Philippe Méléard

Bending elasticities of model membranes: influences of temperature and sterol content.

Giant liposomes obtained by electroformation and observed by phase-contrast video microscopy show spontaneous deformations originating from Brownian motion that are characterized, in the case of quasispherical vesicles, by two parameters only, the membrane tension sigma and the bending elasticity k(c). For liposomes containing dimyristoyl phosphatidylcholine (DMPC) or a 10 mol% cholesterol/DMPC mixture, the mechanical property of the membrane, k(c), is shown to be temperature dependent on approaching the main (thermotropic) phase transition temperature T(m). In the case of DMPC/cholesterol bilayers, we also obtained evidence for a relation between the bending elasticity and the corresponding temperature/cholesterol molecular ratio phase diagram. Comparison of DMPC/cholesterol with DMPC/cholesterol sulfate bilayers at 30 degrees C containing 30% sterol ratio shows that k(c) is independent of the surface charge density of the bilayer. Finally, bending elasticities of red blood cell (RBC) total lipid extracts lead to a very low k(c) at 37 degrees C if we refer to DMPC/cholesterol bilayers. At 25 degrees C, the very low bending elasticity of a cholesterol-free RBC lipid extract seems to be related to a phase coexistence, as it can be observed by solid-state (31)P-NMR. At the same temperature, the cholesterol-containing RBC lipid extract membrane shows an increase in the bending constant comparable to the one observed for a high cholesterol ratio in DMPC membranes.

Giant unilamellar vesicle formation under physiologically relevant conditions

We present an upgrade to the giant unilamellar vesicle (GUV) electroformation method allowing easy GUV production in different buffers and with various membrane compositions. Our experimental results reveal that lipid deposits obtained from aqueous liposome or proteoliposome dispersions are highly efficient for GUV electroformation. This is related to the ability of such dispersions to produce readily well-oriented membrane stacks. Furthermore, we present a protocol for GUV electroformation in various aqueous media, including electrolyte-containing buffers at characteristic concentrations of biological fluids. This work unlocks historical barriers to GUV applications in scientific fields like biology, biochemistry, or biophysics where membrane composition, as well as its aqueous environment, should be adapted to biological significance.

Giant unilamellar vesicle electroformation from lipid mixtures to native membranes under physiological conditions.

Giant unilamellar vesicles (GUVs) are well-known model systems, especially because they are easily observable using optical microscopy. In this chapter, we revisit in detail the versatile GUV electroformation protocol. We demonstrate how GUV electroformation can be adapted to various membrane systems including synthetic lipid mixtures, natural lipid extracts, and bilayers containing membrane proteins. Further, we show how to adjust this protocol to a given aqueous environment and prove that GUVs can be obtained under physiologically relevant conditions, that is, in the presence of electrolytes. Finally, we provide firm evidence that electroformation is a method of choice to produce giant vesicles from native cell membranes. This is illustrated with the example of GUV electroformation from red blood cell ghosts in a physiologically pertinent buffer. GUVs obtained in this manner maintain the native membrane asymmetry, thereby validating the physiological relevance of GUV electroformation.

MECHANICAL PROPERTIES OF MODEL MEMBRANES STUDIED FROM SHAPE TRANSFORMATIONS OF GIANT VESICLES

Membrane deformations occur frequently in cell functioning. From the physical point of view, the understanding of such shape changes requires the introduction of mechanical parameters like bending elasticity. In this article it is shown how this physical property can be obtained from the analysis of small or large shape transformations from giant vesicles. Then it is demonstrated that the bending modulus is strongly dependent on the membrane composition and environmental conditions. This is the case for one-component bilayers (dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine and stearoyloleoyl-phosphatidylcholine (SOPC) and for two-component lipid mixtures (DMPC/cholesterol, DLPC/dilauroylphosphatidic acid). Further it is shown that the bending elasticity of natural lipid extracts (egg phosphatidylcholine, digalactosyl diglyceride and red blood cell lipid extracts) is generally smaller than that of comparable synthetic model membranes. The role of transmembrane proteins is examined by measuring the bending elasticity of SOPC/gramicidin mixtures. Finally, larger scale shape transformations of giant vesicles under an alternative electric field are discussed.

Incorporation of α-tocopherol in marine lipid-based liposomes: In vitro and in vivo studies

Liposomes made from a natural marine lipid extract and containing a high polyunsaturated n−3 fatty lipid ratio were envisaged as oral route vectors and a potential α-tocopherol supplement. The behavior of vesicles obtained by simple filtration and of giant vesicles prepared by electroformation was investigated in gastrointestinal-like conditions. The influence of α-tocopherol incorporation into liposomes was studied on both physical and chemical membrane stability. Propanal, as an oxidation product of n−3 polyunsaturated fatty acids, was quantified by static headspace gas chromatography when α-tocopherol incorporation into liposome ratios ranged from 0.01 to 12 mol%. Best oxidative stability was obtained for liposomes that contained 5 mol% α-tocopherol. Compared to the other formulas, propanal formation was reduced, and time of the oxidation induction phase was longer. Moreover, α-tocopherol induced both liposome structural modifications, evidenced by turbidity, and phospholipid chemical hydrolysis, quantified as the amount of lysophospholipids. This physicochemical liposome instability was even more pronounced in acid storage conditions, i.e., α-tocopherol incorporation into liposome membranes accelerated the structural rearrangements and increased the rate of phospholipid hydrolysis. In particular, giant vesicles incubated at pH 1.5 underwent complex irreversible shape transformations including invaginations. In parallel, the absorption rate of α-tocopherol was measured in lymph-cannulated rats when α-tocopherol was administrated, as liposome suspension or added to sardine oil, through a gastrostomy tube. α-Tocopherol recovery in lymph was increased by almost threefold, following liposome administration. This may be related to phospholipids that should favor α-tocopherol solubilization and to liposome instability in the case of a high amount of α-tocopherol in the membranes. A need to correlate results obtained from in vitro liposome behavior with in vivo lipid absorption was demonstrated by this study.

Softening of POPC membranes by magainin

Magainin 2 belongs to the family of peptides, which interacts with the lipid membranes. The present work deals with the effect of this peptide on the mechanical properties of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine Giant Unilamellar Vesicle, characterized by the bending stiffness modulus. The bending elastic modulus is measured by Vesicle Fluctuation Analysis at biologically relevant pH and physiological buffer conditions and shows a dramatic decrease with increasing peptide concentration. The observed bilayer softening is interpreted in terms of a continuum model describing perturbations on the membrane organization. Our analysis suggests that the adsorbed peptides give rise to considerable local curvature disruptions of the membrane.

Physical and chemical stability of marine lipid-based liposomes under acid conditions

Liposomes made from a marine lipid extract containing a high polyunsaturated fatty lipid ratio were submitted to large pH variations, ranging from 1 to 8. Shape transformations were followed by video microscopy using giant liposomes and micromanipulation experiments. Acidification induced a decrease of the vesicle size simultaneous to the appearance of invaginations. These pH-dependent structural rearrangements were interpreted in terms of osmotic shocks and chemical modifications of the membranes. Liposomes produced by direct filtration were studied using turbidity measurements and optical microscopy observations. A low pH led to an instantaneous vesicle aggregation and to complex supramolecular and/or morphological changes as a function of time. The subsequent buffer neutralization of the liposome suspensions induced a partial reversion of the aggregation phenomenon while the structural membrane rearrangements were persisting. Furthermore, weak chemical degradations (oxidation and hydrolysis) were evidenced when the vesicles were incubated at low pH up to a 24-h incubation time. Thus, although acidification revealed liposome size and shape changes, the bilayer structure was maintained indicating that marine lipid-based liposomes could be used as oral administration vectors.

Advantages of statistical analysis of giant vesicle flickering for bending elasticity measurements

We show how to greatly improve precision when determining bending elasticity of giant unilamellar vesicles. Taking advantage of the well-known quasi-spherical model of liposome flickering, we analyze the full probability distributions of the configurational fluctuations instead of limiting the analysis to the second moment measurements only as usually done in previously published works. This leads to objective criteria to reject vesicles that do not behave according to the model. As a result, the confidence in the bending elasticity determination of individual vesicles that fit the model is improved and, consequently, the reproducibility of this measurement for a given membrane system. This approach uncovers new possibilities for bending elasticity studies like detection of minute influences by solutes in the buffer or into the membrane. In the same way, we are now able to detect the inhomogeneous behavior of giant vesicle systems such as the hazardous production of peroxide in bilayers containing fluorescent dyes.

FURA-2 IMAGING OF SPONTANEOUS AND ELECTRICALLY INDUCED OSCILLATIONS OF INTRACELLULAR FREE CA2+ IN RAT MYOTUBES

Rat myotubes have a resting [Ca2+]i of about 82 nM. Myotubes 3–5 days old (quiescent myotubes) display electrically induced and spontaneous transients in the intracellular concentration of free Ca2+ ions ([Ca2+]i) uncoupled to any detectable contraction. By contrast, 1-to 2-day-old myotubes are insensitive to electrical stimuli and, after 6 days in culture, stimulated myotubes always show [Ca2+]i transients and twitch contractions. The spatial distribution of [Ca2+]i variations in quiescent myotubes is heterogeneous, local increases in [Ca2+]i being mainly observed near the periphery of the cell. The small effect of different external Ca2+ concentrations and of Cd2+ on the amplitude of the [Ca2+]i oscillation indicates that the main source of Ca2+ may be the sarcoplasmic reticulum. This conclusion is supported by the close similarity between electrically induced and caffeine-induced [Ca2+]i maps. These findings suggest that, at an early stage of myotube ontogenesis, a part of the excitation/contraction coupling, as membrane ionic channels, voltage sensors and Ca2+ release and reuptake mechanisms, is functional but, apparently, still uncoupled to the contractile machinery.

THERMAL SHAPE FLUCTUATIONS OF A QUASI SPHERICAL LIPID VESICLE WHEN THE MUTUAL DISPLACEMENTS OF ITS MONOLAYERS ARE TAKEN INTO ACCOUNT

Abstract In most of the previous theories dealing with shape thermal fluctuations of quasi spherical vesicles, the membrane was considered as a homogeneous shell characterized by its bending moduli. Our description considers the fluctuations to be followed by a lateral redistribution of the molecules within the bilayer and by an intermonolayer friction. These phenomena change the expressions for both the mean square values and the dynamic behavior of the fluctuation mode amplitudes. The corrections are expressed through the bending moduli of blocked and free exchange of the molecules within the monolayers. Numerical estimations show that such effects can be observed when shape fluctuations of giant quasi spherical vesicles are analyzed.