Correlated Motion and Complex Formation of Lipid-Raft Components Analyzed by High-Resolution 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. We have been developing the use of secondary ion mass spectrometry imaging to directly visualize molecules of interest within supported lipid bilayers (Lozano et al. JACS 2013). Specifically, we want to address the question which neutral membrane components associate with the well-known lipid-raft marker ganglioside GM1, which has a single negative charge, after GM1 is reorganized by an in-plane electric field on a patterned supported bilayer (SLB). NanoSIMS imaging was used to generate molecule-specific concentration profiles of several SLB compositions following electrophoresis: GM1/DOPC, GM1/CHOL/DOPC, and GM1/CHOL/PSM/DOPC. In a control SLB sample without GM1 it is observed that CHOL, PSM, and DOPC do not reorganize by membrane electrophoresis. However, NanoSIMS images clearly illustrate that PSM and CHOL associate with GM1 as it is transported toward the positive electrode while DOPC is displaced in the opposite direction towards the negative electrode. Further analysis of the concentration profiles for the co-diffusing components (GM1/CHOL/PSM) suggest the formation of a 4:2:1 PSM:GM1:CHOL stoichiometric complex. These results demonstrate that both cholesterol and sphingomyelin do tend to associate with GM1 and we might speculate that similar behavior would be observed for GPI or myristolyated proteins.