Electric Field Induced Co-Localization of Membrane Components in Supported Lipid Bilayers Detected by Secondary Ion Mass Spectrometry

Abstract

It is widely believed that certain membrane lipids and membrane-anchored proteins associate to form clusters with emergent function. While extensive data on phase diagrams are available for a range of lipid compositions, these are most commonly visualized by the partitioning of dyes between different phases, and, especially in the case of dye-labeled lipids, the label may substantially alter the physical properties of the components. For this reason we have been developing secondary ion mass spectrometry (SIMS) to image membrane components, in particular a NanoSIMS instrument, where components are differentiated by isotope or atom labels. We are particularly interested in components that co-localize with the ganglioside GM1, often considered to be a “raft” marker. Here we exploit the single negative charge on GM1 which allows it to be selectively moved in an electric field parallel to a patterned, supported membrane surface and address the question of whether other membrane components, notably cholesterol and sphingomyelin, also reorganize. Supported lipid bilayers were formed by the fusion of giant unilamellar vesicles (8:16:5:71 sphingomyelin:cholesterol:GM1:DOPC) to oxidized silicon substrates. Orthogonal isotopic labeling of lipid bilayer components and mono-fluorination of GM1 allowed generation of molecule-specific images that map the lateral re-distribution of molecules as a result of an electric field. Simultaneous detection of up to seven different ion species, including secondary electrons, allowed generation of ion ratio images whose signal intensity could be correlated to composition through the use of calibration curves from standard samples.