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.     

Insolubility of lipids in triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts)

The insolubility of lipids in detergents is a useful method for probing the structure of biological membranes. Insolubility in detergents like Triton X-100 is observed in lipid bilayers that exist in physical states in which lipid packing is tight. The Triton X-100-insoluble lipid fraction obtained after detergent extraction of eukaryotic cells is composed of detergent-insoluble membranes rich in sphingolipids and cholesterol. These insoluble membranes appear to arise from sphingolipid- and cholesterol-rich membrane domains (rafts) in the tightly packed liquid ordered state. Because the degree of lipid insolubility depends on the stability of lipid-lipid interactions relative to lipid-detergent interactions, the quantitative relationship between rafts and detergent-insoluble membranes is complex, and can depend on lipid composition, detergent and temperature. Nevertheless, when used conservatively detergent insolubility is an invaluable tool for studying cellular rafts and characterizing their composition.     

Both raft- and non-raft proteins associate with CHAPS-insoluble complexes: some APP in large complexes

Components of caveolae and lipid rafts are characterized by their buoyancy after detergent extraction. Using flotations in density gradients, we now show that non-raft membrane molecules are also associated with detergent-insoluble, buoyant assemblies. When Triton X-100 cellular extracts were spun to equilibrium in Nycodenz, only components of classical rafts floated. In contrast, with the zwitterionic detergent CHAPS, non-raft residents such as calnexin and APP also buoyed. When CHAPS extracts were spun in non-equilibrium (velocity) conditions, some raft components rapidly exited the input fractions while other raft markers and non-raft molecules remained relatively immobile. This pointed to size heterogeneities of CHAPS-insoluble complexes. Combined velocity/equilibrium gradients broadly divided CHAPS-insoluble membrane complexes into three size categories, which all contained cholesterol and the glycosphingolipid GM1. Large complexes were enriched in caveolin and ESA. Medium size complexes were enriched in PrP, whereas small complexes contained non-raft proteins, PrP, and some ESA. While Alzheimer's APP was primarily confined to small assemblies, a portion of its glycosylated form did buoy with large complexes. Large CHAPS-insoluble complexes resemble, but are not equal to, classical rafts. These findings extend considerably the range of detergent-insoluble membranal domains.     

Raft-mediated trafficking of apical resident proteins occurs in both direct and transcytotic pathways in polarized hepatic cells: role of distinct lipid microdomains

In polarized hepatic cells, pathways and molecular principles mediating the flow of resident apical bile canalicular proteins have not yet been resolved. Herein, we have investigated apical trafficking of a glycosylphosphatidylinositol-linked and two single transmembrane domain proteins on the one hand, and two polytopic proteins on the other in polarized HepG2 cells. We demonstrate that the former arrive at the bile canalicular membrane via the indirect transcytotic pathway, whereas the polytopic proteins reach the apical membrane directly, after Golgi exit. Most importantly, cholesterol-based lipid microdomains ("rafts") are operating in either pathway, and protein sorting into such domains occurs in the biosynthetic pathway, largely in the Golgi. Interestingly, rafts involved in the direct pathway are Lubrol WX insoluble but Triton X-100 soluble, whereas rafts in the indirect pathway are both Lubrol WX and Triton X-100 insoluble. Moreover, whereas cholesterol depletion alters raft-detergent insolubility in the indirect pathway without affecting apical sorting, protein missorting occurs in the direct pathway without affecting raft insolubility. The data implicate cholesterol as a traffic direction-determining parameter in the direct apical pathway. Furthermore, raft-cargo likely distinguishing single vs. multispanning membrane anchors, rather than rafts per se (co)determine the sorting pathway.     

Heliothis virescens and Manduca sexta lipid rafts are involved in Cry1A toxin binding to the midgut epithelium and subsequent pore formation

Lipid rafts are characterized by their insolubility in nonionic detergents such as Triton X-100 at 4 degrees C. They have been studied in mammals, where they play critical roles in protein sorting and signal transduction. To understand the potential role of lipid rafts in lepidopteran insects, we isolated and analyzed the protein and lipid components of these lipid raft microdomains from the midgut epithelial membrane of Heliothis virescens and Manduca sexta. Like their mammalian counterparts, H. virescens and M. sexta lipid rafts are enriched in cholesterol, sphingolipids, and glycosylphosphatidylinositol-anchored proteins. In H. virescens and M. sexta, pretreatment of membranes with the cholesterol-depleting reagent saponin and methyl-beta-cyclodextrin differentially disrupted the formation of lipid rafts, indicating an important role for cholesterol in lepidopteran lipid rafts structure. We showed that several putative Bacillus thuringiensis Cry1A receptors, including the 120- and 170-kDa aminopeptidases from H. virescens and the 120-kDa aminopeptidase from M. sexta, were preferentially partitioned into lipid rafts. Additionally, the leucine aminopeptidase activity was enriched approximately 2-3-fold in these rafts compared with brush border membrane vesicles. We also demonstrated that Cry1A toxins were associated with lipid rafts, and that lipid raft integrity was essential for in vitro Cry1Ab pore forming activity. Our study strongly suggests that these microdomains might be involved in Cry1A toxin aggregation and pore formation.     

Lipid raft localization of GABA A receptor and Na+, K+-ATPase in discrete microdomain clusters in rat cerebellar granule cells

The microdomain localization of the GABA(A) receptor in rat cerebellar granule cells was studied by subcellular fractionation and fluorescence- and immunogold electron microscopy. The receptor resided in lipid rafts, prepared at 37 degrees C by extraction with the nonionic detergent Brij 98, but the raft fraction, defined by the marker ganglioside GM(1) in the floating fractions following density gradient centrifugation, was heterogeneous in density and protein composition. Thus, another major raft-associated membrane protein, the Na(+), K(+)-ATPase, was found in discrete rafts of lower density, reflecting clustering of the two proteins in separate membrane microdomains. Both proteins were observed in patchy "hot spots" at the cell surface as well as in isolated lipid rafts. Their insolubility in Brij 98 was only marginally affected by methyl-beta-cyclodextrin. In contrast, both the GABA(A) receptor and Na(+), K(+)-ATPase were largely soluble in ice cold Triton X-100. This indicates that Brij 98 extraction defines an unusual type of cholesterol-independent lipid rafts that harbour membrane proteins also associated with underlying scaffolding/cytoskeletal proteins such as gephyrin (GABA(A) receptor) and ankyrin G (Na(+), K(+)-ATPase). By providing an ordered membrane microenvironment, lipid rafts may contribute to the clustering of the GABA(A) receptor and the Na(+), K(+)-ATPase at distinct functional locations on the cell surface.     

Epidermal growth factor receptors are localized to lipid rafts that contain a balance of inner and outer leaflet lipids: a shotgun lipidomics study

The epidermal growth factor (EGF) receptor partitions into lipid rafts made using a detergent-free method, but is extracted from low density fractions by Triton X-100. By screening several detergents, we identified Brij 98 as a detergent in which the EGF receptor is retained in detergent-resistant membrane fractions. To identify the difference in lipid composition between those rafts that harbored the EGF receptor (detergent-free and Brij 98-resistant) and those that did not (Triton X-100-resistant), we used multidimensional electrospray ionization mass spectrometry to perform a lipidomics study on these three raft preparations. Although all three raft preparations were similarly enriched in cholesterol, the EGF receptor-containing rafts contained more ethanolamine glycerophospholipids and less sphingomyelin than did the non-EGF receptor-containing Triton X-100 rafts. As a result, the detergent-free and Brij 98-resistant rafts exhibited a balance of inner and outer leaflet lipids, whereas the Triton X-100 rafts contained a preponderance of outer leaflet lipids. Furthermore, in all raft preparations, the outer leaflet phospholipid species were significantly different from those in the bulk membrane, whereas the inner leaflet lipids were quite similar to those found in the bulk membrane. These findings indicate that the EGF receptor is retained only in rafts that exhibit a lipid distribution compatible with a bilayer structure and that the selection of phospholipids for inclusion into rafts occurs mainly on the outer leaflet lipids.     

The CD20 calcium channel is localized to microvilli and constitutively associated with membrane rafts: antibody binding increases the affinity of the association through an epitope-dependent cross-linking-independent mechanism

CD20 is a B cell-specific membrane protein that functions in store-operated calcium entry and serves as a useful target for antibody-mediated therapeutic depletion of B cells. Antibody binding to CD20 induces a diversity of biological effects, some of which are dependent on lipid rafts. Rafts are isolated as low density detergent-resistant membranes, initially characterized using Triton X-100. We have previously reported that CD20 is soluble in 1% Triton but that antibodies induce the association of CD20 with Triton-resistant rafts. However, by using several other detergents to isolate rafts and by microscopic co-localization with a glycosylphosphatidylinositol-linked protein, we show in this report that CD20 is constitutively raft-associated. CD20 was distributed in a punctate pattern on the cell surface as visualized by fluorescence imaging and was also localized to microvilli by electron microscopy. The mechanism underlying antibody-induced association of CD20 with Triton-resistant rafts was investigated and found not to require cellular ATP, kinase activity, actin polymerization, or antibody cross-linking but was dependent on the epitope recognized. Thus, antibody-induced insolubility in 1% Triton most likely reflects a transition from relatively weak to strong raft association that occurs as a result of a conformational change in the CD20 protein.     

Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane

Membrane cholesterol-sphingolipid 'rafts', which are characterized by their insolubility in the non-ionic detergent Triton X-100 in the cold, have been implicated in the sorting of certain membrane proteins, such as placental alkaline phosphatase (PLAP), to the apical plasma membrane domain of epithelial cells. Here we show that prominin, an apically sorted pentaspan membrane protein, becomes associated in the trans-Golgi network with a lipid raft that is soluble in Triton X-100 but insoluble in another non-ionic detergent, Lubrol WX. At the cell surface, prominin remains insoluble in Lubrol WX and is selectively associated with microvilli, being largely segregated from the membrane subdomains containing PLAP. Cholesterol depletion results in the loss of prominin's microvillus-specific localization but does not lead to its complete intermixing with PLAP. We propose the coexistence within a membrane domain, such as the apical plasma membrane, of different cholesterol-based lipid rafts, which underlie the generation and maintenance of membrane subdomains.     

The low-density lipoprotein receptor-related protein-1 associates transiently with lipid rafts

The low-density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional receptor that undergoes constitutive endocytosis and recycling. To identify LRP-1 in lipid rafts, we biotin-labeled cells using a membrane-impermeable reagent and prepared Triton X-100 fractions. Raft-associated proteins were identified in streptavidin affinity-precipitates of the Triton X-100-insoluble fraction. PDGF beta-receptor was identified exclusively in lipid rafts, whereas transferrin receptor was excluded. LRP-1 distributed partially into rafts in murine embryonic fibroblasts (MEFs) and HT 1080 cells, but not in smooth muscle cells and CHO cells. LRP-1 partitioning into rafts was not altered by ligands, including alpha2-macroglobulin, platelet-derived growth factor-BB, and receptor-associated protein (RAP). To examine LRP-1 trafficking between membrane microdomains, we developed a novel method based on biotinylation and detergent fractionation. Association of LRP-1 with rafts was transient; by 15 min, nearly all of the LRP-1 that was initially raft-associated exited this compartment. LRP-1 in the Triton X-100-soluble fraction, which excludes lipid rafts, demonstrated complex kinetics, with phases reflecting import from rafts, endocytosis, and recycling. Potassium depletion blocked LRP-1 endocytosis but did not inhibit trafficking of LRP-1 from rafts into detergent-soluble microdomains. Our data support a model in which LRP-1 transiently associates with rafts but does not form a stable pool. Fluid movement of LRP-1 between microdomains may facilitate its function in promoting the endocytosis of other plasma membrane proteins, such as the urokinase receptor, which localizes in lipid rafts.     

Membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation

Sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and certain signaling molecules segregate from bulk membrane lipids into lateral domains termed lipid rafts, which are often isolated based on their insolubility in cold nonionic detergents. During immunohistological studies of gangliosides, major sphingolipids of the brain, we found that cold Triton X-100 solubility is bidirectional, leading to histological redistribution from gray to white matter. When brain sections were treated with > or =0.25% Triton X-100 at 4 degrees C, ganglioside GD1a, which is normally enriched in gray matter and depleted in white matter, redistributed into white matter tracts. Incubation of brain sections from knockout mice lacking GD1a with wild-type sections in the presence of cold Triton X-100 resulted in GD1a redistribution from wild-type gray matter to knockout white matter. GM1, which is normally enriched in white matter, remained in white matter after cold detergent treatment and did not migrate to knockout mouse brain sections. However, when gray matter gangliosides were enzymatically converted into GM1 in situ, the newly formed GM1 transmigrated to knockout mouse brain sections in the presence of cold detergent. When purified GD1a was added to knockout mouse brain sections in the presence of cold Triton X-100, it preferentially incorporated into white matter tracts. These data demonstrate that brain white matter is a sink for gangliosides, which redistribute from gray matter in the presence of low concentrations of cold Triton X-100. A GPI-anchored protein, Thy-1, also transmigrated from wild-type to Thy-1 knockout mouse brain sections in the presence of detergent at 4 degrees C, although less efficiently than did gangliosides. These data raise technical challenges for using nonionic detergents in certain histological protocols and for isolation of lipid rafts from brain tissue.     

Effects of freezing and protein cross-linker on isolating membrane raft-associated proteins

Since the discovery of cellular membrane rafts, the defining of these domains has remained ambiguous due to a great number of isolation procedures proposed for the extraction of the rafts from cells. Characterization of membrane rafts using Triton X-100 insolubility is limited by the fact that weak interactions between proteins and lipids within the membrane rafts cannot be detected. In order to study the role of membrane rafts in cell signal transduction, it is crucial that weak membrane raft-associated proteins are detected. In this report, we demonstrate that by incorporating 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) crosslinking and freezing at -80°C into the membrane raft isolation procedure of HaCaT cells, both membrane raft-associated proteins caveolin-1 and Fas receptor are able to be reproducibly isolated into a single fraction containing the membrane rafts of the cells.     

The platelet receptor for type III collagen (TIIICBP) is present in platelet membrane lipid microdomains (rafts)

Platelet interactions with collagen are orchestrated by the presence or the migration of platelet receptor(s) for collagen into lipid rafts, which are specialized lipid microdomains from the platelet plasma membrane enriched in signalling proteins. Electron microscopy shows that in resting platelets, TIIICBP, a receptor specific for type III collagen, is present on the platelet membrane and associated with the open canalicular system, and redistributes to the platelet membrane upon platelet activation. After platelet lysis by 1% Triton X-100 and the separation of lipid rafts on a discontinuous sucrose gradient, TIIICBP is recovered in lipid raft-containing fractions and Triton X-100 insoluble fractions enriched in cytoskeleton proteins. Platelet aggregation, induced by type III collagen, was inhibited after disruption of the lipid rafts by cholesterol depletion, whereas platelet adhesion under static conditions did not require lipid raft integrity. These results indicate that TIIICBP, a platelet receptor involved in platelet interaction with type III collagen, is localized within platelet lipid rafts where it could interact with other platelet receptors for collagen (GP VI and α2β1 integrin) for efficient platelet activation. Pascal Maurice and Ludovic Waeckel have contributed equally to this work.     

Glucose deprivation enhances targeting of GLUT1 to lipid rafts in 3T3-L1 adipocytes

Glucose deprivation dramatically increases glucose transport activity in 3T3-L1 adipocytes without changing the concentration of GLUT1 in the plasma membrane (PM). Recent data suggest that subcompartments within the PM, specifically lipid rafts, may sequester selected proteins and alter their activity. To evaluate this possibility, we examined the distribution of GLUT1 in Triton X-100-soluble and -insoluble fractions. Our data show that 77% of the GLUT1 pool in PMs isolated from control 3T3-L1 adipocytes was extracted by 0.2% Triton X-100. After glucose deprivation for 12 h, only 56% of GLUT1 was extracted by detergent. In contrast, there was a twofold increase in the GLUT1 content of the detergent-resistant fraction. To evaluate whether GLUT1 interacts with a specific protein within lipid rafts, we focused on stomatin, recently shown to interact with and inhibit GLUT1 activity. Stomatin is distributed about equally between the PM and the biosynthetic compartments, and its expression is not affected by glucose deprivation. Nearly 90% of the PM pool of stomatin is in detergent-resistant lipid rafts. In normal 3T3-L1 adipocytes, we were unable to demonstrate an interaction between GLUT1 and stomatin in coimmunoprecipitation experiments. However, in stomatin-overexpressing cells, there was clear coprecipitation of stomatin with GLUT1 antibodies. Glucose deprivation increased this interaction threefold, which may reflect the increase of GLUT1 in lipid rafts. Despite this, there was little change in transport activity in glucose-deprived, stomatin-overexpressing cells vs. that in control cells. Thus GLUT1 interacts with stomatin in lipid rafts, but this interaction per se does not alter transport activity. Rather, stomatin may serve as an anchor for GLUT1 in lipid rafts, the environment of which favors activation.     

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.     

Differential sensitivity to detergents of actin cytoskeleton from nerve endings

Detergent-resistant membranes (DRM), an experimental model used to study lipid rafts, are typically extracted from cells by means of detergent treatment and subsequent ultracentrifugation in density gradients, Triton X-100 being the detergent of choice in most of the works. Since lipid rafts are membrane microdomains rich in cholesterol, depletion of this component causes solubilization of DRM with detergent. In previous works from our group, the lack of effect of cholesterol depletion on DRM solubilization with Triton X-100 was detected in isolated rat brain synaptosomes. In consequence, the aim of the present work is to explore reasons for this observation, analyzing the possible role of the actin cytoskeleton, as well as the use of an alternative detergent, Brij 98, to overcome the insensitivity to Triton X-100 of cholesterol-depleted DRM. Brij 98 yields Brij-DRM that are highly dependent on cholesterol, since marker proteins (Flotillin-1 and Thy-1), as well as actin, appear solubilized after MCD treatment. Pretreatment with Latrunculin A results in a significant increase in Flotillin-1, Thy-1 and actin solubilization by Triton X-100 after cholesterol depletion. Studies with transmission electron microscopy show that combined treatment with MCD and Latrunculin A leads to a significant increase in solubilization of DRM with Triton X-100. Thus, Triton-DRM resistance to cholesterol depletion can be explained, at least partially, thanks to the scaffolding action of the actin cytoskeleton, without discarding differential effects of Brij 98 and Triton X-100 on specific membrane components. In conclusion, the detergent of choice is important when events that depend on the actin cytoskeleton are going to be studied.     

Domain-specific lipid distribution in macrophage plasma membranes

Lipid rafts, defined as cholesterol- and sphingolipid-rich domains, provide specialized lipid environments understood to regulate the organization and function of many plasma membrane proteins. Growing evidence of their existence, protein cargo, and regulation is based largely on the study of isolated lipid rafts; however, the consistency and validity of common isolation methods is controversial. Here, we provide a detailed and direct comparison of the lipid and protein composition of plasma membrane "rafts" prepared from human macrophages by different methods, including several detergent-based isolations and a detergent-free method. We find that detergent-based and detergent-free methods can generate raft fractions with similar lipid contents and a biophysical structure close to that previously found on living cells, even in cells not expressing caveolin-1, such as primary human macrophages. However, important differences between isolation methods are demonstrated. Triton X-100-resistant rafts are less sensitive to cholesterol or sphingomyelin depletion than those prepared by detergent-free methods. Moreover, we show that detergent-based methods can scramble membrane lipids during the isolation process, reorganizing lipids previously in sonication-derived nonraft domains to generate new detergent-resistant rafts. The role of rafts in regulating the biological activities of macrophage plasma membrane proteins may require careful reevaluation using multiple isolation procedures, analyses of lipids, and microscopic techniques.     

Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts

Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSD to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-beta-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption.     

A GFP fluorescence-based approach to determine detergent insolubility of the human serotonin1A receptor

Insolubility in non-ionic detergents such as Triton X-100 is a widely used biochemical criterion for characterization of membrane domains. We report here a novel green fluorescent protein fluorescence-based approach to directly determine detergent insolubility of specific membrane proteins. We have applied this method to explore the detergent resistance of an important G-protein coupled receptor, the serotonin1A (5-HT1A) receptor. Our results show, for the first time, that a small yet significant fraction of the 5-HT1A receptor exhibits detergent insolubility. These results are validated by control experiments involving fluorescent lipid probes and protein markers. Our results assume relevance in the context of localization of the 5-HT1A receptor in membrane domains and its significance in receptor function and signaling.     

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.