Tanja Pott

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.

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.

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.

A comparative study of the action of melittin on sphingomyelin and phosphatidylcholine bilayers

Abstract To investigate whether lipid solubilization is of relevance in describing the interaction between melittin and biological membranes, we studied melittin-induced polymorphism using model membranes composed of the biological lipid sphingomyelin (bovine brain). The behavior of the system was monitored by solid state 31P-NMR and turbidity measurements and compared to the peptides well-characterized action on the synthetic lipid dipalmitoylphosphatidylcholine. It was found that melittin-induced macroscopic changes of sphingomyelin membranes are qualitatively the same as in the case of dipalmitoylphosphatidylcholine bilayers. The sphingomyelin/melittin system is thus proposed to show a reversible vesicle-to-disc transition (fluid-to-gel phase) through an intermediate fusion or aggregation event centered at the main transition temperature, Tm, as reported in the case of saturated phosphatidylcholine. In the case of spontaneous disc formation at 37 °C, the lipid-to-peptide molar ratio in the discoidal objects was determined to be approximately 20 for dipalmitoylphosphatidylcholine and about 12 in the case of natural sphingomyelin. Melittin partition coefficients between membranes and the aqueous medium at 37 °C were found to be 6.1±0.8 mm–1 and 3.7±0.4 mm–1 for sphingomyelin and dipalmitoylphosphatidylcholine, respectively. For very high peptide quantities (lipid-to-peptide molar ratio, Ri≤5) mixed micelles are formed over the entire temperature range (20° to 60 °C) for both kinds of lipids.

Effects of pH and cholesterol on DMPA membranes: a solid state 2H- and 31P-NMR study

The effect of pH and cholesterol on the dimyristoylphosphatidic acid (DMPA) model membrane system has been investigated by solid state 2H- and 31P-NMR. It has been shown that each of the three protonation states of the DMPA molecule corresponds to a 31P-NMR powder pattern with characteristic delta sigma values; this implies additionally that the proton exchange on the membrane surface is slow on the NMR time scale (millisecond range). Under these conditions, the 2H-labeled lipid chains sense only one magnetic environment, indicating that the three spectra detected by 31P-NMR are related to charge-dependent local dynamics or orientations of the phosphate headgroup or both. Chain ordering in the fluid phase is also found to depend weakly on the charge at the interface. In addition, it has also been found that the first pK of the DMPA membrane is modified by changes in the lipid lateral packing (gel or fluid phases or in the presence of cholesterol) in contrast to the second pK. The incorporation of 30 mol% cholesterol affects the phosphatidic acid bilayer in a way similar to what has been reported for phosphatidylcholine/cholesterol membranes, but to an extent comparable to 10-20 mol % sterol in phosphatidylcholines. However, the orientation and molecular order parameter of cholesterol in DMPA are similar to those found in dimyristoylphosphatidylcholine.

The lipid charge density at the bilayer surface modulates the effects of melittin on membranes.

The influence of melittin on two DMPA membrane systems at pH 4.2 and 8.2 has been investigated by solid-state 31P and 2H NMR, as a function of temperature and peptide concentration. Melittin promotes greater morphological changes for both systems in the fluid phase, the effect being larger at pH 4.2. Close inspection of fatty acyl chain dynamics suggests that some parallels can be drawn between the DMPA/melittin at pH 8.2 and PC/melittin systems. In addition, at pH 8.2 a direct neutralization at the interface of one of the lipid negative charges by a positive charge of the peptide occurs, as can be monitored by 31P NMR at the molecular level. For the system at pH 4.2 and at high temperature, a lipid-to-peptide molar ratio of 30 is sufficient to transform the whole system into an isotropic phase, proposed to be inverted micelles. When the system is cooled down towards the gel phase one observes an intermediate hexagonal phase in a narrow range of temperature.

Overview of a Quest for Bending Elasticity Measurement

Abstract We present a brief review of over 30 years of research that led step-by-step to a reproductive method to determine bending elasticity, based on the analysis of thermal shape fluctuations of giant unilamellar vesicles. We also acknowledge the strong contribution of Marin D. Mitov and of a long-lasting French–Bulgarian cooperation in this research. The chapter starts with an introduction of the main theoretical notions necessary to understand this experimental method. Then we review the link between this physical model and the experimental measurable quantities, that is, the amplitudes of the vesicle thermal shape fluctuations. Further, we discuss the technical progress necessary to gradually overcome some principal technical limitations. Finally, we summarize what is currently the most accurate technique for bending elasticity measurements and briefly review published bending elasticity values obtained using thermally induced shape fluctuations of quasi-spherical giant unilamellar vesicles.

31P-NMR methods for investigating phospholipid-based molecular structure and dynamics

Abstract Phospholipids embedded in lamellar, hexagonal or isotropic phases, exhibit very different 31P-NMR spectra. Phase changes induced by temperature, pH or addition of melittin or cholesterol can thus be proben. On the other hand, motions may drastically alter the spectral lineshapes and can be characterized by measuring NMR relaxation rimes. Correlation times and activation energies for intra-, inter-molecular and collective motions present in model membranes can thus be obtained.