GPI-anchored proteins, glycosphingolipids, and sphingomyelin are sequestered to caveolae only after crosslinking

GPI-anchored proteins, glycosphingolipids, and sphingomyelin are all enriched in the detergent-insoluble complex which has been suggested to be purified caveolae. I studied the relationship of the molecules with caveolae in cultured cells by immunocytochemical methods. In cells reacted with antibodies to various membrane proteins and lipids on ice and fixed before applying secondary antibodies, labeling did not show concentration in caveolae. In contrast, when cells were incubated with the primary and secondary antibodies on ice and then transferred to 37 degrees C without fixation, labeled Thy-1.2, beta 2-microglobulin, lactosyl ceramide, ceramide tetrahexose, Forssman antigen, and sphingomyelin became concentrated in caveolae, whereas labeled transferrin receptor did not. Thy-1.2 and sphingolipids formed common patches and were sequestered in the same caveolae when crosslinked with two primary antibodies simultaneously. On the other hand, when either Thy-1.2 alone or lactosyl ceramide alone was crosslinked and sequestered to caveolae, the other antigen remained evenly distributed. Caveolar sequestration of the antigens occurred in the presence of cytochalasin D, nocodazole, or a mixture of the two reagents. The results show that not only GPI-anchored proteins but also glycosphingolipids and sphingomyelin are sequestered in caveolae only after crosslinking, and that the sequestration does not require the intact cytoskeleton.