Caged ceramide analogues (C6-, C16-, C18-, C22- and C24-Cer) have been prepared by introducing a hydrophilic coumarin-based cage bearing a short polyethylene glycol (PEG) chain. (6-Bromo-7-mTEGylated-coumarin-4-yl)methyl (Btc) caged ceramide showed efficient photo-uncaging to release the parent ceramide upon direct exposure to 350 nm UV light; in contrast (7-mTEGylated-coumarin-4-yl) methyl (Tc) caged ceramide was photolysed more slowly. In preliminary experiments, Btc-caged ceramides were taken up by cells and their photolysis led to decreases in cell viability, but not to activation of caspase enzymes, suggesting that either reactive oxygen species or an alternate caspase-independent pathway may be responsible for the decreases in cell viability caused by photolysis of caged ceramides.

No abstract

Multimodal imaging methods that combine multiple contrast mechanisms have been widely applied to study the localization and cooperative rearrangement of membrane lipids and proteins in supported lipid membranes, as a model for the heterogeneity of natural cell membranes. We use a combination of fluorescence spectroscopy and microscopy and atomic force microscopy (AFM) to probe the organization of lipid domains in supported lipid monolayers and bilayers and to examine the distribution of lipids and proteins in these domains. The application of these methods is illustrated with studies of the membrane reorganization promoted by ceramide incorporation. Ceramides are among the most hydrophobic lipids and have distinct effects on the physical properties of membranes, promoting phase separation, non-lamellar phases and membrane permeability [1]. The enzymatic generation of ceramide is believed to cause coalescence of small ordered membrane domains to give larger signaling platforms, thus providing a mechanism to aggregate membrane receptors and enhance signaling efficiency [2]. We have examined the consequences of ceramide incorporation in supported membranes prepared from ternary lipid mixtures that have coexisting fluid and liquid-ordered phases [3]. The direct incorporation of ceramide leads to the formation of a new ceramide-rich ordered phase that is localized in small subdomains within the original ordered domains. Enzymatic generation of ceramide also produces ceramide-enriched islands, but leads to a larger scale reorganization that includes clustering of domains, formation of areas of fluid phase that are devoid of domains and formation of membrane defects.We have used correlated AFM and fluorescence imaging combined with custom-designed NBD-labeled cholesterol and ceramide probes to provide further information on the complex bilayer restructuring induced by enzymatic generation of ceramide. Images obtained using an NBD-cholesterol probe to examine membrane restructuring are shown in Figure 1 A,B. Enzyme treatment results in the rapid growth of large dye-excluded regions that exclude the cholesterol probe, suggesting that these are ceramide-enriched regions of the membrane. This hypothesis was confirmed by using a ceramide probe with an NBD fluorophore attached to the lipid headgroup (NBD-Cer) for polarized total internal reflection fluorescence microscopy experiments. Measuring fluorescence images as a function of the polarization of the excitation beam can be used to obtain the average fluorophore tilt angle and calculate order parameter maps for bilayers labeled with NBD-Cer. The order parameters provide information on the organization of the bilayer and demonstrate that the large dye-excluded patches formed by enzymatic reaction are more ordered than either the liquid-ordered or liquid-disordered regions of the bilayer, consistent with their assignment to a new ceramide-enriched phase (Figure 1 C,D).Difficulties in controlling and reproducing the morphologies of enzyme-restructured bilayers prompted us to ...

No abstract