Asymmetric Lipid Transfer between Zwitterionic Vesicles by Nanoviscosity Measurements.
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ABSTRACT:
SUBMITTER: Bar L
PROVIDER: S-EPMC8145678 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The effect of lipid composition on the rate of cholesterol movement between cellular membranes is investigated using lipid vesicles. The separation of donor and acceptor vesicles required for rate measurement is achieved by differential centrifugation so that the lipid effect can be quantified in the absence of a charged lipid generally used for ion-exchange-based separation. The rate of cholesterol transfer from small unilamellar vesicles (SUVs) containing 50 mol% cholesterol to a common large unilamellar vesicle (LUV) acceptor containing 20 mol% cholesterol decreases with increasing mol% of sphingomyelin in the SUVs, while phosphatidylethanolamine and phosphatidylserine have no appreciable effect at physiologically relevant levels. There is a large decrease in rate when phosphatidylethanolamine constitutes 50 mol% of donor phospholipids. Interestingly, gangliosides which have the same hydrocarbon moiety as sphingomyelin exert an opposite effect. The effect of spingomyelin seems to be mediated by its ability to decrease the fluidity of the lipid matrix, while that of gangliosides may arise from a weakening of phosphatidylcholine-cholesterol interactions or from a more favourable (less polar) microenvironment for the desorption of cholesterol provided by the head-group interactions involving sugar residues. If the effect of asymmetric transbilayer distribution of lipids is taken into consideration, the observed composition-dependent rate changes could partly account for the large difference in the rates of cholesterol desorption from the inner and outer layers of plasma membrane. Such rate differences may be responsible for an unequal steady-state distribution of cholesterol among various cellular membranes and lipoproteins.
| S-EPMC1148963 | biostudies-other
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.
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Project description:We measured the effect of intrinsic lipid curvature, J0, on structural properties of asymmetric vesicles made of palmitoyl-oleoyl-phosphatidylethanolamine (POPE; J0<0) and palmitoyl-oleoyl-phosphatidylcholine (POPC; J0?0). Electron microscopy and dynamic light scattering were used to determine vesicle size and morphology, and x-ray and neutron scattering, combined with calorimetric experiments and solution NMR, yielded insights into leaflet-specific lipid packing and melting processes. Below the lipid melting temperature we observed strong interleaflet coupling in asymmetric vesicles with POPE inner bilayer leaflets and outer leaflets enriched in POPC. This lipid arrangement manifested itself by lipids melting cooperatively in both leaflets, and a rearrangement of lipid packing in both monolayers. On the other hand, no coupling was observed in vesicles with POPC inner bilayer leaflets and outer leaflets enriched in POPE. In this case, the leaflets melted independently and did not affect each other's acyl chain packing. Furthermore, we found no evidence for transbilayer structural coupling above the melting temperature of either sample preparation. Our results are consistent with the energetically preferred location of POPE residing in the inner leaflet, where it also resides in natural membranes, most likely causing the coupling of both leaflets. The loss of this coupling in the fluid bilayers is most likely the result of entropic contributions.
| S-EPMC5773765 | biostudies-literature
Project description:The effect of surface curvature on the spontaneous movement of cholesterol between membranes was investigated by measuring the rates of cholesterol transfer from donor vesicles of various sizes to a common acceptor vesicle. Donor vesicles of size in the range 40-240 nm were prepared by extruding multilamellar dispersions through polycarbonate filters of different pore sizes under pressure. The smallest donor vesicle and the acceptor vesicles were obtained by the normal sonication procedures. The rate of cholesterol transfer, as measured by the movement of [3H]cholesterol, decreases with increasing size of the donor vesicle in an almost linear fashion. The extrapolation of the results gave a half-time (t1/2) of 16-20 h of the desorption of cholesterol from a planar bilayer, and this can be considered as a reference value for most cellular membranes which are characterized by very low curvatures. Our earlier studies have shown that the t1/2 for cholesterol efflux is influenced by the presence of gangliosides and phosphatidylethanolamine, and the asymmetric distribution of these lipids in the plasma membrane could partially account for the large difference in the rates of cholesterol movement from the two sides of the plasma membrane. The small differences in rates arising from asymmetric distribution will be magnified by the longer t1/2 obtained here for membranes of low curvatures, so that the large difference in rates might be a coupled effect of lipid asymmetry and low curvature of the plasma membrane. This, in turn, may have a role in maintaining the large differences in cholesterol/phospholipid molar ratios observed between plasma membrane and intracellular membranes.
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Project description:Lipid membrane asymmetry plays an important role in cell function and activity, being for instance a relevant signal of its integrity. The development of artificial asymmetric membranes thus represents a key challenge. In this context, an emulsion-centrifugation method is developed to prepare giant vesicles with an asymmetric membrane composed of an inner monolayer of poly(butadiene)-b-poly(ethylene oxide) (PBut-b-PEO) and outer monolayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The formation of a complete membrane asymmetry is demonstrated and its stability with time is followed by measuring lipid transverse diffusion. From fluorescence spectroscopy measurements, the lipid half-life is estimated to be 7.5 h. Using fluorescence recovery after photobleaching technique, the diffusion coefficient of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE-rhod, inserted into the POPC leaflet) is determined to be about D = 1.8 ± 0.50 ?m2 s-1 at 25 °C and D = 2.3 ± 0.7 ?m2 s-1 at 37 °C, between the characteristic values of pure POPC and pure polymer giant vesicles and in good agreement with the diffusion of lipids in a variety of biological membranes. These results demonstrate the ability to prepare a cell-like model system that displays an asymmetric membrane with transverse and translational diffusion properties similar to that of biological cells.
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Project description:Recently, effort has been placed into fabricating model free-floating asymmetric lipid membranes, such as asymmetric vesicles. Here, we report on the use of lipid-coated silica nanoparticles to exchange lipids with initially symmetric vesicles to generate composition-controlled asymmetric vesicles. Our method relies on the simple and natural exchange of lipids between membranes through an aqueous medium. Using a selected temperature, time, and ratio of lipid-coated silica nanoparticles to vesicles, we produced a desired highly asymmetric leaflet composition. At this point, the silica nanoparticles were removed by centrifugation, leaving the asymmetric vesicles in solution. In the present work, the asymmetric vesicles were composed of isotopically distinct dipalmitoylphosphatidylcholine lipids. Lipid asymmetry was detected by both small-angle neutron scattering (SANS) and proton nuclear magnetic resonance (1H NMR). The rate at which the membrane homogenizes at 75 °C was also assessed.
| S-EPMC7899156 | biostudies-literature
Project description:Using Förster resonance energy transfer, raft/liquid-ordered-domain formation was assessed in asymmetric vesicles containing outer leaflets composed of high-Tm (melting temperature) saturated phosphatidylcholines (diC18:0PC, diC16:0PC, diC15:0PC, or diC14:0PC), low-Tm unsaturated dioleoylphosphatidylcholine (DOPC) and cholesterol, and inner leaflets composed of lipids that by themselves would not form ordered domains (DOPC and cholesterol). Ordered-domain formation in the outer leaflet was compared to that in symmetric vesicles with the same lipid composition as the asymmetric vesicle outer leaflets. The difference between ordered-domain thermal stability in asymmetric and symmetric vesicles was highly dependent on high-Tm PC acyl-chain length. At one extreme, in diC14:0PC-containing asymmetric vesicles, the outer leaflet did not segregate to form ordered domains over the entire experimental temperature range even though ordered domains formed in the symmetric vesicles, indicating the inner leaflet dominated outer-leaflet physical behavior in the asymmetric vesicles. At the other extreme, in diC18:0PC-containing asymmetric vesicles, ordered domains formed over the entire temperature range at which they were present in symmetric vesicles, indicating the inner leaflet did not dominate outer-leaflet physical behavior. DiC15:0PC- and diC16:0PC-containing vesicles exhibited intermediate behaviors. A different set of vesicles was prepared with high-Tm lipid sphingomyelin (SM) in place of saturated phosphatidylcholine, and the % SM was varied. The thermal stability of outer-leaflet ordered domains in asymmetric vesicles was found to decrease more than in symmetric vesicles as SM levels decreased, indicating that the inner leaflet increasingly dominated outer leaflet physical state as SM levels decreased. Overall, inhibition of outer-leaflet ordered-domain formation in asymmetric vesicles by inner-leaflet lipids decreased as the ability of outer-leaflet lipids to form an ordered state by themselves increased, i.e., when outer-leaflet high-Tm lipid content or acyl-chain length increased. This has implications for how ordered-domain formation may be controlled in vivo.
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Project description:Cellular membranes play host to a wide variety of morphologically and chemically complex processes. Although model membranes, like liposomes, are already widely used to reconstitute and study these processes, better tools are needed for making model bilayers that faithfully mimic cellular membranes. Existing methods for fabricating cell-sized (?m) or organelle-sized (tens to hundreds of nanometers) lipid vesicles have distinctly different requirements. Of particular note for biology, it remains challenging for any technique to efficiently encapsulate fragile cargo molecules or to generate liposomes with stable, asymmetric lipid leaflets within the bilayer. Here a tunable microfluidic device and protocol for fabricating liposomes with desired diameters ranging from ?10 ?m to ?100 nm are described. Lipid vesicle size is templated by the simple inclusion of a polycarbonate filter within the microfluidic system and tuned with flow rate. It is shown that the vesicles made with this device are stable, unilamellar, lipid asymmetric, and capable of supporting transmembrane protein assembly, peripheral membrane protein binding, as well as soluble cargo encapsulation (including designer nanocages for biotechnology applications). These fabricated vesicles provide a new platform for studying the biophysically rich processes found within lipid-lipid and lipid-protein systems typically associated with cellular membranes.
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Project description:Low- and high-resolution models describing the internal transbilayer structure of asymmetric lipid vesicles have been developed. These models can be used for the joint analysis of small-angle neutron and X-ray scattering data. The models describe the underlying scattering length density/electron density profiles either in terms of slabs or through the so-called scattering density profile, previously applied to symmetric lipid vesicles. Both models yield structural details of asymmetric membranes, such as the individual area per lipid, and the hydrocarbon thickness of the inner and outer bilayer leaflets. The scattering density profile model, however, comes at a cost of increased computational effort but results in greater structural resolution, showing a slightly lower packing of lipids in the outer bilayer leaflet of ∼120 nm diameter palmitoyl-oleoyl phosphatidyl-choline (POPC) vesicles, compared to the inner leaflet. Analysis of asymmetric dipalmitoyl phosphatidylcholine/POPC vesicles did not reveal evidence of transbilayer coupling between the inner and outer leaflets at 323 K, i.e. above the melting transition temperature of the two lipids.
| S-EPMC5377341 | biostudies-literature
Project description:A method for the study of conjugated polyelectrolyte (CPE) photophysics in solution at the single-molecule level is described. Extended observation times of single polymer molecules are enabled by the encapsulation of the CPEs within 200-nm lipid vesicles, which are in turn immobilized on a surface. When combined with a molecular-level visualization of vesicles and CPE via cryo-transmission electron microscopy, these single-molecule spectroscopy studies on CPEs enable us to directly correlate the polymer conformation with its spectroscopic features. These studies are conducted with poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylene-vinylene] (MPS-PPV, a negatively charged CPE), when encapsulated in neutral and in negatively charged lipid vesicles. MPS-PPV exists as a freely diffusing polymer when confined in negatively charged vesicles. Individual MPS-PPV molecules adopt a collapsed-chain conformation leading to efficient energy migration over multiple chromophores. Both the presence of stepwise photobleaching in fluorescence intensity-time trajectories and emission from low-energy chromophores along the chain are observed. These results correlate with the amplified sensing potential reported for MPS-PPV in aqueous solution. When confined within neutral vesicles, single MPS-PPV molecules adopt an extended conformation upon insertion in the lipid bilayer. In this case emission arises from multiple chromophores within the isolated polymer chains, leading to an exponential decay of the intensity over time and a broad blue-shifted emission spectrum.
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