Symmetric and asymmetric planar supported bilayers for the study of lipid rafts and proteins involved in membrane fusion

The plasma membrane (PM) of mammalian cells is heterogeneous and asymmetric in composition. One of the possible consequences of lipid diversity is the existence of segregated liquid-ordered (lo) phase domains, sometimes called lipid rafts, in the otherwise liquid-disordered (ld) outer leaflet of the PM, that may play roles in biological processes such as protein sorting and cell signaling. A key question in this field concerns how lipid domains on the outer leaflet of the PM may affect the inner leaflet, where most of the important work of the cell takes place.; We use fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) in planar supported lipid bilayers composed of phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol, to map the composition-dependence of ld-lo phase coexistence. When mixed with PC/SM, cholesterol induces the formation of l o phase domains. The fraction of the membrane in the l o phase is directly proportional to cholesterol concentration. We then examine natural and synthetic mixtures of lipids mimicking the inner leaflet of the PM and find no evidence of ld- lo phase coexistence. We develop a fitting technique to use fluorescence interference contrast (FLIC) microscopy to measure lipid asymmetry in planar supported bilayers. We compare the final degree of asymmetry in various bilayers made by either the Langmuir-Blodgett/Schafer (LB/LS) method or a combined Langmuir-Blodgett/vesicle fusion (LB/VF) technique. We show that only bilayers made by the LB/VF method, and tethered to solid supports with polymer spacers, retain lipid asymmetry while maintaining the integrity of lo phase domains. FRAP is used to estimate transbilayer coupling between lo phase domains in the proximal leaflet and ld phase lipids in the distal leaflet of planar supported bilayers. Finally, transmembrane and peripheral membrane proteins that are involved in synaptic membrane fusion are reconstituted into asymmetric LB/VF bilayers that contain proximal lo phase domains. We find that these domains cluster syntaxin in the ld regions of the bilayer, and that lipid diffusion is impeded in the presence of syntaxin clusters. Additionally, the C2A domain of synaptotagmin I has less affinity for regions of the distal face of a bilayer that are directly opposite lo phase domains.