Evidence that ganglioside enriched domains are distinct from caveolae in MDCK II and human fibroblast cells in culture

Cultures of MDCK II and human fibroblast cells were fed radioactive sphingo sine and a radioactive GM3 ganglioside derivative containing a photoactivab le group. The derived cell homogenates were treated with Triton X-100 and f ractionated by sucrose-gradient centrifugation to prepare a detergent-insol uble membrane fraction known to be enriched in sphingolipid and caveolin-1, i.e. of caveolae. The detergent-insoluble membrane fraction prepared after feeding [1-H-3]sph ingosine to cells, was found to be highly enriched, with respect to protein content, in metabolically radiolabeled sphingomyelin and glycosphingolipid s (about 18-fold). By feeding cells photoactivable radioactive GM3, after 2 h-chase, caveolin-1, CAV1, and proteins of high molecular mass became cros s-linked to GM3, the cross-linking complexes being highly concentrated in t he detergent-insoluble membrane fraction. The interaction between the gangl ioside derivative and CAV1 was a time-dependent, transient process so that CAV1 cross-linking to GM3 was hardly detectable after a 24-h chase followed the pulse time. After a 24-h chase, only the high molecular mass proteins cross-linked to GM3 could be clearly observed. These results suggest that a portion of the GM3 administered to cells enters caveolae and moves to the glycosphingolipid domains, or enters caveolae that are then rapidly catabol ized. Electron microscopy of cells in a culture immunostained with a monoclonal a ntibody to GM3 and a secondary gold-conjugated antibody detected several cl usters of gangliosides on the plasma membranes separate from caveolae; gang liosides located inside the caveolae could not be detected. Scanning confoc al microscopy of cells immunostained with anti-GM3 and anti-CAV1 Ig showed only a very small overlap with the CAV1 and GM3 signals. Thus, the biochemical and microscopic studies suggest that caveolae contain at most a low level of gangliosides and are separate from the GM3 ganglios ide enriched domains.