Subnanometer Structure of an Asymmetric Model Membrane: Interleaflet Coupling Influences Domain Properties

Abstract

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.

1H NMR Shows Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers

“…Still, DPPC flip-flop is slow, even at fluid-phase temperatures; for example, a half time of nearly 6 days was found at 50 °C. Slow translocation is nevertheless consistent with many previous reports for PC lipids in vesicles, 13,20,47−49 including the inability of a SANS study to detect intrinsic POPC flip-flop at 37 °C over a period of 2 days. 21 A significant advantage of our method is the ability to measure the intrinsic flip-flop rate of the host lipid (here, DPPC) as opposed to that of an extrinsic probe molecule.…”

“…20,82 Briefly, extruded 100 nm-diameter acceptor LUVs (10–12 mg/mL) provided lipids for the aLUV inner leaflet, whereas donor MLVs provided different lipids for the outer leaflet. An excess of donor lipid (threefold over acceptor) was used.…”

“…20 Use of asymmetric vesicles having different DPPC isotopes in the inner and outer leaflets enabled measurement of intrinsic flip-flop rates using solution 1 H NMR. We find that DPPC flip-flop in the gel phase is too slow to accurately measure, whereas fluid-phase DPPC undergoes flip-flop with a temperature-dependent half time ranging from days to weeks.…”

Interleaflet Coupling, Pinning, and Leaflet Asymmetry—Major Players in Plasma Membrane Nanodomain Formation

“…In black membranes it has also been demonstrated that ld and lo phase separation in one leaflet can induce phase separation in an opposing ld-phase lipid mix and moreover that ld and lo phase separation can be suppressed by ld-phase lipid mixes (Collins and Keller, 2008). Also in asymmetric vesicles, produced by lipid exchange, registered lo-domains can be induced in a ld lipid mix by an opposing ld and lo phase separated bilayer (Lin and London, 2015) and, conversely, a gel phase becomes less ordered than that in symmetric bilayers when facing ld-phase (Heberle et al, 2016). However, direct evidence of the existence of inner plasma membrane leaflet lo-domains is still missing.…”

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

“…This allowed preparation of artificial vesicles that mimic natural membranes by having both natural lipid composition and asymmetry. The approach is beginning to see applications by other laboratories (19)(20)(21)(22).…”