Influence of Cholesterol on Phase Behavior and Nonrandom Mixing of Membrane Components

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The goal of this project is to study the thermodynamic basis for nonrandom mixing in lipid bilayers that model biomembranes. The project has four main conceptual elements: (i) 3-component bilayer lipid mixtures serve as models for the outer and the inner leaflets of mammalian plasma membranes. These 3-component mixtures are sufficiently complex to model real biomembranes, yet are chemically well-defined; (ii) Several independent, complementary methods are used to thoroughly map and characterize the compositional phase behavior of these model membranes; (iii) Real biomembranes are asymmetric: model bilayers that are also asymmetric will be studied in order to bridge the gap between the chemically-simple models and real biomembranes; (iv) Behavior of the plasma membranes of living cells, including the nature membrane rafts, will be investigated by the methods developed in the model systems and analyzed in terms of the phase diagrams obtained for the model systems. Experimentally, this work involves large data sets of fluorescence resonance energy transfer and perylene excimer/monomer measurements to find the lipid phase boundaries and the partition behavior of fluorescent probes. Confocal fluorescence microscopy will be used to visualize and identify coexisting phases. New procedures will be developed in order to prepare asymmetric lipid bilayers.Cholesterol is the single most abundant lipid species in mammalian plasma membranes. Nonetheless, the way cholesterol interacts with neighboring membrane molecules has remained obscure. Cholesterol is known to mix nonrandomly with other membrane lipids, and yet in most cases a clear understanding is missing for the lateral distribution of cholesterol, and the way cholesterol influences the lateral distribution of other membrane components. A current picture of nonrandom mixing in biomembranes, the "raft model" of cholesterol-rich patches floating within a cholesterol-poor bilayer, is likely to be improved by this research project. In addition, in order to promote scientific education and training, a group of undergraduate students will receive lectures in lipid physical chemistry and fluorescence spectroscopy, followed by systematic training in lipid analytical chemistry, lipid organic synthesis, and spectroscopic and microscope techniques, which they will then use in their assigned independent research.