AFM investigations of phase separation in supported membranes of binary mixtures of POPC and an eicosanyl-based bisphosphocholine bolalipid

Abstract

Supported membranes prepared from binary mixtures of DOPC and the bolalipid C(20)BAS have been examined by atomic force microscopy (AFM). The supported membranes are phase separated to give a thicker DOPC-rich phase and a thinner bolalipid-rich phase for a range of lipid compositions. These results confirm an earlier prediction from mean field theory that phase separation is the thermodynamically stable state for membranes containing approximately equimolar C(20)BAS and double chain monopolar lipids with chain lengths exceeding 15 carbons. Hydrophobic mismatch between the monopolar lipid hydrocarbon chains and the membrane spanning bolalipid chains was suggested to provide the driving force for phase separation. The AFM results also show that the morphology of the mixed POPC:C(20)BAS supported membranes varies significantly with the conditions used to prepare the vesicles and supported membrane samples. The complex membrane morphologies observed are attributed to the interplay of several factors, including a compositionally heterogeneous vesicle population, exchange of lipid between the vesicle solution and solid substrate during formation of the supported membrane, and slow equilibration of domains due to pinning of the lipids to the solid support.

Figure 1

Structures of C₂₀BAS, POPC and DMPC. The cartoon shows phase separation for a mixed C₂₀BAS/PC lipid membrane to give thinner domains that are predominantly transmembrane bolalipids and thicker domains that are enriched in monopolar lipid and have a small fraction of bolalipid U-conformers.

Figure 2

AFM images of POPC and C₂₀BAS supported membranes prepared from sonicated vesicles at room temperature on mica. (A) POPC patches and (B) a continuous bilayer formed by incubating 3 μg and 50 μg total lipid for 1 hour. (C) C₂₀BAS membrane patches produced by incubating 50 μg total lipid for 72 hours; the insert shows an area of continuous membrane for a different sample for which a small region was scanned at high force to create a persistent defect. Cross sections for the lines marked on each image are shown below the images.

Figure 3

(A–E) AFM images of supported membranes prepared from sonicated vesicles with various POPC:C₂₀BAS ratios. Images A–E are for X_B = 0.9, 0.6, 0.5, 0.4 and 0.1, respectively. Image F shows a supported membrane prepared by mixing pure POPC and pure C₂₀BAS sonicated vesicles (1:1 ratio) immediately before exposure to the mica substrate. Cross sections for the lines marked on images B, C, D and F are shown on the right.

Figure 4

(A, B) AFM images of supported membranes prepared from sonicated vesicles (POPC:C₂₀BAS, X_B = 0.4) after incubation at 60°C on mica. (C) AFM images of supported membranes prepared from extruded vesicles (POPC:C₂₀BAS, X_B = 0.4, mean vesicle diameter = 71 nm) on mica at room temperature. (D) AFM images of supported membranes prepared from sonicated vesicles (POPC:C₂₀BAS, X_B = 0.5) on silicon (111) at room temperature. Cross sections for the lines marked on each image are shown below the images.

Figure 5

CryoTEM images at 50,000× magnification of POPC:C₂₀BAS vesicles (X_B = 0.5, 30 mM total lipid) prepared by sonication (A, B) and extrusion through 200 nm filters (C, D). Image B shows one large irregularly shaped vesicle as well as several membrane fragments (outlined in red). Images C and D show vesicles where there are noticeably thicker and thinner membrane regions, indicated with thick black arrows and thin red arrows, respectively.

Figure 6

AFM images for membranes prepared from extruded (A, 100 nm filter) and sonicated (B) DMPC:C₂₀BAS (X_B = 0.4) vesicles on mica. (C) Cross section for the line marked on image B.