Date/Time
Date(s) - 24/10/2012
3:20 pm - 4:20 pm

Title: Multimodal Imaging/Spectroscopy Approaches for Studying Ceramide-induced Reorganization of Lipid Membranes

Speaker: Dr. Linda Johnston

Institute: NRC

Location: ABB 102

Description:

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 will be 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. 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. We have examined the consequences of ceramide incorporation in supported membranes prepared from ternary lipid mixtures that have coexisting fluid and liquid-ordered phases. The direct incorporation of ceramide leads to the formation of a new ceramide-rich ordered phase that is localized in small sub-domains within the original ordered domains. Enzymatic generation of ceramide also produces ceramide-enriched islands, but leads to a larger scale membrane reorganization that includes clustering of individual domains, formation of areas of fluid phase that are devoid of domains and formation of membrane defects. A combination of correlated AFM and fluorescence imaging and the use of custom-designed cholesterol and ceramide probes provide further information on the complex enzyme-mediated bilayer restructuring. These results are compared to those obtained using photochemical generation of ceramide using a caged lipid precursor. The spatial and temporal control provided by photo-uncaging provides a useful alternative to enzymatic methods which can be difficult to control and reproduce.