Free energy potential for aggregation of giant, neutral lipid bilayer vesicles by Van der Waals attraction.

Here, we report the first direct observation of Van der Waals' attraction between biomembrane capsules using measurements of the free energy reduction per unit area of membrane-membrane contact formation. In these studies, the membrane capsules were reconstituted neutral (egg phosphatidylcholine) lipid bilayers of giant (greater than 10(-3) cm diam) vesicles. Micromanipulation methods were used to select and maneuver two vesicles into proximity for contact; after adhesion was allowed to occur, the extent of contact formation was regulated through the vesicle membrane tensions that were controlled by micropipette suction. The free energy reduction per unit area of contact formation was proportional to the membrane tension multiplied by a simple function of the pipette and vesicle dimensions. The free energy potential for Van der Waals attraction between the neutral bilayers in 120 mM NaCl solutions was 1.5 X 10(-2) ergs/cm2. Also, when human serum albumin was added to the medium in the range of 0-1 mg/ml, the free energy potential for bilayer-bilayer adhesion was not affected. Using published values for equilibrium spacing between lipid bilayers in multilamellar lipid-water dispersions and the theoretical equation for van der Waals attraction between continuous dielectric layers, we calculated the value for the Hamaker coefficient of the Van der Waals attraction to be 5.8 X 10(-14) ergs.     

Elasticity, strength, and water permeability of bilayers that contain raft microdomain-forming lipids

Bilayers composed of phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol (CHOL) are commonly used as systems to model the raft-lipid domain structure believed to compartmentalize particular cell membrane proteins. In this work, micropipette aspiration of giant unilamellar vesicles was used to test the elasticities, water permeabilities, and rupture tensions of single-component PC, binary 1:1 PC/CHOL, and 1:1 SM/CHOL, and ternary 1:1:1 PC/SM/CHOL bilayers, one set of measurements with dioleoyl PC (DOPC; C18:1/C18:1 PC) and the other with stearoyloleoyl PC (SOPC; C18:0/C18:1 PC). Defining the elastic moduli (K_A), the initial slopes of the increase in tension (σ) versus stretch in lipid surface area (α_e) were determined for all systems at low (15°C) and high (32-33°C) temperatures. The moduli for the single-component PC and binary phospholipid/CHOL bilayers followed a descending hierarchy of stretch resistance with SM/CHOL > SOPC/CHOL > DOPC/CHOL > PC. Although much more resistant to stretch than the single-component PC bilayers, the elastic response of vesicle bilayers made from the ternary phospholipid/CHOL mixtures showed an abrupt softening (discontinuity in slope), when immediately subjected to a steady ramp of tension at the low temperature (15°C). However, the discontinuities in elastic stretch resistance at low temperature vanished when the bilayers were held at ∼1 mN/m prestress for long times before a tension ramp and when tested at the higher temperature 32-33°C. The elastic moduli of single-component PC and DOPC/CHOL bilayers changed very little with temperature, whereas the moduli of the binary SOPC/CHOL and SM/CHOL bilayers diminished markedly with increase in temperature, as did the ternary SOPC/SM/CHOL system. For all systems, increasing temperature increased the water permeability but decreased rupture tension. Concomitantly, the measurements of permeability exhibited a prominent correlation with the rupture tension across all the systems. Together, these micromechanical tests of binary and ternary phospholipid/CHOL bilayers demonstrate that PC hydrocarbon chain unsaturation and temperature are major determinants of the mechanical and permeation properties of membranes composed of raft microdomain-forming lipids.     

Developing initial conditions for simulations of asymmetric membranes: a practical recommendation

It has been proposed that the surface tension difference between leaflets (or differential stress) in asymmetric bilayers is generally nonvanishing. This implies that there is no unique approach to generate initial conditions for simulations of asymmetric bilayers in the absence of experimentally derived constraints. Current generation methods include individual area per lipid (APL) based, leaflet surface area (SA) matching, and zero leaflet tension based (0-DS). This work adds a bilayer-based approach that aims for achieving partial chemical equilibrium by interleaflet switching of selected lipids via P2₁ periodic boundary conditions. Based on a recently proposed theoretical framework, we obtained expressions for tensions in asymmetric bilayers from both the bending and area strains. We also developed a quantitative measure for the energetic penalty from the differential stress. The impacts of APL-, SA-, and 0-DS-based approaches on mechanical properties are assessed for two different asymmetric bilayers. The lateral pressure profile and its moments differ significantly for each method, whereas the area compressibility modulus is relatively insensitive. Application of P2₁ periodic boundary conditions (APL/P2₁, SA/P2₁, and 0-DS/P2₁) results in better agreement in mechanical properties between asymmetric bilayers generated by APL-, SA-, and 0-DS-based approaches, in which changes are the smallest for bilayers from the SA-based method. The estimated differential stress from the theory shows good agreement with that from the simulations. These simulation results and the good agreement between the predicted and observed differential stress further support the theoretical framework in which bilayer mechanical properties are outcomes of the interplay between intrinsic bending and asymmetric lipid packing. Based on the simulation results and theoretical predictions, the SA/P2₁-based, or at least the SA-based (when the differential stress is small), approach is recommended as a practical method for developing initial conditions for asymmetric bilayer simulations.     

Unusual penetration of phospholipid mono- and bilayers by Quillaja bark saponin biosurfactant

The interactions between a model phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and a biosurfactant Quillaja Bark Saponin (QBS) obtained from the bark of Quillaja saponaria Molina were studied using simple models of biological membranes. QBS is known to interact strongly with the latter, exerting a number of haemolytic, cytotoxic and anti-microbial actions. The interaction of QBS dissolved in the subphase with DPPC monolayers and silicon-supported bilayers was studied above the cmc (10^− 3 M). Surface pressure relaxation and surface dilatational rheology combined with quartz crystal microbalance (QCM) and neutron reflectivity (NR) were employed for this purpose. The DPPC-penetrating abilities of QBS are compared with those of typical synthetic surfactants (SDS, CTAB and Triton X-100). We show that the penetration studies using high surface activity (bio)surfactants should be performed by a subphase exchange, not by spreading onto the surfactant solution. In contrast to the synthetic surfactants of similar surface activity, QBS does not collapse DPPC mono- and bilayers, but penetrates them, improving their surface dilatational elastic properties even in the highly compressed solid state. The dilatational viscoelasticity modulus increases from 204 mN/m for pure DPPC up to 310 mN/m for the QBS-penetrated layers, while it drops to near zero values in the case of the synthetic surfactants. The estimated maximum insertion pressure of QBS into DPPC monolayers exceeds the maximum surface pressure achievable in our setup, in agreement with the surface rheological response of the penetrated layers.     

NaCl-dependent formation of the highly crystalline phase in sufficiently hydrated dimyristoylphosphatidylglycerol bilayers

We investigated the low-temperature phase behavior of dimyristoylphosphatidylglycerol (DMPG) bilayers in the presence of high concentration of NaCl (≥100 mM). Differential scanning calorimetry showed that the highly crystalline (HC) phase grew after an initial delay period when DMPG bilayers were sufficiently hydrated and incubated at 1 °C in the presence of more than 100 mM NaCl. The HC phase formation reached a plateau, the level of which depended on NaCl concentration; all the lipids were unable to be in the HC phase at the plateau stage without a quite high concentration of NaCl. Since electron microscopic observations suggested that the HC phase formed coexists with the precursor phases in a closed vesicle, elastic constrain and/or shortage of free sodium ions in the inside of the closed vesicle may prevent the complete transition into the HC phase.     

Protein chemistry at membrane interfaces: non-additivity of electrostatic and hydrophobic interactions

Non-specific binding of proteins and peptides to charged membrane interfaces depends upon the combined contributions of hydrophobic (DeltaG(HPhi)) and electrostatic (DeltaG(ES)) free energies. If these are simply additive, then the observed free energy of binding (DeltaG(obs)) will be given by DeltaG(obs)=DeltaG(HPhi)+DeltaG(ES), where DeltaG(HPhi)=-sigma(NP)A(NP) and DeltaG(ES)=zFphi. In these expressions, A(NP) is the non-polar accessible area, sigma(NP) the non-polar solvation parameter, z the formal peptide valence, F the Faraday constant, and phi the membrane surface potential. But several lines of evidence suggest that hydrophobic and electrostatic binding free energies of proteins at membrane interfaces, such as those associated with cell signaling, are not simply additive. In order to explore this issue systematically, we have determined the interfacial partitioning free energies of variants of indolicidin, a cationic proline-rich antimicrobial peptide. The synthesized variants of the 13 residue peptide covered a wide range of hydrophobic free energies, which allowed us to examine the effect of hydrophobicity on electrostatic binding to membranes formed from mixtures of neutral and anionic lipids. Although DeltaG(obs) was always a linear function of DeltaG(HPhi), the slope depended upon anionic lipid content: the slope was 1.0 for pure, zwitterionic phosphocholine bilayers and 0.3 for pure phosphoglycerol membranes. DeltaG(obs) also varied linearly with surface potential, but the slope was smaller than the expected value, zF. As observed by others, this suggests an effective peptide valence z(eff) that is smaller than the formal valence z. Because of our systematic approach, we were able to establish a useful rule-of-thumb: z(eff) is reduced relative to z by about 20 % for each 3 kcal mol(-1) (1 kcal=4.184 kJ) favorable increase in DeltaG(HPhi). For neutral phosphocholine interfaces, we found that DeltaG(obs) could be predicted with remarkable accuracy using the Wimley-White experiment-based interfacial hydrophobicity scale.     

Free energy potential for aggregation of mixed phosphatidylcholine/phosphatidylserine lipid vesicles in glucose polymer (dextran) solutions.

The energetics of lipid vesicle-vesicle aggregation in dextran (36,000 mol wt) solutions have been studied with the use of micromechanical experiments. The affinities (free energy reduction per unit area of contact) for vesicle-vesicle aggregation were determined from measurements of the tension induced in an initially flaccid vesicle membrane as it adhered to another vesicle. The experiments involved controlled aggregation of single vesicles by the following procedure: two giant (approximately 20 micron diam) vesicles were selected from a chamber on the microscope stage that contained the vesicle suspension and transferred to a second chamber that contained a dextran (36,000 mol wt) salt solution (120 mM); the vesicles were then maneuvered into position for contact. One vesicle was aspirated with sufficient suction pressure to create a rigid sphere outside the pipette; the other vesicle was allowed to spread over the rigid vesicle surface. The aggregation potential (affinity) was derived from the membrane tension vs. contact area. Vesicles were formed from mixture of egg lecithin (PC) and phosphatidylserine (PS). For vesicles with a PC/PS ratio of 10:1, the affinity showed a linear increase with concentration of dextran; the values were on the order of 10(-1) ergs/cm2 at 10% by weight in grams. Similarly, pure PC vesicle aggregation was characterized by an affinity value of 1.5 X 10(-1) ergs/cm2 in 10% dextran by weight in grams. In 10% by weight in grams solutions of dextran, the free energy potential for vesicle aggregation decreased as the surface charge (PS) was increased; the affinity extrapolated to zero at a PC/PS ratio of 2:1. When adherent vesicle pairs were transferred into a dextran-free buffer, the vesicles did not spontaneously separate. They maintained adhesive contact until forceably separated, after which they would not read here. Thus, it appears that dextran forms a "cross-bridge" between the vesicle surfaces.     

Permeability of acetic acid across gel and liquid-crystalline lipid bilayers conforms to free-surface-area theory.

Solubility-diffusion theory, which treats the lipid bilayer membrane as a bulk lipid solvent into which permeants must partition and diffuse across, fails to account for the effects of lipid bilayer chain order on the permeability coefficient of any given permeant. This study addresses the scaling factor that must be applied to predictions from solubility-diffusion theory to correct for chain ordering. The effects of bilayer chemical composition, temperature, and phase structure on the permeability coefficient (Pm) of acetic acid were investigated in large unilamellar vesicles by a combined method of NMR line broadening and dynamic light scattering. Permeability values were obtained in distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dilauroylphosphatidylcholine bilayers, and their mixtures with cholesterol, at various temperatures both above and below the gel-->liquid-crystalline phase transition temperatures (Tm). A new scaling factor, the permeability decrement f, is introduced to account for the decrease in permeability coefficient from that predicted by solubility-diffusion theory owing to chain ordering in lipid bilayers. Values of f were obtained by division of the observed Pm by the permeability coefficient predicted from a bulk solubility-diffusion model. In liquid-crystalline phases, a strong correlation (r = 0.94) between f and the normalized surface density sigma was obtained: in f = 5.3 - 10.6 sigma. Activation energies (Ea) for the permeability of acetic acid decreased with decreasing phospholipid chain length and correlated with the sensitivity of chain ordering to temperature, [symbol: see text] sigma/[symbol: see text](1/T), as chain length was varied. Pm values decreased abruptly at temperatures below the main phase transition temperatures in pure dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine bilayers (30-60-fold) and below the pretransition in dipalmitoylphosphatidylcholine bilayers (8-fold), and the linear relationship between in f and sigma established for liquid-crystalline bilayers was no longer followed. However, in both gel and liquid-crystalline phases in f was found to exhibit an inverse correlation with free surface area (in f = -0.31 - 29.1/af, where af is the average free area (in square angstroms) per lipid molecule). Thus, the lipid bilayer permeability of acetic acid can be predicted from the relevant chain-packing properties in the bilayer (free surface area), regardless of whether chain ordering is varied by changes in temperature, lipid chain length, cholesterol concentration, or bilayer phase structure, provided that temperature effects on permeant dehydration and diffusion and the chain-length effects on bilayer barrier thickness are properly taken into account.     

Binding,folding and insertion of a β-hairpin peptide at a lipid bilayer surface: Influence of electrostatics and lipid tail packing

Antimicrobial peptides (AMPs) act as host defenses against microbial pathogens. Here we investigate the interactions of SVS-1 (KVKVKVKV^dP^lPTKVKVKVK), an engineered AMP and anti-cancer β-hairpin peptide, with lipid bilayers using spectroscopic studies and atomistic molecular dynamics simulations. In agreement with literature reports, simulation and experiment show preferential binding of SVS-1 peptides to anionic over neutral bilayers. Fluorescence and circular dichroism studies of a Trp-substituted SVS-1 analogue indicate, however, that it will bind to a zwitterionic DPPC bilayer under high-curvature conditions and folds into a hairpin. In bilayers formed from a 1:1 mixture of DPPC and anionic DPPG lipids, curvature and lipid fluidity are also observed to promote deeper insertion of the fluorescent peptide. Simulations using the CHARMM C36m force field offer complementary insight into timescales and mechanisms of folding and insertion. SVS-1 simulated at an anionic mixed POPC/POPG bilayer folded into a hairpin over a microsecond, the final stage in folding coinciding with the establishment of contact between the peptide's valine sidechains and the lipid tails through a “flip and dip” mechanism. Partial, transient folding and superficial bilayer contact are seen in simulation of the peptide at a zwitterionic POPC bilayer. Only when external surface tension is applied does the peptide establish lasting contact with the POPC bilayer. Our findings reveal the influence of disruption to lipid headgroup packing (via curvature or surface tension) on the pathway of binding and insertion, highlighting the collaborative effort of electrostatic and hydrophobic interactions on interaction of SVS-1 with lipid bilayers.     

Location and dynamics of anthracyclines bound to unilamellar phosphatidylcholine vesicles

We have exploited the intrinsic fluorescence properties of the anthracycline antitumor antibiotics to study the dependence on drug structure of relative drug location and dynamics when the anthracyclines were bound to sonicated dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) vesicles at 27.5 degrees C. Iodide quenching experiments at constant ionic strength were used to evaluate the relative accessibilities of the bound fluorophores to membrane-impermeable iodide. Iodide was found to quench the fluorescence of anthracyclines in free solution by both static and dynamic mechanisms, whereas quenching of membrane-bound fluorophores was predominantly due to the dynamic mechanism. Modified Stern-Volmer plots of anthracyclines bound to fluid-phase DMPC bilayers were linear, and the biomolecular rate constant (kq) values ranged from 0.6 X 10(9) to 1.3 X 10(9) M-1 s-1. Modified Stern-Volmer plots of anthracyclines bound to solid-phase DPPC bilayers were curved, indicative of a heterogeneous-bound drug population. A strong correlation between drug hydrophobicity and penetration of the fluorophore into the bilayer was observed for the daunosamine-containing anthracyclines. Steady-state fluorescence anisotropy measurements under iodide quenching conditions were used to investigate the diffusive motions of anthracyclines in isotropic solvent and in fluid-phase DMPC bilayers. Anthracycline derivatives free in solution exhibited limiting anisotropy (alpha infinity) values which decayed to zero at times long compared to the excited-state lifetime, in contrast to anthracyclines bound to fluid-phase DMPC bilayers, which showed nonzero alpha infinity values. Steady-state anisotropies of membrane-bound anthracyclines were found to be governed principally by alpha infinity and not by the mean rotational rate (R).(ABSTRACT TRUNCATED AT 250 WORDS)     

Origin of gradients in lipid density and surface tension between connected lipid droplet and bilayer

We combined theory and experiments to depict physical parameters modulating the phospholipid (PL) density and tension equilibrium between a bilayer and an oil droplet in contiguity. This situation is encountered during a neutral lipid (NL) droplet formation in the endoplasmic reticulum. We set up macroscopic and microscopic models to uncover free parameters and the origin of molecular interactions controlling the PL densities of the droplet monolayer and the bilayer. The established physical laws and predictions agreed with experiments performed with droplet-embedded vesicles. We found that the droplet monolayer is always by a few percent (∼10%) less packed with PLs than the bilayer. Such a density gradient arises from PL-NL interactions on the droplet, which are lower than PL-PL trans interactions in the bilayer, i.e., interactions between PLs belonging to different leaflets of the bilayer. Finally, despite the pseudo-surface tension for the water/PL acyl chains in the bilayer being higher than the water/NL surface tension, the droplet monolayer always has a higher surface tension than the bilayer because of its lower PL density. Thus, a PL density gradient is mandatory to maintain the mechanical and thermodynamic equilibrium of the droplet-bilayer continuity. Our study sheds light on the origin of the molecular interactions responsible for the unique surface properties of lipid droplets compared with cellular bilayer membranes.     

A charge-modified general amber force field for phospholipids: improved structural properties in the tensionless ensemble

Accurately predicting the structural properties of phospholipid with a fully atomistic molecular model is critical for the study of pure phospholipid bilayers, mixed bilayer systems and bilayers containing proteins. The general amber force field (GAFF) has traditionally required the presence of a surface tension parameter to correctly model phospholipid bilayer properties such as area per lipid and order parameters. In this work, the GAFF partial charges for 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphochiline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) were re-parameterised utilising high-level ab initio calculations and the restrained electrostatic potential method. Simulations of pure POPA, POPC and POPG bilayers using the charge-modified GAFF and no applied surface tension are compared with available experimental data, the original GAFF model and the recent Lipid14 variant. The results indicate a significant improvement in the accuracy of the lipid model for reproducing experimental observables without the need for a surface tension parameter. The successful application of modifying the lipid charge distributions represents an alternative to the use of a surface tension parameter within GAFF, and highlights the importance of the partial charge calculations when modelling lipid bilayers.     

Bilayer Mixing,Fusion, and Lysis Following the Interaction of Populations of Cationic and Anionic Phospholipid Bilayer Vesicles

Cationic, O-alkylphosphatidylcholines, recently developed as DNA transfection agents, form bilayers indistinguishable from those of natural phospholipids and undergo fusion with anionic bilayers. Membrane merging (lipid mixing), contents release, and contents mixing between populations of positive vesicles containing O-ethylphosphatidylcholine (EDOPC) and negative vesicles containing dioleolylphosphatidylglycerol (DOPG) have been determined with standard fluorometric vesicle-population assays. Surface-charge densities were varied from zero to full charge. All interactions depended critically on surface-charge density, as expected from the adhesion-condensation mechanism. Membrane mixing ranged from zero to 100%, with significant mixing (>10 <70%) occurring between cationic vesicles that were fully charged and anionic vesicles that had fractional surface charges as low as 0.1. Such mixing with membranes as weakly charged as cell membranes should be relevant to transfection with cationic lipids. Unexpectedly, lipid mixing was higher at high than at low ionic strength when one lipid dispersion was prepared from EDOPC plus DOPG (in different proportions), especially when the other vesicles were of EDOPC; this may somehow be a consequence of the ability of the former mixture to assume non-lamellar phases. Leakage of aqueous contents was also a strong function of charge, with fully charged vesicles releasing essentially all of their contents less than 1 min after mixing. EDOPC was more active in this regard than was DOPG, which probably reflects stronger intermolecular interactions of DOPG. Fusion, as measured by contents mixing, exhibited maximal values of 10% at intermediate surface charge. Reduced fusion at higher charge is attributed to multiple vesicle interactions leading to rupture. The existence of previously published data on individual interactions of vesicles of the same composition made it possible for the first time to compare pairwise with population interactions, confirming the likelihood of population studies to overestimate rupture and hemifusion and underestimate true vesicle fusion.     

Determination of electric parameters of cell membranes by a dielectrophoresis method.

Marszalek, P., J. J. Zielinsky, and M. Fikus (1989. Bioelectrochem. Bioenerg. 22:289-298) have described a novel design for measuring the complete dielectrophoretic spectrum of a single cell. From the analysis of the dielectrophoretic spectrum, the membrane conductivity, sigma membr, and the membrane dielectric permittivity, epsilon membr, of the cell may be determined according to the theory of dielectrophoresis described by Sauer, F. A. (1985. Interactions between Electromagnetic Field and Cells. A. Chiabrera, C. Nicolini, and H.P. Schwan, editors. Plenum Publishing Corp., New York. 181-202). At Fo, the net force experienced by a single shell sphere in a nonuniform periodic field is zero, and the sphere ceases to move in the field. In other words, at Fo, the effective polarizability, chi eff, of the sphere (the polarizability of sphere minus the polarizability of the medium) is equal to zero. For biological cells in high conductivity medium, e.g., the isotonic saline, sigma membr falls below 2 x 10(-6) S m-1, where Fo becomes insensitive to sigma membr, and the method becomes impractical. In a low conductivity medium, 0.3 M sucrose, sigma membr of cells is generally higher and the method may be applied. Assuming a membrane thickness of 9 nm, epsilon membr of Neurospora crassa slime cells was determined to be in the range of 8.3-9.4 epsilon o, and of myeloma Tib9 to be 9.4 epsilon o, epsilon o being the dielectric permittivity of the free space. The values for the slime cells were compared with values obtained by the dielectric spectroscopy method which measures average values for cells in suspension.     

Low concentrations of free hydrophobic amino acids disrupt the Escherichia coli RNA polymerase core-sigma(70) protein-protein interaction.

Studies of the Escherichia coli RNA polymerase subunit sigma-70 employing limited proteolytic digestion and binding by monoclonal antibodies indicate that conserved region 3 is solvent accessible in the free protein and in the RNA polymerase holoenzyme. Conversely, when sigma-70 binds to core RNA polymerase, proteolytic cleavage of region 3 is dramatically reduced. The former set of results seems to indicate the physical presence of region 3 on or near the surface of the holoenzyme while the latter of these results suggest that region 3 is sequestered in a direct protein-protein contact within the RNA holoenzyme which alters its protease sensitivity. To further investigate these possibilities we inserted an internal histidine-tag within region 3 of sigma(70) (sigma(70)-R3-His6) between amino acids 508 and 509. Confirmation that the internal His-tag insertion does not disrupt normal sigma(70) function was verified by genetic complementation. His-tagged protein was immobilized on nickel-agarose and core RNAP was tethered via the sigma-core interaction. Our results are consistent with the localization of region 3 on or near the surface both of free sigma(70) and of RNA polymerase holoenzyme. Furthermore, we find that the sigma(70)-core interaction is resistant to high ionic conditions but is completely disrupted by the presence of the low-molecular-weight hydrophobic amino acids phenylalanine and leucine free in solution. These results demonstrate the general usefulness of this approach to the disruption of protein-protein interactions and its potential application for protein purification.     

Pretransitional effects in dimyristoylphosphatidylcholine vesicle membranes: optical dynamometry study

We used micron-sized latex spheres to probe the phase state and the viscoelastic properties of dimyristoylphosphatidylcholine (DMPC) bilayers as a function of temperature. One or two particles were manipulated and stuck to a DMPC giant vesicle by means of an optical trap. Above the fluid-gel main transition temperature, T(m) congruent with 23.4 degrees C, the particles could move on the surface of the vesicle, spontaneously (Brownian motion) or driven by an external force, either gravity or the laser beam's radiation pressure. From the analysis of the particle motions, we deduced the values of the membrane hydrodynamic shear viscosity, eta(s), and found that it would increase considerably near T(m). Below T(m), the long-distance motion of the particles was blocked. We performed experiments with two particles stuck on the membrane. By optical dynamometry, we measured the elastic resistance of the membrane to a variation in the interparticle distance and found that it would decrease considerably (down to zero) when the temperature was increased to T(m). We propose an interpretation relating the elastic response to the membrane curvature modulus, k(C). In this scheme, the two-bead dynamometry experiments provide a direct measurement of k(C) in the P'(beta) phase of lipid bilayers.     

Analysis of adhesion of large vesicles to surfaces.

An experimental procedure that can be used to measure the interfacial free energy density for the adhesion of membranes of large vesicles to other surfaces is outlined and analyzed. The approach can be used for both large phospholipid bilayer vesicles and red blood cells when the membrane force resultants are dominated by isotropic tension. The large vesicle or red cell is aspirated by a micropipet with sufficient suction pressure to form a spherical segment outside the pipet. The vesicle is then brought into close proximity of the surface to be tested and, the suction pressure reduced to permit adhesion, and the new equilibrium configuration is established. The mechanical analysis of the equilibrium shape provides the interfacial free energy density for the surface affinity. With this approach, the measurable range of membrane surface affinity is 10(-4)-3 erg/cm2 for large phospholipid bilayer vesicles and 10(-2)-10 erg/cm2 for red blood cells.     

Electrostatic interaction between ion-penetrable multi-layered membranes

A theory of the double layer interaction regulated by the Donnan potential between two ion-penetrable membranes in an electrolyte solution developed previously by Ohshima and Kondo is extended to the case in which the membranes consist of many layers having different thickness and densities of membrane-fixed charges. The interaction force is found to be determined mainly by the contributions from layers located within the depth of 1/kappa (kappa, Debye-Hückel parameter) from the membrane surface. It is also predicted that the interaction force may alter its sign with changing electrolyte concentration.