Lipid rafts are an important new concept in biomembranes but one that remains controversial because at this juncture the cell biologist's definitions of rafts--detergent resistant membranes and cholesterol extraction-induced abrogation of raft function--have no unambiguous cellular correlate as determined by microscopic methods. In Specific Aim I, we will compare the results of sophisticated and highly appropriate light microscopic methods applied to living cells to the predictions of three extreme models for lipid microdomains in cell membranes. The methods include fluorescence correlation spectroscopy, single particle tracking, single dye tracing, and single domain imaging. The results of the comparison between models and experiments should lead to a considerable refinement in our views of lipid microdomains on cell surfaces. Specific Aim II is directed towards exploring the physical and chemical properties of rafts that could provide functionality. Transbilayer communication and why certain molecular components reside in rafts are key unanswered questions in the field today. Using asymmetric planar supported bilayers, we will determine how liquid-ordered domains on the outer monolayer communicate with the inner monolayer to facilitate raft-mediated signal transduction processes. The factors that determine partitioning of various raft-preferring and cross-linked membrane components into liquid-ordered domains and the competition between raft-resident components for sites in these domains will also be defined. Because the concept of raft detergent resistance is so crucial to the concept, we will image the action of various detergents on raft domains existing in planar supported membranes using fluorescence microscopy.