Patching of ganglioside GM1 in human erythrocytes

Abstract

Patching of ganglioside GM1 (rafts) induced by cholera toxin subunit B (CTB) plus anti-CTB in human erythrocytes was studied by fluorescence microscopy. A peculiar patching pattern was revealed. The patches were distributed uniformly over the membrane of the discoid human erythrocyte and the number of patches was usually 30–60 per cell. The patches did not coalesce into one large domain, as has been observed in other cell types. This may be due to the absence of major cytoskeletal (motor) proteins in the human erythrocyte. Patching did not affect the discoid shape of erythrocytes and was rather insensitive to pre-fixation (paraformaldehyde 5%) of the cells. In methyl-cyclodextrin pre-treated erythrocytes patching did not occur, indicating that cholesterol is an important structural component in GM1-containing patches. Our study further showed that GM1 patches accumulate in (calcium-induced) echinocytic spiculae. Similarly, in a previous study where we examined the membrane curvature-dependent lateral distribution of raft markers, it was shown that CTB (which may bind GM1 pentavalently) accumulates into (calcium-induced) echinocytic spiculae [Hagerstrand et al., 2006. Mol. Membr. Biol. 23, 277–288]. Since membrane components may or may not accumulate in patched rafts and/or echinocytic spiculae, we use our erythrocyte model system to identify “raftophilic”, “echinophilic” and “immobile” membrane components. In conclusion, our study indicates that the human erythrocyte, small in size but not in complexity, might be a useful cell model for studying by conventional fluorescence microscopic methods membrane microdomain patches.