We aim to understand biomembrane structure/function with the aid of a lab-achieved breakthrough in control of membrane phospholipid and sphingolipid composition via cyclodextrin-catalyzed lipid exchange. Lipid exchange is generally efficient (80-100%), and permits preparation of lipid vesicles mimicking natural membranes closely in terms of lipid asymmetry, the difference in the lipid composition in the inner and outer lipid monolayers (leaflets). The lab has now extended this method to control lipid composition of plasma membrane outer leaflets in mammalian cells (without harming them), and without disturbing membrane sterol. The method is being applied to solve long-standing issues of membrane organization and function. We proposed in 1994 what remains the working model: that membrane domains form in cells due to segregation of sphingolipid-cholesterol rich liquid ordered (Lo) domains from unsaturated lipid rich liquid disordered domains. In the last grant period, using lipid exchange to prepare asymmetric membranes, we found how lipid structure in each leaflet controls when there is overall induction or suppression of Lo domains. With physiologically-relevant lipids, we found that Lo domain formation is greatly enhanced by loss of lipid asymmetry, and confirmed this is also true in natural plasma membrane vesicles. This is important because transient loss of lipid asymmetry occurs during signal transduction. In the next grant period we propose to test the hypothesis that Lo domain formation is induced by loss of lipid asymmetry in intact cells by using red blood cells, which have a number of advantages including only a single membrane, known lipid asymmetry, known methods to control lipid asymmetry in situ, and facile use for lipid exchange. We will then extend studies to metabolically-active cells to see if loss of lipid asymmetry stabilizes Lo domains during antibody-triggered signal transduction in immune RBL-2H3 cells, and defining the importance of Lo domains by exchange using lipids that enhance or inhibit Lo domain formation. In the last grant period we also used lipid exchange to show the ability to form Lo domains is necessary and sufficient for a lipid to support insulin receptor function. We proposed the hypothesis that localization in Lo domain, which are thick, decreases insulin receptor transmembrane helix tilt, activating autophosphorylation. We will test this hypothesis by defining receptor domain localization before and after activation, and by mutations that lengthen or shorten its transmembrane segment, to see if predicted activity changes are observed. We also propose to test a novel competition assay for defining receptor domain localization. Finally, we propose to extend lipid exchange to membrane enveloped viruses. A range of lipids will be exchanged into the envelope of pseudovirus with SARS CoV-2 spike or VSV-G proteins to see how viral lipids/domains modulate cellular uptake. These studies may help design of improved ...