Distributions of proteins and lipids in the erythrocyte membrane

Fluorescence digital imaging microscopy was used to characterize erythrocyte membrane domains. To investigate the role of specific proteins in forming lipid domains, erythrocyte ghosts were double-labeled with fluorescent phospholipids and a fluorescent label specific for band 3, the major integral membrane protein. Areas of enrichment, or domains, were observed for both the protein and lipid components. The size and enrichment of the protein and lipid domains in the membrane depended upon the ionic strength but not the temperature. Also, there was a higher correlation between the areas of band 3 and phosphatidylcholine enrichment than between band 3 and phosphatidylserine enrichment. This is consistent with band 3 playing a role in the formation of the phosphatidylcholine domains. Furthermore, areas of spectrin enrichment also were observed in immunofluorescently labeled ghosts, and the band 3 domains were found to coincide with the spectrin domains. Thus, the distribution of band 3 in ghosts is influenced by interactions with the cytoskeleton. Together, these experiments illustrate the type of membrane heterogeneity present in erythrocyte ghosts and the type of lipid and protein interactions that can affect the domains.