Impact of membrane-anchored fluorescent probes on the mechanical properties of lipid bilayers.

Fluorescent probes are used in membrane biophysics studies to provide information about physical properties such as lipid packing, polarity and lipid diffusion or to visualize membrane domains. However, our understanding of the effects the dyes themselves may induce on the membrane structure and properties are sparse. As mechanical properties like bending elasticity were already shown to be highly sensitive to the addition of "impurities" into the membranes, we have investigated the impact of six different commonly used fluorescent membrane probes (LAURDAN, TR-DPPE, Rh-DPPE, DiIC18, Bodipy-PC and NBD-PC) on the bending elasticity of dye containing POPC GUVs as compared to single component POPC GUVs. Small changes in the membrane bending elasticity compared to single POPC bilayers are observed when 2 mol% of Rh-DPPE, Bodipy-PC or NBD-PC are added in POPC membranes. These binary membranes are showing non reproducible mechanical properties attributed to a photo-induced peroxidation processes that may be controlled by a reduction of the fluorescent dye concentration. For TR-DPPE, a measurable decrease of the bending elasticity is detected with reproducible bending elasticity measurements. This is a direct indication that this dye, when exposed to illumination by a microscope lamp and contrary to Rh-DPPE, does not induce chemical degradation. At last, LAURDAN and DiIC18 probes mixed with POPC do not significantly affect the bending elasticity of pure POPC bilayers, even at 2 mol%, suggesting these latter probes do not induce major perturbations on the structure of POPC bilayers.

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.

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.

Melittin modifies bending elasticity in an unexpected way.

Understanding the molecular mechanism of the interaction of amphipathic and antimicrobial peptides with membranes is of fundamental interest, especially because of the potential of amphipathic peptides as therapeutics. The most studied amphipathic peptides in this context are certainly melittin, magainin and alamethicin, of which melittin is the only one to exhibit a powerful hemolytic and therefore toxic action. Herein we study the effect of the antimicrobial but hemolytic peptide melittin on the bending elasticity of giant unilamellar vesicles (GUVs). The results are compared to the effects of non-hemolytic amphipathic peptides such as alamethicin. We found that monomeric melittin acts very differently on the membrane mechanical properties. Strikingly, the difference is the most pronounced for low peptide concentrations, relevant for the hemolytic action. This gives some insight into the subtle nature of this peptide-membrane interaction. Furthermore, the results show that bending elasticity measurements might be a sensitive way to distinguish between lytic and non-lytic antimicrobial peptides.

DNA intercalation in neutral multilamellar membranes.

We report a small angle X-ray scattering study of the DNA/neutral lipid/water system showing that it is possible to confine DNA into a neutral multilamellar phase at high lipid-to-DNA weight ratio, despite the lack of electrostatic interactions in this system. This phase is characterized by a 2D ordering of the DNA molecules intercalated between the neutral bilayers of a 3D smectic phase as shown from the presence of a DNA-DNA correlation peak and the 1D electron density profile of the multilamellar phase. We further demonstrate that it is possible to disperse this phase as small multilamellar vesicles encapsulating high amounts of DNA.

New insight into the nanostructure of ionic liquids: a small angle X-ray scattering (SAXS) study on liquid tri-alkyl-methyl-ammonium bis(trifluoromethanesulfonyl)amides and their mixtures.

We report a small angle X-ray scattering study on the liquid phase of a series of room temperature ionic liquids and their binary mixtures. The ionic liquids studied belong to the tri-alkyl-methyl-ammonium family with bis(trifluoromethanesulfonyl)amide as the anion and were studied as a function of alkyl chain length. These ionic liquids were found to exhibit marked nanoscale ordering in their isotropic liquid state as judged from the small angle X-ray scattering. The observed structural ordering is of supramolecular order and depends strongly on the length of the cation hydrophobic side chain. Moreover, the data can be analyzed on the basis of a disordered smectic A phase, consisting of strongly interdigitated bilayers that sequester the ionic liquid into polar and hydrophobic regions. These findings were also found to be consistent with density data of these molten salts. Additionally, we demonstrate that this experimentally observed nanostructuring can further be fine-tuned using binary mixtures.

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.

Alamethicin influence on the membrane bending elasticity.

We investigate the bending elasticity of lipid membranes with the increase of the alamethicin concentrations in the membrane via analysis of the thermally induced shape fluctuations of quasi-spherical giant vesicles. Our experimental results prove the strong influence of alamethicin molecules on the bending elasticity of diphytanoyl phosphatidylcholine and dilauroyl phosphatidylcholine membranes even in the range of very low peptide concentrations (less than 10(-3) mol/mol in the membrane). The results presented in this work, testify to the peripheral orientation of alamethicin molecules at low peptide concentrations in the membrane for both types of lipid bilayers. An upper limit of the concentration of the peptide in the membrane is determined below which the system behaves as an ideal two-dimensional solution and the peptide molecules have a planar orientation in the membrane.