Evidence that ganglioside enriched domains are distinct from caveolae in MDCK II and human fibroblast cells in culture
Open Access
- 1 July 2000
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 267 (13), 4187-4197
- https://doi.org/10.1046/j.1432-1327.2000.01454.x
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.Keywords
This publication has 45 references indexed in Scilit:
- Lipid Domains in the Membrane: Thermotropic Properties of Sphingomyelin Vesicles Containing GM1 Ganglioside and CholesterolBiochemistry, 1997
- Metabolic Processing of Gangliosides by Normal and Salla Human Fibroblasts in CultureJournal of Biological Chemistry, 1996
- Preparation of rediolabeled gangliosidesGlycobiology, 1996
- A photo‐reactive derivative of ganglioside GM1 specifically cross‐links VIP21‐caveolin on the cell surfaceFEBS Letters, 1995
- A detergent-free method for purifying caveolae membrane from tissue culture cells.Proceedings of the National Academy of Sciences, 1995
- Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surfaceCell, 1992
- A facile and regiospecific tritiation of sphingosine: Synthesis of (2S,3R,4E)‐2‐amino‐4‐octadecene‐1,3‐diol‐1‐3HJournal of Labelled Compounds and Radiopharmaceuticals, 1991
- Association of gangliosides to fibroblasts in culture: A study performed with GM1 [14C]-labelled at the sialic acid acetyl groupGlycoconjugate Journal, 1985
- Incorporation of ganglioside analogs into fibroblast cell membranes. A spin-label studyBiochemistry, 1983
- A new procedure for the extraction, purification and fractionation of brain gangliosidesBiochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1973