Elastic Area Compressibility Modulus Research Articles

In Silico Measurement of the Mechanical Properties of Realistic Bacterial Inner and Outer Membranes

The cell envelope in Gram-negative bacteria is made of two distinct membranes and a cell wall between them. From a mechanics point of view, the cell maintains a higher concentration of solutes in the cytoplasm than in the external environment and the difference in osmotic pressure, known as the turgor pressure, places a great stress on the envelope, although which element bears the most is unclear. Now, we have used molecular dynamics (MD) simulations of membranes to resolve how lipid membranes respond to changes in lateral tension resulting from the turgor pressure. In this study, we used two models of the inner, cytoplasmic membrane; the first membrane is modeled as a mixed 75%POPE/25% POPG bilayer while the second membrane model consists of saturated, unsaturated, and cycle-containing lipids that more accurately reflect the diverse population of lipids within the E. coli cytoplasmic membrane. Additionally, we looked at the bacterial outer membrane, which has an outer leaflet of lipopolysaccharides that stiffen it. We applied surface tensions of values from 10 to 200 dynes/cm, in increments of 10 dynes/cm, and measured a variety of properties of membranes (area per lipid, thickness, etc.), thus providing a quantitative description of the requirements of membrane rupture. More general mechanical properties of membranes were also characterized, namely the elastic area compressibility modulus, the energy change, and Young's modulus, in order to describe the elasticity of membrane. Our work demonstrates that differences in lipid composition result in a differential response to lateral tension.

Cell Mechanics: Combining Speed with Precision

The mechanical response of cells is a powerful biophysical marker for cell state. Information on a cell’s elasticity can, for instance, be used to distinguish between different cell phenotypes, or between healthy and diseased cells.

Area Per Lipid and Elastic Deformation of Membrane Bilayers under Osmotic Stress

The energetics of membrane proteins and cellular functions are modulated significantly by membrane properties [1]. Accurate knowledge of cross-sectional area/lipid of membrane systems is pertinent for molecular dynamics simulations [2,3]. Here we address the sensitivity of lipid bilayer structure and fluctuations to osmotic pressure and temperature using solid-state 2H NMR spectroscopy. Applied stress allows us to probe intermembrane interactions including collective membrane motions and lipid protrusions [1,3]. Through 2H NMR studies [3] we obtain striking evidence for bilayer deformation due to application of osmotic pressure to model membranes (DMPC-d54) using polyethylene glycol as osmolyte. The average structure of the membrane is manifested in the segmental order parameters (SCD) of the lipids that are measured with 2H NMR spectroscopy. Measurements of membrane structural parameters such as bilayer thickness and area per lipid employ a mean-torque analysis of 2H NMR order parameters (SCD) [4]. These NMR measurements allow us to interpret the free energy cost of bilayer deformation when combined with complementary X-ray observables. NMR results are used to calculate the osmotic coefficient to distinguish the different regimes of intermolecular forces, thereby yielding insights into bilayer separation forces [2]. We calculated the elastic area compressibility modulus that describes the membrane deformation with osmotic pressure. The present NMR study distinguishes between different intermembrane forces, and suggests that the undulations dominate at intermediate intermembrane distances whereas protrusions act at short distances. The thermodynamic descriptions of these experimental measurements shed light on the effect of osmotic pressure on membranes and their implications for protein functions. [1] M.F. Brown et al. (2002) JACS 124,8471-8484. [2] K.J. Mallikarjunaiah et al. (2011) BJ 100, 98-107. [3] H.I. Petrache et al. (2000) BJ79, 3172-3192. [4] K.J. Mallikarjunaiah et al. (2012) to be submitted to PCCP.

Membrane Area Deformation under Osmotic Stress: Deuterium NMR Approach

We address the hypothesis that the sensitivity of lipid bilayers to pressure, temperature, and osmotic stress represents influences of non-specific lipid-protein interactions on functions of cellular membranes [1,2]. Measurements of membrane structural parameters such as bilayer thickness and area per lipid employ a mean-torque analysis [3] of 2H solid-state NMR order parameters (SCD). NMR lipid order parameters are very sensitive to changes in cross-sectional area per molecule. We observed striking (≈20%) changes in structural properties (decrease in area per lipid and increase in bilayer thickness) when ≈200 atmospheres of pressure (dehydration pressure or osmotic pressure) are applied to lipid (DMPC) bilayers in the liquid-crystalline state. We show the equivalence of osmotic and dehydration pressures by NMR experimental measurements as well as thermodynamically [2]. The elastic area compressibility modulus (Ka) of bilayers is determined by employing different pressure techniques in combination with NMR and vapor pressure osmometry methods. Our findings agree well with the reported Ka value for DMPC [4] obtained with a much smaller range of osmotic pressures. However, we observe an additional variation in Ka determined at higher osmotic pressures, where the role of complex dynamics in the bilayer structural changes becomes more evident. We propose that the hierarchy of forces and motions is perturbed by membrane dehydration (osmotic pressure) due to the alteration of interlamellar spacings, with corresponding changes in elastic area compressibility moduli. Our findings have significant implications for the applicability of solid-state 2H NMR spectroscopy together with membrane stress techniques for understanding the mechanisms of action of pressure-sensitive proteins.[1] A.V. Botelho et al. (2006) BJ 91, 4464-4477.[2] K.J. Mallikarjunaiah et al. (2011) BJ 100, 98-107.[3] H.I. Petrache et al. (2000) BJ 79, 3172-3192.[4] H.I. Petrache et al. (1998) CPL 95, 83-94.

Solution pH Alters Mechanical and Electrical Properties of Phosphatidylcholine Membranes: Relation between Interfacial Electrostatics, Intramembrane Potential, and Bending Elasticity

Solution pH affects numerous biological processes and some biological membranes are exposed to extreme pH environments. We utilized micropipette aspiration of giant unilamellar vesicles composed of 1-stearoyl-2-oleoyl-phosphatidylcholine to characterize the effect of solution pH (2–9) on membrane mechanical properties. The elastic area compressibility modulus was unaffected between pH 3 and 9 but was reduced by ∼30% at pH 2. Fluorescence experiments utilizing the phase-sensitive probe Laurdan confirmed gel-phase characteristics at pH 2, explaining the reduction of membrane elasticity. The membrane bending stiffness, k c, increased by ∼40% at pH 4 and pH 9 over the control value at pH 6.5. Electrophoretic mobility measurements indicate that these changes are qualitatively consistent with theoretical models that predict the effect of membrane surface charge density and Debye length on k c, substantiating a coupling between the mechanical and interfacial electrical properties of the membrane. The effect of pH on intramembrane electrical properties was examined by studying the spectral shifts of the potentiometric probe di-8 ANEPPS. The intramembrane (dipole) potential (Ψ d) increased linearly as the solution pH decreased in a manner consistent with the partitioning of hydroxide ions into the membrane. However, changes in Ψ d did not correlate with changes in k c. These mechanical and electrical studies lead to the conclusion that the effect of pH on membrane bending stiffness results from alterations in interfacial, as opposed to intramembrane, electrostatics.

Characterization of Mixed Monolayers of Phosphatidylcholine and a Dicationic Gemini Surfactant SS-1 with a Langmuir Balance: Effects Of DNA

Monolayers of a cationic gemini surfactant, 2,3-dimethoxy-1,4-bis( N-hexadecyl- N; N-dimethyl-ammonium)butane dibromide (abbreviated as SS-1) and its mixtures with 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) were studied using a Langmuir balance. More specifically, we measured the force-area ( π- A) curves and determined the elastic area compressibility modulus ( C s −1) as a function of lateral packing pressure and the mole fraction of the cationic lipid ( X SS-1), with and without DNA in the subphase. Both SS-1 and POPC exhibited smooth compression isotherms, indicating their monolayers to be in the liquid expanded state. Even low contents ( X SS-1 < 0.05) of SS-1 in a POPC monolayer condensed the film dramatically, up to 20% at 30 mN/m. This effect is suggested to reflect reorientation of the P −-N + dipole of the POPC headgroup. Accordingly, the magnitude of the condensing effect diminishes with X SS-1 and is not observed for mixed films of dioleoylglycerol and SS-1. Reorientation of the P −-N + dipole is further supported by the pronounced increase in monolayer dipole potential ψ due to SS-1. The presence of DNA in the subphase affected the mixed POPC/SS-1 monolayers differently depending on the constituent lipid stoichiometry as well as on the DNA/SS-1 charge ratio. At a DNA concentration of 0.63 μM (in base pairs) condensation of neat POPC monolayers was evident, and this effect remained up to X SS-1 < 0.5, corresponding to DNA/SS-1 charge ratio of 1.25. An expansion due to DNA, evident as an increase in Δ A/molecule, was observed at X SS-1 > 0.5. At a higher concentration of DNA (1.88 μM base pairs) in the subphase corresponding to DNA/SS-1 charge ratio of 3.75 at X SS-1 = 0.5, condensation was observed at all values of X SS-1.

Membrane lateral compressibility determined by NMR and x-ray diffraction: effect of acyl chain polyunsaturation

The elastic area compressibility modulus, Ka, of lamellar liquid crystalline bilayers was determined by a new experimental approach using 2H-NMR order parameters of lipid hydrocarbon chains together with lamellar repeat spacings measured by x-ray diffraction. The combination of NMR and x-ray techniques yields accurate determination of lateral area per lipid molecule. Samples of saturated, monounsaturated, and polyunsaturated phospholipids were equilibrated with polyethylene glycol (PEG) 20,000 solutions in water at concentrations from 0 to 55 wt % PEG at 30 degrees C. This procedure is equivalent to applying 0 to 8 dyn/cm lateral pressure to the bilayers. The resulting reductions in area per lipid were measured with a resolution of +/-0.2 A2 and the fractional area decrease was proportional to applied lateral pressure. For 1,2-dimyristoyl(d54)-sn-glycero-3-phosphocholine, 1-stearoyl(d35)-2-oleoyl-sn-glycero-3-phosphocholine (SOPC-d35), and 1-stearoyl(d35)-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC-d35) cross-sectional areas per molecule in excess water of 59.5, 61.4, and 69.2 A2 and bilayer elastic area compressibility moduli of 141, 221, and 121 dyn/cm were determined, respectively. Combining NMR and x-ray results enables the determination of compressibility differences between saturated and unsaturated hydrocarbon chains. In mixed-chain SOPC-d35 both chains have similar compressibility moduli; however, in mixed-chain polyunsaturated SDPC-d35, the saturated stearic acid chain appears to be far less compressible than the polyunsaturated docosahexaenoic acid chain.

Comparison of the fragilities of several animal cell lines

The mean bursting membrane tension, elastic area compressibility modulus, and cell diameter of PQX1/2 murine hybridomas, NS1 myelomas and SF9 insect cells were measured during batch cultures. The fragilities of the cells were characterised by these mechanical properties. In general, the bursting membrane tension and elastic area compressibility modulus rose during rapid growth phase and fell during the death phase. Among the three cell lines, NS1 myelomas seemed to be weakest, but all were weaker than TB/C3 murine hybridomas studied previously.

Effect of Pluronic F-68 on the mechanical properties of mammalian cells

The mechanical properties of TB/C3 hybridoma cells taken from a continuous culture were measured by micromanipulation. The culture conditions were constant except for the presence or absence of Pluronic F-68 in the medium. It was found that the mean bursting membrane tension and the mean elastic area compressibility modulus of the cells were significantly greater (60% and 120%, respectively) in a medium with 0.05% (w/u) Pluronic F-68 compared to that without Pluronic. Pluronic F-68 therefore affected the strength of the membranes when the cells were exposed to it for a long period of time, i.e., in culture. The short-term effect of Pluronic F-68 on cell strength was also tested by its addition at various levels up to 0.2% (w/v) immediately before the mechanical property measurements. The resulting cell strength depended on the Pluronic concentration, but a significant short-term effect could only be detected above a threshold of 0.1% (w/v). Previous reports on the effect of Pluronic F-68 on animal cell culture are evaluated in the light of these observations.

Shape changes of giant liposomes induced by an asymmetric transmembrane distribution of phospholipids

The influence of a phospholipid transmembrane redistribution on the shape of nonspherical flaccid vesicles was investigated at a fixed temperature by optical microscopy. In a first series of experiments, a transmembrane pH gradient was imposed on egg phosphatidylcholine (EPC)-egg phosphatidylglycerol (EPG) (100:1) giant vesicles. The delta pH induced an asymmetric distribution of EPG. Simultaneously, discoid vesicles were transformed into tubular or a series of connected small vesicles. The fraction of phospholipid transfer necessary for a shape change from discoid to two connected vesicles was of the order of 0.1% of the total phospholipids. Additional lipid redistribution was accompanied by a sequence of shape changes. In a second series of experiments, lyso phosphatidylcholine (L-PC) was added to, or subtracted from, the external leaflet of giant EPC vesicles. The addition of L-PC induced a change from discoid to a two-vesicle state without further evolution, suggesting that lipid transfer and lipid addition are not equivalent. L-PC depletion from the outer leaflet generated stomatocyte-like vesicles. Whenever possible, we have determined whether the giant vesicles undergoing shape changes were unilamellar or multilamellar by measuring the elastic area compressibility modulus, K, by the micropipette assay (Kwok and Evans, 1981). Shape transformations triggered by phospholipid modification of the most external bilayer were indeed influenced by the presence of other underlying membranes that played a role comparable to that of a passive cytoskeleton layer. It appears that in real cells, invaginations of the plasma membrane or budding of organelles could be triggered by a phospholipid transfer from one leaflet to the other caused, for instance, by the aminophospholipid translocase which is present in eukaryotic membranes.

Mechanical properties of hybridoma cells in batch culture

Direct measurements throughout a batch culture of bursting force, bursting membrane tension, elastic area compressibility modulus, and the size of single hybridoma cells have been made by a novel micromanipulation technique. It has been found that the bursting membrane tension and compressibility modulus rise significantly in the rapid growth phase, and fall in the death phase. An approach is suggested for relating these mechanical properties to the shear sensitivity of the cells when they are exposed to shear stresses in flow fields. It is shown that reports of changes in hybridoma fragility during batch cultures, as measured using viscometers, might be explained using more fundamental micromanipulation measurements.

A micromanipulation technique with a theoretical cell model for determining mechanical properties of single mammalian cells

Abstract a micromanipulation technique has been developed to squeeze single mammalian cells between two parallel surfaces, and to measure simultaneously the force being imposed on a cell and its deformation. The quantitative relationship between the force and the distance between the surfaces, given directly by the technique, has been modelled so that the bursting membrane tension, bursting pressure and the elastic area compressibility modulus of the cells can be obtained. The tension and compressibility modulus are shown to be essentially independent of cell size for cells from a continuous culture. This test might be used to predict damage to cells in processing equipment and to optimize culture conditions.

Elastic deformation and failure of lipid bilayer membranes containing cholesterol

Giant bilayer vesicles were reconstituted from several lipids and lipid/cholesterol (CHOL) mixtures: stearolyloleoylphosphatidylcholine (SOPC), bovine sphingomyelin (BSM), diarachidonylphosphatidylcholine (DAPC), SOPC/CHOL, BSM/CHOL, DAPC/CHOL, and extracted red blood cell (RBC) lipids with native cholesterol. Single-walled vesicles were manipulated by micropipette suction and several membrane material properties were determined. The properties measured were the elastic area compressibility modulus K, the critical areal strain alpha c, and the tensile strength tau lys, from which the failure energy or membrane toughness Tf was calculated. The elastic area expansion moduli for these lipid and lipid/cholesterol bilayers ranged from 57 dyn/cm for DAPC to 1,734 dyn/cm for BSM/CHOL. The SOPC/CHOL series and RBC lipids had intermediate values. The results indicated that the presence of cholesterol is the single most influential factor in increasing bilayer cohesion, but only for lipids where both chains are saturated, or mono- or diunsaturated. Multiple unsaturation in both lipid chains inhibits the condensing effect of cholesterol in bilayers. The SOPC/CHOL system was studied in more detail. The area expansion modulus showed a nonlinear increase with increasing cholesterol concentration up to a constant plateau, indicating a saturation limit for cholesterol in the bilayer phase of approximately 55 mol% CHOL. The membrane compressibility was modeled by a property-averaging composite theory involving two bilayer components, namely, uncomplexed lipid and a lipid/cholesterol complex of stoichiometry 1/1.22. The area expansion modulus of this molecular composite membrane was evaluated by a combination of the expansion moduli of each component scaled by their area fractions in the bilayer. Bilayer toughness, which is the energy stored in the bilayer at failure, showed a maximum value at approximately 40 mol% CHOL. This breakdown energy was found to be only a fraction of the available thermal energy, implying that many molecules (approximately 50-100) may be involved in forming the defect structure that leads to failure. The area expansion modulus of extracted RBC lipids with native cholesterol was compared with recent measurements of intact RBC membrane compressibility. The natural membrane was also modeled as a simple composite made up to a compressible lipid/cholesterol matrix containing relatively incompressible transmembrane proteins. It appears that the interaction of incompressible proteins with surrounding lipid confers enhanced compressibility on the composite structure.

Membrane elasticity of mouse dorsal root ganglion neurons decreases with aging

Elastic area compressibility modulus (EACM) method was applied to measure the membrane elasticity of cultured dorsal root ganglion neurons from fetal and 3-month-old mice. The values of the EACM were 21.3 dyn cm in 3-month-old neurons and 2.8 dyn cm in fetal neurons. These results indicate that neural cell membrane elasticity decreases with aging.

Thermomechanical and transition properties of dimyristoylphosphatidylcholine/cholesterol bilayers.

Mixtures of dimyristoylphosphatidylcholine (DMPC) and cholesterol (Chol) have been used to examine the effects of cholesterol on the chain crystallization transitions and thermomechanical properties in phospholipid bilayer membranes. The mechanical properties--elastic moduli and level of tension at membrane rupture--were derived from micropipet pressurization of giant single-walled vesicles. Also, the micropipet method allowed temperature-dependent area transitions to be measured at constant membrane tension. X-ray diffraction measurements were made on selected lipid/cholesterol mixtures. Wide-angle patterns and electron density profiles were used to measure bilayer thickness as an indication of chain tilt and fluidity. Vesicle area versus temperature plots showed that the main acyl chain crystallization transition of DMPC broadened and shifted to higher temperatures. Both above and below the broad transition, the elastic area compressibility modulus, K, was greatly increased with cholesterol addition. The value for the 1:1 DMPC/Chol complex was found to be approximately 700 dyn/cm, comparable to that for DMPC in the L beta' phase. However, for all concentrations above 12.5 mol % (which was weakly solid), vesicle bilayers behaved as surface liquids with no surface shear rigidity even at temperatures well below the DMPC phase transition. Area changes over the broadened transitions were reduced by cholesterol and disappeared with the addition of 50 mol % to leave the thermal area expansivity at 1.3 X 10(-3)/degrees C. These area changes are consistent with separate formation of a 1:1 DMPC/Chol complex that does not condense plus residual free lipid and lipid loosely associated with the 1:1 complex that freezes normally.(ABSTRACT TRUNCATED AT 250 WORDS)

Forces between phospholipid bilayers

The measurement of forces between lipid bilayers is complicated by the coupling which exists between bilayer structure and interbilayer interaction. In fact, direct measurements are impracticable and methods must be sought which enable these two factors to be separated. A procedure is developed for calculating the bilayer stress, γ, and the interbilayer force, ƒ, from simultaneous X-ray measurements of the bilayer structure and water layer thickness and the activity of water. The method is applied to existing experimental measurements for the egg-lecithin–water system. The interbilayer ‘hydration force’ is found to be attractive, exponential with a decay length of 0.22 nm and to dominate completely the van der Waals dispersion force for bilayer separations well beyond the swelling limit. The spontaneous uptake of water by the lamellar phase is shown to be a consequence of the decrease in Gibbs energy associated with the lateral expansion of the bilayer. The elastic area compressibility modulus, ka, near to the swelling limits is found to be 140 mN m–1. The implications of these results for the phase behaviour of phospholipid–water mixtures and the stability of vesicle dispersions are discussed.

Giant vesicle bilayers composed of mixtures of lipids, cholesterol and polypeptides. Thermomechanical and (mutual) adherence properties.

Micromechanical tests of giant vesicle bilayer elasticity and bilayer–bilayer adhesivity have been carried out on vesicles made from mixtures of lipids, cholesterol and polypeptides. Mixtures of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) exhibited ideal solution behaviour over a temperature range that covered both liquid-to-gel phase transitions. Addition of cholesterol (CHOL) to a saturated chain lecithin (DMPC) reduced, broadened and shifted to higher temperature the main crystalline-to-liquid acyl chain transition. Cholesterol greatly reduced the membrane area compressibility and increased membrane cohesion to levels exhibited by frozen acyl chain bilayers, but maintained the bilayer in a liquid state. Addition of amphiphilic polypeptides to PC and PC–CHOL mixtures slightly increased bilayer compressibility at high temperatures; when the temperature was lowered, bilayer compressibility for DMPC–CHOL–peptide mixtures was greatly reduced to below that of the single component lipid. Cholesterol appeared to change from an association with the lipid at low temperatures to an association with the protein at high temperatures. Membrane cohesion correlated with a simple fracture energy model where the level of tension required to lyse vesicles is proportional to the square root of the elastic area compressibility modulus. Free-energy potentials for adhesion of mixed lipid (PC and PE) bilayers showed a dramatic increase as the mole fraction of PE approached unity. Based on published values of separation distance between lamellae at full hydration for each pure component, the free energy potentials for adhesion of mixed-lipid bilayers were calculated from theoretical models of the van der Waals attraction and hydration repulsion then compared with the measured values of adhesion energy. The correlation provides strong evidence that the hydration repulsion can be represented by a surface-potential theory.

Molecular packing and area compressibility of lipid bilayers.

Knowledge of the molecular packing of lipids and water in lipid bilayers is important for understanding bilayer mechanics and thermodynamics. Information on packing is most often obtained from x-ray or neutron diffraction measurements. Given the d spacing, composition, and partial specific volumes of the lipid and water, it is a simple matter to calculate the area per lipid molecule, bilayer thickness, and bilayer mass density. The partial specific volumes are commonly assumed to be those of bulk water and of lipid in excess water regardless of the degree of bilayer hydration. We present evidence here that these assumptions should be seriously questioned. At low hydrations, we find the head groups of egg and dioleoyl lecithin to be much less tightly packed than previously thought and the partial specific volume of water to be considerably smaller than 1 ml/g. Because the molecular packing affects the mechanical properties of bilayers, we use the results to reevaluate published experiments concerning the elastic area compressibility modulus of egg lecithin bilayers and the repulsive hydration force between bilayers.

Cell poking. Determination of the elastic area compressibility modulus of the erythrocyte membrane

Cell poking, a new method for measuring mechanical properties of single cells was used to determine the elastic area compressibility modulus of osmotically swollen human erythrocytes. With this method we determined the force required to indent cells attached to a glass coverslip (Petersen, N.O., W. B. McConnaughey , and E. L. Elson , 1982, Proc. Natl. Acad. Sci. USA, 79:5327. Forces on the order of one millidyne and indentations on the order of one micron were detected. An analysis of these data in terms of a simplified mechanical model yielded the elastic area compressibility modulus. This analysis used a variational approach to minimize the isothermal elastic potential energy density function given by E. A. Evans and R. Skalak (Mechanics and Thermodynamics of Biomembranes, 1980, CRC Press, Boca Raton , FL). Measurements on swollen erythrocytes gave a range of values, depending in part on the osmotic conditions, of 17.9 +/- 8.2 to 34.8 +/- 12.0 mdyn /micron for the elastic area compressibility modulus at 25 degrees C. Fractional area expansion greater than 2.6 +/- 0.8% produced rapid cell lysis. These values were not corrected for the reversible movement of water across the cell membrane in response to hydrostatic pressure gradients. Our results agree reasonably with those obtained by Evans et al. (Evans, E.A., R. Waugh , and L. Melnick , 1976, Biophys. J., 16:585-595.) using micropipette aspiration under similar conditions.

Thermoelasticity of large lecithin bilayer vesicles

Micromechanical experiments on large lecithin bilayer vesicles as a function of temperature have demonstrated an essential feature of bilayer vesicles as closed systems: the bilayer can exist in a tension-free state (within the limits of experimental resolution, i.e., less than 10(-2) dyn/cm). Furthermore, because of the fixed internal volume, there is a critical temperature at which the vesicle becomes a tension-free sphere. Below this temperature, thermoelastic tension builds up in the membrane and the vesicle's internal pressure increases while the surface area remains constant. Above this temperature, the vesicle's surface area increases while the tension and internal pressure are negligible. Without mechanical support, the vesicles fragment into small vesicles because they have insufficient surface rigidity. In the upper temperature range we have measured the increase of surface area with temperature. These data established the thermal area expansivity to be 2.4 X 10(-3)/degrees C. At constant temperature, we used either pipet aspiration with suction pressures up to 10(4) dyn/cm2 or compression against a flat surface with forces up to 10(-2) dyn to produce area dilation of the vesicle surface on the order of 1%. The rate of increase of membrane tension with area dilation was calculated, which established the elastic area compressibility modulus to be 140 dyn/cm. The tension limit that produced lysis was observed to be 3-4 dyn/cm (equivalent to 2-3% area increase). The product of the elastic area compressibility modulus, the thermal area expansivity, and the temperature gives the reversible heat of expansion at constant temperature for the bilayer. This value is 100 ergs/cm2 at 25 degrees C, or approximately 5 kcal/mol of lecithin. Similarly, the product of the thermal area expansivity multiplied by the area compressibility modulus determines the rate of increase of thermoelastic tension with decrease in temperature when the area is held constant, i.e., -0.34 dyn/cm/degrees C.