Unbinding-binding transition induced by molecular snaps in model membranes

We have used a lamellar phase made of a nonionic surfactant, dodecane and water, as a model membrane to investigate its interactions with macromolecular inclusions bringing together two membranes, i.e., acting as macromolecular snaps. In systems devoid of inclusions, the interlamellar distance depends on the total volume fraction of membranes Phi. We show that, in presence of a transmembrane protein, or of several de novo designed peptides of different length and composition, the lamellar phase undergoes a binding transition. Under such conditions, the interlamellar distance is no longer proportional to Phi(-1), but rather to the surface concentration of snaps within the membrane. It also appears that, in the presence of the hydrophobic segment of peptide snaps, the length of the inclusions must be at least equal to the hydrophobic length of the membrane to be active. Experimental results have been precisely fitted to a model of thermally stabilized membranes, decorated with snaps. However, in the presence of inclusions, the parameter describing the interactions between membranes, has to take into account the length of the inclusion to preserve good predictive capabilities.     

Ion channel behavior of amphotericin B in sterol-free and cholesterol- or ergosterol-containing supported phosphatidylcholine bilayer model membranes investigated by electrochemistry and spectroscopy

Amphotericin B (AmB) is a popular drug frequently applied in the treatment of systemic fungal infections. In the presence of ruthenium (II) as the maker ion, the behavior of AmB to form ion channels in sterol-free and cholesterol- or ergosterol-containing supported phosphatidylcholine bilayer model membranes were studied by cyclic votammetry, AC impedance spectroscopy, and UV/visible absorbance spectroscopy. Different concentrations of AmB ranging from a molecularly dispersed to a highly aggregated state of the drug were investigated. In a fixed cholesterol or ergosterol content (5 mol %) in glassy carbon electrode-supported model membranes, our results showed that no matter what form of AmB, monomeric or aggregated, AmB could form ion channels in supported ergosterol-containing phosphatidylcholine bilayer model membranes. However, AmB could not form ion channels in its monomeric form in sterol-free and cholesterol-containing supported model membranes. On the one hand, when AmB is present as an aggregated state, it can form ion channels in cholesterol-containing supported model membranes; on the other hand, only when AmB is present as a relatively highly aggregated state can it form ion channels in sterol-free supported phosphatidylcholine bilayer model membranes. The results showed that the state of AmB played an important role in forming ion channels in sterol-free and cholesterol-containing supported phosphatidylcholine bilayer model membranes.     

Polyene--sterol interaction and selective toxicity

From permeability experiments carried out with series of amphotericin B derivatives in both biological and model membranes, it was concluded that derivatives, whose carboxyl group at the C18 position is blocked by substitution, are much more efficient at inducing permeability in ergosterol-containing than in cholesterol-containing membranes, whereas derivatives whose carboxyl group is free and ionizable are equally efficient in both membranes types. Binding measurements on erythrocyte membranes showed that all amphotericin B derivatives simply partition between membrane lipids and aqueous medium, according to their lipid solubility. There is no relationship between binding and efficiency in inducing permeability. Permeability studies carried out on lipidic vesicles containing various sterols showed that: 1) derivatives having their carboxyl free induced permeability of the 'channel' type, regardless of the sterol present, and no detectable permeability in sterol-free membranes; 2) derivatives whose carboxyl group is blocked induce channels only in membranes containing ergosterol or sterols having an alkyl side chain identical to that of ergosterol. In the presence of other sterols or in sterol-free membranes, their ionophoric activity is poor and always of the 'mobile-carrier' type. A model of polyene-sterol interaction is proposed, accounting for the data obtained with both biological and model membranes.     

In vitro binding of isolated synaptic vesicles to presynaptic plasma membranes: activation by Ca2+ and protein kinase C.

An in vitro model to study the molecular control of binding of highly purified synaptic vesicles to presynaptic plasma membranes has been developed. Presynaptic plasma membranes were immobilized by dotting onto nitrocellulose, and binding of iodinated synaptic vesicle membranes was studied under varying experimental conditions. Synaptic vesicles bind to presynaptic plasma membranes in the presence of Ca2+ and ATP. Binding is reduced in the presence of EGTA and abolished by the calmodulin antagonist trifluoperazine. Vesicle binding is stimulated 5-fold after incubation--prior to dotting--of presynaptic plasma membranes with ATP in the presence of the phorbol-ester 12-O-tetradecanoylphorbol-13-acetate (1 microM) and 2.5-fold after preincubation with Ca2+ (50 microM). Pretreatment of plasma membranes with alkaline phosphatase strongly reduces vesicle binding. Microsomes prepared from bovine liver did not bind to presynaptic plasma membranes. Our results suggest that activation of protein kinase C and Ca2+ stimulate binding of synaptic vesicles to the presynaptic membrane. In the intact nerve terminal this interaction may represent an initial step in synaptic vesicle exocytosis.     

Galactose-Specific Lectins Protect Isolated Thylakoids against Freeze-Thaw Damage

We have measured freeze-thaw damage to isolated spinach (Spinacia oleracea L.) chloroplast thylakoid membranes in the presence of different galactose-specific seed lectins to determine whether the binding of proteins to the membrane surface can lead to cryoprotection. Of the seven lectins investigated, five were protective to different degrees and two showed no measurable effect. Protection was afforded by a reduction of the solute permeability of the membranes. This reduced the solute influx during freezing and thereby osmotic rupture of the thylakoid vesicles during thawing. Using model membranes and fluorescently labeled lectins, we could show that the proteins bound exclusively to the digalactosyl lipids in the membranes. Binding was a prerequisite for the protective effect, because the presence of up to 5 mM galactose in the samples completely inhibited both binding of the lectins to thylakoid and model membranes and cryoprotection. The degree of binding was, in contrast, not related to the cryoprotective efficiency of different lectins; cryoprotection was a function of the hydrophobicity of the proteins.     

Electromechanical stresses and the effect of pH on membrane structure.

The presence of an electric field in a membrane creates stresses which lead to a mechanical compression of the membrane material. It is shown that considerations of this effect yield results consistent with the observed differential effect of pH on the plasma membrane substructure if the central electron-lucent layer in OsO4-fixed membranes is identified with the ionic depletion in the double-fixed charge model of cell membranes.     

Evolution and development of model membranes for physicochemical and functional studies of the membrane lateral heterogeneity

One of the main questions in the membrane biology is the functional roles of membrane heterogeneity and molecular localization. Although segregation and local enrichment of protein/lipid components (rafts) have been extensively studied, the presence and functions of such membrane domains still remain elusive. Along with biochemical, cell observation, and simulation studies, model membranes are emerging as an important tool for understanding the biological membrane, providing quantitative information on the physicochemical properties of membrane proteins and lipids. Segregation of fluid lipid bilayer into liquid-ordered (Lo) and liquid-disordered (Ld) phases has been studied as a simplified model of raft in model membranes, including giant unilamellar vesicles (GUVs), giant plasma membrane vesicles (GPMVs), and supported lipid bilayers (SLB). Partition coefficients of membrane proteins between Lo and Ld phases were measured to gauze their affinities to lipid rafts (raftophilicity). One important development in model membrane is patterned SLB based on the microfabrication technology. Patterned Lo/Ld phases have been applied to study the partition and function of membrane-bound molecules. Quantitative information of individual molecular species attained by model membranes is critical for elucidating the molecular functions in the complex web of molecular interactions. The present review gives a short account of the model membranes developed for studying the lateral heterogeneity, especially focusing on patterned model membranes on solid substrates.     

Interactions of small nonpolar molecules with biological membranes: an exactly solvable model

An exactly solvable model of the interaction of small nonpolar molecules with biological membranes is developed. This model, which is based upon a “decorated dimer model” extension of Nagle's membrane model, is demonstrated to qualitatively reproduce many of the changes in the order-disorder phase transition seen when biological membranes are exposed to anesthetic gases. The decorated dimer model is itself interesting because it provides an example of an exactly solvable monomer-dimer model in which phase transitions can occur in the presence of monomers.     

Interaction of the eukaryotic pore-forming cytolysin equinatoxin II with model membranes: 19F NMR studies

Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, which lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. As a means of characterising membrane binding by the actinoporin equinatoxin II (EqTII), we have used 19F NMR to probe the environment of Trp residues in the presence of micelles and bicelles. Trp was chosen as previous data from mutational studies and truncated analogues had identified the N-terminal helix of EqTII and the surface aromatic cluster including tryptophan residues 112 and 116 as being important for membrane interactions. The five tryptophan residues were replaced with 5-fluorotryptophan and assigned by site-directed mutagenesis. The 19F resonance of W112 was most affected in the presence of phospholipid micelles or bicelles, followed by W116, with further change induced by the addition of sphingomyelin. Although binding to phosphatidylcholine is not sufficient to enable pore formation in bilayer membranes, this interaction had a greater effect on the tryptophan residues in our studies than the subsequent interaction with sphingomyelin. Furthermore, sphingomyelin had a direct effect on EqTII in both model membranes, so its role in EqTII pore formation involves more than simply an indirect effect mediated via bulk lipid properties. The lack of change in chemical shift for W149 even in the presence of sphingomyelin indicates that, at least in the model membranes studied here, interaction with sphingomyelin was not sufficient to trigger dissociation of the N-terminal helix from the beta-sandwich, which forms the bulk of the protein.     

Virioimmunoassay for studying the interaction of diphtheria toxin with cell membranes]

A model for studying the interaction of diphtheria toxin with cell membranes includes immobilization of purified cell membranes on Sephadex G-25, adsorbtion of toxin on the membranes in the presence of protective colloid, and subsequent detection of adsorbed toxin by means of virioimmunoassay. Diphtheria toxin adsorbed rapidly on membranes of both sensitive (HeLa, macrophages) and resistent to tis action cells, but not on stromaof human erythrocytes. The rate of interaction depends on the concentration of toxin and the temperature of incubation. Adsorbed toxin may be eluted by acidic buffer, 8 M area and 4 M guanidin. HCl, but not by triton X-100, tween 20 and 80, sodium dodecylaulfate and basic buffer.     

How principles of domain formation in model membranes may explain ambiguities concerning lipid raft formation in cells

Sphingolipid and cholesterol-rich liquid ordered lipid domains (lipid rafts) have been studied in both eukaryotic cells and model membranes. However, while the coexistence of ordered and disordered liquid phases can now be easily demonstrated in model membranes, the situation in cell membranes remains ambiguous. Unlike the usual situation in model membranes, under most conditions, cell membranes rich in sphingolipid and cholesterol may have a "granular" organization in which the size of ordered and/or disordered domains is extremely small and domains may be of borderline stability. This review attempts to explain the origin of the divergence between of our understanding of rafts in model membranes and in cells, and how the physical properties of model membranes can help explain many of the ambiguities concerning raft formation and properties in cells. How physical principles of ordered domain formation relate to limitations of detergent insolubility and cholesterol depletion methods used to infer the presence of rafts in cells is also discussed. Possible modifications of these techniques that may increase their reliability are considered. It will be necessary to study model membrane systems more closely approximating cell membranes in order gain a complete understanding of raft properties in cells. Very high concentrations of membrane cholesterol and proteins may explain key physical characteristics of domains in cellular membranes, and are the two of the most obvious factors requiring additional study.     

Electromechanical stresses and the effect of pH on membranes structure

The presence of an electric field in a membrane creates stresses which lead to a mechanical compression of the membrane material.It is shown that considerations of this effect yield results consistent with the observed differential effect of pH on the plasma membrane substructure if the central electron-lucent layer in OsO₄-fixed membranes is identified with the ionic depletion in the double-fixed charge model of cell membranes.     

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

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

ATP-dependent membrane assembly of F-actin facilitates membrane fusion

We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macrophage cytosol (). Here, we show that different reagents affecting the actin cytoskeleton can either inhibit or stimulate this fusion process. Because the membranes of purified phagosomes can assemble F-actin de novo from pure actin with ATP (), we focused here on the ability of membranes to nucleate actin in the presence of J774 cytosolic extracts. For this, we used F-actin sedimentation, pyrene actin assays, and torsional rheometry, a biophysical approach that could provide kinetic information on actin polymerization and gel formation. We make two major conclusions. First, under our standard in vitro conditions (4 mg/ml cytosol and 1 mM ATP), the presence of membranes actively catalyzed the assembly of cytosolic F-actin, which assembled into highly viscoelastic gels. A model is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP depletion than under high-ATP conditions, even in the absence of membranes; we discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.     

Vibrational circular dichroism of gramicidin D in vesicles and micelles

The vibrational circular dichroism (VCD) and absorption spectra of gramicidin D in three model membranes (dioctadecyldimethylammonium chloride vesicles, dimyristoyl-phosphatidylcholine vesicles, and sodium dodecyl sulfate micelles) are presented. The absorption and VCD spectra suggest that the stable gramicidin D conformation in the model membranes is different from those in organic solvents. The presence of cations does not change the membrane-bound conformation of gramicidin D.     

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

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

t-butyl hydroperoxide-induced perturbations of human erythrocytes as a model for oxidant stress

Erythrocytes were incubated with t-butyl hydroperoxide in the presence and absence of hemoglobin as a model system for oxidative stress and the alterations in the structure and integrity of the membranes were investigated. The results showed that in the presence of hemoglobin a significant modification in the membrane surface charge was induced but no such alteration was observed in peroxidized hemoglobin-free membranes. As increased hemoglobin oxidation occurred in the erythrocytes, membrane lipid peroxidation diminished, suggesting a protective role for methemoglobin in t-butyl hydroperoxide-induced lipid peroxidation. Electrophoresis on polyacrylamide gels showed modification of the cytoplasmic protein region but no high molecular weight aggregates formed at the concentrations of the hydroperoxide used in this work. The results suggest that the t-butyl hydroperoxide/normal erythrocyte system seems to be an instructive model for membrane perturbations characteristic of oxidative disorders.     

Model Hydrophobic Ion Exchange Membrane

Two models of hydrophobic ion exchange membranes were examined theoretically with regard to the characteristics of cellulose acetate-nitrate membranes saturated with hydrophobic solvents. The first model, consisting of fixed negative sites dispersed in a homogeneous medium of low dielectric constant, was shown to be invalid for the experimental membranes. The second model, consisting of fixed negative sites in an aqueous channel surrounded by a medium of low dielectric constant, explains many properties of the cellulose acetate-nitrate hydrophobic membranes and was analyzed in some detail. Organic cations can enter the membranes through the hydrophobic phase as well as through the aqueous channels. The mechanism of counterion movement in such a model is assumed to consist of exchange of vacancies and or double-occupied sites positions. The presence of the medium of low dielectric constant around the aqueous channel increases the “self”-energy of the ions in the channel and the electrostatic interaction between a fixed site and a counterion in the membrane. Both these factors can account for the marked dependence of ion mobility in the aqueous channels on the dielectric constant of the surrounding medium. The model predicts membrane preference for monovalent counterions over divalent ones.     

Attempts to relate enzyme inactivation to degradation in vivo

Inactivation of liver cytosol proteins has been measured in vitro in the presence of various membranes and disulphides. Inactivation rates correlate with the known degradation rate constants of the enzymes in the intact liver. More extensive studies were carried out with glucose-6-phosphate dehydrogenase (G6PD) and phosphoenolpyruvate carboxykinase (PEPCK) using either cytosol as a source of these enzymes or alternatively highly purified preparations of each enzyme. All membranes purified from liver had a considerable capacity to inactivate the enzymes with higher activity found in the hepatocyte plasma membrane. Various lipid preparations or plasma membranes from other tissues were virtually ineffective. Inactivation was dependent on disulphides in the membranes as shown by the inhibition of activity if membranes were pretreated with thiols. Preliminary experiments of the fate of inactivated G6PD or PEPCK show binding to membranes and subsequent proteolysis. A model is proposed for the degradation of labile enzymes.     

Formation of age pigment-like fluorescent substances during peroxidation of lipids in model membranes

The formation of age pigment-like fluorescent substances during the lipid peroxidation of model membranes has been studied. Ferrous ion and ascorbate-induced lipid peroxidation of liposomal membranes containing phosphatidylethanolamine led to the formation of fluorescent substances which have characteristics similar to those of compounds derived from the reaction of phosphatidylethanolamine with purified fatty acid hydroperoxides. The fluorescent substances were accumulated in liposomal membranes, whereas thiobarbituric acid-reactive substances formed during lipid preoxidation were immediately released from the liposomal membranes. The thiobarbituric acid-reactive substances free from the membranes were not reactive with amino compounds such as phosphatidylethanolamine in liposomes or glycine in aqueous phase. It was suggested that the products reacting with amino compounds are short-lived, and may be rapidly inactivated after released into aqueous phase. The formation of fluorescent products was inefficient when phosphatidylethanolamine incorporated into the liposomes insensitive to lipid preoxidation was incubated with ferrous ion and ascorbate in the presence of liposomes sensitive to the peroxidation. The results suggest that some products generated from peroxidation-sensitive lipids react with the amino group of phosphatidylethanolamine molecules which are located on the same membranes, forming fluorescent substances. The presence of phosphatidylethanolamine in the membrane suppressed the formation of thiobarbituric acid-reactive substances, suggesting that phosphatidylethanolamine may react with radicals formed and terminate the propagation.