Membrane domains and the "lipid raft" concept.

The bulk structure of biological membranes consists of a bilayer of amphipathic lipids. According to the fluid mosaic model proposed by Singer and Nicholson, the glycerophospholipid bilayer is a two-dimensional fluid construct that allows the lateral movement of membrane components. Different types of lateral interactions among membrane components can take place, giving rise to multiple levels of lateral order that lead to highly organized structures. Early observations suggested that some of the lipid components of biological membranes may play active roles in the creation of these levels of order. In the late 1980s, a diverse series of experimental findings collectively gave rise to the lipid raft hypothesis. Lipid rafts were originally defined as membrane domains, i.e., ordered structures created as a consequence of the lateral segregation of sphingolipids and differing from the surrounding membrane in their molecular composition and properties. This definition was subsequently modified to introduce the notion that lipid rafts correspond to membrane areas stabilized by the presence of cholesterol within a liquid-ordered phase. During the past two decades, the concept of lipid rafts has become extremely popular among cell biologists, and these structures have been suggested to be involved in a great variety of cellular functions and biological events. During the same period, however, some groups presented experimental evidence that appeared to contradict the basic tenets that underlie the lipid raft concept. The concept is currently being re-defined, with greater consistency regarding the true nature and role of lipid rafts. In this article we will review the concepts, criticisms, and the novel confirmatory findings relating to the lipid raft hypothesis.

Ganglioside GM1 forces the redistribution of cholesterol in a biomimetic membrane.

Neutron reflectivity has been applied to investigate different mixed asymmetric lipid systems, in the form of single "supported+floating" bilayers, made of phospholipids, cholesterol and GM1 ganglioside (Neu5Acα2-3(Galß1-3GalNAcß1-4)Galß1-4Glcß1Cer)) in bio-similar mole ratios. Bilayer preparation was carried out layer-by-layer with the Langmuir-Blodgett Langmuir-Schaefer techniques, allowing for compositional asymmetry in the system buildup. It is the first time that such a complex model membrane system is reported. Two important conclusions are drawn. First, it is experimentally shown that the presence of GM1 enforces an asymmetry in cholesterol distribution, opposite to what happens for a GM1-free membrane that, submitted to a similar procedure, results in a full symmetrization of cholesterol distribution. We underline that natural cholesterol has been used. Second, and most interesting, our results suggest that a preferential asymmetric distribution of GM1 and cholesterol is attained in a model membrane with biomimetic composition, revealing that a true coupling between the two molecular species occurs.

Sialic acid and GM3 ganglioside expression in papillomavirus-associated urinary bladder tumours of cattle with chronic enzootic haematuria.

This study was based on 30 papillomavirus-associated urinary bladder tumours from cattle with chronic haematuria, the animals having been kept since birth on pasture rich in bracken fern. The ganglioside content was assessed and compared with that of normal bovine urinary bladders, which was shown to be 28.6+/-3.3 (mean+/-SD) microg of lipid-bound sialic acid per gram of fresh tissue. In neoplastic bladder samples this value was higher but variable (120.9+/-80.6 in benign tumours, and 94.7+/-45.7 in malignant tumours). The main ganglioside, GM3, represented ca 75% of the total ganglioside mixture in normal tissues and 50-80% in tumour samples. GM1, GM2, GD1a, GD3 and FucGM1 were found as minor components. The study suggested that GM3 ganglioside may have a crucial role in "downregulation" of the metastatic potential of bovine urothelial cancers.

Association of rat8 with Fyn protein kinase via lipid rafts is required for rat mammary cell differentiation in vitro.

We previously identified rat8 in the pathway involved in epithelial cell differentiation that occurs in the rat mammary gland at pregnancy when tubules and alveoli are formed. rat8, which encodes an IFN-inducible membrane protein, is the rat homologue of the mouse gene fragilis. By differential detergent extraction and isopycnic sucrose density gradients, we show that rat8 protein is associated to lipid membrane domains together with Lyn and Fyn, members of the Src tyrosine kinase family. We also show that recruitment of rat8 to lipid membrane domains is a necessary step in mammary epithelial cell differentiation. Immunoprecipitation analysis, performed with an anti-Fyn protein antibody, shows that rat8 was present in the Fyn immunoprecipitate. Antisense oligonucleotides, used to inhibit Fyn protein expression, block mammary cell differentiation. Taken together, these results suggest that the functional interaction, via lipid membrane domains, of rat8 and Fyn proteins is required for mammary cell differentiation. Therefore, rat8, like fragilis, may be involved in developmental decisions and the demarcation of a subset of cells in the mammary gland that cause epithelial cells to develop into a network of tubuloalveolar structures involved in secretion.

Restoration of the GM2 ganglioside metabolism in bone marrow-derived stromal cells from Tay-Sachs disease animal model.

The therapeutic potential of bone marrow-derived stromal cells for the therapy of Tay-Sachs disease is primarily related to the restoration of their own GM2 ganglioside storage. With this aim, we produced bone marrow-derived stromal cells from the adult Tay-Sachs animal model and transduced them with a retroviral vector encoding for the alpha-subunit of the lysosomal enzyme beta-hexosaminidase A (E.C. 3.2.1.52). Our results demonstrate that transduced Tay-Sachs bone marrow-derived stromal cells have beta-hexosaminidase A comparable to that of bone marrow-derived stromal cells from wild-type mice. Moreover, beta-hexosaminidase A in transduced Tay-Sachs bone marrow-derived stromal cells was able to hydrolyze the GM2 ganglioside in a feeding experiment, thus demonstrating the correction of the altered phenotype.

Immunoseparation of sphingolipid-enriched membrane domains enriched in Src family protein tyrosine kinases and in the neuronal adhesion molecule TAG-1 by anti-GD3 ganglioside monoclonal antibody.

Rat cerebellar granule cells differentiated in culture were fed [1-(3)H]sphingosine, allowing the metabolic radiolabelling of all cell sphingolipids and phosphatidylethanolamine. A detergent-insoluble sphingolipid-enriched membrane fraction, containing about 60% of cell sphingolipids, but only trace amounts of phosphatidylethanolamine, was prepared from [1-(3)H]sphingosine-fed cells by sucrose gradient centrifugation. This fraction was enriched in the Src family protein tyrosine kinases c-Src, Lyn and Fyn and in the GPI-anchored neuronal adhesion molecule TAG-1. The cell lysate and the sphingolipid-enriched membrane fraction were subjected to immunoprecipitation with anti-GD3 ganglioside monoclonal antibody R24, under experimental conditions designed to preserve the integrity of the domain. The radioactive lipid composition of the immunoprecipitates obtained from the cell lysate and from the sphingolipid-enriched fraction were very similar, and closely resembled the sphingolipid composition of the whole sphingolipid-enriched membrane fraction. In fact, the immunoprecipitates contained, together with GD3 ganglioside, all cell glycosphingolipids and sphingomyelin, whereas they did not contain phosphatidylethanolamine. Moreover, cholesterol and phosphatidylcholine were detected in the immunoprecipitates by qualitative TLC analysis followed by colourimetric visualization. c-Src, Lyn, Fyn and TAG-1 were associated with the anti-GD3 antibody immunoprecipitate. These proteins were not detected in the immunoprecipitates obtained under experimental conditions different from those designed to preserve the integrity of the domain. These data suggest that a membrane domain containing cholesterol, phosphatidylcholine, sphingolipids and proteins can be separated from the total cell membranes by anti-GD3 antibody immunoprecipitation, and that the association of c-Src, Fyn, Lyn, and TAG-1 with the sphingolipid-enriched domain is mediated by the interaction with a complex lipid environment, rather than by specific interactions with a single sphingolipid species.

Preparation of deacetyl-, lyso-, and deacetyl-lyso-GM(3) by selective alkaline hydrolysis of GM3 ganglioside.

Three methods (using GM3 quantities ranging from a few milligrams to grams) have been developed to prepare, in high yield, the three derivatives of ganglioside GM3 [alpha-Neu5Ac-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-ceramide]: deacetyl-GM3 [alpha-Neu-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-ceramide], lyso-GM3 [alpha-Neu5Ac-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-sphingosine], and deacetyl-lyso-GM3 [alpha-Neu-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-sphingosine]. This is the first report of the preparation of lyso-GM3 by a one-pot reaction. We can now define the optimal conditions for the different preparations. Preparation of deacetyl-GM3: alkaline reagent, 2 M KOH in water; GM3 concentration, 33 mg/ml; reaction temperature, 90 degrees C; reaction time, 3.5 h; nitrogen atmosphere. Preparation of deacetyl-lyso-GM3: alkaline reagent, 8 M KOH in water; GM3 concentration, 10 mg/ml; reaction temperature, 90 degrees C; reaction time, 18 h; nitrogen atmosphere. Preparation of lyso-GM(3): alkaline reagent, 1 M sodium tert-butoxide in methanol; GM3 concentration, 10 mg/ml; reaction temperature, 80 degrees C; reaction time, 18 h; anhydrous conditions. The percentage yield of deacetyl-GM3 was 70;-75%, that of deacetyl-lyso-GM3 100%, and of lyso-GM3 36;-40%.Deacetyl-GM3, deacetyl-lyso-GM3, and lyso-GM3 were purified by column chromatography, and chemical structures were confirmed by electron spray-mass spectrometry.

GM3 ganglioside inhibits CD9-facilitated haptotactic cell motility: coexpression of GM3 and CD9 is essential in the downregulation of tumor cell motility and malignancy.

A cooperative inhibitory effect of GM3, together with CD9, on haptotactic cell motility was demonstrated by a few lines of study as described below. (i) Haptotactic motility of colorectal carcinoma cell lines SW480, SW620, and HRT18, which express CD9 at a high level, is inhibited by exogenous GM3, but not by GM1. (ii) Motility of gastric cancer cell line MKN74, which expresses CD9 at a low level, was not affected by exogenous GM3. Its motility became susceptible to and inhibited by exogenous GM3, but not GM1, when the CD9 level of MKN74 cells was converted to a high level by transfection with CD9 cDNA. Findings i and ii suggest that haptotactic tumor cell motility is cooperatively inhibited by coexpression of CD9 and GM3. (iii) This possibility was further demonstrated using cell line ldlD 14, and its derivative expressing CD9 through transfection of its gene (termed ldlD/CD9). Both of these cell lines are defective in UDP-Gal 4-epimerase and cannot synthesize GM3 unless cultured in the presence of galactose (Gal(+)), whereas GM3 synthesis does not occur when cells are cultured in the absence of Gal (Gal(-)). Haptotactic motility of parental ldlD cells is low, and shows no difference in the presence and absence of Gal. In contrast, the motility of ldlD/CD9 cells is very high in Gal(-) whereby endogenous GM3 synthesis does not occur, and is very reduced in Gal(+) whereby endogenous GM3 synthesis occurs. (iv) Photoactivatable (3)H-labeled GM3 added to HRT18 cells, followed by UV irradiation, causes cross-linking of GM3 to CD9, as evidenced by (3)H labeling of CD9, which is immunoprecipitated with anti-CD9 antibody. These findings suggest that CD9 is a target molecule interacting with GM3, and that CD9 and GM3 cooperatively downregulate tumor cell motility.

Changes in the lipid turnover, composition, and organization, as sphingolipid-enriched membrane domains, in rat cerebellar granule cells developing in vitro.

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.

Exogenous sphingosine enters Xenopus laevis embryos grown in petri dishes and it is metabolized.

Xenopus embryos of different developmental stages were exposed to 0.1 microM [1-3H]sphingosine. Labeled sphingosine was quickly absorbed by Xenopus embryos. The amount of radioactivity absorbed increased with embryo age and appeared to be linearly correlated (R = 0.97) to the embryo surface area. About 45% of the total radioactivity associated to the embryos was found in the skin, 22% in the intestine, 15% in the heart, 12% in the liver and 6% in the brain. A portion of [1-3H]sphingosine entered very rapidly the biosynthetic pathway of sphingolipids; after 30 min of incubation, in fact, only a small amount of free radioactive sphingosine could be detected. Sphingomyelin was the main radioactive sphingolipid synthesized; radioactive ceramide, galactosylceramide and lactosylceramide could also be recognized and quantified. On the contrary, the amount of radioactive gangliosides was hardly detectable. A portion of [1-3H]sphinogosine absorbed by Xenopus embryos (30 to 60% depending on the developmental stage) entered the catabolic pathway producing radioactive phosphoethanolamine that was recycled for the biosynthesis of radioactive phosphatidylethanolamine. This phospholipid was produced mainly in the intestine and in the skin, while sphingomyelin was the main radioactive lipid in the heart, liver and brain.

Ganglioside molecular species containing C18- and C20-sphingosine in mammalian nervous tissues and neuronal cell cultures.

Gangliosides exist as a very complex mixture of species differing in both the hydrophilic and hydrophobic moieties. They are particularly abundant in the central nervous system (CNS), where they have been associated with development and maturation of the brain, neuritogenesis, synaptic transmission, memory formation and synaptic aging. Today, many data suggest that some of the effects exerted by gangliosides are due to interactions with proteins that participate in the transduction of signals through the membrane in membrane microdomains. A specific characteristic of CNS gangliosides is the structure of their long-chain base (LCB). In fact, considering all the mammalian cell sphingolipids, gangliosides, sulphatides, neutral glycosphingolipids, sphingomyelin and ceramides, it would seem that while the LCB with 18 carbons is the main component of all sphingolipids, only CNS gangliosides contain significant amounts of LCB with 20 carbons. C18-Sphingosine is always present in cell gangliosides; the individual ganglioside species containing C18-sphingosine increase during cell differentiation then remain constant during cell aging. Gangliosides containing C20-sphingosine are absent, or present only in traces, in undifferentiated cells but with the onset of cell differentiation they appear, their content slowly but continuously increasing throughout the life span. In this review we discuss the chemistry, physico-chemistry and metabolism of ganglioside species differing in LCB length and introduce the hypothesis that the varying ratio between C18- and C20-gangliosides during CNS development and aging can be instrumental in modulating membrane domain organisation and cell properties.

The alphabeta T cell response to self-glycolipids shows a novel mechanism of CD1b loading and a requirement for complex oligosaccharides.

The structural basis for the T cell recognition of lipoglycans remains to be elucidated. We have described autoreactive T cells responsive to GM1 ganglioside presented by CD1b. We show that glycosphingolipids bind to CD1b on the cell surface at neutral pH and are recognized without internalization or processing. Furthermore, soluble GM-CD1b complexes stimulate specific T cells. Oligosaccharide groups containing five or more sugars are required to build a minimal epitope for TCR recognition. This suggests a mechanism for T cell recognition of glycosphingolipids in which much of the CD1b-bound ligand is exposed. Binding to CD1b is a highly reversible process and other ceramide-containing glycosphingolipids displace GM1. These nonantigenic compounds act as blockers and may prevent harmful autoreactivity in vivo.

Second generation mimics of ganglioside GM1 as artificial receptors for cholera toxin: replacement of the sialic acid moiety.

In a program directed towards the design and synthesis of mimics of ganglioside GM1, the NeuAc recognition domain was replaced by simple hydroxy acids, and the affinity of the new ligands to the cholera toxin was determined by fluorescence spectroscopy. The (R)-lactic acid derivative 4 was found to display the highest affinity of the series (KD = 190 microM).

Linalool modifies the nicotinic receptor-ion channel kinetics at the mouse neuromuscular junction.

Linalool is a monoterpene compound reported to be a major component of essential oils in various aromatic species. Several linalool-producing species are used in traditional medical systems. Among these is Aeolanthus suaveolens G. Dom (Labiatae) which is used as an anticonvulsant in the Brazilian Amazon. Psychopharmacological in vivo evaluation of linalool showed that this compound has dose-dependent marked sedative effects at the central nervous system (CNS), including hypnotic, anticonvulsant and hypothermic properties. It has been suggested that these neurochemical effects might be ascribed to the local anaesthetic activity of linalool. The present study reports an inhibitory effect of linalool on the acetylcholine (ACh) release and on the channel open time in the mouse neuromuscular junction. These findings could provide a rational basis to confirm the traditional medical use of linalool-producing plant species. Indeed, our data demonstrate some interactions in the modulation of the ACh release at the mouse neuromuscular junction, which are well correlated with the suggested molecular mechanisms. Linalool induced a reduction of the ACh-evoked release. The possibility that this effect could be ascribed to some interaction with pre-synaptic function is noteworthy. Moreover, the inhibitory effect induced on the kinetics of the miniature end-plate current decay demonstrates a local anaesthetic action, either on the voltage or on the receptor-activated channels.

Evidence that ganglioside enriched domains are distinct from caveolae in MDCK II and human fibroblast cells in culture.

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.

Sphingolipid-enriched membrane domains from rat cerebellar granule cells differentiated in culture. A compositional study.

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-phosphorylated proteins and was enriched in two proteins with apparent molecular masses of 135 and 15 kDa. 12% of cellular glycerophospholipids were located in the fraction, with phosphatidylcholine having the highest enrichment. The molar ratio between proteins, glycerophospholipids, cholesterol, sphingomyelin, ceramide and gangliosides in cerebellar granule cells was 1.6:41.6:6. 1:1.3:0.3:1 in the cell homogenate and 0.04:8.3:4.0:1.4:0.2:1 in the sphingolipid-enriched membrane fraction. These data indicate that selected proteins segregate with sphingolipids in specialized domains in the membrane of cultured neurons.

Interaction of human substantia nigra neuromelanin with lipids and peptides.

Neuromelanin was isolated from human substantia nigra using different procedures. In the pigment isolated by any of these procedures a peptide component covalently bound to the melanic structure was found, as shown by treatment with reagents known to eliminate noncovalently bound proteins. The amino acid content of such a peptide component was reproducible and corresponded to approximately 15% of the neuromelanin weight. Neuromelanin also showed the ability to absorb specifically lipid molecules, approximately 20% of its weight, and among these lipids cholesterol was identified, constituting approximately 5% of the total lipid mixture. A synthetic melanin, incubated with putamen homogenate, bound tissue peptides with an amino acid content quite close to that of neuromelanin. The same synthetic melanin adsorbed a lower amount of lipids from the putamen homogenate compared with neuromelanin. The sulfur content of neuromelanin was also reproducible even using different isolation procedures. A nonpigmented tissue like corpus callosum was used as a control and extracted by the method used for neuromelanin isolation; a total elimination of tissue components was found, thus demonstrating the capability of the reported procedures to isolate neuromelanin alone. The presence of a peptide component in the neuromelanin structure and the selective affinity for lipid molecules suggest new aspects of the functional role and metabolic pathway of neuromelanin.

Association of Src-family protein tyrosine kinases with sphingolipids in rat cerebellar granule cells differentiated in culture.

Src family kinases play a relevant role in the development and differentiation of neuronal cells. They are abundant in sphingolipid-enriched membrane domains of many cell types, and these domains are hypothesized to function in bringing together molecules important to signal transduction. We studied the association of Src family tyrosine kinases and their negative regulatory kinase, Csk, with sphingolipids in sphingolipid-enriched domains of rat cerebellar granule cells differentiated in culture. We find that c-Src, Lyn and Csk are enriched in the sphingolipid-enriched fraction prepared from these cells. Coimmunoprecipitation experiments show that these and sphingolipids are part of the same domain. Cross-linking experiments with a photoactivable, radioactive GD1b derivative show that c-Src and Lyn, which are anchored to the membrane via a myristoyl chain, associate directly with GD1b. Csk, which is not inserted in the hydrophobic core of the membrane, is not photolabeled by this ganglioside. These results suggest that lipid-lipid, lipid-protein, and protein-protein interactions cooperate to maintain domain structure. We hypothesize that such interactions might play a role in the process of neuronal differentiation.

New approaches to the study of sphingolipid enriched membrane domains: the use of electron microscopic autoradiography to reveal metabolically tritium labeled sphingolipids in cell cultures.

This paper is the first report on the use of the electron microscopy autoradiography technique to detect metabolically tritium labeled sphingolipids in intact cells in culture. To label cell sphingolipids, human fibroblasts in culture were fed by a 24 hours pulse, repeated 5 times, of 3 x 10(-7) M [1-(3)H]sphingosine. [1-(3)H]sphingosine was efficently taken up by the cells and very rapidly used for the biosynthesis of complex sphingolipids, including neutral glycolipids, gangliosides, ceramide and sphingomyelin. The treatment with [1-(3)H]sphingosine did not induce any morphological alteration of cell structures, and well preserved cells, plasma membranes, and intracellular organelles could be observed by microscopy. Ultrathin sections from metabolic radiolabeled cells were coated with autoradiographic emulsion. One to four weeks of exposition resulted in pictures where the location of radioactive sphingolipids was evidenced by the characteristic appearance of silver grains as irregular coiled ribbons of metallic silver. Radioactive sphingolipids were found at the level of the plasma membranes, on the endoplasmic reticulum and inside of cytoplasmic vesicles. Thus, electron microscopy autoradiography is a very useful technique to study sphingolipid-enriched membrane domain organization and biosynthesis.