Controlled Protein Confinement in Phase-Separated Membranes Tethered onto Micro-Patterned Supports

Phase-separation of lipid membranes into liquid-disordered (ld) and liquid-ordered (lo) domains has been recognized as a key principle for the functional organization of the plasma membrane. In classic model systems such as GUVs, the spatial organization of phase separated membranes is a stochastic, time-dependent process, which depends on the lipid composition and often leads to a complete coalescence of the lipid phases. We have here established an approach for a spatial control of lipid phase separation in tethered polymer-supported membranes (PSM). On a dense poly(ethylene glycol) polymer brush functionalized with hydrophobic tethers, contiguous, highly fluid PSM were obtained by means of fusion of SUVs.1 Free diffusion of lipids and reconstituted transmembrane proteins in these PSM was confirmed by FRAP, FCS and single molecule tracking. Strikingly, phase separation of ternary lipid mixtures (DOPC/SM/cholesterol) in PSM into ld and lo phases was dependent on the properties of the anchoring group. We exploited these features for assembly of lo domains into defined structures using micropatterned tethers. Within isolated micropatterns, ld and lo phases self-assembled into stable, reproducible membrane architectures. By binary micro-patterning of different tethering groups into complementary areas, ternary lipids mixtures separated into lo and ld phases controlled by the geometry of the underlying tethers. Transmembrane proteins reconstituted in these phase-separated PSM strictly partitioned into the ld phase. Hence, the lo phase could be used for confining transmembrane proteins into microscopic and submicroscopic domains. The permeability of these barriers for lipids and proteins and thus their exchange between adjacent ld compartments can be globally and locally controlled by the temperature. These features have been exploited for probing interactions and diffusion of a transmembrane receptor in the context of ld and lo phases.1) Roder, F.; et al. Anal Chem 2011, 83, 6792-6799.