Caveolin-1-dependent and -independent membrane domains
Lipid rafts defi ned as cholesterol- and sphingomyelin-rich domains have been isolated from different cell types that vary greatly in their lipid profi les. Here, we investigated the contribution of the structural protein caveolin-1 (Cav1) to the overall lipid composition and domain abundance in mouse embryonic fi broblasts (MEFs) from wildtype (WT) or Cav1-defi cient (Cav1 / ) animals. Our fi ndings show that Cav1 expression had no effect on free (membrane-associated) cholesterol levels. However, Cav1 / -defi cient cells did have a higher proportion of sphingomyelin, decreased abundance of unsaturated phospholipids, and a trend toward shorter fatty acid chains in phosphatidylcholine. We isolated detergent-resistant membranes (DRMs), nondetergent raft domains (NDR), and cholesterol oxidase (CO)-sensitive domains and assessed the abundance of ordered domains in intact cells using the fl uorescent dye Laurdan. Despite differences in phospholipid composition, we found that cholesterol levels in DRMs, NDR, and CO-sensitive domains were similar in both cell types. The data suggest that Cav1 is not required to target cholesterol to lipid rafts and that CO does not specifi cally oxidize caveolar cholesterol.In contrast, the abundance of ordered domains in adherent cells is reduced in Cav1 / compared with WT MEFs, suggesting that cell architecture is critical in maintaining Cav1-induced lipid rafts.