Nonequilibrium Behavior in Supported Lipid Membranes Containing Cholesterol

Abstract

We investigate lateral organization of lipid domains in vesicles versus supported membranes and monolayers. The lipid mixtures used are predominantly DOPC/DPPC/Chol and DOPC/BSM/Chol, which have been previously shown to produce coexisting liquid phases in vesicles and monolayers. In a monolayer at an air-water interface, these lipids have miscibility transition pressures of approximately 12-15 mN/m, which can rise to 32 mN/m if the monolayer is exposed to air. Lipid monolayers can be transferred by Langmuir-Schäfer deposition onto either silanized glass or existing Langmuir-Blodgett supported monolayers. Micron-scale domains are present in the transferred lipids only if they were present in the original monolayer before deposition. This result is valid for transfers at 32 mN/m and also at lower pressures. Domains transferred to glass supports differ from liquid domains in vesicles because they are static, do not align in registration across leaflets, and do not reappear after temperature is cycled. Similar static domains are found for vesicles ruptured onto glass surfaces. Although supported membranes on glass capture some aspects of vesicles in equilibrium (e.g., gel-liquid transition temperatures and diffusion rates of individual lipids), the collective behavior of lipids in large liquid domains is poorly reproduced.

Tuning lipid mixtures to induce or suppress domain formation across leaflets of unsupported asymmetric bilayers

“…Domains on supported bilayers are typically captured from a deposited monolayer (10), and supported bilayers often reflect the phase behavior of the two lipid monolayers from which they are made, rather than of a bilayer (11). Lipids in monolayers are usually oxidized to exhibit domains above the surface pressure of 32 mN/m before deposition as a supported bilayer (11).…”

“…Moreover, Chol compositions in the two liquid phases differ widely in monolayers but are comparable in bilayers, so that tie-lines run oppositely in the two systems (11). Domains successfully induced from one supported leaflet to another can produce counterintuitive dye distributions (7,10). Finally, the bilayer's proximity to the substrate often means that domain diffusion is too slow to measure on experimental time scales (10).…”

“…Domains successfully induced from one supported leaflet to another can produce counterintuitive dye distributions (7,10). Finally, the bilayer's proximity to the substrate often means that domain diffusion is too slow to measure on experimental time scales (10). In contrast, planar bilayers produce domains in asymmetric, unsupported, unoxidized model membranes.…”

“…Bilayers on solid supports have previously been used to study interleaflet interactions (10,19), but the proximity of the substrate prevents domains from coming into registration (and reaching equilibrium) when leaflets are deposited from monolayers. Inserting a Ն58-Å polymer cushion between a supported bilayer and its substrate brings domains into registration but still produces results distinct from our unsupported bilayers.…”

“…Some researchers argue that only proteins should be able to induce domains across leaflets (9). Other groups, including our own, have observed what seems to be domain induction across leaflets of protein-free membranes deposited on solid surfaces, but generally these efforts have been frustrated by experimental difficulties (7,(10)(11)(12).…”