Characterization and comparison of raft-like membranes isolated by two different methods from rat submandibular gland cells

Lipid rafts are defined as cholesterol and sphingolipid enriched domains in biological membranes. Their role in signalling and other cellular processes is widely accepted but the methodology used for their biochemical isolation and characterization remains controversial. Raft-like membranes from rat submandibular glands were isolated by two different protocols commonly described in the literature; one protocol was based on selective solubilization by Triton X-100 at low temperature and the other protocol consisted in extensive sonication. In both cases a low density vesicular fraction was obtained after ultracentrifugation in a sucrose density gradient. These fractions contained about 20% of total cholesterol but less than 8% of total proteins, and were more rigid than bulk membranes. Fatty acid analyses revealed a similar composition of raft-like membranes isolated by the two different methods, which was characterized by an enrichment in saturated fatty acids in detriment of polyunsaturated acids when compared with the whole cell membranes. Protein profile of detergent resistant membranes or raft-like membranes prepared by sonication was assessed by silver staining after SDS-PAGE and by MALDI-TOF. Both analyses provided evidence of a different protein composition of the Triton X-100 and sonication preparations. Immunoblot experiments revealed that raft-like membranes prepared by detergent extraction or sonication were free of Golgi apparatus or endoplasmic reticulum protein markers (β-COP and calnexin, respectively) and that they were not substantially contaminated by transferrin receptor (a non-raft protein). While caveolin-1 was highly enriched in raft-like membranes prepared by the two methods, the P2X₇ receptor was enriched in raft-like membrane fractions prepared by sonication, but almost undetectable in the detergent resistant membranes. It can be concluded that both methods can be used to obtain raft-like membranes, but that detergent may affect protein interactions responsible for their association with different membrane domains.     

Resistance of Human Erythrocyte Membranes to Triton X-100 and C<Subscript>12</Subscript>E<Subscript>8</Subscript>

Lipid rafts are microdomains enriched in cholesterol and sphingolipids that contain specific membrane proteins. The resistance of domains to extraction by nonionic detergents at 4°C is the commonly used method to characterize these structures that are operationally defined as detergent-resistant membranes (DRMs). Because the selectivity of different detergents in defining membrane rafts has been questioned, we have compared DRMs from human erythrocytes prepared with two detergents: Triton X-100 and C₁₂E₈. The DRMs obtained presented a cholesterol/protein mass ratio three times higher than in the whole membrane. Flotillin-2 was revealed in trace amounts in DRMs obtained with C₁₂E₈, but it was almost completely confined within the DRM fraction with Triton X-100. Differently, stomatin was found distributed in DRM and non-DRM fractions for both detergents. We have also measured the order parameter (S) of nitroxide spin labels inserted into DRMs by means of electron paramagnetic resonance. The 5- and 16-stearic acid spin label revealed significantly higher S values for DRMs obtained with either Triton X-100 or C₁₂E₈ in comparison to intact cells, while the difference in the S values between Triton X-100 and C₁₂E₈ DRMs was not statistically significant. Our results suggest that although the acyl chain packing is similar in DRMs prepared with either Triton X-100 or C₁₂E₈ detergent, protein content is dissimilar, with flotillin-2 being selectively enriched in Triton X-100 DRMs.     

Association of a photoreceptor-specific tetraspanin protein,ROM-1, with triton X-100-resistant membrane rafts from rod outer segment disk membranes

This study reports the isolation and characterization of a Triton X-100-resistant membrane fraction from homogenates of rod outer segment (ROS) disk membranes purified free of the surrounding plasma membrane. A portion of the ROS disk membrane was found to be resistant to Triton X-100 extraction at 4 degrees C. This detergent-resistant fraction was isolated as a low buoyant density band on sucrose density gradients and exhibited an increase in light scattering detected at 600 nm. Biochemical analysis of the Triton X-100-resistant fraction showed it to be enriched in cholesterol and sphingomyelin relative to phospholipid and in phospholipid relative to protein compared with the soluble fraction. The Triton X-100-resistant membranes described herein did not arise simply from partial solubilization of the ROS disk membranes because detergent-treated low buoyant density fractions isolated from homogenates with octyl glucopyranoside had cholesterol and sphingomyelin content indistinguishable from that of solubilized ROS disk homogenates. Analysis of proteins associated with the Triton X-100-resistant fraction showed it to be enriched in the rim-specific protein ROM-1 and caveolin; surprisingly, the fusion protein peripherin/rds (where rds is retinal degeneration slow), also localized to the disk rim, was entirely absent from the membrane raft domain. The lipid profiles of the Triton X-100-resistant membranes were virtually identical in preparations homogenized in either the light or dark. Slightly more ROM-1 was recovered from samples prepared in the light (23%) than from samples prepared in the dark (13%), but peripherin/rds could not be detected in either preparation. When the Triton X-100-resistant membranes were treated with methyl-beta-cyclodextran to deplete membrane cholesterol, the resultant membranes contained slightly lower levels of ROM-1, specifically in the dimeric form. Cholesterol depletion also resulted in the collapse of the large caveolin complex to monomeric caveolae. The results presented herein characterize a pool of ROM-1, a photoreceptor tetraspanin protein, that may play a regulatory role in peripherin/rds-dependent fusion.     

The γ-aminobutyric acid receptor B, but not the metabotropic glutamate receptor type-1, associates with lipid rafts in the rat cerebellum

Recent evidence suggests that specialized microdomains, called lipid rafts, exist within plasma membranes. These domains are enriched in cholesterol and sphingolipids and are resistant to non-ionic detergent-extraction at 4 degrees C. They contain specific populations of membrane proteins, and can change their size and composition in response to cellular signals, resulting in activation of signalling cascades. Here, we demonstrate that both the metabotropic gamma-aminobutyric acid receptor B (GABA(B) receptor) and the metabotropic glutamate receptor-1 from rat cerebellum are insoluble in the non-ionic detergent Triton X-100. However, only the GABA(B) receptor associates with raft fractions isolated from rat brain by sucrose gradient centrifugation. Moreover, increasing the stringency of isolation by decreasing the protein : detergent ratio caused an enrichment of the GABA(B) receptor in raft fractions. In contrast, depletion of cholesterol from cerebellar membranes by either saponin or methyl-beta-cyclodextrin treatment, which solubilize known raft markers, also increased the solubility of the GABA(B) receptor. These properties are all consistent with an association of the GABA(B) receptor with lipid raft microdomains.     

The secretory pathway Ca-ATPase 1 is associated with cholesterol-rich microdomains of human colon adenocarcinoma cells

Lipid rafts are often considered as microdomains enriched in sphingomyelin and cholesterol, predominantly residing in the plasma membrane but which originate in earlier compartments of the cellular secretory pathway. Within this pathway, the membranes of the Golgi complex represent a transition stage between the cholesterol-poor membranes of the endoplasmic reticulum (ER) and the cholesterol-rich plasma membrane. The rafts are related to detergent-resistant membranes, which because of their ordered structure are poorly penetrated by cold non-ionic detergents and float in density gradient centrifugation. In this study the microdomain niche of the Golgi-resident SPCA Ca²⁺/Mn²⁺ pumps was investigated in HT29 cells by Triton X-100 detergent extraction and density-gradient centrifugation. Similarly to cholesterol and the raft-resident flotillin-2, SPCA1 was found mainly in detergent-resistant fractions, while SERCA3 was detergent-soluble. Furthermore, cholesterol depletion of cells resulted in redistribution of flotillin-2 and SPCA1 to the detergent-soluble fractions of the density gradient. Additionally, the time course of solubilization by Triton X-100 was investigated in live COS-1 and HT29 cells expressing fluorescent SERCA2b, SPCA1d or SPCA2. In both cell types, the ER-resident SERCA2b protein was gradually solubilized, while SPCA1d resisted to detergent solubilization. SPCA2 was more sensitive to detergent extraction than SPCA1d. To investigate the functional impact of cholesterol on SPCA1, ATPase activity was monitored. Depletion of cholesterol inhibited the activity of SPCA1d, while SERCA2b function was not altered. From these results we conclude that SPCA1 is associated with cholesterol-rich domains of HT29 cells and that the cholesterol-rich environment is essential for the functioning of the pump.     

Smooth muscle raft-like membranes

We developed a method for extracting raft-like, liquid-ordered membranes from the particulate fraction prepared from porcine trachealis smooth muscle. This fraction, which contains most of the plasma membrane in this tissue, was homogenized in the presence of cold 0.5% Triton X-100. After centrifugation, membranes containing high contents of sphingomyelin (SM) and cholesterol and low phosphatidylcholine (PC) contents remained in the pellet. Thirty-five millimolar octyl glucoside (OG) extracted 75% of these membranes from the Triton X-100-resistant pellet. These membranes had low buoyant densities and accounted for 28% of the particulate fraction lipid. Their lipid composition, 22% SM, 60% cholesterol, 11% phosphatidylethanolamine, 8% PC, <1% phosphatidylinositol, and coisolation with 5'-nucleotidase and caveolin-1 suggest that they are liquid-ordered membranes. We compared characteristics of OG and Triton X-100 extractions of the particulate fraction. In contrast to Triton X-100 extractions, membranes released from the particulate fraction by OG were mainly collected in low buoyant fractions at densities ranging from 1.05 to 1.11 g/ml and had phospholipid and cholesterol contents consistent with a mixture of liquid-ordered and liquid-disordered membranes. Thus, OG extraction of apparent liquid-ordered membranes from Triton X-100-resistant pellets was not due to selective extraction of these membranes. Low buoyant density appears not to be unique for liquid-ordered membranes.     

Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 degrees C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.     

Detergent-free domain isolated from Xenopus egg plasma membrane with properties similar to those of detergent-resistant membranes

Microdomains known as "rafts" have been isolated from many cell types as detergent-resistant membranes (DRMs) and are enriched in sphingolipids and cholesterol. However, there has been considerable controversy over whether such domains are found in native membranes or are artificially generated by the purification procedure. This controversy is based at least in part on the fact that raft membranes were first detected following detergent extraction in the cold. We isolated two plasma membrane fractions, without detergent treatment, using a discontinuous sucrose density gradient. One fraction was designated "light" and the other "heavy." These fractions were compared with DRMs, which were isolated in the presence of 1% Triton X-100. We found that Xenopus DRMs are enriched with sphingomyelin and cholesterol and exhibit a phase state similar to the liquid-ordered phase. Comparison of DRM complexes with the light and heavy plasma membrane fractions revealed some physical and biochemical similarities between the light fraction of the plasma membrane and the DRM complexes, based on (1) the phosphatidylcholine/sphingomyelin ratio and (2) the protein composition visualized on a two-dimensional gel. These two fractions are also quite similar in their thermotropic phase behavior, and their high levels of ganglioside GM1. We conclude that the light membrane fraction isolated in a detergent-free environment has many of the characteristics normally associated with DRMs.     

Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 °C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.     

Distribution of macular xanthophylls between domains in a model of photoreceptor outer segment membranes

A model of photoreceptor outer segment (POS) membranes has been proposed, consisting of an equimolar ternary mixture of 1-palmitoyl-2-docosahexaenoylphosphatidylcholine/distearoylphosphatidylcholine/cholesterol. It was shown that, as in membranes made from the raft-forming mixture, in the model of POS membranes, two domains are formed: the raft domain (detergent resistant membranes, DRM), and the bulk domain (detergent soluble membranes, DSM). Saturation-recovery EPR discrimination by oxygen transport method also demonstrated the presence of two domains in this model system in situ at a wide range of temperatures (10-55 degrees C), showing additionally that neither lutein nor zeaxanthin at 1 mol% affect the formation of these domains. These membrane domains have been separated using cold Triton X-100 extraction from a model of POS membranes containing 1 mol% of either lutein or zeaxanthin. The results indicated that the macular xanthophylls lutein and zeaxanthin are substantially excluded from DRM and remain concentrated in DSM, a domain enriched in highly unsaturated docosahexaenoyl acid which is abundant in retina membranes. The concentration of xanthophylls in DRM and DSM calculated as the mol ratio of either xanthophyll to total lipid (phospholipid+cholesterol) was 0.0028 and 0.0391, respectively. Thus, xanthophylls are about 14 times more concentrated in DSM than in DRM. No significant difference in the distribution of lutein and zeaxanthin was found. The obtained results suggest that in POS membranes macular xanthophylls should also be concentrated in domains enriched in polyunsaturated chains.     

Differential localization of glucose transporter isoforms in non-polarized mammalian cells: distribution of GLUT1 but not GLUT3 to detergent-resistant membrane domains

The hexose transporter family, which mediates a facilitated uptake in mammalian cells, consists of more than 10 members containing 12 membrane-spanning segments with a single N-glycosylation site. However, it remains unknown how these isoforms are functionally organized in the membrane domains. In this report, we describe a differential distribution of the glucose transporter isoforms GLUT1 and GLUT3 to detergent-resistant membrane domains (DRMs) in non-polarized mammalian cells. Whereas more than 80% of cellular proteins containing GLUT3 in HeLa cell lines was solubilized by a non-ionic detergent (either Triton X-100 or Lubrol WX) at 4 degrees C, GLUT1 remained insoluble together with the DRM-associated proteins, such as caveolin-1 and intestinal alkaline phosphatase (IAP). These DRM-associated proteins and the ganglioside GM1 were shown to float to the upper fractions when Triton X-100-solubilized cell extracts were centrifuged on a density gradient. In contrast, GLUT3 as well as most soluble proteins remained in the lower layers. Furthermore, perturbations of DRMs due to depletion of cholesterol by methyl-beta-cyclodextrin (m beta CD) rendered GLUT1 soluble in Triton X-100. Immunostaining patterns for these isoforms detected by confocal laser scanning microscopy in a living cell were also distinctive. These results suggest that in non-polarized mammalian cells, GLUT1 can be organized into a raft-like DRM domain but GLUT3 may distribute to fluid membrane domains. This differential distribution may occur irrespective of the N-glycosylation state or cell type.     

T cell receptor can be recruited to a subset of plasma membrane rafts,independently of cell signaling and attendantly to raft clustering

The constitutive/inducible association of the T cell receptor (TCR) with isolated detergent-resistant, lipid raft-derived membranes has been studied in Jurkat T lymphocytes. Membranes resistant to 1% Triton X-100 contained virtually no CD3epsilon, part of the TCR complex, irrespective of cell stimulation. On the other hand, membranes resistant either to a lower Triton X-100 concentration (i.e. 0.2%) or to the less hydrophobic detergent Brij 58 (1%) contained (i) a low CD3epsilon amount (approximate 2.7% of total) in resting cells and (ii) a several times higher amount of the TCR component, after T cell stimulation with either antigen-presenting cells or with phytohemagglutinin. It appeared that CD3/TCR was constitutively associated with and recruited to a raft-derived membrane subset because (i) all three membrane preparations contained a similar amount of the raft marker tyrosine kinase Lck but no detectable amounts of the conventional membrane markers, CD45 phosphatase and transferrin receptor; (ii) a larger amount of particulate membranes were resistant to solubilization with 0.2% Triton X-100 and Brij 58 than to solubilization with 1% Triton X-100; and (iii) higher cholesterol levels were present in membranes resistant to either the lower Triton X-100 concentration or to Brij 58, as compared with those resistant to 1% Triton X-100. The recruitment of CD3 to the raft-derived membrane subset appeared (i) to occur independently of cell signaling events, such as protein-tyrosine phosphorylation and Ca(2+) mobilization/influx, and (ii) to be associated with clustering of plasma membrane rafts induced by multiple cross-linking of either TCR or the raft component, ganglioside GM(1). We suggest that during T cell stimulation a lateral reorganization of rafts into polarized larger domains can determine the recruitment of TCR into these domains, which favors a polarization of the signaling cascade.     

Solubilization and fractionation of glycoproteins and glycolipids of KB cell membranes

1. A fraction enriched in plasma membranes of human tumour KB cell line, a permissive cell for adenovirus type 5, was obtained. 2. Electrophoresis of the membranes in polyacrylamide gels with buffers containing sodium dodecyl sulphate showed that the membranes after reduction with 2-mercaptoethanol contained over 20 polypeptide species. Three polypeptides were glycosylated and had apparent mol.wts. of 92000, 72000 and 62000. 3. The glycoproteins and the specific receptors responsible for adenovirus adsorption to the membranes were readily extracted into solutions containing low concentrations of Triton X-100. Glycolipids and proteins were also made soluble. A membranous residue obtained after Triton X-100 extraction was enriched in several proteins that appeared to consist of polypeptides of lower molecular weight than the average of KB membrane polypeptides. 4. Sphingomyelin, cholesterol and triglycerides were similarly concentrated in the insoluble residue remaining after successive extractions of KB membranes with Triton X-100. Further, ceramide trihexoside was significantly less easily extracted from KB membranes than lactosyl ceramide. 5. The differences noted in the ease of extraction of membrane components are discussed. 6. The components of membranes made soluble by detergent extraction and containing the large part of the KB membrane glycoproteins were subjected to chromatography on Sepharose 6B and DEAE-cellulose and to isoelectric focusing in the presence of buffers containing Triton X-100. In general, the degree of separation into fractions enriched in individual glycoproteins was disappointing. Possible reasons for the poor fractionation of membrane components by chromatographic systems conveniently used for purification of proteins and glycoproteins of non-membranous origin are briefly discussed.     

Membrane localization of Junín virus glycoproteins requires cholesterol and cholesterol rich membranes

Arenavirus morphogenesis and budding occurs at cellular plasma membrane; however, the nature of membrane assembly sites remains poorly understood. In this study we examined the effect of different cholesterol-lowering agents on Junín virus (JUNV) multiplication. We found that cholesterol cell depletion reduced JUNV glycoproteins (GPs) membrane expression and virus budding. Analysis of membrane protein insolubility in Triton X-100 suggested that JUNV GPs associate with cholesterol enriched membranes. Rafts dissociation conditions as warm detergent extraction and cholesterol removal by methyl-β-cyclodextrin compound showed to impair GPs cholesterol enriched membrane association. Analysis of GPs transfected cells showed similar results suggesting that membrane raft association is independent of other viral proteins.     

Annexin A6 is recruited into lipid rafts of Niemann–Pick type C disease fibroblasts in a Ca-dependent manner

Niemann–Pick type C (NPC) disease is characterized by excessive accumulation of cholesterol in the late endosome/lysosome compartment. Some members of the annexin family of proteins such as annexin A2 (AnxA2) and annexin A6 (AnxA6) follow the same route as cholesterol during the endocytic pathway and are found, as AnxA6, attached to the membranes of the cholesterol storage compartment in NPC disease fibroblasts. Therefore, the purpose of this work was to test the hypothesis that AnxA6 participates in the NPC-induced changes in the organization of membrane microdomains resistant to solubilization by a nonionic detergent, Triton X-100, i.e., detergent-resistant microdomains (DRMs). Using cellular fractionation, fluorescence microscopy and specific antibodies we observed that in the absence of calcium AnxA6 was found in the DRM-depleted membrane fractions isolated from NPC and control fibroblasts. In the presence of calcium, AnxA6 re-located to the fractions enriched in DRMs only in the NPC cells, suggestive of AnxA6 participation in organization of these microdomains.     

Annexin A6 is recruited into lipid rafts of Niemann-Pick type C disease fibroblasts in a Ca2+-dependent manner

Niemann-Pick type C (NPC) disease is characterized by excessive accumulation of cholesterol in the late endosome/lysosome compartment. Some members of the annexin family of proteins such as annexin A2 (AnxA2) and annexin A6 (AnxA6) follow the same route as cholesterol during the endocytic pathway and are found, as AnxA6, attached to the membranes of the cholesterol storage compartment in NPC disease fibroblasts. Therefore, the purpose of this work was to test the hypothesis that AnxA6 participates in the NPC-induced changes in the organization of membrane microdomains resistant to solubilization by a nonionic detergent, Triton X-100, i.e., detergent-resistant microdomains (DRMs). Using cellular fractionation, fluorescence microscopy and specific antibodies we observed that in the absence of calcium AnxA6 was found in the DRM-depleted membrane fractions isolated from NPC and control fibroblasts. In the presence of calcium, AnxA6 re-located to the fractions enriched in DRMs only in the NPC cells, suggestive of AnxA6 participation in organization of these microdomains.     

Assembly of glycoprotein-80 adhesion complexes in Dictyostelium. Receptor compartmentalization and oligomerization in membrane rafts

The phospholipid-anchored membrane glycoprotein (gp)-80 mediates cell-cell adhesion through a homophilic trans-interaction mechanism during Dictyostelium development and is enriched in a Triton X-100-insoluble floating fraction. To elucidate how gp80 adhesion complexes assemble in the plasma membrane, gp80-gp80 and gp80-raft interactions were investigated. A low density raft-like membrane fraction was isolated using a detergent-free method. It was enriched in sterols, the phospholipid-anchored proteins gp80, gp138, and ponticulin, as well as DdCD36 and actin, corresponding to components found in the Triton X-100-insoluble floating fraction. Chemical cross-linking revealed that gp80 oligomers were enriched in the raft-like membrane fraction, implicating stable oligomer-raft interactions. However, gp80 oligomers resisted sterol sequestration and were partially dissociated with Triton X-100, suggesting that compartmentalization in rafts was not solely responsible for their formation. The trans-dimer known to mediate adhesion was identified, but cis-oligomerization predominated and displayed greater accumulation during development. In fact, oligomerization was dependent on the level of gp80 expression and occurred among isolated gp80 extracellular domains, indicating that it was mediated by direct gp80-gp80 interactions. Rafts existed in gp80-null cells and such pre-existent membrane domains may provide optimal microenvironments for gp80 cis-oligomerization and the assembly of adhesion complexes.     

Drug resistance-associated changes in sphingolipids and ABC transporters occur in different regions of membrane domains

We have recently shown that two ATP binding cassette (ABC) transporters are enriched in Lubrol-resistant noncaveolar membrane domains in multidrug-resistant human cancer cells [Hinrichs, J. W. J., K. Klappe, I. Hummel, and J. W. Kok. 2004. ATP-binding cassette transporters are enriched in non-caveolar detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in human multidrug-resistant cancer cells. J. Biol. Chem. 279: 5734-5738]. Here, we show that aminophospholipids are relatively enriched in Lubrol-resistant membrane domains compared with Triton X-100-resistant membrane domains, whereas sphingolipids are relatively enriched in the latter. Moreover, Lubrol-resistant membrane domains contain more protein and lipid mass. Based on these results, we postulate a model for detergent-insoluble glycosphingolipid-enriched membrane domains consisting of a Lubrol-insoluble/Triton X-100-insoluble region and a Lubrol-insoluble/Triton X-100-soluble region. The latter region contains most of the ABC transporters as well as lipids known to be necessary for their efflux activity. Compared with drug-sensitive cells, the detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in drug-resistant cells differ specifically in sphingolipid content and not in protein, phospholipid, or cholesterol content. In drug-resistant cells, sphingolipids with specific fatty acids (especially C24:1) are enriched in these membrane domains. Together, these data show that multidrug resistance-associated changes in both sphingolipids and ABC transporters occur in DIGs, but in different regions of these domains.     

Detergent-insoluble glycosphingolipid/cholesterol-rich membrane domains, lipid rafts and caveolae (review).

Within the cell membrane glycosphingolipids and cholesterol cluster together in distinct domains or lipid rafts, along with glycosyl-phosphatidylinositol (GPI)-anchored proteins in the outer leaflet and acylated proteins in the inner leaflet of the bilayer. These lipid rafts are characterized by insolubility in detergents such as Triton X-100 at 4 degrees C. Studies on model membrane systems have shown that the clustering of glycosphingolipids and GPI-anchored proteins in lipid rafts is an intrinsic property of the acyl chains of these membrane components, and that detergent extraction does not artefactually induce clustering. Cholesterol is not required for clustering in model membranes but does enhance this process. Single particle tracking, chemical cross-linking, fluorescence resonance energy transfer and immunofluorescence microscopy have been used to directly visualize lipid rafts in membranes. The sizes of the rafts observed in these studies range from 70-370 nm, and depletion of cellular cholesterol levels disrupts the rafts. Caveolae, flask-shaped invaginations of the plasma membrane, that contain the coat protein caveolin, are also enriched in cholesterol and glycosphingolipids. Although caveolae are also insoluble in Triton X-100, more selective isolation procedures indicate that caveolae do not equate with detergent-insoluble lipid rafts. Numerous proteins involved in cell signalling have been identified in caveolae, suggesting that these structures may function as signal transduction centres. Depletion of membrane cholesterol with cholesterol binding drugs or by blocking cellular cholesterol biosynthesis disrupts the formation and function of both lipid rafts and caveolae, indicating that these membrane domains are involved in a range of biological processes.     

Measurement of lipid nanodomain (raft) formation and size in sphingomyelin/POPC/cholesterol vesicles shows TX-100 and transmembrane helices increase domain size by coalescing preexisting nanodomains but do not induce domain formation

Mixtures of unsaturated lipids, sphingolipids, and cholesterol form coexisting liquid-disordered and sphingolipid and cholesterol-rich liquid-ordered (Lo) phases in water. The detergent Triton X-100 does not readily solubilize Lo domains, but does solubilize liquid-disordered domains, and is commonly used to prepare detergent-resistant membranes from cells and model membranes. However, it has been proposed that in membranes with mixtures of sphingomyelin (SM), 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC), and cholesterol Triton X-100 may induce Lo domain formation, and therefore detergent-resistant membranes may not reflect the presence of preexisting domains. To examine this hypothesis, the effect of Triton on Lo domain formation was measured in SM/POPC/cholesterol vesicles. Nitroxide quenching methods that can detect ordered nanodomains with radii >12 Å showed that in the absence of Triton X-100 this mixture formed ordered state domains that melt with a midpoint (= T_mid) at ∼45°C. However, T_mid was lower when detected using various fluorescence resonance energy transfer (FRET) pairs. Furthermore, the T_mid value was Ro dependent, and decreased as Ro increased. Because FRET can only readily detect domains with radii >Ro, this result can be explained by domain radii that are close to Ro and decrease as temperature increases. An analysis of FRET and quenching data suggests that nanodomain radius gradually decreases from ≥150 Å to <40 Å as temperature increases from 10 to 45°C. Interestingly, the presence of Triton X-100 or a transmembrane-type peptide did not stabilize ordered state formation when detected by nitroxide quenching, i.e., did not increase T_mid. However, FRET-detected T_mid did increase in the presence of Triton X-100 or a transmembrane peptide, indicating that both increased domain size. Controls showed that the results could not be accounted for by probe-induced perturbations. Thus, SM/POPC/cholesterol, a mixture similar to that in the outer leaflet of plasma membranes, forms nanodomains at physiological temperatures, and TX-100 does not induce domain formation or increase the fraction of the bilayer in the ordered state, although it does increase domain size by coalescing preexisting domains.