Colocalization of the Ganglioside G_M1 and Cholesterol Detected by Secondary Ion Mass Spectrometry

Abstract

The characterization of the lateral organization of components in biological membranes and the evolution of this arrangement in response to external triggers remains a major challenge. The concept of lipid rafts is widely invoked, however, direct evidence of the existence of these ephemeral entities remains elusive. We report here the use of Secondary Ion Mass Spectrometry (SIMS) to image the cholesterol-dependent cohesive phase separation of the ganglioside GM1 into nano and micro-scale assemblies in a canonical lipid raft composition of lipids. This assembly of domains was interrogated in a model membrane system composed of palmitoyl sphingomyelin (PSM), cholesterol, and an unsaturated lipid (dioleoylphosphatidylcholine, DOPC). Orthogonal isotopic labeling of every lipid bilayer component and monofluorination of GM1 allowed generation of molecule specific images using a NanoSIMS. Simultaneous detection of six different ion species in SIMS, including secondary electrons, was used to generate ion ratio images whose signal intensity values could be correlated to composition through the use of calibration curves from standard samples. Images of this system provide the first direct, molecule specific, visual evidence for the co-localization of cholesterol and GM1 in supported lipid bilayers and further indicate the presence of three compositionally distinct phases: (1) the interdomain region; (2) micrometer-scale domains (d>3 μm); and, (3) nanometer-scale domains (d=100 nm − 1 μm) localized within the micrometer-scale domains and the interdomain region. PSM-rich, nanometer-scale domains prefer to partition within the more ordered, cholesterol-rich/DOPC-poor/GM1-rich micrometer-scale phase, while GM1-rich, nanometer-scale domains prefer to partition within the surrounding, disordered, cholesterol-poor/PSM-rich/DOPC-rich interdomain phase.

A comparative study on fluorescent cholesterol analogs as versatile cellular reporters

“…While GUVs are fully artifi cial membrane vesicles, usually comprised of a ternary mixture of Chol and unsaturated and saturated lipids, GPMVs are derived from the plasma membrane of living cells, thus containing its lipid and protein diversity ( 32,46 ). Despite several recent contradictory reports ( 20,(47)(48)(49), Chol has been given an essential role in this lipid raft concept ( 2 ). This is mainly because Chol not only fl uidizes the ordered environment, but also orders the disordered environment, and Choldependent density fl uctuations are supposed to manifest at the nanoscale, i.e., beyond the resolution limit of classical imaging approaches ( 50,51 ).…”

Nonenzymatic biomimetic remodeling of phospholipids in synthetic liposomes

“…Model membranes composed of well-defined synthetic lipids are useful tools for studying questions related to membrane biology (10,11). The importance of lipid remodeling in biology underscores the need for straightforward methods to remodel lipid composition in artificial model membranes.…”

Sphingomyelin distribution in lipid rafts of artificial monolayer membranes visualized by Raman microscopy

“…Although this technique requires destructive imaging conditions, including ultrahigh vacuum, and suffers from a matrix effect that prevents quantitative analysis, it has been applied to molecule-specific imaging of raft mixtures (38). Quantitative analysis with SIMS was recently achieved by using a magnetic sector detection system (39,40). This imaging modality achieves chemical specificity by using isotopic labeling of target molecules and affords high spatial resolution up to 50 nm.…”