Cell membrane components are organized as specialized domains involved in membrane-associated events such as cell signaling, cell adhesion, and protein sorting. These membrane domains are enriched in sphingolipids and cholesterol but display a low protein content. Theoretical considerations and experimental data suggest that some properties of gangliosides play an important role in the formation and stabilization of specific cell lipid membrane domains. Gangliosides are glycolipids with strong amphiphilic character and are particularly abundant in the plasma membranes, where they are inserted into the external leaflet with the hydrophobic ceramide moiety and with the oligosaccharide chain protruding into the extracellular medium. The geometry of the monomer inserted into the membrane, largely determined by the very large surface area occupied by the oligosaccharide chain, the ability of the ceramide amide linkage to form a network of hydrogen bonds at the water-lipid interface of cell membranes, the Delta(4) double bond of sphingosine proximal to the water-lipid interface, the capability of the oligosaccharide chain to interact with water, and the absence of double bonds into the double-tailed hydrophobic moiety are the ganglioside features that will be discussed in this review, to show how gangliosides are responsible for the formation of cell lipid membrane domains characterized by a strong positive curvature.

Sphingolipid-enriched membrane domains, characterized by a particular protein and lipid composition, have been detected in a variety of cells. However, limited data are available concerning these domains in neuronal cells. We analyzed the lipid and protein composition of a sphingolipid-enriched membrane fraction prepared from primary rat cerebellar granule cells differentiated in culture. Although the protein content of this fraction was only 1.4% of total cellular protein, 60% of the gangliosides, 67% of the sphingomyelin, 50% of the ceramide, and 40% of the cholesterol were located in this fraction. The protein pattern of the sphingolipid-enriched domain fraction was dramatically different from that associated with the cell homogenate. This fraction contained 25% of the tyrosine- The increasing body of evidence, obtained by several different experimental approaches from both artificial and cellular models (1-23), suggests that lipid and protein components in the cell membrane are not randomly or homogeneously distributed but rather organized in domains with peculiar physicochemical and functional properties, different from those of the surrounding membrane environment, confirming the original prediction of Singer and Nicholson (24).Sphingolipid-enriched domains that are reported to be enriched in gangliosides, sphingomyelin, and cholesterol (1, 17-23) are emerging as membrane compartments with relevant biological functions. They are rich in proteins involved in the mechanisms of signal transduction (1, 18, 21-23, 25-31) and cell adhesion molecules (34). Thus, sphingolipid-enriched microdomains could represent a site within the plasma membrane where different molecules (both lipids and proteins) involved in signal transduction and/or cell adhesion and cell-cell interactions are specifically sorted and concentrated, allowing reciprocal interactions of functional significance. Recent studies have revealed that gangliosides in membrane sphingolipidenriched domains associate closely and specifically with single or multiple signal transducer molecules. Ganglioside GM3 1 is closely associated with c-Src, Rho, FAK, and Ras in B16 melanoma cells (1, 21), with c-Src and Csk in neuroblastoma Neuro2a cells (22), and GD3 is associated with Src-family kinase Lyn and the neural cell adhesion molecule TAG-1 in rat brain (33,35). Such structural units seem to be involved in signal transduction in response to glycosphingolipid-mediated stimulation; GM3-mediated cell adhesion of melanoma B16 cells induces c-Src and FAK phosphorylation and Rho and Ras activation (1); treatment of neuroblastoma Neuro2a cells with exogenous gangliosides induces c-Src and mitogen-activated protein kinase activation, leading to neuronal differentiation (22); and treatment of primary cultured rat cerebellar neurons with anti-GD3 antibody induces Lyn activation with consequent phosphorylation of mitogen-activated protein kinases (33). Neurotrophin-induced p75 NTR -dependent sphingomyelin hydrolysis is also localized in a caveolar domain (27).Procedures...

In the present paper, we report on the properties of sphingolipid-enriched domains of rat cerebellar granule cells in culture at different stages of neuronal development. The major lipid components of these domains were glycerophospholipids and cholesterol. Glycerophospholipids were 45-75% and cholesterol 15-45% of total lipids of the domains. This corresponded to 5-17% of total cell glycerophospholipids and 15-45% of total cell cholesterol. Phosphatidylcholine, mainly dipalmitoylphosphatidylcholine, was 66 -85% of all the glycerophospholipids associated with these domains. Consequently, the palmitoyl residue was significantly enriched in the domains. The surface occupied by these structures increased during development. 40 -70% of cell sphingolipids segregated in sphingolipid-enriched membrane domains, with the maximum ganglioside density in fully differentiated neurons. A high content of ceramide was found in the domains of aging neurons. Then, the sphingolipid/glycerophospholipid molar ratio was more than doubled during the initial stage of development, whereas the cholesterol/glycerophospholipid molar ratio gradually decreased during in vitro differentiation. Phosphorylated phosphoinositides, which were scant in the domains of undifferentiated cells, dramatically increased during differentiation and aging in culture. Proteins were minor components of the domains (0.1-2.8% of all domain components). Phosphotyrosine-containing proteins were selectively recovered in the sphingolipid-enriched domain. Among these, Src family protein-tyrosine kinases, known to participate to the process of neuronal differentiation, were associated with the sphingolipid-enriched domains in a way specific for the type of kinase and for the developmental stage of the cell. Proteins belonging to other signaling pathways, such as phosphoinositide 3-kinase and its downstream target, Akt, were not associated with the domains.