DYNAMIQUE DES MEMBRANES HETEROGENES ET EFFETS DES MOLECULES D'ASYMETRIE STERIQUE POSITIVE. ETUDES SUR DES VESICULES GEANTES

The model systems (giant unilamellar vesicles, GUVs) represent an essential tool to study the formation, stability, dynamics and the functions of rafts in biological membranes. We developed a methodology for studding domain budding and fission from a heterogeneous GUV. That made it possible: i) to visualize vesicle budding and fission events induced by biological agents (sPLA2, Lyso PC), and, ii) to directly visualize the effects of detergents (Triton X100, Brij 98) on GUVs - the selective solubilization of the Ld/Lo membrane and the budding and fission of Lo domains. These experiments support the idea that DRM extraction from cell membranes was not an artefact. We suggested as well a possible mechanism for the Lo domain vesicle fission induced by molecules having positive steric asymmetry. Finally, our results show that giant heterogeneous vesicles present an essential progress in the development of model membrane systems mimicking biological membrane structure and functions.Les systèmes modèles (vésicules géantes unilamellaires) représentent un outil indispensable pour étudier la formation, la stabilité, la dynamique et les fonctions des rafts dans les membranes biologiques. L'originalité de notre étude réside : i) dans la visualisation du bourgeonnement et de la fission induits par des agents biologiques (sPLA2 ou Lyso PC) et ii) dans la visualisation directe de la solubilisation d'une membrane modèle de type Ld/Lo ainsi que le bourgeonnement et la fission des domaines Lo traitée avec des détergents (Triton X-100, Brij 98). Ces expériences appuient l'idée que l'isolement de DRM à partir des membranes cellulaires n'est probablement pas un artefact. Nous avons décrit un mécanisme possible pour l'expulsion d'une vésicule en phase Lo déclenché par des molécules d'asymétrie stérique positive. Enfin, les VGU hétérogènes peuvent présenter un modèle assez proche des phénomènes observés sur les membranes biologiques

Dynamique des membranes hétérogènes et effets des molécules d'asymétrie stérique positive (étude sur des vésicules géantes)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

SEGREGATIVE CLUSTERING OF Lo AND Ld MEMBRANE MICRODOMAINS INDUCED BY LOCAL pH GRADIENTS IN GM1-CONTAINING GIANT VESICLES A LIPID MODEL FOR CELLULAR POLARIZATION

International audienceSeveral cell polarization processes are coupled to local pH gradients at the membrane surface. We have investigated the involvement of a lipid-mediated effect in such coupling. The influence of lateral pH gradients along the membrane surface on lipid microdomain dynamics in giant unilamellar vesicles containing phosphatidylcholine, sphingomyelin, cholesterol, and the ganglioside GM1 was studied. Lo/Ld phase separation was generated by photosensitization. A lateral pH gradient was established along the external membrane surface by acid local microinjection. The gradient promotes the segregation of microdomains: Lo domains within an Ld phase move toward the higher pH side, whereas Ld domains within an Lo phase move toward the lower pH side. This results in a polarization of the vesicle membrane into Lo and Ld phases poles in the axis of the proton source. A secondary effect is inward tubulation in the Ld phase. None of these processes occurs without GM1 or with the analog asialo-GM1. These are therefore related to the acidic character of the GM1 headgroup. LAURDAN fluorescence experiments on large unilamellar vesicles indicated that, with GM1, an increase in lipid packing occurs with decreasing pH, attributed to the lowering of repulsion between GM1 molecules. Packing increase is much higher for Ld phase vesicles than for Lo phase vesicles. It is proposed that the driving forces for domain vectorial segregative clustering and vesicle polarization are related to such differences in packing variations with pH decrease between the Lo and Ld phases. Such pH-driven domain clustering might play a role in cellular membrane polarization processes in which local lateral pH gradients are known to be important, such as migrating cells and epithelial cells

Tuning of membrane electrostatic properties by single chain sphingolipids sphingosine and sphingosine-1-phosphate: The effect on bilayer dipole potential

International audienceAn important question in membrane biological chemistry is whether bioactive signaling lipids act only as second messenger ligands or also through an effect on bilayer physical properties. Sphingosine (Sph) and sphingosine-1-phosphate (S1P) are single-chained charged sphingolipids that have antagonistic functions in the "sphingolipid rheostat" which determines cell fate. Sph and S1P respectively promote apoptosis and cell growth. In the present study, the effects of these bioactive lipids on the dipole potential of the lipid bilayer were evaluated. We have investigated the effect of both sphingolipids, incorporated separately or together, in large egg phosphadidylcholine (EPC) unilamellar vesicles on the fluorescence of di-8-ANEPPS, a probe of dipole potential. Both sphingolipids promote a decrease of the dipole potential which is *Manuscript Click here to view linked Reference

MAKING A TOOL OF AN ARTIFACT: THE APPLICATION OF PHOTOINDUCED Lo DOMAINS IN GIANT UNILAMELLAR VESICLES TO THE STUDY OF Lo/Ld PHASES SPINODAL DECOMPOSITION AND ITS MODULATION BY THE GANGLIOSIDE GM1

International audienceElectroformed giant unilamellar vesicles containing liquid-ordered Lo domains are important tools for the modeling of the physicochemical properties and biological functions of lipid rafts. Lo domains are usually imaged using fluorescence microscopy of differentially phase-partionioning membrane-embedded probes. Recently, it has been shown that these probes also have a photosensitizing effect that leads to lipid chemical modification during the fluorescence microscopy experiments. Moreover, the lipid reaction products are able as such to promote Lo microdomain formation, leading to potential artifacts. We show here that this photoinduced effect can also purposely be used as a new approach to study Lo microdomain formation in giant unilamellar vesicles. Photosensitized lipid modification can promote Lo microdomain appearance and growth uniformly and on

Antagonism and Synergy of Single Chain Sphingolipids Sphingosine and Sphingosine-1-phosphate toward Lipid Bilayer Properties. Consequences for Their Role as Cell Fate Regulators

International audienceA recurring question in membrane biological chemistry is whether bioactive signaling lipids act only as second messenger ligands or also through an effect on bilayer physical properties. Sphingosine (Sph) and sphingosine-1-phosphate (S1P) are single-chained charged sphingolipids that have antagonistic functions in the "sphingolipid rheostat" which determines cell fate. Sph and S1P respectively promote apoptosis and cell growth. In the present study, potential effects of these bioactive lipids on physicochemical properties of the lipid bilayer of cell membranes were evaluated. We have investigated the effect of both sphingolipids, incorporated separately or, for the first time, together, in large or giant phosphadidylcholine (PC) unilamellar vesicles. Three bilayer properties were examined: membrane surface charge, lipid packing and formation of membrane microdomains. Sph and S1P appear to have distinct, when not inverse, effects on all three properties. Besides, when both sphingolipids are mixed together, their effect

On the possible structural role of single chain sphingolipids Sphingosine and Sphingosine 1-phosphate in the amyloid-β peptide interactions with membranes. Consequences for Alzheimer’s disease development

International audienceA strong interplay between the neurodegerative effects of the amyloid β (Aβ) peptides and the cell lipid composition or metabolism has been evidenced in Alzheimer’s disease. This appears to be, in part, related to Aβ-membrane interactions. Recently, an influence of the two cell fate-modulating single-chain sphingolipids, sphingosine (Sph) and sphingosine 1-phosphate (S1P), on AD-related mechanisms has been reported. We have investigated the influence of Sph and S1P on the interaction of Aβ(1–42) with lipid model membranes. A fluorescent Aβ(1–42) binds to egg phosphatidylcholine (EPC) giant unilamellar vesicles containing Sph or S1P. With Sph, gel microdomains are present at low temperature and Aβ(1–42) binds preferentially to these domains, especially at their boundaries. With S1P, which displays single lipid phase morphology, Aβ(1–42) binding is uniform. The binding of Aβ(1–42) to EPC/sphingolipid large unilamellar vesicles was investigated by spectrofluorimetry using the 2 probes Laurdan and di-ANEPPS. With most lipid compositions the binding of Aβ(1–42) to LUVs appears superficial. However, with Sph, a deeper membrane penetration is observed. This deeper interaction is reversed to superficial by the simultaneous presence of S1P. It is suggested that the influence of single-chain sphingolipids in AD might be related to a selective interaction of Aβ(1–42) with sphingosine in membranes, that is antagonized by S1P. Such interaction might occur intracellularly for Aβ(1–42) monomers or oligomers and/or extracellularly for Aβ still part of APP. Aβ(1–42) might also influence microdomains by binding to their boundaries. The influence of the Sph/S1P balance on the Alzheimer pathology might be related in part to the differential interactions of Aβ(1–42) with Sph and S1P and their effects on membrane domains

The Alzheimer's disease amyloid-β peptide affects the size-dynamics of raft-mimicking Lo domains in GM1-containing lipid bilayers

International audience† Electronic supplementary information (ESI) available: Films illustrating the effect of A(1-42) on photoinduced spinodal decomposition in GUVs, speed acceletated x 12: Movie_1_GUVs_PC-SM-Chol_10%GM1_withoutAb_20Cx12.avi Movie_2_GUVs_PC-SM-Chol_10%GM1_withAb_20Cx12.avi, Movie_3_GUVs_PC-SM-Chol_10%GM1_withoutAb_14Cx12.avi Movie_4_GUVs_PC-SM-Chol_10%GM1_withAb_14Cx12.avi Alzheimer's disease (AD) is characterized by the overproduction of the amyloid- peptide (A) which forms fibrils under the influence of raft microdomains containing the ganglioside GM1. Raft-mimicking artificial liquid ordered (Lo) domains containing GM1 enhance amyloid- polymerization. Other experiments suggest that A binds preferably to the non-raft liquid disordered (Ld) phase rather than to the Lo phase in the presence of GM1. Here, the interaction of A(1-42) with GM1-containing biphasic Lo-Ld giant vesicles was investigated. Fluorescence colocalisation experiments confirm that A(1-42) binds preferentially to the Ld phase. The effect of A(1-42) on Lo-Ld size dynamics was studied using photoinduced spinodal decomposition which mimics the nanodomain-microdomain raft coalescence. A affects the kinetics of the coarsening phase and the size of the resulting microdomains. The effect depends on which phase is majoritary: when Lo microdomains are formed inside an Ld phase, their growth rate becomes slower and their final size smaller in the presence of A(1-42), whereas when Ld microdomains are formed inside an Lo phase, growth rate becomes faster and final size larger. Fluorimetric measurements on large vesicles using the probe LAURDAN indicate that A(1-42) binding respectively increases or decreases the packing of the Ld phase in the presence or absence of GM1. The differential effects of A on spinodal decomposition are accordingly interpreted as resulting from distinct effects of the peptide on Lo-Ld line tension modulated by GM1. Such modulating effect of A on domain dynamics could be important for lipid rafts in signaling disorders in AD as well as in A fibrillation

Dielectric Properties of Phosphatidylcholine Membranes and the Effect of Sugars

Simple carbohydrates are associated with the enhanced risk of cardiovascular disease and adverse changes in lipoproteins in the organism. Conversely, sugars are known to exert a stabilizing effect on biological membranes, and this effect is widely exploited in medicine and industry for cryopreservation of tissues and materials. In view of elucidating molecular mechanisms involved in the interaction of mono- and disaccharides with biomimetic lipid systems, we study the alteration of dielectric properties, the degree of hydration, and the rotational order parameter and dipole potential of lipid bilayers in the presence of sugars. Frequency-dependent deformation of cell-size unilamellar lipid vesicles in alternating electric fields and fast Fourier transform electrochemical impedance spectroscopy are applied to measure the specific capacitance of phosphatidylcholine lipid bilayers in sucrose, glucose and fructose aqueous solutions. Alteration of membrane specific capacitance is reported in sucrose solutions, while preservation of membrane dielectric properties is established in the presence of glucose and fructose. We address the effect of sugars on the hydration and the rotational order parameter for 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) and 1-stearoyl-2-oleoyl-sn-glycero-3- phosphocholine (SOPC). An increased degree of lipid packing is reported in sucrose solutions. The obtained results provide evidence that some small carbohydrates are able to change membrane dielectric properties, structure, and order related to membrane homeostasis. The reported data are also relevant to future developments based on the response of lipid bilayers to external physical stimuli such as electric fields and temperature changes