Rafts are specialized regions of membranes that consist of phase-separated domains of cholesterol and sphingolipids enriched in particular proteins. A large number of cellular processes - such as signal transduction and intracellular trafficking - are thought to be controlled by raft behavior. The wide-ranging importance of rafts has also linked them to many diseases, and some viruses even appear to fuse and/or bud at raft sites. But specific structures and dynamics of raft formation, growth, and composition are as yet unknown. The planar lipid bilayer model system has many advantages for discovering the physical chemical principles that govern these aspects of rafts; lipid phase separation, partitioning of proteins into cholesterol/sphingolipid domains, and control of formation of these domains by proteins can all be investigated in bilayer membranes. By including cholesterol, sphingomyelin, and fluorescent probes in bilayers, kinetic aspects of phase-separated cholesterol/sphingomyelin domains will be studied by fluorescence microscopy with selectivity and sensitivity not possible using cells. Fluorescent and non-fluorescent (quencher) probes will be constructed to partition into selected domains and placed in raft forming bilayers at high concentrations. These techniques will allow small lipid-microdomain rafts to be detected, their growth and dissolution to be quantified, and their stability to be characterized. Rafts within a single monolayer leaflet will be studied and any coupling to a liquid-ordered domain in the opposite monolayer will be investigated. The extent to which contact between acyl chains of lipids in opposite monolayers controls coupling will be determined. Among the proteins thought to partition into rafts, GPI anchored proteins are prominent; GPI-GFP will be used as a model protein to assess relationships between proteins and rafts under varying conditions. The hypothesis that cholesterol-binding protein can serve as a center for nucleation of rafts will be tested. The results of experimental aims will be used to adapt theory developed for phase creation and growth in other systems, so that an integrated understanding of rafts can be based on fundamental physical principles.
