Treatment with methyl-␤ -cyclodextrin (MCD) induced a time-and dose-dependent efflux of cholesterol, sphingolipids, and phosphatidylcholine (PC) from cerebellar neurons differentiated in culture. With a "mild" treatment, the loss of cell lipids induced a deep reorganization of the remaining membrane lipids. In fact, the amount of PC associated with a Triton X-100-insoluble membrane fraction (highly enriched in sphingolipids and cholesterol in nontreated cells) was lowered by the treatment. This suggested a reduction of the lipid domain area. However, the cholesterol and sphingolipid enrichment of this fraction remained substantially unchanged, suggesting the existence of dynamic processes aimed at preserving the segregation of cholesterol and sphingolipids in membrane domains. Under these conditions, the lipid membrane domains retained the ability to sort signaling proteins, such as Lyn and c-Src, but cells displayed deep alterations in their membrane permeability. However, normal membrane permeability was restored by loading cells with cholesterol. When MCD treatment was more stringent, a large loss of cell lipids occurred, and the lipid domains were much less enriched in cholesterol and lost the ability to sort specific proteins.The loss of the integrity and properties of lipid domains was accompanied by severe changes in the membrane permeability, distress, and eventually cell death.-Ottico, E., A. Prinetti, S. Prioni, C. Giannotta, L. Basso, V. Chigorno, and S. Sonnino.

We studied the interactions between gangliosides and proteins at the exoplasmic surface of the sphingolipid-enriched membrane domains by ganglioside photolabeling combined with cell surface biotin labeling. After cell photolabeling with radioactive photoactivable derivatives of GM3, GM1 and GD1b gangliosides, followed by cell surface biotin labeling, sphingolipid-enriched domains were prepared and immunoprecipitated with streptavidin-coupled beads, under experimental conditions preserving the integrity of the lipid domain. About 50% of the total radioactivity linked to proteins was associated with acylated tubulin, about 10% with a 135-kDa protein present as a series of species with pI ranging from 6.5 to 8.0, about 5% with a protein of about 70 kDa and with pI near to 6.5. By immunoprecipitation with streptavidin-coupled beads under conditions disrupting the integrity of the lipid domain, the 135 kDa protein was recovered in the immunoprecipitate, that did not contain tubulin. Thus, the 135 kDa protein has an exoplasmic domain, and it was then identified as the GPI-anchored neural cell adhesion molecule TAG-1. Remarkably, TAG-1 was cross-linked in a similar extent by the photoactivated ganglioside GM3, GM1 and GD1b. The three gangliosides bear different oligosaccharide chains, suggesting that the ganglioside/TAG)1 interaction is not specifically associated with the ganglioside oligosaccharide structure. Keywords: gangliosides, lipid domains, neuronal cells, photolabeling, TAG-1. Abbreviations used: Cer, ceramide, N-acyl-sphingosine; GM3, II[11-3 H-(Neu5Ac)]GM1-N 3 (position 11 of sialic acid is the acetyl carbon), b-Gal-2-nitro-4-azidophenyl)amino-dodecanoyl]amino-3-hydroxyoctadec-4-ene}; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; SEMF, sphingolipid-enriched membrane fraction; SNIP, supernatant obtained after separation of immunoprecipitate.

Human fibroblasts, rat neurons, and murine neuroblastoma cells, cultured in the presence of fetal calf serum, were fed with [1-3 H]sphingosine to radiolabel sphingolipids. The fate of cell sphingolipids, the release of sphingolipids in the culture medium, the interaction of sphingolipids with the proteins and lipoproteins of fetal calf serum, and the fate of sphingolipids taken up by the cells were investigated. For this latter purpose, the culture medium containing radioactive sphingolipids was delivered to nonlabeled cells. The presence of tritium at position 1 of sphingosine allowed us to follow the extent of sphingolipid catabolism by measuring the production of radioactive phosphatidylethanolamine and proteins by recycling the radioactive ethanolamine formed during sphingosine catabolism and the production of tritiated water. We confirmed that in cells the recycling of sphingosine occurred to a high extent and that only a minor portion of cell sphingolipids was catabolized to the small fragments of ethanolamine and water. Cell sphingolipids were released in the culture medium, where they formed large lipoproteic aggregates at a rate of about 12% per day. Released sphingolipids were taken up by the cells and catabolized to the sphingosine and then to ethanolamine, and recycling of sphingosine was not observed. This suggests that in the presence of fetal calf serum in the culture medium, exogenous sphingolipids directly reach the lysosomes, were they are entirely catabolized. Thus, the trafficking of sphingolipids from cells to the extracellular environment and from this to other cells does not allow the modification of the plasma membrane composition.Membrane sphingolipids are complex lipids displaying a strong amphiphilic character (1). This implies an equilibrium between plasma membrane molecules and free monomers present in the extracellular environment. The critical aggregative concentration of sphingolipids is in the nano-or picomolar range (1, 2), thus the amount of free monomers in solution is low. Nevertheless, the quantity of free monomers should increase when a favorable equilibrium leads to the formation of stable lipoprotein complexes (3-5). This process should also occur in living organisms, where significant amounts of sphingolipids are detectable in serum and cerebrospinal fluid from normal and pathological subjects (6 -8), as components of serum lipoproteins or associated with albumin (4).The presence of sphingolipids in biological fluids leads to the following considerations. Exogenous sphingolipids administered to cells are taken up by the cells, thus entering the sphingolipid metabolic pathway. Several studies have been carried out by administering sphingolipids to cells (9). Gangliosides administered to cells that are cultured in the absence of fetal calf serum (FCS) 1 or proteins have been shown to become components of the cell membranes, modifying in some cases the quantity and the quality of plasma membrane gangliosides, and to enter into the endogenous sphingolipid metabolic pathways ...