Dynamic force spectroscopy on supported lipid bilayers: effect of temperature and sample preparation

Biological membranes are constantly exposed to forces. The stress-strain relation in membranes determines the behavior of many integral membrane proteins or other membrane related-proteins that show a mechanosensitive behavior. Here, we studied by force spectroscopy the behavior of supported lipid bilayers (SLBs) subjected to forces perpendicular to their plane. We measured the lipid bilayer mechanical properties and the force required for the punch-through event characteristic of atomic force spectroscopy on SLBs as a function of the interleaflet coupling. We found that for an uncoupled bilayer, the overall tip penetration occurs sequentially through the two leaflets, giving rise to two penetration events. In the case of a bilayer with coupled leaflets, penetration of the atomic force microscope tip always occurred in a single step. Considering the dependence of the jump-through force value on the tip speed, we also studied the process in the context of dynamic force spectroscopy (DFS). We performed DFS experiments by changing the temperature and cantilever spring constant, and analyzed the results in the context of the developed theories for DFS. We found that experiments performed at different temperatures and with different cantilever spring constants enabled a more effective comparison of experimental data with theory in comparison with previously published data.     

Studying membrane fusion using supported lipid bilayers on superparamagnetic beads

The fusion between two lipid membranes is a ubiquitous mechanism in cell traffic and pathogens invasion. Yet it is not well understood how two distinct bilayers overcome the energy barriers towards fusion and reorganize themselves to form a unique continuous bilayer. The magnitudes and numbers of these energy barriers are themselves an open question. To tackle these issues, we developed a new tool that allows to control the forces applied between two supported lipid bilayers (SLBs) deposited on superparamagnetic beads. By applying a magnetic field, the beads self-organize along field lines in chains of beads and compress the two membranes on the contact zone. Using the diffusion of fluorescently labelled lipids from one bilayer to the other allows us to identify fusion of the bilayers in contact. We applied increasing forces on SLBs and increased the occurrence of fusion. This experimental system allows the simultaneous study of tens of facing bilayers in a single experiment and mitigates the stochasticity of the fusion process. It is thus a powerful tool to test the various parameters involved in the membrane fusion process.     

Decrease of elastic moduli of DOPC bilayers induced by a macrolide antibiotic, azithromycin

The elastic properties of membrane bilayers are key parameters that control its deformation and can be affected by pharmacological agents. Our previous atomic force microscopy studies revealed that the macrolide antibiotic, azithromycin, leads to erosion of DPPC domains in a fluid DOPC matrix [A. Berquand, M. P. Mingeot-Leclercq, Y. F. Dufrene, Real-time imaging of drug-membrane interactions by atomic force microscopy, Biochim. Biophys. Acta 1664 (2004) 198-205.]. Since this observation could be due to an effect on DOPC cohesion, we investigated the effect of azithromycin on elastic properties of DOPC giant unilamellar vesicles (GUVs). Microcinematographic and morphometric analyses revealed that azithromycin addition enhanced lipid membranes fluctuations, leading to eventual disruption of the largest GUVs. These effects were related to change of elastic moduli of DOPC, quantified by the micropipette aspiration technique. Azithromycin decreased both the bending modulus (k(c), from 23.1+/-3.5 to 10.6+/-4.5 k(B)T) and the apparent area compressibility modulus (K(app), from 176+/-35 to 113+/-25 mN/m). These data suggested that insertion of azithromycin into the DOPC bilayer reduced the requirement level of both the energy for thermal fluctuations and the stress to stretch the bilayer. Computer modeling of azithromycin interaction with DOPC bilayer, based on minimal energy, independently predicted that azithromycin (i) inserts at the interface of phospholipid bilayers, (ii) decreases the energy of interaction between DOPC molecules, and (iii) increases the mean surface occupied by each phospholipid molecule. We conclude that azithromycin inserts into the DOPC lipid bilayer, so as to decrease its cohesion and to facilitate the merging of DPPC into the DOPC fluid matrix, as observed by atomic force microscopy. These investigations, based on three complementary approaches, provide the first biophysical evidence for the ability of an amphiphilic antibiotic to alter lipid elastic moduli. This may be an important determinant for drug: lipid interactions and cellular pharmacology.     

Bending and puncturing the influenza lipid envelope

Lysosomes, enveloped viruses, as well as synaptic and secretory vesicles are all examples of natural nanocontainers (diameter ≈ 100 nm) which specifically rely on their lipid bilayer to protect and exchange their contents with the cell. We have applied methods primarily based on atomic force microscopy and finite element modeling that allow precise investigation of the mechanical properties of the influenza virus lipid envelope. The mechanical properties of small, spherical vesicles made from PR8 influenza lipids were probed by an atomic force microscopy tip applying forces up to 0.2 nN, which led to an elastic deformation up to 20%, on average. The liposome deformation was modeled using finite element methods to extract the lipid bilayer elastic properties. We found that influenza liposomes were softer than what would be expected for a gel phase bilayer and highly deformable: Consistent with previous suggestion that influenza lipids do not undergo a major phase transition, we observe that the stiffness of influenza liposomes increases gradually and weakly (within one order of magnitude) with temperature. Surprisingly, influenza liposomes were, in most cases, able to withstand wall-to-wall deformation, and forces >1 nN were generally required to puncture the influenza envelope, which is similar to viral protein shells. Hence, the choice of a highly flexible lipid envelope may provide as efficient a protection for a viral genome as a stiff protein shell.     

Formation and maintenance of tubular membrane projections require mechanical force, but their elongation and shortening do not require additional force

Living cells develop their own characteristic shapes depending on their physiological functions, and their morphologies are based on the mechanical characteristics of the cytoskeleton and of membranes. To investigate the role of lipid membranes in morphogenesis, we constructed a simple system that can manipulate liposomes and measure the forces required to transform their shapes. Two polystyrene beads (1 microm in diameter) were encapsulated in giant liposomes and were manipulated using double-beam laser tweezers. Without any specific interaction between the lipid membrane and beads, mechanical forces could be applied to the liposome membrane from the inside. Spherical liposomes transformed into a lemon shape with increasing tension, and tubular membrane projections were subsequently generated in the tips at either end. This process is similar to the liposomal transformation caused by elongation of encapsulated cytoskeletons. In the elongation stage of lemon-shaped liposomes, the force required for the transformation became larger as the end-to-end length increased. Just before the tubular membrane was generated, the force reached the maximum strength (approximately 11 pN). However, immediately after the tubular membrane developed, the force suddenly decreased and was maintained at a constant strength (approximately 4 pN) that was independent of further tube elongation or shortening, even though there was no excess membrane reservoir as occurs in living cells. When the tube length was shortened to approximately 2 microm, the liposome reversed to a lemon shape and the force temporarily increased (to approximately 7 pN). These results indicate that the simple application of mechanical force is sufficient to form a protrusion in a membrane, that a critical force and length is needed to form and to maintain the protrusion, and suggest that the lipid bilayer itself has the ability to buffer the membrane tension.     

High resolution nanomechanical characterization of multi‐domain model membranes by fast Force Volume

Plasma membrane is a complex structure, mainly composed by lipids and proteins, which plays a pivotal role in cell metabolism by regulating its selective permeability to ions and molecules. According to the “raft hypothesis”, lipids in the bilayer are not forming a structurally passive solvent, but are rather organized in specific domains, which present different structural and functional characteristics. The mechanical properties of the lipid part of plasma membrane have been recently characterized through Atomic Force Microscopy, by analyzing the features of force vs distance curves collected on supported lipid bilayers (SLBs). In case of lipid domains sizing from tens to hundreds of nanometers, which mimic in a good way the lateral organization of real membranes, a high lateral resolution and a large number of curves are often required for properly expressing the complexity of the system, with a consequent exponential growth of acquisition and processing time. In this paper we propose a method, based on a recently developed high speed Force Volume technique and on home‐built data processing software, for the mechanical characterization of nanostructured SLBs. With our software we have been able to process data set composed by tens of thousands of curves, collected with a spatial resolution ranging from 8 to 40 nm/pixel. Multiparametric maps and distribution histograms produced by our analysis allowed identifying a specific behavior for each lipid phase in the investigated model membranes, even in presence of nanosized features. Copyright © 2015 John Wiley & Sons, Ltd.     

Single-molecule adhesion forces and attachment lifetimes of myosin-I phosphoinositide interactions

Phosphoinositides regulate the activities and localization of many cytoskeletal proteins involved in crucial biological processes, including membrane-cytoskeleton adhesion. Yet little is known about the mechanics of protein-phosphoinositide interactions, or about the membrane-attachment mechanics of any peripheral membrane proteins. Myosin-Ic (myo1c) is a molecular motor that links membranes to the cytoskeleton via phosphoinositide binding, so it is particularly important to understand the mechanics of its membrane attachment. We used optical tweezers to measure the strength and attachment lifetime of single myo1c molecules as they bind beads coated with a bilayer of 2% phosphatidylinositol 4,5-bisphosphate and 98% phosphatidylcholine. Adhesion forces measured under ramp-load ranged between 5.5 and 16 pN at loading rates between 250 and 1800 pN/s. Dissociation rates increased linearly with constant force (0.3-2.5 pN), with rates exceeding 360 s⁻¹ at 2.5 pN. Attachment lifetimes calculated from adhesion force measurements were loading-rate-dependent, suggesting nonadiabatic behavior during pulling. The adhesion forces of myo1c with phosphoinositides are greater than the motors stall forces and are within twofold of the force required to extract a lipid molecule from the membrane. However, attachment durations are short-lived, suggesting that phosphoinositides alone do not provide the mechanical stability required to anchor myo1c to membranes during multiple ATPase cycles.     

Force measurements on myelin basic protein adsorbed to mica and lipid bilayer surfaces done with the atomic force microscope.

The mechanical and adhesion properties of myelin basic protein (MBP) are important for its function, namely the compaction of the myelin sheath. To get more information about these properties we used atomic force microscopy to study tip-sample interaction of mica and mixed dioleoylphosphatidylserine (DOPS) (20%)/egg phosphatidylcholine (EPC) (80%) lipid bilayer surfaces in the absence and presence of bovine MBP. On mica or DOPS/EPC bilayers a short-range repulsive force (decay length 1.0-1.3 nm) was observed during the approach. The presence of MBP always led to an attractive force between tip and sample. When retracting the tip again, force curves on mica and on lipid layers were different. While attached to the mica surface, the MBP molecules exhibited elastic stretching behavior that agreed with the worm-like chain model, yielding a persistence length of 0.5 +/- 0.25 nm and an average contour length of 53 +/- 19 nm. MBP attached to a lipid bilayer did not show elastic stretching behavior. This shows that the protein adopts a different conformation when in contact with lipids. The lipid bilayer is strongly modified by MBP attachment, indicating formation of MBP-lipid complexes and possibly disruption of the original bilayer structure.     

Single molecule studies of antibody-antigen interaction strength versus intra-molecular antigen stability

We investigated molecular recognition of antibodies to membrane-antigens and extraction of the antigens out of membranes at the single molecule level. Using dynamic force microscopy imaging and enzyme immunoassay, binding of anti-sendai antibodies to sendai-epitopes genetically fused into bacteriorhodopsin molecules from purple membranes were detected under physiological conditions. The antibody/antigen interaction strength of 70-170 pN at loading rates of 2-50 nN/second yielded a barrier width of x = 0.12 nm and a kinetic off-rate (corresponding to the barrier height) of k(off) = 6s(-1), respectively. Bacteriorhodopsin unfolding revealed a characteristic intra-molecular force pattern, in which wild-type and sendai-bacteriorhodopsin molecules were clearly distinguishable in their length distributions, originating from the additional 13 amino acid residues epitope in sendai purple membranes. The inter-molecular antibody/antigen unbinding force was significantly lower than the force required to mechanically extract the binding epitope-containing helix pair out of the membrane and unfold it (126 pN compared to 204 pN at the same loading rate), meeting the expectation that inter-molecular unbinding forces are weaker than intra-molecular unfolding forces responsible for stabilizing native conformations of proteins.     

Protein shape change has a major effect on the gating energy of a mechanosensitive channel

Increasing experimental evidence has shown that membrane protein functionality depends on molecular composition of cell membranes. However, the origin of this dependence is not fully understood. It is reasonable to assume that specific lipid-protein interactions are important, yet more generic effects due to mechanical properties of lipid bilayers likely play a significant role too. Previously it has been demonstrated using models for elastic properties of membranes and lateral pressure profiles of lipid bilayers that the mechanical properties of a lipid bilayer can contribute as much as ∼10 k_BT to the free energy difference associated with a change in protein conformational state. Here, we extend those previous approaches to a more realistic model for a large mechanosensitive channel (MscL). We use molecular dynamics together with the MARTINI model to simulate the open and closed states of MscL embedded in a DOPC bilayer. We introduce a procedure to calculate the mechanical energy change in the channel gating using a three-dimensional pressure distribution inside a membrane, computed from the molecular dynamics simulations. We decompose the mechanical energy to terms associated with area dilation and shape contribution. Our results highlight that the lateral pressure profile of a lipid bilayer together with the shape change in gating can induce a contribution of ∼30 k_BT on the gating energy of MscL. This contribution arises largely from the interfacial tension between hydrophobic and hydrophilic regions in a lipid bilayer.     

Material Properties of Lipid Microdomains: Force-Volume Imaging Study of the Effect of Cholesterol on Lipid Microdomain Rigidity

The effect of cholesterol (CHOL) on the material properties of supported lipid bilayers composed of lipid mixtures that mimic the composition of lipid microdomains was studied by force-volume (FV) imaging under near-physiological conditions. These studies were carried out with lipid mixtures of dioleoylphosphatidylcholine, dioleoylphosphatidylserine, and sphingomyelin. FV imaging enabled simultaneous topology and force measurements of sphingomyelin-rich domains (higher domain (HD)) and phospholipid-rich domains (lower domain (LD)), which allowed quantitative measurement of the force needed to puncture the lipid bilayer with or without CHOL. The force required to penetrate the various domains of the bilayer was probed using high- and low-ionic-strength buffers as a function of increasing amounts of CHOL in the bilayer. The progressive addition of CHOL also led to a decreasing height difference between HD and LD. FV imaging further demonstrated a lack of adhesion between the atomic force microscope tip and the HD or LD at loads below the breakthrough force. These results can lead to a better understanding of the role that CHOL plays in the mechanical properties of cellular membranes in modulating membrane rigidity, which has important implications for cellular mechanotransduction.     

Structural impact of cations on lipid bilayer models: Nanomechanical properties by AFM-force spectroscopy

Atomic Force Microscopy (AFM) has become an invaluable tool for studying the micro- and nanoworlds. As a stand-alone, high-resolution imaging technique and force transducer, it defies most other surface instrumentation in ease of use, sensitivity and versatility. The main strength of AFM relies on the possibility to operate in an aqueous environment on a wide variety of biological samples, from single molecules – DNA or proteins – to macromolecular assemblies like biological membranes. Understanding the effect of mechanical stress on membranes is of primary importance in biophysics, since cells are known to perform their function under a complex combination of forces. In the later years, AFM-based Force-Spectroscopy (AFM-FS) has provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Lipid membranes are electrostatically charged entities that physiologically coexist with electrolyte solutions. Thus, specific interactions with ions are a matter of considerable interest. The distribution of ions in the solution and their interaction with the membranes are factors that substantially modify the structure and dynamics of the cell membranes. Furthermore, signaling processes are modified by the membrane capability of retaining ions. Supported Lipid Bilayers (SLBs) are a versatile tool to investigate phospholipid membranes mimicking biological surfaces. In the present contribution, we review selected experiments on the mechanical stability of SLBs as models of lipid membranes by means of AFM-FS, with special focus on the effect of cations and ionic strength in the overall nanomechanical stability.     

Measuring the viscoelastic properties of human platelets with the atomic force microscope.

We have measured force curves as a function of the lateral position on top of human platelets with the atomic force microscope. These force curves show the indentation of the cell as the tip loads the sample. By analyzing these force curves we were able to determine the elastic modulus of the platelet with a lateral resolution of approximately 100 nm. The elastic moduli were in a range of 1-50 kPa measured in the frequency range of 1-50 Hz. Loading forces could be controlled with a resolution of 80 pN and indentations of the platelet could be determined with a resolution of 20 nm.     

Membrane elasticity in giant vesicles with fluid phase coexistence

Biological membranes are known to contain compositional heterogeneities, often termed rafts, with distinguishable composition and function, and these heterogeneities participate in vigorous transport processes. Membrane lipid phase coexistence is expected to modulate these processes through the differing mechanical properties of the bulk domains and line tension at phase boundaries. In this contribution, we compare the predictions from a shape theory derived for vesicles with fluid phase coexistence to the geometry of giant unilamellar vesicles with coexisting liquid-disordered (L(d)) and liquid-ordered (L(o)) phases. We find a bending modulus for the L(o) phase higher than that of the L(d) phase and a saddle-splay (Gauss) modulus difference with the Gauss modulus of the L(o) phase being more negative than the L(d) phase. The Gauss modulus critically influences membrane processes that change topology, such as vesicle fission or fusion, and could therefore be of significant biological relevance in heterogeneous membranes. Our observations of experimental vesicle geometries being modulated by Gaussian curvature moduli differences confirm the prediction by the theory of Juelicher and Lipowsky.     

Young modulus of supported lipid membranes containing milk sphingomyelin in the gel,fluid or liquid-ordered phase,determined using AFM force spectroscopy

The biological membrane surrounding milk fat globules (MFGM) exhibits lateral phase separation of lipids, interpreted as gel or liquid-ordered phase sphingomyelin-rich (milk SM) domains dispersed in a fluid continuous lipid phase. The objective of this study was to investigate whether changes in the phase state of milk SM-rich domains induced by temperature (T < Tm or T > Tm) or cholesterol affected the Young modulus of the lipid membrane. Supported lipid bilayers composed of MFGM polar lipids, milk SM or milk SM/cholesterol (50:50 mol) were investigated at 20 °C and 50 °C using atomic force microscopy (AFM) and force spectroscopy. At 20 °C, gel-phase SM-rich domains and the surrounding fluid phase of the MFGM polar lipids exhibited Young modulus values of 10–20 MPa and 4–6 MPa, respectively. Upon heating at 50 °C, the milk SM-rich domains in MFGM bilayers as well as pure milk SM bilayers melted, leading to the formation of a homogeneous membrane with similar Young modulus values to that of a fluid phase (0–5 MPa). Upon addition of cholesterol to the milk SM to reach 50:50 mol%, membranes in the liquid-ordered phase exhibited Young modulus values of a few MPa, at either 20 or 50 °C. This indicated that the presence of cholesterol fluidized milk SM membranes and that the Young modulus was weakly affected by the temperature. These results open perspectives for the development of milk polar lipid based vesicles with modulated mechanical properties.     

Elastic curvature constants of lipid monolayers and bilayers

Bending elasticity is an important property of lipid vesicles, non-lamellar lipid phases and biological membranes. Experimental values of the mean curvature moduli, k(c), of lipid bilayers and of the monolayer leaflets of inverted hexagonal (H(II)) phases of lipids are tabulated here for easy reference. Experimental estimates of the Gaussian curvature modulus, k (c), are also included. Consideration is given to the relation between the bending moduli of bilayers and the constituent monolayer leaflets. Useful mathematical relations involving the bending moduli and spontaneous curvature are summarized.     

The phase behavior of cationic lipid-DNA complexes

We present a theoretical analysis of the phase behavior of solutions containing DNA, cationic lipids, and nonionic (helper) lipids. Our model allows for five possible structures, treated as incompressible macroscopic phases: two lipid-DNA composite (lipoplex) phases, namely, the lamellar (L(alpha)(C)) and hexagonal (H(II)(C)) complexes; two binary (cationic/neutral) lipid phases, that is, the bilayer (L(alpha)) and inverse-hexagonal (H(II)) structures, and uncomplexed DNA. The free energy of the four lipid-containing phases is expressed as a sum of composition-dependent electrostatic, elastic, and mixing terms. The electrostatic free energies of all phases are calculated based on Poisson-Boltzmann theory. The phase diagram of the system is evaluated by minimizing the total free energy of the three-component mixture with respect to all the compositional degrees of freedom. We show that the phase behavior, in particular the preferred lipid-DNA complex geometry, is governed by a subtle interplay between the electrostatic, elastic, and mixing terms, which depend, in turn, on the lipid composition and lipid/DNA ratio. Detailed calculations are presented for three prototypical systems, exhibiting markedly different phase behaviors. The simplest mixture corresponds to a rigid planar membrane as the lipid source, in which case, only lamellar complexes appear in solution. When the membranes are "soft" (i.e., low bending modulus) the system exhibits the formation of both lamellar and hexagonal complexes, sometimes coexisting with each other, and with pure lipid or DNA phases. The last system corresponds to a lipid mixture involving helper lipids with strong propensity toward the inverse-hexagonal phase. Here, again, the phase diagram is rather complex, revealing a multitude of phase transitions and coexistences. Lamellar and hexagonal complexes appear, sometimes together, in different regions of the phase diagram.     

Effects of chemical modification, tropomyosin, and myosin subfragment 1 on the yield strength and critical concentration of F-actin

The effects of coupling with tetramethylrhodamine-5-iodoacetamide and of the decoration with tropomyosin and with myosin subfragment 1 on the elastic properties of F-actin filament are investigated. At 22 degrees C, in 15 mM orthophosphate and 3 mM MgCl2, tetramethylrhodamine F-actin displays a yield strength of 3.69 +/- 0.213 pN and an elastic modulus by stretching of 0.91 MPa. Decoration with tropomyosin increases the yield strength of tetramethylrhodamine F-actin to 10.51 +/- 0.24 pN and the elastic modulus by stretching to 23-75 MPa. Mixtures of myosin subfragment 1 and tetramethylrhodamine F-actin at the 0.2:1, 0.4:1, 0.6:1, 0.8:1, and 1:1 molar ratios are also studied. Both yield strength and the elastic modulus by stretching are found to increase progressively with the ratio. At the 1:1 molar ratio, the yield strength is 15.81 +/- 0.26 pN and the elastic modulus by stretching is 13.45 to 40 MPa. Decoration of tetramethylrhodamine F-actin with both tropomyosin and myosin subfragment 1, at the 1:1 molar ratio with the actin monomer, produces filaments with an yield strength of 22.3 +/- 0.48 pN.     

Cell Visco-Elasticity Measured with AFM and Optical Trapping at Sub-Micrometer Deformations

The measurement of the elastic properties of cells is widely used as an indicator for cellular changes during differentiation, upon drug treatment, or resulting from the interaction with the supporting matrix. Elasticity is routinely quantified by indenting the cell with a probe of an AFM while applying nano-Newton forces. Because the resulting deformations are in the micrometer range, the measurements will be affected by the finite thickness of the cell, viscous effects and even cell damage induced by the experiment itself. Here, we have analyzed the response of single 3T3 fibroblasts that were indented with a micrometer-sized bead attached to an AFM cantilever at forces from 30–600 pN, resulting in indentations ranging from 0.2 to 1.2 micrometer. To investigate the cellular response at lower forces up to 10 pN, we developed an optical trap to indent the cell in vertical direction, normal to the plane of the coverslip. Deformations of up to two hundred nanometers achieved at forces of up to 30 pN showed a reversible, thus truly elastic response that was independent on the rate of deformation. We found that at such small deformations, the elastic modulus of 100 Pa is largely determined by the presence of the actin cortex. At higher indentations, viscous effects led to an increase of the apparent elastic modulus. This viscous contribution that followed a weak power law, increased at larger cell indentations. Both AFM and optical trapping indentation experiments give consistent results for the cell elasticity. Optical trapping has the benefit of a lower force noise, which allows a more accurate determination of the absolute indentation. The combination of both techniques allows the investigation of single cells at small and large indentations and enables the separation of their viscous and elastic components.     

Thermotropic and barotropic phase transitions of N-methylated dipalmitoylphosphatidylethanolamine bilayers

In order to understand the effect of polar head group modification on the thermotropic and barotropic phase behavior of phospholipid bilayer membranes, the phase transitions of dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidyl-N-methylethanolamine (DPMePE), dipalmitoylphosphatidyl-N,N-dimethylethanolamine (DPMe2PE) and dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were observed by differential scanning calorimetry and high-pressure optical methods. The temperatures of the so-called main transition from the gel (L(beta)) or ripple gel (P(beta)') phase to the liquid crystalline (L(alpha)) phase were almost linearly elevated by applying pressure. The slope of the temperature-pressure boundary, dT/dp, was in the range of 0.220-0.264 K MPa(-1) depending on the number of methyl groups in the head group of lipids. The main-transition temperatures of N-methylated DPPEs decreased with increasing size of head group by stepwise N-methylation. On the other hand, there was no significant difference in thermodynamic quantities of the main transition between the phospholipids. With respect to the transition from the subgel (L(c)) phase to the lamellar gel (L(beta) or L(beta)') phase, the transition temperatures were also elevated by applying pressure. In the case of DPPE bilayer the L(c)/L(beta) transition appeared at a pressure higher than 21.8 MPa. At a pressure below 21.8 MPa the L(c)/L(alpha) transition was observed at a temperature higher than the main-transition temperature. The main (L(beta)/L(alpha)) transition can be recognized as the transformation between metastable phases in the range from ambient pressure to 21.8 MPa. Polymorphism in the gel phase is characteristic of DPPC bilayer membrane unlike other lipid bilayers used in this study: the L(beta)', P(beta)' and pressure-induced interdigitated gel (L(beta)I) phases were observed only in the DPPC bilayer. Regarding the bilayers of DPPE, DPMePE and DPMe2PE, the interdigitation of acyl chain did not appear even at pressures as high as 200 MPa.