The toolbox of vesicle sidedness determination

Vesicles prepared from cellular plasma membranes are widely used in science for different purposes. The outer membrane leaflet differs from the inner membrane leaflet of the vesicle, and during vesicle preparation procedures two types of vesicles will be generated: right-side-out vesicles, of which the outer leaflet is topologically equivalent to the outer monolayer of the cellular plasma membrane, and inside-out vesicles. Because two populations of vesicles exist, sidedness information of the vesicle preparation is indispensable. This note focuses on the ins and outs of sidedness determination of vesicles and compares various methodologies used to establish this ratio.     

The curvature and cholesterol content of phospholipid bilayers alter the transbilayer distribution of specific molecular species of phosphatidylethanolamine

The curvature, cholesterol content, and transbilayer distribution of phospholipids significantly influence the functional properties of cellular membranes, yet little is known of how these parameters interact. In this study, the transbilayer distribution of phosphatidylethanolamine (PE) is determined in vesicles with large (98 nm) and small (19 nm) radii of curvature and with different proportions of PE, phosphatidylcholine, and cholesterol. It was found that the mean diameters of both types of vesicles were not influenced by their lipid composition, and that the amino-reactive compound 2,4,6-trinitrobenzenesulphonic acid (TNBS) was unable to cross the bilayer of either type of vesicle. When large vesicles were treated with TNBS, approximately 40% of the total membrane PE was derivatized; in the small vesicles 55% reacted. These values are interpreted as representing the percentage of total membrane PE residing in the outer leaflet of the vesicle bilayer. The large vesicles likely contained approximately 20% of the total membrane lipid as internal membranes. Therefore, in both types of vesicles, PE as a phospholipid class was randomly distributed between the inner and outer leaflets of the bilayer. The proportion of total PE residing in the outer leaflet was unaffected by changes in either the cholesterol or PE content of the vesicles. However, the transbilayer distributions of individual molecular species of PE were not random, and were significantly influenced by radius of curvature, membrane cholesterol content, or both. For example, palmitate- and docosahexaenoate-containing species of PE were preferentially located in the outer leaflet of the bilayer. Membrane cholesterol content affected the transbilayer distributions of stearate-, oleate-, and linoleate-containing species. The transbilayer distributions of palmitate-, docosahexaenoate-, and stearate-containing species were significantly influenced by membrane curvature, but only in the presence of high levels of cholesterol. Thus, differences in membrane curvature and cholesterol content alter the array of PE molecules present on the surfaces of phospholipid bilayers. In cells and organelles, these differences could have profound effects on a number of critical membrane functions and processes.     

The Curvature and cholesterol content of phospholipid bilayers alter the transbilayer distribution of specific molecular species of phosphatidylethanolamine

The curvature, cholesterol content,and transbilayer distribution of phospholipids significantly influence the functional properties of cellular membranes, yet little is known of how these parameters interact. In this study, the transbilayer distribution of phosphatidylethanolamine (PE) is determined in vesicles with large (98 nm) and small (19 nm)radii of curvature and with different proportions of PE, phosphatidylcholine, and cholesterol. It was found that the mean diameters of both types of vesicles were not influenced by their lipid composition, and that the amino-reactive compound 2,4,6-trinitrobenzenesulphonic acid (TNBS) was unable to cross the bilayer of either type of vesicle. When large vesicles were treated with TNBS, ~40% of the total membrane PE was derivatized; in the small vesicles 55% reacted. These values are interpreted as representing the percentage of total membrane PE residing in the outer leaflet of the vesicle bilayer. The large vesicles likely contained ~20% of the total membrane lipid as internal membranes. Therefore, in both types of vesicles, PE as a phospholipid class was randomly distributed between the inner and outer leaflets ofthe bilayer. The proportion oftotal PE residing in the outer leaflet was unaffected by changes in either the cholesterol orPE content of the vesicles. However, the transbilayer distributions of individual molecular species of PE were not random, and were significantly influenced by radius of curvature, membrane cholesterol content, or both. For example, palmitate and docosahexaenoate-containing species of PE were preferentially located in the outer leaflet of the bilayer. Membrane cholesterol content affected the transbilayer distributions of stearate-, oleate-, and linoleate-containing species. The transbilayer distributions ofpalmitate-, docosahexaenoate-, and stearate-containing species were significantly influenced by membrane curvature, but only in the presence of high levels of cholesterol. Thus, differences in membrane curvature and cholesterol content alter the array of PE molecules present on the surfaces of phospholipid bilayers. In cells and organelles, these differences could have profound effects on a number of critical membrane functions and processes.     

Real-time Observation of Lipoplex Formation and Interaction with Anionic Bilayer Vesicles

A novel development has allowed for the direct observation of single, pairwise interactions of linear DNA with cationic vesicles and of DNA-cationic lipid complexes with anionic vesicles. A new cationic phospholipid derivative, l,2-dioleoyl-sn-glycero-3-ethylphosphocholine, was used to prepare giant bilayer vesicles and to form DNA-cationic lipid complexes (lipoplexes). The cationic vesicles were electrophoretically maneuvered into contact with DNA, and similarly, complexes were brought into contact with anionic phospholipid vesicles composed of dioleoylphosphatidylglycerol (DOPG; 100%), DOPG/dioleoylphosphatidylethanolamine (DOPE; 1:1) or DOPG/dioleoylphosphatidylcholine (DOPC; 1:1). Video fluorescence microscopy revealed that upon contact with phospholipid anionic vesicles, lipoplexes exhibited four different types of behavior: adhesion, vesicle rupture, membrane perforation (manifested as vesicle shrinkage and/or content loss), and expansion of DNA (which was always concomitant with membrane perforation.) In one instance, the lipoplex was injected into the target vesicle just prior to DNA expansion. In all other instances, the DNA expanded over the outer surface of the vesicle, and expansion was faster, the larger the area of vesicle over which it expanded. Given the likelihood of incorporation of cellular anionic lipids into lipoplexes, the expansion of the DNA could be important in DNA release during cell transfection. Upon contact with naked DNA, giant cationic vesicles usually ruptured and condensed the DNA into a small particle. Contact of cationic vesicles that were partially coated with DNA usually caused the DNA to wrap around the vesicle, leading to vesicle rupture, vesicle fusion (with other attached vesicles or lipid aggregates), or simply cessation of movement. These behaviors clearly indicated that both DNA and vesicles could be partly or fully covered by the other, thus modifying surface charges, which, among others, allowed adhesion of DNA-coated vesicles with uncoated vesicles and of lipid-coated DNA with uncoated DNA.     

Structural changes in membranes of large unilamellar vesicles after binding of sodium cholate

The interaction of the bile salt cholate with unilamellar vesicles was studied. At low cholate content, equilibrium binding measurements with egg yolk lecithin membranes suggest that cholate binds to the outer vesicle leaflet. At increasing concentrations, further bile salt binding to the membrane is hampered. Before the onset of membrane solubilization, diphenylhexatriene fluorescence anisotropy decreases to a shallow minimum. It then increases to the initial value in the cholate concentration range of membrane solubilization. At still higher cholate concentrations, a drop in fluorescence anisotropy indicates the transformation of mixed disk micelles into spherical micelles. Perturbation of the vesicle membranes at molar ratios of bound cholate/lecithin exceeding 0.15 leads to a transient release of oligosaccharides from intravesicular space. The cholate concentrations required to induce the release depend on the size of the entrapped sugars. Cholesterol stabilizes the membrane, whereas, in spite of enhanced membrane order, sphingomyelin destabilizes the membrane against cholate. Freeze-fracture electron microscopy and phosphorus-31 nuclear magnetic resonance (31P NMR) also reflect a change in membrane structure at maximal cholate binding to the vesicles. In 31P NMR spectra, superimposed on the anisotropic line typically found in phospholipid bilayers, an isotropic peak was found. This signal is most probably due to the formation of smaller vesicles after addition of cholate. The results were discussed with respect to bile salt/membrane interactions in the liver cell. It is concluded that vesicular bile salt transport in the cytoplasm is unlikely and that cholate binding is restricted to the outer leaflet of the canalicular part of the plasma membrane.     

Effects of hemagglutinin fusion peptide on poly(ethylene glycol)-mediated fusion of phosphatidylcholine vesicles.

The effects of hemagglutinin (HA) fusion peptide (X-31) on poly(ethylene glycol)- (PEG-) mediated vesicle fusion in three different vesicle systems have been compared: dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV) and palmitoyloleoylphosphatidylcholine (POPC) large unilamellar perturbed vesicles (pert. LUV). POPC LUVs were asymmetrically perturbed by hydrolyzing 2.5% of the outer leaflet lipid with phospholipase A(2) and removing hydrolysis products with BSA. The mixing of vesicle contents showed that these perturbed vesicles fused in the presence of PEG as did DOPC SUV, but unperturbed LUV did not. Fusion peptide had different effects on the fusion of these different types of vesicles: fusion was not induced in the absence of PEG or in unperturbed DOPC LUV even in the presence of PEG. Fusion was enhanced in DOPC SUV at low peptide surface occupancy but hindered at high surface occupancy. Finally, fusion was hindered in proportion to peptide concentration in perturbed POPC LUV. Contents leakage assays demonstrated that the peptide enhanced leakage in all vesicles. The peptide enhanced lipid transfer between both fusogenic and nonfusogenic vesicles. Peptide binding was detected in terms of enhanced tryptophan fluorescence or through transfer of tryptophan excited-state energy to membrane-bound diphenylhexatriene (DPH). The peptide had a higher affinity for vesicles with packing defects (SUV and perturbed LUV). Quasi-elastic light scattering (QELS) indicated that the peptide caused vesicles to aggregate. We conclude that binding of the fusion peptide to vesicle membranes has a significant effect on membrane properties but does not induce fusion. Indeed, the fusion peptide inhibited fusion of perturbed LUV. It can, however, enhance fusion between highly curved membranes that normally fuse when brought into close contact by PEG.     

Asymmetric disposition of detergents within vesicle bilayer and its effect on ion permeation through the membrane

Phospholipid vesicles were prepared by detergent removal using hydrophobic porous beads, Amberlite XAD-2, or dialysis from detergent-phospholipid mixed micelles. The liposomes formed were found to be mostly unilammellar vesicles. The vesicle diameter was estimated, by both quasi-elastic light-scattering and gel-exclusion chromatography on Sephacryl S-1000, to be 80 nm for the vesicles formed by removal of octaethylene glycol monododecyl ether by the bead method. The effect of detergents within a bilayer on ion permeation was demonstrated. When the content of octaethylene glycol monododecyl ether reached a molar ratio of 0.2, the intrinsic ion selectivity of the phospholipid membrane between anion and cation was diminished. The ion permeability measured for vesicles with detergent incorporated into initially detergent-free vesicles was about 10-times greater than that for vesicles with detergent remaining following the process of detergent removal. This observation was explained by the different disposition of the detergent in the bilayer, that is, when vesicles were formed by the removal of detergent from mixed micelles, the residual detergent became distributed in both the outer and inner leaflets, and when the detergent was incorporated into initially detergent-free vesicles, the detergent became distributed only in the outer leaflet within the experimental time limits. This idea was supported by the NMR studies. It was also found that, as a detergent, octaethylene glycol monododecyl ether has a stronger effect on ion permeation than octyl glucoside.     

The Influence of Natural Lipid Asymmetry upon the Conformation of a Membrane-inserted Protein (Perfringolysin O)

Eukaryotic membrane proteins generally reside in membrane bilayers that have lipid asymmetry. However, in vitro studies of the impact of lipids upon membrane proteins are generally carried out in model membrane vesicles that lack lipid asymmetry. Our recently developed method to prepare lipid vesicles with asymmetry similar to that in plasma membranes and with controlled amounts of cholesterol was used to investigate the influence of lipid composition and lipid asymmetry upon the conformational behavior of the pore-forming, cholesterol-dependent cytolysin perfringolysin O (PFO). PFO conformational behavior in asymmetric vesicles was found to be distinct both from that in symmetric vesicles with the same lipid composition as the asymmetric vesicles and from that in vesicles containing either only the inner leaflet lipids from the asymmetric vesicles or only the outer leaflet lipids from the asymmetric vesicles. The presence of phosphatidylcholine in the outer leaflet increased the cholesterol concentration required to induce PFO binding, whereas phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a novel deeply inserted conformation that does not form pores, even though it contains transmembrane segments. This conformation may represent an important intermediate stage in PFO pore formation. These studies show that lipid asymmetry can strongly influence the behavior of membrane-inserted proteins.     

Lysophosphatidylcholine stabilizes small unilamellar phosphatidylcholine vesicles. Phosphorus-31 NMR evidence for the "wedge" effect

Sonication of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-sn-glycero-3-phosphocholine (lysoPC, up to approximately 30 mol %) produces small unilamellar vesicles (SUV, 250-265 A diameter). Phosphorus-31 NMR of the POPC/lysoPC vesicles gives rise to four distinct peaks for POPC and lysoPC in the outer and in the inner bilayer leaflet which can be used to localize and quantify the phospholipids in both vesicle shells. Addition of paramagnetic ions (3 mM Pr3+) enhances outside/inside chemical shift differences and allows monitoring of membrane integrity by the absence of Pr3+ in the vesicle interior. 31P NMR shows that lysoPC in these highly curved POPC/lysoPC vesicles prefers the outer bilayer leaflet. LysoPC incorporation into POPC SUV furthermore causes a substantial and concentration-dependent decrease in spin-spin relaxations (T*2) of the outside POPC phosphorus signals from 55 ms for pure POPC vesicles (v1/2, 5.8 Hz) to 29.5 ms (v1/2, 10.8 Hz) for POPC/lysoPC vesicles containing 25 mol % lysoPC. Our findings are consistent with the idea of a cone-shaped lysoPC molecule which, for geometric reasons, is preferentially accommodated in the outer bilayer leaflet. LysoPC incorporation into POPC SUV restricts POPC headgroup motion and tightens phospholipid packing, but only in the outer bilayer shell.     

Isothermal titration calorimetry studies of the binding of the antimicrobial peptide gramicidin S to phospholipid bilayer membranes

The binding of the amphiphilic, positively charged, cyclic beta-sheet antimicrobial decapeptide gramicidin S (GS) to various lipid bilayer model membrane systems was studied by isothermal titration calorimetry. Large unilamellar vesicles composed of the zwitterionic phospholipid 1-palmitoyl-2-oleoylphosphatidylcholine or the anionic phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, or a binary mixture of the two, with or without cholesterol, were used to mimic the lipid compositions of the outer monolayers of the lipid bilayers of mammalian and bacterial membranes, respectively. Dynamic light scattering results suggest the absence of major alterations in vesicle size or appreciable vesicle fusion upon the binding of GS to the lipid vesicles under our experimental conditions. The binding isotherms can be reasonably well described by a one-site binding model. GS is found to bind with higher affinity to anionic phosphatidylglycerol than to zwitterionic phosphatidylcholine vesicles, indicating that electrostatic interactions in the former system facilitate peptide binding. However, the presence of cholesterol reduced binding only slightly, indicating that the binding of GS is not highly sensitive to the order of the phospholipid bilayer system. Similarly, the measured positive endothermic binding enthalpy (DeltaH) varies only modestly (2.6 to 4.4 kcal/mol), and the negative free energy of binding (DeltaG) also remains relatively constant (-10.9 to -12.1 kcal/mol). The relatively large but invariant positive binding entropy, reflected in relatively large TDeltaS values (13.4 to 16.4 kcal/mol), indicates that GS binding to phospholipid bilayers is primarily entropy driven. Finally, the relative binding affinities of GS for various phospholipid vesicles correlate relatively well with the relative lipid specificity for GS interactions with bacterial and erythrocyte membranes observed in vivo.     

Localization of cyanine dye binding to brush-border membranes by quenching of n-(9-anthroyloxy) fatty acid probes

The location and orientation of 3,3'-dipropylthiodicarbocyanine (diS-C3-(5)) binding sites in renal brush-border membrane vesicles was examined from the quenching of n-(9-anthroyloxy) fatty acid (n-AS) fluorescence. Based on previous kinetic studies (Cabrini, G. and Verkman, A.S. (1986) J. Membrane Biol. 90, 163-175) monomeric aqueous diS-C3-(5) binds to brush-border membrane vesicles (BBMV) by an initial 6 ms association to form bound monomer, a 30-40 ms equilibrium between bound monomer (M) and bound dimer (D), and a 1-1.3 s translocation of D from the outer to inner membrane leaflet. Based on Stern-Volmer and lifetime analyses, M and D quench the fluorescence of the n-AS probes by a collisional mechanism. At low [diS-C3-(5)]/[BBMV] (R), where M predominates, the n-AS quenching efficiencies (Q) are similar (n = 2-16); at high R, where D predominates, Q increases with n (16 greater than 12 much much greater than 6 greater than 2), suggesting that M is oriented parallel, and D perpendicular, to the phospholipid chains deep within the membrane. Mixture of diS-C3-(5) with brush-border membrane vesicles containing n-AS in a stopped-flow apparatus gave a biexponential fluorescence decrease (excitation 390 nm, emission above 450 nm) with time constants 30-40 ms and 1-1.5 s; there was no 6 ms quenching process. These findings are incorporated into a model in which diS-C3-(5) adheres loosely to the outer membrane surface in 6 ms, binds parallel to the membrane phospholipid in 30-40 ms, dimerizes and rotates by 90 degrees in much less than 30 ms, and translocates to the opposite half of the bilayer in 1-15 s.     

Ion gradient-induced membrane translocation of model peptides.

The K+ diffusion potential-induced association of synthetic model peptides carrying a single positive charge originating from the NH2-terminal amino function with large unilamellar vesicles (LUV) consisting of phosphatidylcholine (PC) has been reported previously (de Kroon, A. I. P. M., J. de Gier, and B. de Kruijff. 1989. Biochim. Biophys. Acta. 981:371-373). To determine the vesicle localization of the associated peptides, fluorescence measurements utilizing the peptides' tryptophan residue as intrinsic fluorescent probe were performed. The application in these measurements, of vesicles that exhibit an asymmetric transbilayer distribution of brominated PC which is a quencher of tryptophan fluorescence, unequivocally demonstrated that the peptide H3N(+)-AIMLWA-Ome (AIXme+) is accumulated in the interface of the inner leaflet of the vesicle membrane in response to the valinomycin-induced K+ diffusion potential (negative inside). The relative contributions of the membrane potential (delta psi) and the pH gradient (delta pH, acidic inside) induced by the K+ diffusion potential, to the process have been assessed. An analysis of the pH and delta pH dependencies of the process demonstrated that the K+ diffusion potential-induced peptide accumulation is largely determined by a redistribution of peptide according to the transbilayer pH gradient, in agreement with a translocation across the vesicle membrane of the neutral, deprotonated form of the peptide. The general validity of the mechanism proposed for the vesicle-uptake of AIXme+ has been examined by extending the experiments to peptide analogues with a single negative charge and to peptides with two positive charges, and by investigating the effect of incorporating the acidic phospholipid cardiolipin (CL) into the LUV. The incorporation of CL appeared not to affect the K+ diffusion, potential-induced vesicle uptake of AIXme+. The peptide H3N(+)-RMLWA-Ome (RXme2+) showed a small delta pH independent fluorescence response to the delta psi upon raising the CL content of the vesicles to 25%.     

Charge-dependent translocation of the Trojan peptide penetratin across lipid membranes

We studied the interaction of the cell-penetrating peptide penetratin with mixed dioleoylphosphatidylcholine/dioleoylphoshatidylglycerol (DOPC/DOPG) unilamellar vesicles as a function of the molar fraction of anionic lipid, X(PG), by means of isothermal titration calorimetry. The work was aimed at getting a better understanding of factors that affect the peptide binding to lipid membranes and its permeation through the bilayer. The binding was well described by a surface partitioning equilibrium using an effective charge of the peptide of z(P) approximately 5.1 +/- 0.5. The peptide first binds to the outer surface of the vesicles, the effective binding capacity of which increases with X(PG). At X(PG) approximately 0.5 and a molar ratio of bound peptide-to-lipid of approximately 1/20 the membranes become permeable and penetratin binds also to the inner monolayer after internalization. The results were rationalized in terms of an "electroporation-like" mechanism, according to which the asymmetrical distribution of the peptide between the outer and inner surfaces of the charged bilayer causes a transmembrane electrical field, which alters the lateral and the curvature stress acting within the membrane. At a threshold value these effects induce internalization of penetratin presumably via inversely curved transient structures.     

On the binding preference of human groups IIA and X phospholipases A2 for membranes with anionic phospholipids

Mammals contain 9-10 secreted phospholipases A(2) (sPLA(2)s) that display widely different affinities for membranes, depending on the phospholipid composition. The much higher enzymatic activity of human group X sPLA(2) (hGX) compared with human group IIA sPLA(2) (hGIIA) on phosphatidylcholine (PC)-rich vesicles is due in large part to the higher affinity of the former enzyme for such vesicles; this result also holds when vesicles contain cholesterol and sphingomyelin. The inclusion of anionic phosphatidylserine in PC vesicles dramatically enhances interfacial binding and catalysis of hGIIA but not of hGX. This is the result of the large number of lysine and arginine residues scattered over the entire surface of hGIIA, which cause the enzyme to form a supramolecular aggregate with multiple vesicles. Thus, high affinity binding of hGIIA to anionic vesicles is a complex process and cannot be attributed to a few basic residues on its interfacial binding surface, as is also evident from mutagenesis studies. The main reason hGIIA binds poorly to PC-rich vesicles is that it lacks a tryptophan residue on its interfacial binding surface, a residue that contributes to the high affinity binding of hGX to PC-rich vesicles. Results show that the lag in the onset of hydrolysis of PC vesicles by hGIIA is due in part to the poor affinity of this enzyme for these vesicles. Binding affinity of hGIIA, hGX, and their mutants to PC-rich vesicles is well correlated to the ability of these enzymes to act on the PC-rich outer plasma membrane of mammalian cells.     

Asymmetry of lipid organization in cholinergic synaptic vesicle membranes.

The lipid composition of purified Torpedo cholinergic synaptic vesicles was determined and their distribution between the inner and outer leaflets of the vesicular membrane was investigated. The vesicles contain cholesterol and phospholipids at a molar ratio of 0.63. The vesicular phospholipids are (mol% of total phospholipids): phosphatidylcholine (40.9); phosphatidylethanolamine (24.6); plasmenylethanolamine (11.5); sphingomyelin (12); phosphatidylserine (7.3); phosphatidylinositol (3.7). The asymmetry of the synaptic vesicle membranes was investigated by two independent approaches: (a) determining accessibility of the amino lipids to the chemical label trinitrobenzenesulphonic acid (TNBS); (b) determining accessibility of the vesicular glycerophospholipids to phospholipase C (Bacillus cereus). TNBS was found to render the vesicles leaky and thus cannot be used reliably to determine the asymmetry of Torpedo synaptic vesicle membranes. Incubation of the vesicles with phospholipase C (Bacillus cereus) results in biphasic hydrolysis of the vesicular glycerophospholipids. About 45% of the phospholipids are hydrolysed in less than 1 min, during which no vesicular acetylcholine is released. In the second phase, the hydrolysis of the phospholipids slows down markedly and is accompanied by loss of all the vesicular acetylcholine. These findings suggest that the lipids hydrolysed during the first phase are those comprising the outer leaflet. Analysis of the results thus obtained indicate that the vesicular membrane is asymmetric: all the phosphatidylinositol, 77% of the phosphatidylethanolamine, 47% of the plasmenylethanolamine and 58% of the phosphatidylcholine were found to reside in the outer leaflet. Since phosphatidylserine is a poor substrate for phospholipase C (B. cereus), its distribution between the two leaflets of the synaptic vesicle membrane is only suggestive.     

Intramembrane electrostatic interactions destabilize lipid vesicles

Membrane stability is of central concern in many biology and biotechnology processes. It has been suggested that intramembrane electrostatic interactions play a key role in membrane stability. However, due primarily to a lack of supporting experimental evidence, they are not commonly considered in mechanical analyses of lipid membranes. In this paper, we use the micropipette aspiration technique to characterize the elastic moduli and critical tensions of lipid vesicles with varying surface charge. Charge was induced by doping neutral phosphatidylcholine vesicles with anionic lipids phosphatidylglycerol and phosphatidic acid. Measurements were taken in potassium chloride (moderate ion-lipid binding) and tetramethylammonium chloride (low ion-lipid binding) solutions. We show that inclusion of anionic lipid does not appreciably alter the areal dilation elasticity of lipid vesicles. However, the tension required for vesicle rupture decreases with increasing anionic lipid fraction and is a function of electrolyte composition. Using vesicles with 30% charged (i.e., unbound) anionic lipid, we measured critical tension reductions of 75%, demonstrating the important role of electrostatic interactions in membrane stability.     

A novel leaflet-selective fluorescence labeling technique reveals differences between inner and outer leaflets at high bilayer curvature

Understanding the differences in the physical properties of the inner and outer leaflet of membranes and how the leaflets are coupled to each other requires methods that can selectively label both the outer and inner leaflets. In this report we introduce a combined chromatography/cyclodextrin method for selective labeling of the inner leaflet. Combining this method with selective labeling of the outer leaflet, we are able to show that there is a distinct difference in polar headgroup physical properties of the inner and outer leaflet headgroups in small unilamellar vesicles composed of a wide variety of phosphatidylcholines and a phosphaticylcholine/sphingomyelin mixture. It appears that the inner leaflet headgroups are more tightly packed than those of the outer leaflet. This differential packing disappears when vesicle size increases, showing that it is a consequence of membrane curvature. Differential packing is also reduced as acyl chain length is decreased. In the future, selective leaflet labeling is likely to be a powerful tool for investigating the properties of asymmetric lipid vesicles.     

Annexin V and vesicle membrane electroporation

The method of membrane electroporation (ME) has been used as an analytical tool to quantify the effect of membrane curvature on transient electric pore for-mation, and on the adsorption of the protein annexin V (M_r = 35,800) to the outer surface of unilamellar lipid vesicles (of radii 25 ≤ a/nm ≤ 200). Relaxation kinetic studies using optical membrane probes of the diphenylhexatriene type suggest that electric pore formation is induced by ionic interfacial polarization causing entrance of the (more polarizable) water into the lipid bilayer membrane yielding (hydrophobic and hydrophilic) pore states with a mean stationary pore radius r_p=0.35 (±0.05) nm. Extent and rate of ME, compared at the same induced transmembrane voltage, were found to decrease both with increasing vesicle radius and with increasing protein concentration. This `inhibitory' effect of annexin V is apparently allosteric and saturates at about [AN_T]_sat = 4 μm annexin V for vesicles of a = 100 nm at 1 mm total lipid concentration, 0.13 mm total Ca²⁺ concentration and at T = 293 K. Data analysis in terms of Gibbs area-difference-elasticity energy suggests that the bound annexin V reduces the gradient of the lateral pressure across the membrane. At [AN_T]_sat, about 20% of the vesicle surface is covered by the bound protein, but it is only 0.01% of the surface of the outer lipid leaflet in which a part of the protein, perhaps the aromatic residue of the tryptophan (W 187), is inserted. Insertion leads to a denser packing of the lipid molecules in the outer membrane leaflet. As a consequence, the radius of the electropores in the remaining membrane part, not covered by annexin V decreases (r_p/nm = 0.37, 0.36 and 0.27) with increasing adsorption of the protein ([AN_T] = 0, 2 and 4 μm, respectively). Received: 9 January 1997 / Accepted: 21 April 1997     

REGULAR STRUCTURES IN MEMBRANES : I. Membranes in the Endocytic Complex of Ileal Epithelial Cells

An "apical endocytic complex" in the ileal lining cells of suckling rats is described. The complex consists of a continuous network of membrane-limited tubules which originate as invaginations of the apical plasma membrane at the base of the microvilli, some associated vesicles, and a giant vacuole. The lumenal surface of this tubular network of membranes and associated vesicles is covered with a regular repeating particulate structure. The repeating unit is an ~7.5-nm diameter particle which has a distinct subunit structure composed of possibly nine smaller particles each ~3 nm in diameter. The ~7.5-nm diameter particles are joined together with a center-to-center separation of ~15 nm to form long rows. These linear aggregates, when arranged laterally, give rise to several square and oblique two-dimensional lattice arrangements of the particles which cover the surface of the membrane. Whether a square or oblique lattice is generated depends on the center-to-center separation of the rows and on the relative displacement of the particles in adjacent rows. Four membrane faces are revealed by fracturing frozen membranes of the apical tubules and vesicles: two complementary inner membrane faces exposed by the fracturing process and the lumenal and cytoplasmic membrane surfaces revealed by etching. The outer membrane face reveals a distinct array of membrane particles. This array also sometimes can be seen on the outer (B) fracture face and is sometimes faintly visible on the inner (A) fracture face. Combined data from sectioned, negatively stained, and freeze-etched preparations indicate that this regular particulate structure is a specialization that is primarily localized in the outer half of the membrane mainly in the outer leaflet.     

Indirect evidence of submicroscopic pores in giant unilamellar [correction of unilamelar] vesicles

Formation of pore-like structures in cell membranes could participate in exchange of matter between cell compartments and modify the lipid distribution between the leaflets of a bilayer. We present experiments on two model systems in which major lipid redistribution is attributed to few submicroscopic transient pores. The first kind of experiments consists in destabilizing the membrane of a giant unilamellar vesicle by inserting conic-shaped fluorescent lipids from the outer medium. The inserted lipids (10% of the vesicle lipids) should lead to membrane rupture if segregated on the outer leaflet. We show that a 5-nm diameter pore is sufficient to ease the stress on the membrane by redistributing the lipids. The second kind of experiments consists in forcing giant vesicles containing functionalized lipids to adhere. This adhesion leads to hemifusion (merging of the outer leaflets). In certain cases, the formation of pores in one of the vesicles is attested by contrast loss on this vesicle and redistribution of fluorescent labels between the leaflets. The kinetics of these phenomena is compatible with transient submicroscopic pores and long-lived membrane defects.