Phase separation in pore-spanning membranes induced by differences in surface adhesion

Lipid domains in plasma membranes act as molecular sorting platforms for e.g. , signalling processes. In model membranes, such as freestanding or supported bilayers, some lipid domains with defined chemical composition, lipid packing and physical behaviour can be reproduced. However, in vivo , the plasma membrane experiences a proteinaceous scaffold underneath, which can sort, compartmentalize and recruit components within the membrane. The influence of such scaffolds on the phase behaviour of lipid membranes has been barely studied. Here, we investigated the partial attachment of a membrane to a support and its influence on the phase behaviour using pore-spanning membranes (PSMs). PSMs were prepared on SiO x =1-2 functionalized silicon substrates with 1.2 μm-sized pores by spreading giant unilamellar vesicles (GUVs) composed of DOPC/sphingomyelin (1 : 1) with different cholesterol concentrations. Using two different fluorophores, PSMs were visualized by fluorescence microscopy allowing us to distinguish between different membrane phases, a gel ( l β ), a liquid ordered ( l o ), and a liquid disordered ( l d ) phase. At low cholesterol concentrations, coexistence of l β and l d was found, while at higher cholesterol concentrations, coexistence of l o and l d was predominant. Below the mixing temperature, determined by temperature scans, the more ordered phase was always found in the freestanding PSMs, whereas the l d -phase was present in the supported PSMs. We attribute this lipid sorting to a stronger adhesion of the l d -phase lipids to the underlying scaffold. The difference in adhesion alters the phase behaviour from a nominal DOPC/sphingomyelin (1 : 1) mixture to a DOPC/sphingomyelin (1 : 2-1 : 4) mixture compared to phase diagrams obtained from GUVs highlighting the importance of differential adhesive surfaces on lipid domain formation. A porous scaffold providing different adhesion energies alters the behaviour of coexisting phases in lipid membranes considerably.