Asymmetric Structural Features in Single Supported Lipid Bilayers Containing Cholesterol and GM1 Resolved with Synchrotron X-Ray Reflectivity

Abstract

The cell membrane comprises numerous protein and lipid molecules capable of asymmetric organization between leaflets and liquid-liquid phase separation. We use single supported lipid bilayers (SLBs) to model cell membranes, and study how cholesterol and asymmetrically oriented ganglioside receptor G(M1) affect membrane structure using synchrotron x-ray reflectivity. Using mixtures of cholesterol, sphingomyelin, and 1,2-dioleoyl-sn-glycero-3-phosphocholine, we characterize the structure of liquid-ordered and liquid-disordered SLBs in terms of acyl-chain density, headgroup size, and leaflet thickness. SLBs modeling the liquid-ordered phase are 10 A thicker and have a higher acyl-chain electron density (rho(chain) = 0.33 e(-)/A(3)) compared to SLBs modeling the liquid-disordered phase, or pure phosphatidylcholine SLBs (rho(chain) = 0.28 e(-)/A(3)). Incorporating G(M1) into the distal bilayer leaflet results in membrane asymmetry and thickening of the leaflet of 4-9 A. The structural effect of G(M1) is more complex in SLBs of cholesterol/sphingomyelin/1,2-dioleoyl-sn-glycero-3-phosphocholine, where the distal chains show a high electron density (rho(chain) = 0.33 e(-)/A(3)) and the lipid diffusion constant is reduced by approximately 50%, as measured by fluorescence microscopy. These results give quantitative information about the leaflet asymmetry and electron density changes induced by receptor molecules that penetrate a single lipid bilayer.

Model cell membranes: Techniques to form complex biomimetic supported lipid bilayers via vesicle fusion

“…Once the SLB is formed, the deionized water can be replaced with standard buffer to maintain SLB stability. [50, 51]…”

Functional liposomes and supported lipid bilayers: towards the complexity of biological archetypes

“…Also, the presence of raft-like domains in SLB systems formed by ULV fusion was imaged by AFM by Kane and coworkers exclusively after thermal annealing treatment of the sample. 63 Conversely, Ra¨dler characterized by synchrotron X-ray reflectivity the structure of liquid-ordered and liquid-disordered SLB formed upon insertion of the membrane receptor GM1 on preformed SLB; 64 asymmetric structural changes across lipid bilayer leaflets were shown to be induced by the incorporation of GM1 into the outer leaflet layers.…”

The GM1 Ganglioside Forms GM1-Rich Gel Phase Microdomains within Lipid Rafts

“…Atomic force microscopy (AFM) at mica-supported lipid bilayers may detect changes in bilayer thickness and domain size [11][12][13][14]. X-ray reflectivity may provide useful information in terms of acyl chain and headgroup electron densities and bilayer thicknesses [15]. SIMS may provide distinct images of all components of a lipid mixture via isotopic labeling of each of them [16].…”

“…All surface sensitive techniques use lipid mono-or bilayers deposited on smooth supports, such as mica [11,12,14], silicon wafers [15], or silanized glass [10]. Lipid domains in the above solid supports may lack the reversible phase behavior characteristic of fluid membranes, and the domains can be pinned to the supports [15,17,18]. Chart 1.…”