On cell membranes lipids and proteins form specialized regions, """"""""rafts"""""""", that are too small to be seen by conventional light microscopy. The raft lipids constitute an ordered liquid phase physically distinct-more tightly packed and viscous-from the disordered surrounding lipids. The selective incorporation of specific proteins into rafts is important for membrane trafficking, signaling, and assembly of specialized structures, e.g., in virus budding and immune responses. Little is known about the factors that regulate the formation and dynamic properties of membrane rafts. The heterogeneity and complexity of cell membranes makes study of the basic biophysical properties of these structures difficult. Lipid bilayer membranes formed in vitro from selected lipid components serve as simplified models in which to study phase behavior relevant to membrane rafts. The long range goals of the proposed work are to understand the factors that determine the sizes, stabilities, and dynamic properties of submicroscopic lipid """"""""nanodomains"""""""" (representing rafts) in controlled models of biological cell membranes. The first specific aim is to measure the distribution of sizes and dynamic properties of lipid nanodomains in Giant Unilamellar Vesicles (GUVs) that model the physical properties of cell membranes. Fluorescence fluctuation methods, including Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Intensity Distribution Analysis (FIDA) will provide information about dynamic properties and sizes of the lipid nanodomains. These measurements will rely on fluorescent lipid analogs that partition selectively into different lipid phases.
The second aim i s to determine how selected proteins bind to nanodomains. FIDA measurements will determine the correlation between the extent of protein binding and nanodomain size;FCS will yield information about the kinetics of protein-nanodomain interaction.
The third aim i s to develop a theoretical model explain the structural basis of nanodomain formation and refine imaging techniques. The experimental results both motivate and test the theoretical model. The experimental methods developed in this work and the measured data and theoretical models will be applicable to cell membranes and so could yield basic biophysical information about biological raft structure and function.
Protein-lipid structures called """"""""rafts"""""""" regulate signaling and structure formation on the cell surface, and are believed to be a central player in the replication of viruses and in several immune responses. The goal of this work is to refine existing imaging techniques and develop computer simulations that will provide a mechanistic understanding of how such rafts form in a model system.
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