extensive theoretical framework is given and an application to a specific biological issue, namely the structure and stability of mitochondrial cristae, is described. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We have investigated the dynamics of phospholipid vesicles composed of 1,2-dioleoyl- sn-glycero-3-phosphocholine triggered by ionic stimuli using electrolytes such as CaCl 2 , NaCl, and NaOH. The ionic stimuli induce two characteristic vesicle dynamics, deformation due to the ion binding to the lipids in the outer leaflet of the vesicle and migration due to the concentration gradient of ions, that is, diffusiophoresis or the interfacial energy gradient mechanism. We examined the deformation pathway for each electrolyte as a function of time and analyzed it based on the surface dissociation model and the area difference elasticity model, which reveals the change of the cross-sectional area of the phospholipid by the ion binding. The metal ions such as Ca 2+ and Na + encourage inward budding deformation by decreasing the cross-sectional area of a lipid, whereas the hydroxide ion (OH - ) encourages outward budding deformation by increasing the cross-sectional area of a lipid. When we microinjected these electrolytes toward the vesicles, a strong coupling between the deformation and the migration of the vesicle was observed for CaCl 2 and NaOH, whereas for NaCl, the coupling was very weak. This difference probably originates from the binding constants of the ions.

We have investigated the migrations of phospholipid vesicles under the concentration gradients of metal ions. We microinjected metal chloride solutions, monovalent (NaCl and KCl), divalent (CaCl 2 and MgCl 2 ), and trivalent (LaCl 3 ) salts, toward phospholipid giant vesicles (GVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). For NaCl, CaCl 2 , and MgCl 2 solutions, the GVs migrated straight toward the tip of the micropipette in response to the concentration gradients, whereas for KCl and LaCl 3 , GVs moved to the opposite direction. Our motion tracking of lipid domains in a vesicle membrane showed no unidirectional flow in the membrane during the vesicle migration, indicating that the Marangoni mechanism is not responsible for the observed vesicle migration. We calculated the diffusiophoretic velocities for symmetric and asymmetrical electrolytes by solving the Stokes' equation numerically. The theoretical diffusiophoretic velocities described the observed migration velocities well. Thus, we can control the migration of vesicle in response to the concentration gradient by adapting the electrolytes and the lipids.

We demonstrate migration of phospholipid vesicles in response to a pH gradient. Upon simple micro-injection of a NaOH solution, the vesicles linearly moved to the tip of the micro-pipette and the migration velocity was proportional to the gradient of OH(-) concentration. Vesicle migration was characteristic of OH(-) ions and no migration was observed for monovalent salts or nonionic sucrose solutions. The migration of vesicles is quantitatively described by the surface tension gradient model where the hydrolysis of the phospholipids by NaOH solution decreases the surface tension of the vesicle. The vesicles move toward a direction where the surface energy decreases. Thus the chemical modification of lipids produces a mechanical force to drive vesicles.

A recurring question in membrane biological chemistry is whether bioactive signaling lipids act only as second messenger ligands or also through an effect on bilayer physical properties. Sphingosine (Sph) and sphingosine-1-phosphate (S1P) are single-chained charged sphingolipids that have antagonistic functions in the "sphingolipid rheostat" which determines cell fate. Sph and S1P respectively promote apoptosis and cell growth. In the present study, potential effects of these bioactive lipids on physicochemical properties of the lipid bilayer of cell membranes were evaluated. We have investigated the effect of both sphingolipids, incorporated separately or, for the first time, together, in large or giant phosphadidylcholine (PC) unilamellar vesicles. Three bilayer properties were examined: membrane surface charge, lipid packing, and formation of membrane microdomains. Sph and S1P appear to have distinct, when not inverse, effects on all three properties. Besides, when both sphingolipids are mixed together, their effects on lipid packing are synergistic, whereas their effects on microdomain formation and zeta-potential are mostly antagonistic. These results are interpreted as arising from different electrostatic interactions between lipid headgroups. In particular, Sph and S1P may interact together electrostatically and form a complex. These mostly inverse and opposing effects of both single-chain phospholipids on membrane physical properties might be involved in their antagonistic role in regulating cell fate. Particularly, the mutual interaction between Sph and S1P as a complex might be able to sequester both molecules in a biologically inactive form and therefore to promote a mutual regulation of their biological activities, depending on their ratio, consistent with the sphingolipid rheostat.

In a previous work, we have shown that a spatially localized transmembrane pH gradient, produced by acid micro-injection near the external side of cardiolipin-containing giant unilamellar vesicles, leads to the formation of tubules that retract after the dissipation of this gradient. These tubules have morphologies similar to mitochondrial cristae. The tubulation effect is attributable to direct phospholipid packing modification in the outer leaflet, that is promoted by protonation of cardiolipin headgroups. In this study, we compare the case of cardiolipin-containing giant unilamellar vesicles with that of giant unilamellar vesicles that contain phosphatidylglycerol (PG). Local acidification also promotes formation of tubules in the latter. However, compared with cardiolipin-containing giant unilamellar vesicles the tubules are longer, exhibit a visible pearling, and have a much longer lifetime after acid micro-injection is stopped. We attribute these differences to an additional mechanism that increases monolayer surface imbalance, namely inward PG flip-flop promoted by the local transmembrane pH gradient. Simulations using a fully nonlinear membrane model as well as geometrical calculations are in agreement with this hypothesis. Interestingly, among yeast mutants deficient in cardiolipin biosynthesis, only the crd1-null mutant, which accumulates phosphatidylglycerol, displays significant mitochondrial activity. Our work provides a possible explanation of such a property and further emphasizes the salient role of specific lipids in mitochondrial function., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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" Lo domain size and the search for Lo nanodomains as precursors in Lo 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 Lo/Ld lateral phase separation in PC/SM/Chol/GM1 bilayers. GM1 above 1mol % abolishes the formation of the micrometer-scale Lo domains observed in GUVs. However, the apparently homogeneous phase observed in optical microscopy corresponds in fact, within a certain temperature range, to a Lo/Ld 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 Lo phase domains over an Ld 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 Lo/Ld micrometric phase separation, Lo/Ld nanometric phase separation, and a homogeneous Ld 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 Lo nanodomains., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Several cell polarization processes are coupled to local pH gradients at the membrane surface. We have investigated the involvement of a lipid-mediated effect in such coupling. The influence of lateral pH gradients along the membrane surface on lipid microdomain dynamics in giant unilamellar vesicles containing phosphatidylcholine, sphingomyelin, cholesterol, and the ganglioside GM1 was studied. Lo/Ld phase separation was generated by photosensitization. A lateral pH gradient was established along the external membrane surface by acid local microinjection. The gradient promotes the segregation of microdomains: Lo domains within an Ld phase move toward the higher pH side, whereas Ld domains within an Lo phase move toward the lower pH side. This results in a polarization of the vesicle membrane into Lo and Ld phases poles in the axis of the proton source. A secondary effect is inward tubulation in the Ld phase. None of these processes occurs without GM1 or with the analog asialo-GM1. These are therefore related to the acidic character of the GM1 headgroup. LAURDAN fluorescence experiments on large unilamellar vesicles indicated that, with GM1, an increase in lipid packing occurs with decreasing pH, attributed to the lowering of repulsion between GM1 molecules. Packing increase is much higher for Ld phase vesicles than for Lo phase vesicles. It is proposed that the driving forces for domain vectorial segregative clustering and vesicle polarization are related to such differences in packing variations with pH decrease between the Lo and Ld phases. Such pH-driven domain clustering might play a role in cellular membrane polarization processes in which local lateral pH gradients are known to be important, such as migrating cells and epithelial cells.

Electroformed giant unilamellar vesicles containing liquid-ordered Lo domains are important tools for the modeling of the physicochemical properties and biological functions of lipid rafts. Lo domains are usually imaged using fluorescence microscopy of differentially phase-partionioning membrane-embedded probes. Recently, it has been shown that these probes also have a photosensitizing effect that leads to lipid chemical modification during the fluorescence microscopy experiments. Moreover, the lipid reaction products are able as such to promote Lo microdomain formation, leading to potential artifacts. We show here that this photoinduced effect can also purposely be used as a new approach to study Lo microdomain formation in giant unilamellar vesicles. Photosensitized lipid modification can promote Lo microdomain appearance and growth uniformly and on a faster time scale, thereby yielding new information on such processes. For instance, in egg phosphatidylcholine/egg sphingomyelin/cholesterol 50:30:20 (mol/mol) giant unilamellar vesicles, photoinduced Lo microdomain formation appears to occur by the rarely observed spinodal decomposition process rather than by the common nucleation process usually observed for Lo domain formation in bilayers. Moreover, temperature and the presence of the ganglioside GM1 have a profound effect on the morphological outcome of the photoinduced phase separation, eventually leading to features such as bicontinuous phases, phase percolation inversions, and patterns evoking double phase separations. GM1 also has the effect of destabilizing Lo microdomains. These properties may have consequences for Lo nanodomains stability and therefore for raft dynamics in biomembranes. Our data show that photoinduced Lo microdomains can be used to obtain new data on fast raft-mimicking processes in giant unilamellar vesicles., (© 2011 American Chemical Society)

Cardiolipin is a four-tailed acidic lipid found predominantly within the inner membrane of mitochondria, and is thought to be a key component in determining inner membrane properties and potential. Thus, cardiolipin may be involved in the dynamics of the inner membrane characteristic invaginations (named cristae) that protrude into the matrix space. In previous studies, we showed the possibility to induce, by localized proton flow, a macroscopic cristae-like shape remodeling of an only-lipid model membrane mimicking the inner mitochondrial membrane. In addition, we reported a theoretical model describing the dynamics of a chemically driven membrane shape instability caused by a modification of the plane-shape equilibrium density of the lipids in the membrane. In the present work, we focus on the lipid-packing modifications observed in a model cardiolipin-containing lipid membrane submitted to pH decrease because this is the driving force of the instability. Laurdan fluorescence and ζ-potential measurements show that under pH decrease, membrane surface charge decreases, but that significant modification of the lipid packing is observed only for CL-containing membranes. Our giant unilamellar vesicle experiments also indicate that cristae-like morphologies are only observed for CL-containing lipid membranes. Taken together, these results highlight the fact that only a strong modulation of the lipid packing of the exposed monolayer leads to membrane shape instability and suggest that mitochondrial lipids, in particular the cardiolipin, play a specific role under pH modulation in inner mitochondrial membrane morphology and dynamics., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We study a dynamical curvature instability caused by a local chemical modification of a phospholipid membrane. In our experiments, a basic solution is microinjected close to a giant unilamellar vesicle, which induces a local chemical modification of some lipids in the external monolayer of the membrane. This modification causes a local deformation of the vesicle, which then relaxes. We present a theoretical description of this instability, taking into account both the change of the equilibrium lipid density and the change of the spontaneous membrane curvature induced by the chemical modification. We show that these two types of changes of the membrane properties yield different dynamics. In contrast, it is impossible to distinguish them when studying the equilibrium shape of a vesicle subjected to a global modification. In our model, the longest relaxation timescale is related to the intermonolayer friction, which plays an important part when there is a change in the equilibrium density in one monolayer. We compare our experimental results to the predictions of our model by fitting the measured time evolution of the deformation height to the solution of our dynamical equations. We obtain good agreement between theory and experiments. Our fits enable us to estimate the intermonolayer friction coefficient, yielding values that are consistent with previous measurements.

Mitochondria are cell substructures (organelles) critical for cell life, because biological fuel production, the ATP synthesis by oxidative phosphorylation, occurs in them driven by acidity (pH) gradients. Mitochondria play a key role as well in the cell death and in various fatigue and exercise intolerance syndromes. It is clear now that mitochondria present an astonishing variety of inner membrane morphologies, dynamically correlated with their functional state, coupled with the rate of the ATP synthesis, and characteristic for normal as well as for pathological cases. Our work offers some original insights into the factors that determine the dynamical tubular structures of the inner membrane cristae. We show the possibility to induce, by localized proton flow, a macroscopic cristae-like shape remodeling of an only-lipid membrane. We designed a minimal membrane system (GUV) and experimentally showed that the directional modulation of local pH gradient at membrane level of cardiolipin-containing vesicles induces dynamic cristae-like membrane invaginations. We propose a mechanism and theoretical model to explain the observed tubular membrane morphology and suggest the underlying role of cardiolipin. Our results support the hypothesis of localized bioenergetic transduction and contribute to showing the inherent capacity of cristae morphology to become self-maintaining and to optimize the ATP synthesis.

We examine the time-dependent distortion of a nearly circular viscous domain in an infinite viscous sheet when suction occurs. Suction, the driving force of the instability, can occur everywhere in the two phases separated by an interface. The model assumes a two-dimensional Stokes flow; the selection of the wavelength at short times is determined by a variational procedure. Contrary to the viscous fingering instability, undulations of the boundary may be observed for enough pumping, whatever the sign of the viscosity contrast between the two fluids involved. We apply our model to the suction by lipoproteins of cholesterol-enriched domains in giant unilamellar vesicles. Comparison of the number of undulations given by the model and by the experiments gives reasonable values of physical quantities such as the viscosities of the domains.

The liquid-ordered/disordered-phase domain co-existence in large unilamellar vesicle membranes consisting of phosphatidylcholine:sphingomyelin (2:1) with different amounts of cholesterol has been examined using a concentration-dependent self-quenching of a single reporter molecule, C12NBD-PC. A temperature-dependent decrease of fluorescence intensity was associated with the expected formation and increase of l(o)-phase membrane fraction in the vesicles. The result is consistent with exclusion of the fluorescent probe from the liquid-ordered phase which partitions preferentially into the liquid-disordered phase membrane domains. This leads to an increase of the local concentration of fluorophore in the liquid-disordered phase and a decrease of the quantum yield. This effect was used to obtain a quantitative estimation of the fraction of the vesicle membrane occupied by the liquid-ordered phase, Phi(o), as a function of temperature and cholesterol content between 0 and 45 mol%. The value of Phi(o) was related to the assumed partition coefficient k(p) of probe between liquid-ordered/disordered phases. For large unilamellar vesicles containing 20 and 4 mol% cholesterol and probe, respectively, with k(p) = 0 (probe completely excluded from liquid-ordered phase), Phi(o) = 0.16 and with k(p) = 0.2, Phi(o) = 0.2. The results are relevant to the action of detergent in the fractionation of detergent-resistant membrane from living cells.

Cholesterol efflux from the plasma membrane to HDLs is essential for cell cholesterol homeostasis. Recently, cholesterol-enriched ordered membrane domains, i.e. lipid rafts have been proposed to play an important role in this process. Here we introduce a new method to investigate the role of HDL interactions with the raft lipid phase and to directly visualize the effects of HDL-induced cholesterol efflux on rafts in model membranes. Addition of HDLs to giant lipid vesicles containing raft-type domains promoted decrease in size and disappearance of such domains as visualized by fluorescence microscopy. This was interpreted as resulting from cholesterol efflux from the vesicles to the HDLs. The raft vanishing rate was directly related to the HDL concentration. Evidence for a direct interaction of HDLs with the membrane was obtained by observing mutual adhesion of vesicles. It is suggested that the present method can be used to study the selective role of the bilayer lipid phase (raft and non-raft) in cholesterol efflux and membrane-HDL interaction and their underlying mechanisms. Such mechanisms may contribute to cholesterol efflux in vivo.

The adsorption of beta-casein at the air-solution interface has been monitored in equilibrium conditions by neutron reflectivity. It was observed that for a bulk concentration of 100 mg/L, the amount of protein adsorbed per unit surface increases from 2.8 to 4.4 mg/m2 when the ethanol concentration in the bulk changes from 0 to 20% (v/v). Surface pressure measurements on aqueous solutions indicate that the surface pressure is higher when both protein and alcohol are added than when a single substance is in the solution. The addition of protein has an effect when the alcohol concentration is less than 20%. These results are consistent with the occurrence at the interface of a protein network leaving a surface fraction available for ethanol. A thermodynamic model has been developed using scaling law arguments to model the surface pressure and dilational modulus measurements. It introduces an exponent which is characteristic of the solvent "quality" and of the structure of the interfacial layer. The results are interpreted as showing that ethanol modifies the solvent properties, the interactions between the protein and the solvent, and the structure of the adsorption layer. The main transition seems to occurr at 6% ethanol. Copyright 1998 Academic Press.