Glycosphingolipids Are Not Essential for Formation of Detergent-resistant Membrane Rafts in Melanoma Cells

Recent data suggest that membrane microdomains or rafts that are rich in sphingolipids and cholesterol are important in signal transduction and membrane trafficking. Two models of raft structure have been proposed. One proposes a unique role for glycosphingolipids (GSL), suggesting that GSL-head-group interactions are essential in raft formation. The other model suggests that close packing of the long saturated acyl chains found on both GSL and sphingomyelin plays a key role and helps these lipids form liquid-ordered phase domains in the presence of cholesterol. To distinguish between these models, we compared rafts in the MEB-4 melanoma cell line and its GSL-deficient derivative, GM-95. Rafts were isolated from cell lysates as detergent-resistant membranes (DRMs). The two cell lines had very similar DRM protein profiles. The yield of DRM protein was 2-fold higher in the parental than the mutant line, possibly reflecting cytoskeletal differences. The same amount of DRM lipid was isolated from both lines, and the lipid composition was similar except for up-regulation of sphingomyelin in the mutant that compensated for the lack of GSL. DRMs from the two lines had similar fluidity as measured by fluorescence polarization of diphenylhexatriene. Methyl-β-cyclodextrin removed cholesterol from both cell lines with the same kinetics and to the same extent, and both a raft-associated glycosyl phosphatidylinositol-anchored protein and residual cholesterol showed the same distribution between DRMs and the detergent-soluble fraction after cholesterol removal in both cell lines. Finally, a glycosyl phosphatidylinositol-anchored protein was delivered to the cell surface at similar rates in the two lines, even after cholesterol depletion with methyl-β-cyclodextrin. We conclude that GSL are not essential for the formation of rafts and do not play a major role in determining their properties. Recent data suggest that membrane microdomains or rafts that are rich in sphingolipids and cholesterol are important in signal transduction and membrane trafficking. Two models of raft structure have been proposed. One proposes a unique role for glycosphingolipids (GSL), suggesting that GSL-head-group interactions are essential in raft formation. The other model suggests that close packing of the long saturated acyl chains found on both GSL and sphingomyelin plays a key role and helps these lipids form liquid-ordered phase domains in the presence of cholesterol. To distinguish between these models, we compared rafts in the MEB-4 melanoma cell line and its GSL-deficient derivative, GM-95. Rafts were isolated from cell lysates as detergent-resistant membranes (DRMs). The two cell lines had very similar DRM protein profiles. The yield of DRM protein was 2-fold higher in the parental than the mutant line, possibly reflecting cytoskeletal differences. The same amount of DRM lipid was isolated from both lines, and the lipid composition was similar except for up-regulation of sphingomyelin in the mutant that compensated for the lack of GSL. DRMs from the two lines had similar fluidity as measured by fluorescence polarization of diphenylhexatriene. Methyl-β-cyclodextrin removed cholesterol from both cell lines with the same kinetics and to the same extent, and both a raft-associated glycosyl phosphatidylinositol-anchored protein and residual cholesterol showed the same distribution between DRMs and the detergent-soluble fraction after cholesterol removal in both cell lines. Finally, a glycosyl phosphatidylinositol-anchored protein was delivered to the cell surface at similar rates in the two lines, even after cholesterol depletion with methyl-β-cyclodextrin. We conclude that GSL are not essential for the formation of rafts and do not play a major role in determining their properties. glycosphingolipid(s) detergent-resistant membrane glycosyl phosphatidylinositol liquid-ordered placental alkaline phosphatase Dulbecco's modified Eagle's medium methyl-β-cyclodextrin dipalmitoyl phosphatidylcholine dioleoyl phosphatidylcholine diphenylhexatriene phosphate-buffered saline Tris-NaCl-EDTA polyacrylamide gel electrophoresis melting temperature Recent studies suggest that plasma membrane lipids do not always mix homogeneously but that membranes may contain microdomains or rafts that are rich in sphingolipids and cholesterol (1Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8117) Google Scholar, 2Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2551) Google Scholar, 3Brown D.A. London E. J. Membr. Biol. 1998; 164: 103-114Crossref PubMed Scopus (837) Google Scholar). 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Full Text Full Text PDF PubMed Scopus Google suggesting that of cholesterol with rafts We that GSL have a on the of raft the or kinetics of cholesterol we compared cholesterol removal by from MEB-4 and cells removal from the two cell lines with very similar kinetics and to the same To in raft structure with we the of the on DRM formation in the two cell We on the DRM of MEB-4 or cells were with for and at a of of from cell lysates was after were to and of by of from DRMs the was or of was in the in both cell were after of but the not with cell We conclude that the for DRM of in MEB-4 and cells We to the of cholesterol depletion on the of raft To do we the DRM of the cholesterol that in cells after were with and with for at a of were to and and in the was The kinetics and of removal by with removal of cholesterol as by that was a for cell cholesterol. The fraction of in DRMs with in in The at the same in both cell that raft lipid to cholesterol depletion with and of on DRM of in MEB-4 and in in or cells with were with for the and at a of were to The protein of was and were for protein to for cell were and the in of fraction was measured in a The of in for in the The of in DRMs in in and of the are for for in a MEB-4 or cells with were with for the and at a of were to The protein of was and were for protein to for cell were and the in of fraction was measured in a The of in for in the The of in DRMs in in and of the are for for We of from the to the plasma membrane in MEB-4 and cells a surface in The were a for the amount of at The for the amount of cell surface at in both cell was at and the at other are as of in of with similar kinetics in the two cell of for depletion was to of with rafts and in epithelial from the to the cell surface in a and epithelial cell line P. Simons K. J. 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