(121a) Detection and Characterization of Lipid Rafts by Fluorescence Spectroscopy

?Lipid rafts? are the ordered regions of the cell membrane, enriched in cholesterol, sphingolipids, and saturated phospholipids, that are hypothesized to allow a number of cellular processes such as lipid and protein sorting, endocytic transport, protein signaling, and pathogen entry to occur. This work uses fluorescence spectroscopic techniques to detect and estimate the size of lipid rafts in model membrane systems and to study the effect of membrane components, specifically cholesterol and lineactants, on raft formation. The study was initially performed on a simple two-component phospholipid and cholesterol system to allow for comparison to a well established phase diagram before being extended to more complex systems. In this simple 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC)-cholesterol system, membrane heterogeneity was observed at temperatures ranging from 30^oC to 60^oC using two steady-state techniques, polarity-induced spectral shift and Forster Resonance Energy Transfer (FRET). It was found that these two techniques used in tandem provide the most effective characterization of membrane heterogeneity. Upon moving to more complex ternary systems, the effect of lineactants on domain size was systematically studied by varying the types of phospholipids present in the membrane. The change in domain size was investigated both qualitatively and quantitatively, leading to insights into the influence of line tension on domain size and presence. Additionally, the effect of cholesterol on the kinetics of raft growth and depletion was determined as cholesterol was added to and removed from the system. In addition to domain size estimation, these fluorescent techniques allow for the study of the functional nature of lipid rafts. For example, the raft-dependent binding of a particular bacterial toxin, Actinobacillus actinomycetemcomitans, to both binary and ternary model membranes was investigated with a FRET assay. Likewise, FRET was used to determine the influence of estrogen on raft size and possible cholesterol crystallization in a study of gallstone pathogenesis.