title: Evidence that ganglioside enriched domains are distinct from caveolae in MDCK II and human fibroblast cells in culture.
abstract: Cultures of MDCK II and human fibroblast cells were fed radioactive sphingosine and a radioactive GM3 ganglioside derivative containing a photoactivable group. The derived cell homogenates were treated with Triton X-100 and fractionated by sucrose-gradient centrifugation to prepare a detergent-insoluble membrane fraction known to be enriched in sphingolipid and caveolin-1, i.e. of caveolae. The detergent-insoluble membrane fraction prepared after feeding [1-3H]sphingosine to cells, was found to be highly enriched, with respect to protein content, in metabolically radiolabeled sphingomyelin and glycosphingolipids (about 18-fold). By feeding cells photoactivable radioactive GM3, after 2 h-chase, caveolin-1, CAV1, and proteins of high molecular mass became cross-linked to GM3, the cross-linking complexes being highly concentrated in the detergent-insoluble membrane fraction. The interaction between the ganglioside 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 catabolized. Electron microscopy of cells in a culture immunostained with a monoclonal antibody to GM3 and a secondary gold-conjugated antibody detected several clusters of gangliosides on the plasma membranes separate from caveolae; gangliosides located inside the caveolae could not be detected. Scanning confocal 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 ganglioside enriched domains.
authors.affiliation: Study Center for the Functional Biochemistry of Brain Lipids, Department of Medical Chemistry and Biochemistry - LITA-Segrate, University of Milan, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Chigorno
authors.forename: P
authors.initials: P
authors.lastname: Palestini
authors.forename: M
authors.initials: M
authors.lastname: Sciannamblo
authors.forename: V
authors.initials: V
authors.lastname: Dolo
authors.forename: A
authors.initials: A
authors.lastname: Pavan
authors.forename: G
authors.initials: G
authors.lastname: Tettamanti
authors.forename: S
authors.initials: S
authors.lastname: Sonnino
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000737:chemistry*; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000737:chemistry; D005732:Gangliosides / Q000032:analysis*; D006801:Humans; D008970:Molecular Weight; D013110:Sphingosine / Q000378:metabolism
chemical_list: D005732:Gangliosides; D013110:Sphingosine