Evidence that ganglioside enriched domains are distinct from caveolae in MDCK II and human fibroblast cells in culture
Articolo
Data di Pubblicazione:
2000
Citazione:
Evidence that ganglioside enriched domains are distinct from caveolae in MDCK II and human fibroblast cells in culture / V. Chigorno, P. Palestini, M. Sciannamblo, V. Dolo, A. Pavan, G. Tettamanti, S. Sonnino. - In: EUROPEAN JOURNAL OF BIOCHEMISTRY. - ISSN 0014-2956. - 267:13(2000), pp. 4187-4197.
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-H-3]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.
Tipologia IRIS:
01 - Articolo su periodico
Keywords:
Caveolae; Caveolin-1; Gangliosides; GM3; Sphingolipid domains
Elenco autori:
V. Chigorno, P. Palestini, M. Sciannamblo, V. Dolo, A. Pavan, G. Tettamanti, S. Sonnino
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