Dataset Information

Effect of sterol structure on ordered membrane domain (raft) stability in symmetric and asymmetric vesicles.

ABSTRACT: Sterol structure influences liquid ordered domains in membranes, and the dependence of biological functions on sterol structure can help identify processes dependent on ordered domains. In this study we compared the effect of sterol structure on ordered domain formation in symmetric vesicles composed of mixtures of sphingomyelin, 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol, and in asymmetric vesicles in which sphingomyelin was introduced into the outer leaflet of vesicles composed of DOPC and cholesterol. In most cases, sterol behavior was similar in symmetric and asymmetric vesicles, with ordered domains most strongly stabilized by 7-dehydrocholesterol (7DHC) and cholesterol, stabilized to a moderate degree by lanosterol, epicholesterol and desmosterol, and very little if at all by 4-cholesten-3-one. However, in asymmetric vesicles desmosterol stabilized ordered domain almost as well as cholesterol, and to a much greater degree than epicholesterol, so that the ability to support ordered domains decreased in the order 7-DHC?>?cholesterol?>?desmosterol?>?lanosterol?>?epicholesterol?>?4-cholesten-3-one. This contrasts with values for intermediate stabilizing sterols in symmetric vesicles in which the ranking was cholesterol?>?lanosterol?~?desmosterol?~?epicholesterol or prior studies in which the ranking was cholesterol?~?epicholesterol?>?lanosterol?~?desmosterol. The reasons for these differences are discussed. Based on these results, we re-evaluated our prior studies in cells and conclude that endocytosis levels and bacterial uptake are even more closely correlated with the ability of sterols to form ordered domains than previously thought, and do not necessarily require that a sterol have a 3?-OH group.

SUBMITTER: St Clair JW

PROVIDER: S-EPMC6525066 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Effect of sterol structure on ordered membrane domain (raft) stability in symmetric and asymmetric vesicles.

St Clair Johnna Wellman JW London Erwin E

Biochimica et biophysica acta. Biomembranes 20190320 6

Sterol structure influences liquid ordered domains in membranes, and the dependence of biological functions on sterol structure can help identify processes dependent on ordered domains. In this study we compared the effect of sterol structure on ordered domain formation in symmetric vesicles composed of mixtures of sphingomyelin, 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol, and in asymmetric vesicles in which sphingomyelin was introduced into the outer leaflet of vesicles co ...[more]

PMID: 30904407

Similar Datasets

Project description:Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Nonenzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels, which have well-defined structures and known functions, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small angle x-ray scattering, circular dichroism, and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling, we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time, suggestive of scrambling, and the effect of the channel on the transbilayer lipid distribution in both symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and asymmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process, we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the extent of membrane deformation. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.

Project description:How lipid asymmetry impacts ordered lipid domain (raft) formation may yield important clues to how ordered domain formation is regulated in vivo. Under some conditions, a sphingomyelin (SM) and cholesterol-rich ordered domain in one leaflet induces ordered domain formation in the corresponding region of an opposite leaflet composed of unsaturated phosphatidylcholine (PC) and cholesterol. In other conditions, the formation of ordered domains in a SM and cholesterol-rich leaflet can be suppressed by an opposite leaflet containing unsaturated PC and cholesterol. To explore how PC unsaturation influences the balance between these behaviors, domain formation was studied in asymmetric and symmetric lipid vesicles composed of egg SM, cholesterol, and either unsaturated dioleoyl PC (DOPC) or 1-palmitoyl 2-oleoyl PC (POPC). The temperature dependence of ordered domain formation was measured using Förster resonance energy transfer, which detects nanodomains as well as large domains. In cholesterol-containing asymmetric SM+PC outside/PC inside vesicles, the PC-containing inner leaflet tended to destabilize ordered domain formation in the SM+PC-containing outer leaflet relative to ordered domain stability in cholesterol-containing symmetric SM/PC vesicles. Residual ordered domain formation was detected in cholesterol-containing asymmetric SM+DOPC outside/DOPC inside vesicles, but ordered domain formation was completely or almost completely suppressed by asymmetry in cholesterol-containing SM+POPC outside/POPC inside vesicles over the entire temperature range measured. Suppression of ordered domain formation in the latter vesicles was confirmed by fluorescence anisotropy measurements. Because mixtures of SM, POPC, and cholesterol form domains in symmetric vesicles, and this lipid composition mimics plasma membranes to a significant degree, it is possible that under some conditions in vivo the loss of lipid asymmetry could trigger ordered domain formation.

Project description:Cell membranes possess a complex three-dimensional architecture, including nonrandom lipid lateral organization within the plane of a bilayer leaflet, and compositional asymmetry between the two leaflets. As a result, delineating the membrane structure-function relationship has been a highly challenging task. Even in simplified model systems, the interactions between bilayer leaflets are poorly understood, due in part to the difficulty of preparing asymmetric model membranes that are free from the effects of residual organic solvent or osmotic stress. To address these problems, we have modified a technique for preparing asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange in order to produce tensionless, solvent-free aLUVs suitable for a range of biophysical studies. Leaflet composition and structure were characterized using isotopic labeling strategies, which allowed us to avoid the use of bulky labels. NMR and gas chromatography provided precise quantification of the extent of lipid exchange and bilayer asymmetry, while small-angle neutron scattering (SANS) was used to resolve bilayer structural features with subnanometer resolution. Isotopically asymmetric POPC vesicles were found to have the same bilayer thickness and area per lipid as symmetric POPC vesicles, demonstrating that the modified exchange protocol preserves native bilayer structure. Partial exchange of DPPC into the outer leaflet of POPC vesicles produced chemically asymmetric vesicles with a gel/fluid phase-separated outer leaflet and a uniform, POPC-rich inner leaflet. SANS was able to separately resolve the thicknesses and areas per lipid of coexisting domains, revealing reduced lipid packing density of the outer leaflet DPPC-rich phase compared to typical gel phases. Our finding that a disordered inner leaflet can partially fluidize ordered outer leaflet domains indicates some degree of interleaflet coupling, and invites speculation on a role for bilayer asymmetry in modulating membrane lateral organization.

Dataset Information

Effect of sterol structure on ordered membrane domain (raft) stability in symmetric and asymmetric vesicles.

Publications

Effect of sterol structure on ordered membrane domain (raft) stability in symmetric and asymmetric vesicles.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets