Probing the Structural and Signalling Roles of Ceramide using 6-Bromo-7-Hydroxycoumarin Caged Derivatives

Abstract

A number of critical cell functions including apoptosis are regulated by the sphingomyelin-ceramide pathway, a ubiquitous signal transduction system that has been evolutionarily conserved. Several different sphingomyelinases can hydrolyze sphingomyelin to generate ceramide in distinct intracellular compartments; however, the specific functions of these dynamic ceramide sub-populations are not completely understood. Ceramide has also attracted interest for its effects on the physical properties of biological membranes. In model membrane studies ceramide has been shown to promote phase separation and domain formation, non-lamellar phases, and enhanced membrane permeability. We recently reported the synthesis and photochemical characterization of long-chain ceramides caged with a 6-bromo-7-hydroxycoumarin group. These derivatives were readily dispersed in aqueous solution, successfully delivered into cells and uncaged therein. Caged compounds such as these can be released in their biologically active form with a high degree of spatial and temporal control. These characteristics are attractive in applications where well-defined amounts of ceramide must be generated. Previous work has largely relied on exogenous delivery of synthetic short-chain ceramides or sphingomyelinase treatments where enzyme activity can be difficult to control. Further studies are currently under way in HL-60 cells using the caged compounds to probe the role of long-chain ceramides in pro-apoptotic pathways. Concurrently, we have incorporated caged ceramides into supported lipid bilayers as part of ongoing work to model ceramide's mechanical and morphological effects in membranes.