Preparation and Properties of Asymmetric Large Unilamellar Vesicles: Interleaflet Coupling in Asymmetric Vesicles Is Dependent on Temperature but Not Curvature

Abstract

Asymmetry of inner and outer leaflet lipid composition is an important characteristic of eukaryotic plasma membranes. We previously described a technique in which methyl-β-cyclodextrin-induced lipid exchange is used to prepare biological membrane-like asymmetric small unilamellar vesicles (SUVs). Here, to mimic plasma membranes more closely, we used a lipid-exchange-based method to prepare asymmetric large unilamellar vesicles (LUVs), which have less membrane curvature than SUVs. Asymmetric LUVs in which sphingomyelin (SM) or SM + 1-palmitoyl-2-oleoyl-phosphatidylcholine was exchanged into the outer leaflet of vesicles composed of 1,2-dioleoyl-phosphatidylethanolamine (DOPE) and 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS) were prepared with or without cholesterol. Approximately 80-100% replacement of outer leaflet DOPE and POPS was achieved. At room temperature, SM exchange into the outer leaflet increased the inner leaflet lipid order, suggesting significant interleaflet interaction. However, the SM-rich outer leaflet formed an ordered state, melting with a midpoint at ∼37°C. This was about the same value observed in pure SM vesicles, and was significantly higher than that observed in symmetric vesicles with the same SM content, which melted at ∼20°C. In other words, ordered state formation by outer-leaflet SM in asymmetric vesicles was not destabilized by an inner leaflet composed of DOPE and POPS. These properties suggest that the coupling between the physical states of the outer and inner leaflets in these asymmetric LUVs becomes very weak as the temperature approaches 37°C. Overall, the properties of asymmetric LUVs were very similar to those previously observed in asymmetric SUVs, indicating that they do not arise from the high membrane curvature of asymmetric SUVs.

Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids

“…The dependence of solubilization on phospholipid structure was modest, and was similar for MαCD and MβCD. To confirm a lack of phospholipid specificity during exchange, a protocol for lipid exchange in model membrane vesicles analogous to that developed previously for MβCD (12) was devised. Efficient exchange was observed using egg SM (eSM) to donate the lipid exchanged into the outer leaflet of acceptor vesicles composed of 1:1:1 (mol:mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS)/1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) with 40 mol% cholesterol (SI Appendix, Fig.…”

“…We previously developed use of CDs to carry out efficient outer leaflet lipid exchange in model membrane vesicles, defining conditions such that the entire outer lipid monolayer (leaflet) of a membrane lipid bilayer can be replaced with exogenous lipids in situ, using MβCD or hydroxypropyl (HP)αCD without disturbing the lipid composition of the inner leaflet (12)(13)(14)(15)(16)(17)(18). This allowed preparation of artificial vesicles that mimic natural membranes by having both natural lipid composition and asymmetry.…”

The dependence of lipid asymmetry upon polar headgroup structure

“…The DPH/lipid molar ratio was 0.1:100 . Samples were scanned stepwise with 4°C steps, between 16°C and by M ␤ CD-induced exchange have great potential for various applications because the procedure is not unduly complicated, they can be prepared with a variety of lipid compositions, and they can be used to create asymmetric vesicles with various sizes as well as reconstituted vesicles with membrane-inserted peptides (43)(44)(45) and proteins. We recently extended this method by generating asymmetric vesicles containing SMs as outer leafl et lipids and a large variety of PCs with different acyl chain structures as inner leafl et lipids ( 46 ).…”

“…In the lipid exchange method, the lipids in the outer leaflet from an excess of donor vesicles are transferred to the outer leafl et of acceptor SUVs using M ␤ CD, which functions as a carrier that exchanges lipids between two different vesicle populations (43)(44)(45)(46). After exchange and isolation of the acceptor vesicles (exchange vesicles), they should have a different lipid composition in their inner and outer leafl ets if asymmetric .…”

“…Lipids were extracted with chloroform/methanol (1:1 v/v) from ordinary and exchange vesicles as described in ( 43 ). The extracted lipids or pure lipid standards were applied to HP-TLC (Silica Gel 60) plates which were heated at 100°C for 30 min and cooled down to room temperature.…”