Inter-Leaflet Coupling and Domain Formation in Asymmetric Giant Unilamellar Vesicles

Abstract

Cellular membranes exhibit an asymmetric distribution of lipids across the two leaflets of the bilayer. While the sphingolipid-rich outer (exoplasmic) leaflet has a lipid composition prone to lateral segregation and domain formation, the situation in the sphingolipid-poor inner (cytosolic) leaflet is unclear. The interaction between the two leaflets (i.e. the inter-leaflet coupling), although involved in several biological processes, is not well understood. We recently developed a highly reproducible solvent-free method for preparing asymmetric giant unilamellar vesicles (GUV). The method is fast, has a high yield, can be used with a wide variety of lipids and is compatible with the reconstitution of transmembrane proteins.The asymmetry of the GUVs was investigated using fluorescence correlation spectroscopy (FCS) in order to probe dynamics of lipids in the two leaflets. We have shown that introduction of sphingomyelin exclusively into the outer leaflet of GUVs composed of an unsaturated phosphatidylcholine (PC) results in a much larger decrease in outer leaflet lateral diffusion compared to the effect on the inner leaflet. Furthermore, by systematically comparing different PCs, we found that the degree to which the inner leaflet dynamics is coupled to that in the outer leaflet depends on the length and saturation of the acyl chains and, possibly, the interaction between the lipids’ terminal methyl groups at the bilayer mid-plane. In more detail, preliminary studies indicate that the degree of coupling increases both when: i) the PC has one saturated acyl chain (independently from its Tm) or ii) the SM is asymmetric and interdigitates into the opposing leaflet. These findings suggest how cells might modulate the inter-leaflet coupling - and thus the flow of information through the membrane - by simply varying its lipid composition.