Rapid Transfer of Transmembrane Proteins for Single Molecule Dimerization Assays in Polymer-Supported Membranes

Dimerization of transmembrane receptors is a key regulatory factor in cellular communication, which has remained challenging to study under well-defined conditions <i>in vitro</i>. We developed a novel strategy to explore membrane protein interactions in a controlled lipid environment requiring minute sample quantities. By rapid transfer of transmembrane proteins from mammalian cells into polymer-supported membranes, membrane proteins could be efficiently fluorescence labeled and reconstituted with very low background. Thus, differential ligand-induced dimerization of the type I interferon (IFN) receptor subunits IFNAR1 and IFNAR2 could be probed quantitatively at physiologically relevant concentrations by single molecule imaging. These measurements clearly support a regulatory role of the affinity of IFNs toward IFNAR1 for controlling the level of receptor dimerization

Spatial Organization of Lipid Phases in Micropatterned Polymer-Supported Membranes

We have established an approach for the spatial control of lipid phase separation in tethered polymer-supported membranes (PSMs), which were obtained by vesicle fusion on a poly­(ethylene glycol) polymer brush functionalized with fatty acid moieties. Phase separation of ternary lipid mixtures (1,2-dioleoyl-<i>sn</i>-glycero-3-phosphocholine/sphingomyelin/cholesterol) into liquid-disordered (l_d) and liquid-ordered (l_o) phases within both leaflets was obtained with palmitic acid as the anchoring group. In contrast, tethering of the PSM with oleic acid interfered with the phase separation in the surface-proximal leaflet. We exploited this feature for the assembly of l_o domains within PSMs into defined structures by binary micropatterning of palmitic and oleic acid into complementary areas. Ternary lipid mixtures spontaneously separated into l_o and l_d phases controlled by the geometry of the underlying tethers. Transmembrane proteins reconstituted in these phase-separated PSMs strictly partitioned into the l_d phase. Hence, the l_o phase could be used for confining transmembrane proteins into microscopic and submicroscopic domains

Spatial Organization of Lipid Phases in Micropatterned Polymer-Supported Membranes

We have established an approach for the spatial control of lipid phase separation in tethered polymer-supported membranes (PSMs), which were obtained by vesicle fusion on a poly­(ethylene glycol) polymer brush functionalized with fatty acid moieties. Phase separation of ternary lipid mixtures (1,2-dioleoyl-<i>sn</i>-glycero-3-phosphocholine/sphingomyelin/cholesterol) into liquid-disordered (l_d) and liquid-ordered (l_o) phases within both leaflets was obtained with palmitic acid as the anchoring group. In contrast, tethering of the PSM with oleic acid interfered with the phase separation in the surface-proximal leaflet. We exploited this feature for the assembly of l_o domains within PSMs into defined structures by binary micropatterning of palmitic and oleic acid into complementary areas. Ternary lipid mixtures spontaneously separated into l_o and l_d phases controlled by the geometry of the underlying tethers. Transmembrane proteins reconstituted in these phase-separated PSMs strictly partitioned into the l_d phase. Hence, the l_o phase could be used for confining transmembrane proteins into microscopic and submicroscopic domains

Spatial Organization of Lipid Phases in Micropatterned Polymer-Supported Membranes

We have established an approach for the spatial control of lipid phase separation in tethered polymer-supported membranes (PSMs), which were obtained by vesicle fusion on a poly­(ethylene glycol) polymer brush functionalized with fatty acid moieties. Phase separation of ternary lipid mixtures (1,2-dioleoyl-<i>sn</i>-glycero-3-phosphocholine/sphingomyelin/cholesterol) into liquid-disordered (l_d) and liquid-ordered (l_o) phases within both leaflets was obtained with palmitic acid as the anchoring group. In contrast, tethering of the PSM with oleic acid interfered with the phase separation in the surface-proximal leaflet. We exploited this feature for the assembly of l_o domains within PSMs into defined structures by binary micropatterning of palmitic and oleic acid into complementary areas. Ternary lipid mixtures spontaneously separated into l_o and l_d phases controlled by the geometry of the underlying tethers. Transmembrane proteins reconstituted in these phase-separated PSMs strictly partitioned into the l_d phase. Hence, the l_o phase could be used for confining transmembrane proteins into microscopic and submicroscopic domains

Spatial Organization of Lipid Phases in Micropatterned Polymer-Supported Membranes

We have established an approach for the spatial control of lipid phase separation in tethered polymer-supported membranes (PSMs), which were obtained by vesicle fusion on a poly­(ethylene glycol) polymer brush functionalized with fatty acid moieties. Phase separation of ternary lipid mixtures (1,2-dioleoyl-<i>sn</i>-glycero-3-phosphocholine/sphingomyelin/cholesterol) into liquid-disordered (l_d) and liquid-ordered (l_o) phases within both leaflets was obtained with palmitic acid as the anchoring group. In contrast, tethering of the PSM with oleic acid interfered with the phase separation in the surface-proximal leaflet. We exploited this feature for the assembly of l_o domains within PSMs into defined structures by binary micropatterning of palmitic and oleic acid into complementary areas. Ternary lipid mixtures spontaneously separated into l_o and l_d phases controlled by the geometry of the underlying tethers. Transmembrane proteins reconstituted in these phase-separated PSMs strictly partitioned into the l_d phase. Hence, the l_o phase could be used for confining transmembrane proteins into microscopic and submicroscopic domains