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.

FIGURE 1

A mixture of 1:1:1 DOPC/BSM/Chol (a) oxidized at free air-water interface at ∼30 mN/m, (b) deposited after oxidation on a coverslip previously coated with a monolayer of eggPC, or (c) deposited on a coverslip previously coated with a monolayer of eggPC and exposed to as little oxidation as possible. The difference in domain sizes between the two images is insignificant; similar regions were found in both systems. Similar results are observed for 1:1:1 DOPC/DPPC/Chol. The scale bar is 50 μm.

FIGURE 2

Individual lipids diffuse in a monolayer of 1:1:1 DOPC/BSM/Chol deposited on a monolayer of eggPC on an acid-cleaned glass coverslip. (a) Supported bilayer before photobleach, (b) immediately after being photobleached for 16 min, and (c) after a recovery for another 16 min. The scale bar is 20 μm.

FIGURE 3

(a–d) Temperature cycle of 1:1:1 DOPC/BSM/Chol monolayer deposited on a layer of eggPC on an acid-cleaned glass coverslip. The temperature series is 22°C, 39°C, 51°C, and 22°C, respectively. Micrographs were taken at ∼0, 7, 10, and 15 min. The scale bar is 50 μm.

FIGURE 4

Domains are not in registration in the top and bottom leaflets of a supported LB-LS bilayer. (a) LB-LS bilayer of 2:2:1 DOPC/BSM/Chol deposited on a soap-cleaned glass coverslip. (b) Sketch of domains in each leaflet to aid visualization. Thin solid lines trace domains in the top layer and dashed thick lines trace domains in the bottom layer. The scale bar is 20 μm.

FIGURE 5

Monolayers deposited on silanized coverslips demonstrate a range of behaviors. (a and b) A mixture of 2:2:1 DOPC/BSM/Chol is raised from 22°C to 50°C. The domains appear stable. (c and d) The same mixture of 2:2:1 DOPC/BSM/Chol on a simultaneously prepared coverslip shows greater mobility of lipids at both 22°C and 50°C, respectively. The scale bar is 20 μm.

FIGURE 6

A single GUV of DOPC/BSM/Chol (2:2:1) deposited on a glass coverslip cleaned with 7X. The series is at temperatures 22°C, 32°C, 42°C, and 22°C. Micrographs were taken at ∼0, 5, 6, and 18 min, respectively. Note the speckle pattern in d. White spots are small vesicles in solution that are not connected to the plane of the membrane. The scale bar is 20 μm.

FIGURE 7

(a) Overlap of domains is observed when the first layer contains no dye and oxidized 2:2:1 DOPC/BSM/Chol. The second layer contains the same composition, with dye. Almond-shaped domains characteristic of the first layer are observed even when there is no overlap of domains. (b–d) Supported bilayers as in (a) except the second layer contains dye and either (b) unoxidized 1:1:1 DOPC/BSM/Chol, (c) unoxidized 70:30 DOPC/Chol, or (d) unoxidized DOPC. The scale bar is 50 μm.