Local and nonlocal curvature elasticity in bilayer membranes by tether formation from lecithin vesicles.

Bilayer membranes exhibit an elastic resistance to changes in curvature. This resistance depends both on the intrinsic stiffness of the constituent monolayers and on the curvature-induced expansion or compression of the monolayers relative to each other. The monolayers are constrained by hydrophobic forces to remain in contact, but they are capable of independent lateral redistribution to minimize the relative expansion or compression of each leaflet. Therefore, the magnitude of the expansion and compression of the monolayers relative to each other depends on the integral of the curvature over the entire membrane capsule. The coefficient characterizing the membrane stiffness resulting from relative expansion is the nonlocal bending modulus kr. Both the intrinsic (local) bending modulus (kc) and the nonlocal bending modulus (kr) can be measured by the formation of thin cylindrical membrane strands (tethers) from giant phospholipid vesicles. Previously, we reported measurements of kc based on measurements of tether radius as a function of force (Song and Waugh, 1991, J. Biomech. Engr. 112:233). Further analysis has revealed that the contribution from the nonlocal bending stiffness can be detected by measuring the change in the aspiration pressure required to establish equilibrium with increasing tether length. Using this approach, we obtain a mean value for the nonlocal bending modulus kr of approximately 4.1 x 10(-19)J. The range of values is broad (1.1-10.1 x 10(-19)J) and could reflect contributions other than simple mechanical equilibrium. Inclusion of the nonlocal bending stiffness in the calculation of kc results in a value for that modulus of approximately 1.20 +/- 0.17 x 10(-19)J, in close agreement with values obtained by other methods.     

Transversal mobility in bilayer membrane vesicles: Use of pyrene lecithin as optical probe

Pyrene lecithin, a new excimer-forming lipid molecule, has been synthesized to examine the transversal mobility of probe molecules in lecithin bilayer vesicles. The rate of the lipid exchange is obtained by following the excimer yield as a function of time after mixing of fluorescence doped and undoped vesicles. A rapid exchange ($\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 11}\text{s}$) is followed by a slow transfer ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 8}\text{h}$). Above the lipid phase transition the fast transfer can be attributed to an exchange of lipid molecules from the outer layer of one vesicle to the outer layer of another one. The slow exchange is interpreted in terms of the ‘flip-flop’ process between the two layers of a single bilayer vesicle.Using pyrene and pyrene decanoic acid as probe molecules only the fast transfer through the water phase is observed ($\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4}\text{s}$ for pyrene and $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 7}\text{s}$ for pyrene decanoic acid). This indicates that molecules like fatty acids or apolar membrane constituents must equilibrate very rapidly in a single bilayer vesicle.The water solubility or the critical micelle concentration of the probe molecules is determined and related to the transfer rates. An exchange process through the water phase via a monomeric state can be excluded.     

Incorporation of membrane proteins into large single bilayer vesicles. Application to rhodopsin

A general procedure to incorporate membrane proteins in a native state into large single bilayer vesicles is described. The results obtained with rhodopsin from vertebrate and invertebrate retinas are presented. The technique involves: (a) the direct transfer of rhodopsin-lipid complexes from native membranes into ether or pentane, and (b) the sonication of the complex in apolar solvent with aqueous buffer followed by solvent evaporation under reduced pressure. The spectral properties of rhodopsin in the large vesicles are similar to those of rhodopsin in photoreceptors; furthermore, bleached bovine rhodopsin is chemically regenerable with 9-cis retinal. These results establish the presence of photochemically functional rhodopsin in the large vesicles. Freeze-fracture replicas of the vesicles reveal that both internal and external leaflets contain numerous particles approximately 80 A in diameter, indicating that rhodopsin is symmetrically distributed within the bilayer. More than 75% of the membrane area is incorporated into vesicles larger than 0.5 micron and approximately 40% into vesicles larger than 1 micron.     

Perturbation of lecithin bilayer structure by globoside.

The ultrastructure of aggregates formed by mixtures of pig erythrocyte lecithin, cholesterol and globoside in aqueous systems was studied by electron microscopy and X-ray diffraction. Globoside and lecithin in up to equimolar amounts formed a lamellar mesophase, although the structure of the lamellae was perturbed. Mixtures containing excess globoside formed complex tubular or reticular aggregates. Cholesterol appeared to promote mixing of lecithin and globoside. The flexibility gradient of the hydrocarbon (hc) region of the lipid bilayers was studied using electron spin resonance (esr) spectroscopy of various nitroxide-labelled stearic acid probes. Globoside in equimolar amounts greatly perturbed the order parameters of lecithin bilayers, reducing the fluidity of the hc region and flattening the flexibility gradient near the polar (p) surface. The effect of globoside on lecithin-cholesterol bilayers was not so pronounced, since the latter was already more ordered than lecithin bilayers. A phase transition of pure globoside at 55 degrees C, involving 'melting' of the hc chains was also detected using X-ray and esr spectroscopic techniques. The interbilayer spacing, dw, of equimolar lecithin-globoside lamellar phase increased by 42% from that of lecithin bilayers, indicating that the glycolipid p group may increase the net repulsive force between bilayers, as was previously predicted theoretically.     

Phospholipid exchange between bilayer membrane vesicles.

The turbidity of lipid vesicles, freshly prepared by sonicating purified dimyristoyllecithin (DML) in dilute KCl solutions, was measured as a function of time at various temperatures. A sharp maximum in the rate of increase of turbidity is found just above the crystal:liquid-crystal phase transition temperature (Tm). The initial rate of turbidity increase is first order with respect to DML concentration. Electron and light microscopy reveal large vesicles which are not present before incubation or after incubation at temperatures far from the Tm. When temperature, rather than time, is the independent variable, a sharp drop in turbidity is seen at the Tm. The magnitude of this drop and the temperature at which it occurs were used to measure the rate of lipid transfer between vesicles composed of different lipids. A mixture of DML vesicles and dipalmitoyllecithin (DPL) vesicles exhibits sharp drops in turbidity at 24 and 41 degrees, the corresponding Tm's. With time, the magnitude of the transition at 24 degrees decreases while that which was originally at 41 degrees moves to lower temperatures and increases in magnitude. At equilibrium there is a single transition at 32.5 degrees characteristic of vesicles composed of equimolar DPL and DML. The rate at which equilibrium is approached increases at around 24 degrees and again around 41 degrees. These observations indicate that vesicles are in equilibrium with monomolecular lipid, the concentration of the latter being higher the shorter the lipid acyl group or the smaller the vesicle. DML molecules are therefore lost from small vesicles to large vesicles (DML system) or lost from DML vesicles to DML-DPL vesicles (mixed system). When DML vesicles containing a few percent brain gangliosides were studied, different behavior was observed; the initial rate of increase of turbidity becomes second order in lipid concentration, and the rate constant increases with increasing concentrations of KCl. The kinetic order, coupled with the fact that electrolyte reduces intervesicle electrostatic repulsion, argues that in this situation the mechanism of vesicle growth requires vesicle collision.     

Glycophorin-induced cholesterol-phospholipid domains in dimyristoylphosphatidylcholine bilayer vesicles.

Glycophorin has been incorporated into unilamellar cholesterol-containing dimyristoylphosphatidylcholine vesicles that were reconstituted by the freeze and thaw technique. Evidence was obtained for a protein-induced structural reorganization of these mixed membranes. By differential scanning calorimetry, we were able to construct a phase diagram for the phospholipid/cholesterol mixture consisting of a liquid-ordered, a solid-ordered, and a liquid-disordered phase. Glycophorin at low molar fractions (XG less than 3 X 10(-3)) increases the relative amount of lipid in the liquid-ordered phase, which is interpreted as an enrichment of cholesterol in the vicinity of the protein. The formation of such steroid-enriched domains could be demonstrated directly by electron paramagnetic resonance using a spin-labeled cholesterol analogue. A drastic increase of the spin-spin interaction of the labeled steroid was observed in the presence of glycophorin.     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca2+ concentration was increased to a threshold of 3--5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 micrometer diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca2+-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca2+ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca2+ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca2+ concentration.     

The lateral diffusion coefficient of lecithin molecules in single bilayer vesicles studied by 14N NMR

The ¹⁴N nuclear relaxation times T₁ and T₂ in egg yolk phosphatidylcholine have been observed in single bilayer vesicles dispersed in the media of different viscosities, ¹H₂O and ²H₂O. The lateral diffusion coefficient of lipid molecule D has been calculated according to the method reported earlier: D = 2.2 × 10^?8cm²s^?1 in ¹H₂O and 2.1 × 10^?8cm²s^?1 in ²H₂O at 20°C. They are in excellent agreement. This result gives a strong basis of usefulness of ¹⁴N NMR method in the evaluation of D without introducing any system perturbation.     

Single bilayer vesicles prepared without sonication. Physico-chemical properties.

Single shelled lecithin vesicles of uniform size (diameter = 300 A) are prepared without sonication by solubilizing unsonicated lecithin dispersions with sodium cholate and removing the detergent from the mixed lecithin - cholate micelles by gel filtration on Sephadex G-50. A homogeneous population of pure lecithin single-bilayer vesicles free of multilamellar structures is obtained. The vesicle diameter is somewhat larger than the average diameter of sonicated vesicles. The curvature of the bilayer seems to be sufficiently large to allow for similar packing densities (areas/molecule) on the outer and inner layer of the bilayer. The morphology and some physico-chemical properties of these vesicles are described and compared with those of sonicated vesicles.     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca²⁺ concentration was increased to a threshold of 3–5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 μm diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca²⁺-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca²⁺ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca²⁺ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca²⁺ concentration.     

Effect of small molecules on the dipalmitoyl lecithin liposomal bilayer: III. Phase transition in lipid bilayer

Summary The effect of more than ninety lipid-soluble compounds on the phase transition behavior ofdl--dipalmitoyl lecithin bilayer has been examined by differential scanning calorimetry. The type of effect on the phase transition profile depends on the nature of the additive, whereas the extent of the effect depends on the concentration. The compounds examined include uncouplers, alkanols, fatty acids, detergents, organic solvents, ionophores, inorganic ions, and some commonly used spin-labelled and fluorescent membrane probes. A qualitatively distinct effect of several of these additives on the phase transition behavior of bilayer provides a method of determining the nature of the perturbation they induce in the bilayer organization. The observations are consistent with the hypothesis that the type of effect induced by an additive on the phase transition profile of the bilayer is related to the position of localization of the additive along the thickness of the bilayer. At least four different types of modified transition profiles that are related to changes in bilayer fluidity can be distinguished. These correspond to the localization of the additive in phosphorylcholine (type D), glycerol backbone (type B), C₁–C₈ methylene (type A), C₉–C₁₆ methylene (type C) region of the bilayer. A possible relationship between the type of phase transition profiles of modified liposomes and the physiological effects of drugs is also discussed.     

Cholesterol stabilizes hemifused phospholipid bilayer vesicles

Cholesterol was found to inhibit full fusion of oppositely charged phospholipid bilayer vesicles by stabilizing the contacting membranes at the stage of the hemifused intermediate. Vesicles of opposite charge containing different amounts of cholesterol were prepared using cationic (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine) and anionic (dioleoylphosphatidylglycerol) phospholipids. Pairwise interactions between such vesicles were observed by fluorescence video microscopy in real time after electrophoretically maneuvering the vesicles into contact. Hemifusion accounted for more than 80% of the observed events when the vesicles contained 33-50 mole% cholesterol. In contrast, vesicles containing only a small proportion of cholesterol (相似文献   

Phase separation of miscible phospholipids by sonication of bilayer vesicles.

Sonication of phospholipid vesicles may result, according to their liquid or solid crystal state, in the generation of unilamellar vesicles or structural defects within their bilayers, respectively. The transition temperature Tm of the phospholipid bilayer is usually the threshold temperature delineating the physical effects of ultrasound. However, for vesicles made from a mixture of two miscible phospholipids, this threshold temperature was not found to be the intermediate Tm of the phospholipid mixture bilayers, but the Tm of the lowest melting component. This was due to a simultaneous lateral phase separation of the two phospholipids induced by the sonication as demonstrated by differential scanning calorimetry analysis.     

X-ray scattering of vesicles of N-acyl sphingomyelins. Determination of bilayer thickness.

A series of N-acyl sphingomyelins (C16:0, C18:0, C20:0, C22:0, and C24:0) have been synthesized and single bilayer vesicles formed by sonication and ultracentrifugation. X-ray scattering data have been collected from the sphingomyelin vesicles at 50 degrees C in the melted-chain state. The x-ray scattering data have been transformed to the corresponding Patterson functions and Fourier electron density profiles; analysis of these functions has provided the intrabilayer phosphate-phosphate separation dp-p, a measure of the lipid bilayer thickness. The bilayer thickness increases linearly with increasing chain length (increment 1.3-1.4 A) and the intercept, 14.3-15.0 A, suggests a contribution of 7.0-7.5 A for each phosphorylcholine group to the bilayer thickness. The electron-density profiles have features suggestive of chain interdigitation when the length of the N-acyl chain (C20:0, C22:0, and C24:0) exceeds significantly the length of the invariant sphingosine chain.     

Chloride flux in bilayer membranes: chloride permeability in aqueous dispersions of single-walled, bilayer vesicles.

Aqueous dispersions of phosphatidylcholine vesicles were utilized to determine bilayer permeability to 36-Cl as a function of pH and temperature. These dispersions were comprised of single-walled vesicles, homogeneous in size, prepared by sonication of purified egg phosphatidylcholine under argon followed by fractionation on a molecular sieve. Permeability constants calculated from the inward flux of 36-Cl and the geometric parameters of these vesicles proved to be dependent on both pH and temperature. Analysis of these dependences leads to the conclusion that 36-Cl permeation in the presence of KCl is due principally to a carrier mediated exchange process involving a phospholipid-HCL complex. Net permeation by H-36-Cl may make a small contribution to the 36-Cl flux, however, studies carried out at very low chloride concentrations show that this flux is much smaller than the exchange flux. Thus chloride permeability for the exchange process is 1.5 times 10- minus 11 cmsec- minus 1 while the corresponding coefficient for the net flux of H-36-Cl is 1.0 times 10- minus 12 cm sec- minus 1 at pH 7. The activation energy for the 36-Cl exchange flux was found to be 19 plus or minus 2 kcal/mol. This value is similar to that obtained for the transbilayer "flip-flop" of phosphatidylcholine molecules in a similar system (Kornberg and McConnell, 1971). This correspondence together with the fact that the experimentally determined flux of 36-Cl agrees well with that calculated from the "flip-flop" parameters, strongly suggests that the flux of 36-Cl and "flip-flop" of phosphatidylcholine may be the same process.     

Tilted hydrocarbon chains of dipalmitoyl lecithin become perpendicular to the bilayer before melting.

Differential scanning calorimetry studies of dipalmitoyl lecithin show two reversible transitions as the temperature is changed between 20 and 50 degrees C. A pretransition endotherm occurs at 35 degrees C prior to the main chain melting endotherm which occurs at 42 degrees C. X-ray diffraction studies show that below 33 degrees C the chains of the lecithin are fully extended, packed in a hexagonal crystalline lattice but tilted with respect to the plane of the bilayer. Between 35 and 42 degrees C the chains are similarly packed but oriented perpendicular to the bilayer plane. Above 44 degrees C the chains are "melted" or disordered. Monolayer studies of dipalmitoyl lecithin using continuous recording of pressure with molecular area reveal the existence of two solid condensed phases corresponding to these tilted and verticle chain structures. The tilted to perpendicular transition would account for the pretransition endotherm of the lipid; the crystalline to melted change corresponds to the larger transition observed at 42 degrees C.     

Interaction of d-β-hydroxybutyrate dehydrogenase with lecithin vesicles

Rat liver mitochondrial d-β-hydroxybutyrate dehydrogenase has an absolute requirement for lecithin. The nature of the interaction between the enzyme and phospholipid has been investigated. Single bilayer lecithin liposomes of shell-like structure bring about maximal enzyme activation, whereas the interaction with larger vesicles leads to enzyme inactivation. The strong binding of the enzyme to lecithin confers great stability to the enzyme activity as compared with the nonlipid-activated enzyme, and permits the isolation of a lipoprotein complex by chromatography on Sephadex G-200. Only 20% of the proteins solubilized with d-β-hydroxybutyrate dehydrogenase from mitochondrial membranes bind to lecithin liposomes, thus a 5-fold purification of the enzyme is achieved. The liposome-bound proteins had a significantly lower polarity than the remaining 80% of solubilized mitochondrial membrane proteins.     

Shapes of bilayer vesicles with membrane embedded molecules

The interdependence of the lateral distribution of molecules which are embedded in a membrane (such as integral membrane proteins) and the shape of a cell with no internal structure (such as phospholipid vesicles or mammalian erythrocytes) has been studied. The coupling of the lateral distribution of the molecules and the cell shape is introduced by considering that the energy of the membrane embedded molecule at a given site of the membrane depends on the curvature of the membrane at that site. Direct interactions between embedded molecules are not considered. A simple expression for the interaction of the membrane embedded molecule with the local membrane curvature is proposed. Starting from this interaction, the consistently related expressions for the free energy and for the distribution function of the embedded molecules are derived. The equilibrium cell shape and the corresponding lateral distribution of the membrane embedded molecules are determined by minimization of the membrane free energy which includes the free energy of the membrane embedded molecules and the membrane elastic energy. The resulting inhomogeneous distribution of the membrane embedded molecules affects the cell shape in a nontrivial manner. In particular, it is shown that the shape corresponding to the absolute energy minimum at given cell volume and membrane area may be elliptically non-axisymmetric, in contrast to the case of a laterally homogeneous membrane where it is axisymmetric.     

Formation and properties of cell-size lipid bilayer vesicles

Hydration of single or mixed phospholipids or lipid protein mixtures at low ionic strength results in the formation of a population of large, solvent free, single bilayer vesicles with included volumes of up to 300 microliters/mumol lipid. Their size ranges from 0.1 to 300 microns and they can be sorted out according to size by centrifugation. When formed in distilled water their internal solution has a conductivity of 20-50 microseconds/cm-1, an osmolarity of 0.5-5 mOsM, and a density of 1.0005-1.001. The osmotic pressure produced by the internal solutes cause a surface stress of 25 dyn/cm for a 20-microns vesicle. Their elastic constant ranges from 75-150 dyn/cm. During formation they can internalize particles such as latex beads or cell nuclei. They can be impaled with microelectrodes, or patch clamped. They can also be sealed to a small Vaseline-treated hole in a thin partition between two aqueous compartments. Sealing occurs in two stages. In the first stage sealing resistance is similar to that seen with patch-clamp pipettes. In the second stage, a much tighter seal is obtained. After sealing, the smaller portion of the sealed vesicle can be selectively broken by an electric shock leaving a single membrane across the hole. The capacitance and resistance of such membranes, in the presence of 10 mM NaCl, are approximately 0.7 microF/cm2 and 10(8) omega cm2 for pure lipid vesicles. Gramicidin increases the membrane conductance and monazomycin induces voltage-dependent gating thus providing further evidence that the vesicles are bounded by a single bilayer.     

Apolipoprotein-induced conversion of phosphatidylcholine bilayer vesicles into nanodisks

Apolipoprotein mediated formation of nanodisks was studied in detail using apolipophorin III (apoLp-III), thereby providing insight in apolipoprotein-lipid binding interactions. The spontaneous solubilization of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles occured only in a very narrow temperature range at the gel-liquid-crystalline phase transition temperature, exhibiting a net exothermic interaction based on isothermal titration calorimetry analysis. The resulting nanodisks were protected from proteolysis by trypsin, endoproteinase Glu-C, chymotrypsin and elastase. DMPC solubilization and the simultaneous formation of nanodisks were promoted by increasing the vesicle diameter, protein to lipid ratio and concentration. Inclusion of cholesterol in DMPC dramatically enhanced the rate of nanodisk formation, presumably by stabilization of lattice defects which form the main insertion sites for apolipoprotein α-helices. The presence of fully saturated acyl chains with a length of 13 or 14 carbons in phosphatidylcholine allowed the spontaneous vesicle solubilization upon apolipoprotein addition. Nanodisks with C13:0-phosphatidylcholine were significantly smaller with a diameter of 11.7 ± 3.1nm compared to 18.5 ± 5.6 nm for DMPC nanodisks determined by transmission electron microscopy. Nanodisk formation was not observed when the phosphatidylcholine vesicles contained acyl chains of 15 or 16 carbons. However, using very high concentrations of lipid and protein (>10mg/ml), 1,2,-dipalmitoyl-sn-glycero-3-phosphocholine nanodisks could be produced spontaneously although the efficiency remained low.