Unraveling of Lipid Raft Organization in Cell Plasma Membranes by Single-Molecule Imaging of Ganglioside Probes

Ganglioside s are involved in a variety of physiological roles and particularly in the formation and function of lipid rafts in cell membranes. However, the dynamic behaviors of gangliosides have not been investigated in living cells owing to the lack of fluorescent probes that behave like their parental molecules. This has recently been resolved by developing new fluorescent ganglioside analogues that act similarly to their parental molecules, synthesized by only chemical methods. We performed single fluorescent-molecule imaging and revealed that ganglioside probes dynamically enter and exit rafts containing CD59, a glycosylphosphatidylinositol (GPI)-anchored protein, both before and after stimulation. The residency time of our ganglioside probes in CD59 oligomers was 48 ms after stimulation. The residency times in CD59 homodimer and monomer rafts were 40 and 12 ms, respectively. These results reveal the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they demonstrate that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner and at a much higher frequency than expected. In this chapter, we review methods for the development and single-molecule imaging of new fluorescent ganglioside analogues and discuss how raft domains are formed, both before and after receptor engagement.