Lipid Structure And Control Of Membrane Ordered Domain Formation And Size By Lipid Composition And Asymmetry In Vitro And In Vivo

We have studied the properties and formation of ordered lipid domains (“lipid rafts”) in model membrane vesicles and cells. For example, recent FRET studies on vesicles with sphingomyelin/phospholipid/cholesterol mixtures found that mammalian lipids have structures and compositions favoring “ultrananodomain” formation, i.e. formation of very small nanodomains 5 nm or less in size. To study domain formation in more biologically-relevant vesicles, we developed methods to prepare vesicles with an asymmetric lipid distributions using cyclodextrin-catalzyed lipid exchange. Recent studies in asymmetric giant vesicles containing raft domains large enough to see with microscopy revealed interleaflet coupling to sphingomyelin- and cholesterol-rich outer leaflet domains induces ordered domains in the portion of the DOPC and cholesterol containing inner leaflets with which they are in contact. Analogous studies using smaller asymmetric vesicles and FRET-based detection may reveal whether nanodomains can form and exhibit coupling between inner and outer leaflets ordered domains. Methods to prepare a wider range of asymmetric membrane compositions and make more facile asymmetry measurements are being developed. In addition, cyclodextrin-catalyzed lipid exchange is being extended to studies of membrane domain structure and function in living cells. Substituting for cholesterol shows the subset of sterols having the ability to support the formation of lipid rafts are necessary and sufficient for them to support membrane domain formation in Borrelia burgdorferi, a bacteriumcontaining fatty acyl cholesterol glycosides.Interestingly,we find fatty acyl cholesterol glycosides are able to form ordered membrane domains without sphingolipids. Studies with cyclodextrin-catalyzed sterol exchange in mammalian cells show that sterols having an ability to promote the formation of ordered membrane domains are necessary and sufficient for them to support both clathrin-dependent and independent endocytosis provided they also contain a 3-beta OH. Efforts to carry out similar experiments with phospholipid and sphingolipid exchange are underway.