Segregation of gangliosides GM1 and GD3 on cell membranes, isolated membrane rafts, and defined supported lipid monolayers

Lateral assemblies of sphingolipids, glycosphingolipids and cholesterol, te rmed rafts, are postulated to be present in biological membranes and to fun ction in important cellular phenomena. We probed whether rafts are heteroge neous by determining the relative distribution of two gangliosides, GM1 and GD3, in artificial supported monolayers, in intact rat primary cerebellar granule neurones, and in membrane rafts isolated from rat cerebellum. Fluor escence resonance energy transfer (FRET) using fluorophore-labelled cholera toxin B subunit (which binds GM1) and mAb R24 (which binds GD3) revealed t hat GM1 spontaneously self-associates but does not co-cluster with GD3 in s upported monolayers and on intact neurones. Cholera toxin and immunocytoche mical labelling of isolated membrane rafts from rat cerebellum further demo nstrated that GM1 does not co-localise with GD3. Furthermore, whereas the m embrane raft resident proteins Lyn and caveolin both co-localise with GD3 i n isolated membrane rafts, GM1 appears in separate and distinct aggregates. These data support prior reports that membrane rafts are heterogeneous, al though the mechanisms for establishing and maintaining such heterogeneity r emain to be determined.