Lipid Phase Separation and Protein-Ganglioside Clustering in Supported Bilayers Are Induced by Photorelease of Ceramide.

Photolysis of 6-bromo-7-hydroxycoumarinyl-caged ceramide was used to generate ceramide with spatial and temporal control in supported lipid bilayers prepared from mixtures of caged ceramide and phospholipids. The caged ceramide molecules are randomly distributed in fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, and upon photolysis with long wavelength UV light small ordered ceramide domains are formed that phase separate from the bulk fluid membrane. Irradiation of a spatially restricted area leads to the transient formation of ceramide-enriched gel phase domains that equilibrate via lipid diffusion with the surrounding unirradiated membrane. Photorelease of C16-ceramide in supported bilayers prepared from POPC, caged ceramide and the ganglioside GM1 (90:10:1 molar ratio) results in partitioning of a ganglioside-protein complex into the ceramide-enriched domains, modeling some aspects of ceramide's behavior in cells. The photo-uncaging strategy used here for delivery of ceramide in bilayers provides a novel and useful alternative to the enzymatic generation of ceramide in sphingomyelin-containing membranes. The ability to control membrane phase separation behavior and the clustering of membrane-anchored proteins illustrates the potential of photo-uncaging for studying the compartmentalization of ceramide in cellular membranes.