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Journal of Lipid Research, Vol. 44, 2142-2151, November 2003
Copyright © 2003 by American Society for Biochemistry and Molecular Biology

Dynamics of membrane lipid domains in neuronal cells differentiated in culture¹

Elena Ottico, Alessandro Prinetti², Simona Prioni, Claudia Giannotta, Luisa Basso, Vanna Chigorno and Sandro Sonnino

Department of Medical Chemistry, Biochemistry, and Biotechnology, Center of Excellence on Neurodegenerative Diseases, University of Milan, Segrate, Italy

² To whom correspondence should be addressed. e-mail: alessandro.prinetti{at}unimi.it

Treatment with methyl-ß-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.

Abbreviations: BME, basal modified Eagle's medium; HPTLC, high-performance thin-layer chromatography; [1-³H]sphingosine, (2S,3R,4E)-2-amino-1,3-dihydroxy-[1-³H]octadecene; MCD, methyl-ß-cyclodextrin; MTT, 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide; PC, phosphatidylcholine; SM, sphingomyelin

Supplementary key words cholesterol • sphingomyelin • ganglioside • sphingolipid-enriched membrane domain • cyclodextrin

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