Spatio-temporal localization of membrane lipid rafts in mouse oocytes and cleaving preimplantation embryos

We report for the first time the detection of membrane lipid rafts in mouse oocytes and cleaving preimplantation embryos. Cholera toxin β (CTβ), which binds to the raft-enriched ganglioside GM1, was selected to label rafts. In a novel application a Qdot reagent was used to detect CTβ labeling. This is the first reported use of nanocrystals in mammalian embryo imaging. Comparative membrane labeling with CTβ and lipophilic membrane dyes containing saturated or unsaturated aliphatic tails showed that the detection of GM1 in mouse oocytes and embryo membranes was consistent with the identification of cholesterol- and sphingolipid-enriched rafts in the cell membrane. Distribution of the GM1 was compared with the known distribution of non-raft membrane components, and disruption of membrane rafts with detergents confirmed the cholesterol dependence of GM1 on lipid raft labeling. Complementary functional studies showed that cholesterol depletion using methyl-β-cyclodextrin inhibited preimplantation development in culture. Our results show that the membranes of the mouse oocyte and zygote are rich in lipid rafts, with heterogeneous and stage-dependent distribution. In dividing embryos, the rafts were clearly associated with the cleavage furrow. At the morula stage, rafts were also apically enriched in each blastomere. In blastocysts, rafts were detectable in the trophectoderm layer, but could not be detected in the inner cell mass without prior fixation and permeabilization of the embryo. Lipid rafts and their associated proteins are, therefore, spatio-temporally positioned to a play a critical role in preimplantation developmental events.     

Segregation of gangliosides GM1 and GD3 on cell membranes, isolated membrane rafts, and defined supported lipid monolayers

Lateral assemblies of sphingolipids, glycosphingolipids and cholesterol, termed rafts, are postulated to be present in biological membranes and to function in important cellular phenomena. We probed whether rafts are heterogeneous by determining the relative distribution of two gangliosides, GM1 and GD3, in artificial supported monolayers, in intact rat primary cerebellar granule neurones, and in membrane rafts isolated from rat cerebellum. Fluorescence resonance energy transfer (FRET) using fluorophore-labelled cholera toxin B subunit (which binds GM1) and mAb R24 (which binds GD3) revealed that GM1 spontaneously self-associates but does not co-cluster with GD3 in supported monolayers and on intact neurones. Cholera toxin and immunocytochemical labelling of isolated membrane rafts from rat cerebellum further demonstrated that GM1 does not co-localise with GD3. Furthermore, whereas the membrane raft resident proteins Lyn and caveolin both co-localise with GD3 in isolated membrane rafts, GM1 appears in separate and distinct aggregates. These data support prior reports that membrane rafts are heterogeneous, although the mechanisms for establishing and maintaining such heterogeneity remain to be determined.     

SCP-2/SCP-x gene ablation alters lipid raft domains in primary cultured mouse hepatocytes

Although reverse cholesterol transport from peripheral cell types is mediated through plasma membrane microdomains termed lipid rafts, almost nothing is known regarding the existence, protein/lipid composition, or structure of these putative domains in liver hepatocytes, cells responsible for the net removal of cholesterol from the body. Lipid rafts purified from hepatocyte plasma membranes by a nondetergent affinity chromatography method were: i) present at 33 +/- 3% of total plasma membrane protein; ii) enriched in key proteins of the reverse cholesterol pathway [scavenger receptor class B type I (SR-B1), ABCA1, P-glycoprotein (P-gp), sterol carrier protein-2 (SCP-2)]; iii) devoid of caveolin-1; iv) enriched in cholesterol, sphingomyelin, GM1, and phospholipids low in polyunsaturated fatty acid and double bond index; and v) exhibited an intermediate liquid-ordered lipid phase with significant transbilayer fluidity gradient. Ablation of the gene encoding SCP-2 significantly altered lipid rafts to: i) increase the proportion of lipid rafts present, thereby increasing raft total content of ABCA1, P-gp, and SR-B1; ii) increase total phospholipids while decreasing GM1 in lipid rafts; iii) decrease the fluidity of lipid rafts, consistent with the increased intermediate liquid-ordered phase; and iv) abolish the lipid raft transbilayer fluidity gradient. Thus, despite the absence of caveolin-1 in liver hepatocytes, lipid rafts represented nearly one-third of the mouse hepatocyte plasma membrane proteins and displayed unique protein, lipid, and biophysical properties that were differentially regulated by SCP-2 expression.     

Thermodynamic properties and characterization of proteoliposomes rich in microdomains carrying alkaline phosphatase

Tissue-nonspecific alkaline phosphatase (TNAP) is associated to the plasma membrane via a GPI-anchor and plays a key role in the biomineralization process. In plasma membranes, most GPI-anchored proteins are associated with "lipid rafts", ordered microdomains enriched in sphingolipids, glycosphingolipids and cholesterol. In order to better understand the role of lipids present in rafts and their interactions with GPI-anchored proteins, the insertion of TNAP into different lipid raft models was studied using dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), sphingomyelin (SM) and ganglioside (GM1). Thus, the membrane models studied were binary systems (9:1 molar ratio) containing DPPC:Chol, DPPC:SM and DPPC:GM1, ternary systems (8:1:1 molar ratio) containing DPPC:Chol:SM, DPPC:Chol:GM1 and DPPC:SM:GM1 and finally, a quaternary system (7:1:1:1 molar ratio) containing DPPC:Chol:SM:GM1. Calorimetry analysis of the liposomes and proteoliposomes indicate that lateral phase segregation could be noted only in the presence of cholesterol, with the formation of cholesterol-rich microdomains centered above Tc=41.5°C. The presence of GM1 and SM into DPPC-liposomes influenced mainly ΔH and Δt(1/2) values. The gradual increase in the complexity of the systems decreased the activity of the enzyme incorporated. The presence of the enzyme also fluidifies the systems, as seen by the intense reduction in ?H values, but do not alter Tc values significantly. Therefore, the study of different microdomains and its biophysical characterization may contribute to the knowledge of the interactions between the lipids present in MVs and its interactions with TNAP.     

Proteomic of lipid rafts in the exocrine pancreas from diet-induced obese rats

In the present work, we induced obesity in rats with high-energy-starch diet and studied exocrine pancreas response. The zymogen granule (ZG) or purified plasma membrane (PM) from the exocrine pancreas was used for the isolation of the detergent-resistant membranes (DRMs). Based on high content of cholesterol, GM1, the bile salt dependent lipase (BSDL), and GP2 enrichment, the low-density fractions were defined as lipid rafts. Additionally, the rafts vesicles were determined by immunogold labeling with anti BSDL. By combining MALDI-TOF/MS and nano-LC ESI Q-TOF MS/MS proteomic identification we have selected 33 proteins from the lipid rafts which were classified into at least four functional families. Our data suggest that the acinar PM from the diet-induced obesity rats may be organized into lipid rafts, and characterization of rafts proteome can contribute to improve our understanding of food digestion under obesity.     

N-Terminal Protein Acylation Confers Localization to Cholesterol, Sphingolipid-enriched Membranes But Not to Lipid Rafts/Caveolae

When variably fatty acylated N-terminal amino acid sequences were appended to a green fluorescent reporter protein (GFP), chimeric GFPs were localized to different membranes in a fatty acylation-dependent manner. To explore the mechanism of localization, the properties of acceptor membranes and their interaction with acylated chimeric GFPs were analyzed in COS-7 cells. Myristoylated GFPs containing a palmitoylated or polybasic region colocalized with cholesterol and ganglioside GM(1), but not with caveolin, at the plasma membrane and endosomes. A dipalmitoylated GFP chimera colocalized with cholesterol and GM(1) at the plasma membrane and with caveolin in the Golgi region. Acylated GFP chimeras did not cofractionate with low-density caveolin-rich lipid rafts prepared with Triton X-100 or detergent-free methods. All GFP chimeras, but not full-length p62(c-yes) and caveolin, were readily solubilized from membranes with various detergents. These data suggest that, although N-terminal acylation can bring GFP to cholesterol and sphingolipid-enriched membranes, protein-protein interactions are required to localize a given protein to detergent-resistant membranes or caveolin-rich membranes. In addition to restricting acceptor membrane localization, N-terminal fatty acylation could represent an efficient means to enrich the concentration of signaling proteins in the vicinity of detergent-resistant membranes and facilitate protein-protein interactions mediating transfer to a detergent-resistant lipid raft core.     

Selective cholesterol dynamics between lipoproteins and caveolae/lipid rafts

Although low-density lipoprotein (LDL) receptor-mediated cholesterol uptake through clathrin-coated pits is now well understood, the molecular details and organizing principles for selective cholesterol uptake/efflux (reverse cholesterol transport, RCT) from peripheral cells remain to be resolved. It is not yet completely clear whether RCT between serum lipoproteins and the plasma membrane occurs primarily through lipid rafts/caveolae or from non-raft domains. To begin to address these issues, lipid raft/caveolae-, caveolae-, and non-raft-enriched fractions were resolved from purified plasma membranes isolated from L-cell fibroblasts and MDCK cells by detergent-free affinity chromatography and compared with detergent-resistant membranes isolated from the same cells. Fluorescent sterol exchange assays between lipoproteins (VLDL, LDL, HDL, apoA1) and these enriched domains provided new insights into supporting the role of lipid rafts/caveolae and caveolae in plasma membrane/lipoprotein cholesterol dynamics: (i) lipids known to be translocated through caveolae were detected (cholesteryl ester, triacylglycerol) and/or enriched (cholesterol, phospholipid) in lipid raft/caveolae fractions; (ii) lipoprotein-mediated sterol uptake/efflux from lipid rafts/caveolae and caveolae was rapid and lipoprotein specific, whereas that from non-rafts was very slow and independent of lipoprotein class; and (iii) the rate and lipoprotein specificity of sterol efflux from lipid rafts/caveolae or caveolae to lipoprotein acceptors in vitro was slower and differed in specificity from that in intact cells-consistent with intracellular factors contributing significantly to cholesterol dynamics between the plasma membrane and lipoproteins.     

Compositional changes in lipid microdomains of air-blood barrier plasma membranes in pulmonary interstitial edema.

We evaluated in anesthetized rabbits the compositional changes of plasmalemmal lipid microdomains from lung tissue samples after inducing pulmonary interstitial edema (0.5 ml/kg for 3 h, leading to approximately 5% increase in extravascular water). Lipid microdomains (lipid rafts and caveolae) were present in the detergent-resistant fraction (DRF) obtained after discontinuous sucrose density gradient. DRF was enriched in caveolin-1, flotillin, aquaporin-1, GM1, cholesterol, sphingomyelin, and phosphatidylserine, and their contents significantly increased in interstitial edema. The higher DRF content in caveolin, flotillin, and aquaporin-1 and of the ganglioside GM1 suggests an increase both in caveolar domains and in lipid rafts, respectively. Compositional changes could be ascribed to endothelial and epithelial cells that provide most of plasma membrane surface area in the air-blood barrier. Alterations in lipid components in the plasma membrane may reflect rearrangement of floating lipid platforms within the membrane and/or lipid translocation from intracellular stores. Lipid traffic could be stimulated by the marked increase in hydraulic interstitial pressure after initial water accumulation, from approximately -10 to 5 cmH2O, due to the low compliance of the pulmonary tissue, in particular in the basement membranes and in the interfibrillar substance. Compositional changes in lipid microdomains represent a sign of cellular activation and suggest the potential role of mechanotransduction in response to developing interstitial edema.     

P-Glycoprotein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains

P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) superfamily responsible for the ATP-driven extrusion of diverse hydrophobic molecules from cells, is a cause of multidrug resistance in human tumours. Pgp can also operate as a phospholipid and glycosphingolipid flippase, and has been functionally linked to cholesterol, suggesting that it might be associated with sphingolipid-cholesterol microdomains in cell membranes. We have used nonionic detergent extraction and density gradient centrifugation of extracts from the multidrug-resistant Chinese hamster ovary cell line, CH(R)B30, to address this question. Our data indicate that Pgp is localized in intermediate-density membrane microdomains different from classical lipid rafts enriched in Src-family kinases. We demonstrate that Brij-96 can selectively isolate the Pgp domains, separating them from the caveolar and classical lipid rafts. Pgp was found entirely in the Brij-96-insoluble domains, and only partially in the Triton X-100-insoluble membrane microdomains. We studied the sensitivity of these domains to cholesterol removal, as well as their relationship to GM(1) ganglioside- and caveolin-1-enriched caveolar domains. We found that the buoyant density of the Brij-96-based Pgp-containing microdomains was sensitive to cholesterol removal by methyl-beta-cyclodextrin. The Brij-96 domains retained their structural integrity after cholesterol depletion while, in contrast, the Triton X-100-based caveolin-1/GM(1) microdomains did not. Using confocal fluorescence microscopy, we determined that caveolin-1 and GM(1) colocalized, while Pgp and caveolin-1, or Pgp and GM(1), did not. Our results suggest that Pgp does not interact directly with caveolin-1, and is localized in intermediate-density domains, distinct from classical lipid rafts and caveolae, which can be isolated using Brij-96.     

Lipid rafts are involved in SARS-CoV entry into Vero E6 cells

Lipid rafts often serve as an entry site for certain viruses. Here, we report that lipid rafts in Vero E6 cells are involved in the entry of severe acute respiratory syndrome coronavirus (SARS-CoV). Infectivity assay showed the integrity of lipid rafts was required for productive infection of pseudotyped SARS-CoV. Depletion of plasma membrane cholesterol with MβCD relocalized raft-resident marker caveolin-1 as well as SARS-CoV receptor ACE2 to a nonraft environment, but did not significantly change the surface expression of ACE2. MβCD-treatment inhibited infectivity of pseudotyped SARS-CoV by 90%. Biochemical fractionation and confocal imaging confirmed that ACE2 colocalized with raft-resident markers. Furthermore, an ectodomain of SARS-CoV S protein (S1188HA) could associate with lipid rafts after binding to its receptor, and colocalize with raft-resident marker ganglioside GM1. The binding of S1188HA was not affected by depleting plasma membrane cholesterol. Taken together, our results support that lipid rafts serve as an entry port for SARS-CoV.     

A protective role for lipid raft cholesterol against amyloid-induced membrane damage in human neuroblastoma cells

Increasing evidence supports the idea that the initial events of Aβ oligomerization and cytotoxicity in Alzheimer's disease involve the interaction of amyloid Aβ-derived diffusible ligands (ADDLs) with the cell membrane. This also indicates lipid rafts, ordered membrane microdomains enriched in cholesterol, sphingolipids and gangliosides, as likely primary interaction sites of ADDLs. To shed further light on the relation between ADDL-cell membrane interaction and oligomer cytotoxicity, we investigated the dependence of ADDLs binding to lipid rafts on membrane cholesterol content in human SH-SY5Y neuroblastoma cells. Confocal laser microscopy showed that Aβ1-42 oligomers markedly interact with membrane rafts and that a moderate enrichment of membrane cholesterol prevents their association with the monosialoganglioside GM1. Moreover, anisotropy fluorescence measurements of flotillin-1-positive rafts purified by sucrose density gradient suggested that the content of membrane cholesterol and membrane perturbation by ADDLs are inversely correlated. Finally, contact mode atomic force microscope images of lipid rafts in liquid showed that ADDLs induce changes in raft morphology with the appearance of large cavities whose size and depth were significantly reduced in similarly treated cholesterol-enriched rafts. Our data suggest that cholesterol reduces amyloid-induced membrane modifications at the lipid raft level by altering raft physicochemical features.     

Perfringolysin O, a cholesterol-binding cytolysin, as a probe for lipid rafts

Gaining an understanding of the structural and functional roles of cholesterol in membrane lipid rafts is a critical issue in studies on cellular signaling and because of the possible involvement of lipid rafts in various diseases. We have focused on the potential of perfringolysin O (theta-toxin), a cholesterol-binding cytolysin produced by Clostridium perfringens, as a probe for studies on membrane cholesterol. We prepared a protease-nicked and biotinylated derivative of perfringolysin O (BCtheta) that binds selectively to cholesterol in cholesterol-rich microdomains of cell membranes without causing membrane lesions. Since the domains fulfill the criteria of lipid rafts, BCtheta can be used to detect cholesterol-rich lipid rafts. This is in marked contrast to filipin, another cholesterol-binding reagent, which binds indiscriminately to cell cholesterol. Using BCtheta, we are now searching for molecules that localize specifically in cholesterol-rich lipid rafts. Recently, we demonstrated that the C-terminal domain of perfringolysin O, domain 4 (D4), possesses the same binding characteristics as BCtheta. BIAcore analysis showed that D4 binds specifically to cholesterol with the same binding affinity as the full-size toxin. Cell-bound D4 is recovered predominantly from detergent-insoluble, low-density membrane fractions where raft markers, such as cholesterol, flotillin and Src family kinases, are enriched, indicating that D4 also binds selectively to lipid rafts. Furthermore, a green fluorescent protein-D4 fusion protein (GFP-D4) was revealed to be useful for real-time monitoring of cholesterol in lipid rafts in the plasma membrane. In addition, the expression of GFP-D4 in the cytoplasm might allow the investigations of intracellular trafficking of lipid rafts. The simultaneous visualization of lipid rafts in plasma membranes and inside cells might help in gaining a total understanding of the dynamic behavior of lipid rafts.     

肌动蛋白结合脂筏促进巨噬细胞摄入土拉弗朗西斯菌

观察土拉弗朗西斯菌LVS借助脂筏以肌动蛋白为动力被鼠巨噬细胞摄入的过程。细胞胆固醇用菲律平Ⅲ染色,结合神经节苷酯GM1的霍乱毒素B亚基用键合了Alexa 594的兔抗霍乱毒素B亚基二抗显色;肌动蛋白用键合了Alexa 594的鬼笔环肽显色。免疫荧光显微镜观察到脂筏成分中的胆固醇、神经节苷酯GM1均可与细菌共定位;胆固醇可与肌动蛋白共定位。随着感染时间的延长,细菌可离开脂筏。离开脂筏的细菌囊泡可与肌动蛋白共定位。这些发现提示肌动蛋白与脂筏结合,在弗朗西斯菌早期进入巨噬细胞期间发挥重要作用。     

More ordered, convex ganglioside-enriched membrane domains: the effects of GM1 on sphingomyelin bilayers containing a low level of cholesterol

The special physical state of the sphingolipid-enriched membranes with characteristic lipid composition, presently one of the most controversial foci in cell biology, provides the essential environment for the proteins inside to be involved in the related physiological processes. The role of gangliosides, an important component of the membranes, deserves attention. The present investigation using several biophysical techniques indicates that ganglioside GM(1) induces the phase separation in the sphingomyelin membrane with 5 mol% cholesterol and regulates the membrane structure. The results of differential scanning calorimetry show that a higher T(m), GM(1)-rich phase emerges behind the lower T(m), sphingomyelin-rich phase with the incorporation of GM(1) into the sphingomyelin/cholesterol bilayers; and the GM(1)-rich phase dominates the membrane when the proportion of GM(1) reaches about 20 mol%. Fluorescence quenching further shows that the separation of the two domains is independent of temperature, occurring both in the gel phase and in the liquid phase. Laser Raman spectroscopy and fluorescence polarization suggest that the order of hydrocarbon chains increases and membrane fluidity decreases with increase in GM(1) content. Use of the fluorescence probe merocyanine-540 and electron microscopy reveals that the insertion of GM(1) leads to an increase in the spatial density of the lipid headgroups and a decrease in the curvature of the sphingomyelin/cholesterol bilayers. In sums, both the hydrophilic sugar heads and the hydrophobic hydrocarbon chains of GM(1) contribute to the regulation of membrane architecture. We suggest that the convex curvature of ganglioside-enriched membrane could be involved in forming and maintaining the characteristic flask-shaped invagination of caveolae.     

The plasma membrane Ca2+-ATPase isoform 4 is localized in lipid rafts of cerebellum synaptic plasma membranes

Here we describe the association of the synaptosomal plasma membrane Ca2+-ATPase (PMCA) from pig cerebellum with cholesterol/sphingomyelin-rich membrane domains (rafts). The PMCA4 was localized exclusively in rafts prepared by flotation in Nycodenz density gradients of ice-cold Brij 96 extracts. This was corroborated by its colocalization with the raft markers cholesterol, ganglioside GM1, and PrP(C). The remaining PMCA isoforms were found in the detergent-soluble fractions, with the majority of the membrane proteins. Activity assays confirmed the bimodal distribution of the PMCA isoforms in the density gradient, with a lower activity for PMCA4 and greater stimulation by calmodulin than for the other isoforms. By providing an ordered membrane microenvironment, lipid rafts may contribute to the interaction of PMCA4 with proteins involved in Ca2+ signaling at discrete functional positions on the synaptic nerve terminals.     

Cholesterol: Coupling between membrane microenvironment and ABC transporter activity

Lipid composition of biological membranes is closely related to the function of the ATP-binding cassette (ABC) transporter P-Glycoprotein (Pgp). Herein, we studied how membrane physico-chemical properties affect Pgp-activity. We effectively modulated the cellular cholesterol content using methyl-beta-cyclodextrin (MbetaCD) and MbetaCD-cholesterol-inclusion complex. Pgp was not liberated from the plasma membrane during cholesterol modulation and functional inhibition of Pgp was related to varying cholesterol levels in the plasma membrane. Our data indicate that membrane fluidity does not solely account for cholesterol dependent modifications of Pgp-activity. Therefore, we isolated lipid rafts and examined distinct membrane microdomains. Both depletion and cholesterol enrichment induces a disassembly of lipid rafts. In cholesterol-depleted cell membranes a shift in the Pgp localisation to detergent soluble fractions was observed. Enrichment of membrane cholesterol changed lipid raft distribution but not the localisation of Pgp. From our data we conclude that Pgp-transport capacity depends on accurate lipid raft properties.     

The size of lipid rafts: an atomic force microscopy study of ganglioside GM1 domains in sphingomyelin/DOPC/cholesterol membranes

Atomic force microscopy has been used to study the distribution of ganglioside GM1 in model membranes composed of ternary lipid mixtures that mimic the composition of lipid rafts. The results demonstrate that addition of 1% GM1 to 1:1:1 sphingomyelin/dioleoylphosphatidylcholine/cholesterol monolayers leads to the formation of small ganglioside-rich microdomains (40-100 nm in size) that are localized preferentially in the more ordered sphingomyelin/cholesterol-rich phase. With 5% GM1 some GM1 microdomains are also detected in the dioleoylphosphatidylcholine-rich phase. A similar preferential localization of GM1 in the ordered phase is observed for bilayers with the same ternary lipid mixture in the upper leaflet. The small GM1-rich domains observed in these experiments are similar to the sizes for lipid rafts in natural membranes but considerably smaller than the ordered bilayer domains that have been shown to be enriched in GM1 in recent fluorescence microscopy studies of lipid bilayers. The combined data from a number of studies of model membranes indicate that lateral organization occurs on a variety of length scales and mimics many of the properties of natural membranes.     

Sulfatide is expressed in neurons and astrocytes and accumulates at degradation deficiency

Few studies of lipid rafts have investigated gangliosides in brain tissue. This study focus on analyses of lipids and the major brain gangliosides (GM1, GD1a, GD1b, GT1b) in human cortex (frontal, temporal) and corresponding detergent resistant membranes (DRMs), i.e. rafts. A high proportion of the gangliosides (18–26%) as well as of cholesterol (21%) and sphingomyelin (38%) was found in rafts, while lower yields was observed for ganglioside GM2 (9%), phospholipids (8%) and in particular proteins (2%). Significant alterations in lipid composition was noticed in rafts from Alzheimer brain tissue. These results show that sphingolipids and cholesterol are major constituents of rafts also in the human brain and that the main brain gangliosides are distributed in rafts to a similar degree. Moreover, lipid rafts might be considered in the pathology of Alzheimer's disease.