Partitioning of Cholesterol and Ganglioside GM1 in Phase Separated Lipid Bilayers Imaged by Secondary Ion Mass Spectrometry

Abstract

Secondary ion mass spectrometry has been used to image the distribution of cholesterol and ganglioside GM1 in model membranes composed of palmitoyl sphingomyelin (PSM) and dioleoylphosphatidylcholine (DOPC) best described as the canonical lipid-raft composition. Isotopic labeling or fluorination of each lipid bilayer component allowed the generation of molecule specific images using the NanoSIMS50L instrument at Stanford University. Simultaneous detection of six different ion species, including secondary electrons, were used to generate ratio images whose signal intensity values could be correlated to composition through the use of calibration curves from standard samples. Images of this system indicate the presence of three compositionally distinct phases corresponding to: 1) the interdomain region; 2) large domains (d>3μm); and, 3) small domains (d=200nm-1μm) localized within the large domains. Although semi-quantitative compositional analysis of these distinct phases suggests that both the small and large domains have similar cholesterol content, the large domains were also GM1 and DOPC-rich while the small domains were also PSM-rich. Since the interdomain region is primarily PSM-rich, this seems to suggest that the small domains do not correspond to kinetically trapped interdomain regions and that their more than doubled cholesterol content makes them instead a completely different phase. Furthermore, the average diameter (d = 380nm ± 230nm) of these small domains suggests they might correspond to the formation of nanometer scale domains thus supporting the lipid raft hypothesis.