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.

Photouncaging of Ceramides Promotes Reorganization of Liquid-Ordered Domains in Supported Lipid Bilayers

“…47 Recently, we have concluded that the selection for a subset of vesicles from a heterogeneous vesicle population during bilayer formation may contribute to unexpected trends in morphology for bolalipid/POPC mixtures. 48 In the present study, the presence of the bromohydroxycoumarin moiety, which is partially deprotonated at the pH used to form SUVs and supported bilayers in our experiments, may be an important factor. Variations in the net negative charge on the SUVs due to different fractions of 1 coupled with electrostatic repulsion between the vesicles and the negatively charged surface could lead to the formation of a supported bilayer with a lower mole fraction of 1 than in the bulk vesicle suspension.…”

Phase Composition Control in Microsphere-Supported Biomembrane Systems

“…47 Thus, the composition of MLVs formed during hydration of dry films depends on the region of the film hydrated. One might contend that sonication would robustly homogenize the mixture during SUV formation, but this has been shown not to be the case, 46 suggesting that MLVs have tremendously de-mixed compositions prior to SUV formation. Finally, one might suppose that in the last step of PLB preparation, where vesicles fuse to particle surfaces, dispersion of SUVs and coordinated fusion would better mix lipid components, but since only ~1000 SUVs of 100 nm diameter are required for fusion to a 5 µm-sized sphere, where suspensions of sonicated vesicles contain on the order of 10 12 – 10 13 SUVs.…”

“…Furthermore, the kinetics of fusion are dependent on vesicle composition and size, where SUVs with more mobile components and greater elasticity will rupture and fuse rapidly after adsorption, but less mobile components require a significant amount of vesicle adsorption before rupture is initiated. 22, 46 This was probed in a recent spherically-supported biomembrane study using fusion with different sizes of starting lipid vesicles that resulted in differences in organization of lipids and shapes of co-existing domains. 39 Taken together, SUVs here are considerably heterogeneous and a difference in the rate of vesicle fusion further deters homogenization, leading to PLBs with different compositions within the L d /L o co-existence region and fractions of L d /L o content.…”

“…During proteoliposome preparation, lipids and peptide are mixed in organic solvent that is then evaporated away to allow for hydration and MLV formation. While lipid components are highly soluble in chloroform, they are found to demix upon solvent evaporation, inducing macroscopic phase segregation. , In addition, high cholesterol content of bulk mixtures can cause cholesterol crystallization during drying . Thus, the composition of MLVs formed during hydration of dry films depends on the region of the film hydrated.…”

Role of Transmembrane Proteins for Phase Separation and Domain Registration in Asymmetric Lipid Bilayers

“…Independently of their length, however, all WALP peptides were observed to increase domain alignment. The structural coupling mechanism is not confined to transmembrane proteins; it also applies to bolalipids [39] and even to lipids with long tails such as monosialotetrahexosylganglioside (GM1) [40] and other long saturated acyl chains [11] that interact with the lipids in the apposed leaflet.…”