Preparation and Properties of Asymmetric Vesicles That Mimic Cell Membranes

Abstract

A methyl-␤-cyclodextrin-induced lipid exchange technique was devised to prepare small unilamellar vesicles with stable asymmetric lipid compositions. Asymmetric vesicles that mimic biological membranes were prepared with sphingomyelin (SM) or SM mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) as the predominant lipids in the outer leaflet and dioleoylphosphatidylcholine (DOPC), POPC, 1-palmitoyl-2-oleoyl-phosphatidyl-L-serine (POPS), or POPS mixed with 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) in the inner leaflet. Fluorescence-based assays were developed to confirm lipid asymmetry. Cholesterol was introduced into these vesicles using a second methyl-␤-cyclodextrin exchange step. In asymmetric vesicles composed of SM outside, DOPC inside (SMo/ DOPCi) or SM outside, 2:1 mol:mol POPE:POPS inside (SMo/ 2:1 POPE:POPSi) the outer leaflet SM formed an ordered state with a thermal stability similar to that in pure SM vesicles and significantly greater than that in symmetric vesicles with the same overall lipid composition. Analogous behavior was observed in vesicles containing cholesterol. This shows that an asymmetric lipid distribution like that in eukaryotic plasma membranes can be conducive to ordered domain (raft) formation. Furthermore asymmetric vesicles containing ϳ25 mol % cholesterol formed ordered domains more thermally stable than those in asymmetric vesicles lacking cholesterol, showing that the crucial ability of cholesterol to stabilize ordered domain formation is likely to contribute to ordered domain formation in cell membranes. Additional studies demonstrated that hydrophobic helix orientation is affected by lipid asymmetry with asymmetry favoring formation of the transmembrane configuration. The ability to form asymmetric vesicles represents an important improvement in model membrane studies and should find many applications in the future.Over the last few decades artificial lipid bilayers of various types have been successfully used as models for biological membranes, yielding many important insights into the architecture of cell membranes. Vesicle dispersions (liposomes) have perhaps been the most useful model membrane system. However, commonly used preparation procedures do not provide control over differences in lipid composition between inner and outer leaflets (lipid asymmetry). This is a troubling limitation because biological membranes are highly asymmetric. In mammalian cells the plasma membrane outer leaflet (exofacial monolayer) is enriched in sphingolipids and phosphatidylcholine (PC), 2 whereas the inner leaflet (cytofacial monolayer) is enriched in phosphatidylethanolamine (PE) and phosphatidylserine (PS) (1).The subject of lipid asymmetry has become all the more important because of its potential role in the structure and function of lipid rafts. Lipid rafts are defined as sphingolipid and sterol-rich lipid domains that exist in the liquid-ordered (Lo) state. Rafts are thought to co-exist in many eukaryotic cell membranes with liquid-disordered (Ld) state domains ...

Decoding the Membrane Activity of the Cyclotide Kalata B1

“…6) reveals that there is expansion of the outer layer (44) and redistribution of phospholipids whereby PE becomes more exposed to the outer layer, whereas PC and SM are redistributed to the inner layer (45). Although only a small percentage of PE is initially exposed, the presence of lo domains rich in Chol and SM (so-called raft domains) in the outer leaflet (54,55) seems to facilitate the initial binding and insertion of kB1 into the outer layer. The increased concentration of PE in the outer membrane promotes the binding of more peptide molecules, leading to pore formation and lysis of RBCs.…”

Lateral organization, bilayer asymmetry, and inter-leaflet coupling of biological membranes

“…To incorporate compositional bilayer asymmetry, London and co-workers developed a technique, in which cyclodextrin-mediated lipid exchange is used to prepare ULVs with different compositions across the lipid bilayers ( Figure 3). 29 In this method, two populations of vesicles are prepared, ULVs with the desired inner leaflet composition, and MLV with the desired outer leaflet composition. These two populations are mixed and incubated with cyclodextrin.…”

“…These two populations are mixed and incubated with cyclodextrin. The choice of cyclodextrin depends on the presence or lack of cholesterol, with methyl-β-cyclodextrin useful for pure lipid systems 29,30,63,80 and hydroxypropyl-α-cyclodextrin used when cholesterol is present in the acceptor ULVs [80][81][82][83] . This approach has proven to be flexible, producing model systems that closely mimic the mammalian plasma membranes (SM and/or PC outside and PE/PS inside), and in which cholesterol content can be readily varied between 0 and 50 mol%.…”

“…Cholesterol is found in both bilayer leaflets, but distributed asymmetrically, with a greater concentration suggested for the inner leaflet 28 . This asymmetry impacts a range of bilayer properties [29][30][31] and is tied to numerous biological functions, including signaling of apoptosis [32][33][34][35] , thrombosis 25,36 , phagocytosis 32,[37][38][39] , and as an indicator of tumorigenic cells 40,41 . In vivo, bilayer asymmetry is maintained by adenosine triphosphate (ATP)-dependent enzymes that transport lipids between the two leaflets 42,43 .…”

Yeast Lipids Can Phase-separate into Micrometer-scale Membrane Domains

“…DPH fluorescence anisotropy was measured with a FluoroMax-3 fluorescence spectrometer (Jobin Yvon Horiba). The samples were excited with 345 nm light, and the fluorescence at 427 nm was detected with an integration time of 0.2 s. The main phase transition temperature was defined by the midpoint of a sigmoid fit to the anisotropy versus temperature curve (35).…”