Creating and Modulating Membrane Microdomains in Pore-Spanning Bilayers

The architecture of the plasma membrane is not only determined by the lipid and protein composition, but is also influenced by its attachment to the underlying cytoskeleton. We show that microscopic phase separation of “raft-like” lipid mixtures in pore-spanning bilayers is strongly determined by the underlying highly ordered porous substrate. In detail, pore-spanning membranes composed of DOPC/sphingomyelin/cholesterol were prepared on ordered pore-arrays in silicon with different pore diameters by spreading and fusion of giant unilamellar vesicles. To induce vesicle rupture and fusion, the top part of a gold-covered silicon substrate was functionalized with a thiol-bearing cholesterol derivative that renders the surface hydrophobic. Confocal laser scanning fluorescence microscopy was used to investigate the phase behavior of the obtained pore-spanning membranes. Coexisting liquid-ordered- and liquid-disordered domains were visualized for DOPC/sphingomyelin/cholesterol (40:40:20) membranes. The same result was obtained for lipid mixtures, in which 5 mol% of sphingomyelin was replaced by 5 mol% of the glycolipid Gb3. Videomicroscopy on these domains demonstrated their lateral mobility on the surface. The size of the lo-phase domains was strongly affected by the underlying pore size of the silicon substrate and could be controlled by temperature, and the cholesterol content in the membrane, which was modulated by the addition of methyl-β-cyclodextrin. Gb3 served as receptor for Shiga toxin B-pentamers, which bind to the membranes and thus considerably modulate the phase behavior of the pore-spanning membranes.