Triglyceride-rich lipoprotein lipolysis releases neutral and oxidized FFAs that induce endothelial cell inflammation

Triglyceride-rich lipoprotein (TGRL) lipolysis products provide a pro-inflammatory stimulus that can alter endothelial barrier function. To probe the mechanism of this lipolysis-induced event, we evaluated the pro-inflammatory potential of lipid classes derived from human postprandial TGRL by lipoprotein lipase (LpL). Incubation of TGRL with LpL for 30 min increased the saturated and unsaturated FFA content of the incubation solutions significantly. Furthermore, concentrations of the hydroxylated linoleates 9-hydroxy ocatadecadienoic acid (9-HODE) and 13-HODE were elevated by LpL lipolysis, more than other measured oxylipids. The FFA fractions elicited pro-inflammatory responses inducing TNFalpha and intracellular adhesion molecule expression and reactive oxygen species (ROS) production in human aortic endothelial cells (HAECs). The FFA-mediated increase in ROS was blocked by both the cytochrome P450 2C9 inhibitor sulfaphenazole and NADPH oxidase inhibitors. Compared with linoleate, 13-HODE was found to be a more potent inducer of ROS production in HAECs, an activity that was insensitive to both NADPH oxidase and cytochrome P450 inhibitors. Therefore, although the oxidative metabolism of FFA in endothelial cells can produce inflammatory responses, TGRL lipolysis can also release preformed mediators of oxidative stress (e.g., HODEs) that may influence endothelial cell function in vivo by stimulating intracellular ROS production.     

Reactive oxygen species promote raft formation in T lymphocytes

Lipid rafts are involved in many cell biology events, yet the molecular mechanisms on how rafts are formed are poorly understood. In this study we probed the possible requirement of reactive oxygen species (ROS) for T-cell receptor (TCR)-induced lipid raft formation. Microscopy and biochemical analyses illustrated that blockage of ROS production, by superoxide dismutase-mimic MnTBAP, significantly reduced partitioning of LAT, phospho-LAT, and PLC-gamma in lipid rafts. Another antioxidant N-acetylcysteine (NAC) displayed a similar suppressive effect on the entry of phospho-LAT into raft microdomains. The involvement of ROS in TCR-mediated raft assembly was observed in T-cell hybridomas, T leukemia cells, and normal T cells. Removal of ROS was accompanied by an attenuated activation of LAT and PKCtheta, with reduced production of IL-2. Consistently, treating T cells with the ROS-producer tert-butyl hydrogen peroxide (TBHP) greatly enhanced membrane raft formation, distribution of phospho-LAT into lipid rafts, and increased IL-2 production. Our results indicate for the first time that ROS contribute to TCR-induced membrane raft formation.     

TNF-alpha potentiates protein-tyrosine nitration through activation of NADPH oxidase and eNOS localized in membrane rafts and caveolae of bovine aortic endothelial cells

A major source of reactive oxygen species (ROS) in endothelial cells is the NADPH oxidase enzyme complex. The selective distributions of any enzyme within cells have important implications in regulating enzyme effectiveness through facilitation of access to local substrates and/or product targets. Because membrane rafts provide a spatially preferable environment for a variety of enzyme systems, we sought to determine whether NADPH oxidase is present and functional in this plasma membrane compartment in endothelial cells. We found that, in resting endothelial cells, NADPH oxidase subunits were preassembled and the enzyme functional in membrane rafts, specifically in caveolae. Stimulation with TNF-alpha induced additional recruitment of the p47(phox) regulatory subunit to raft-localized NADPH oxidase and enhanced ROS production within raft domains. TNF-alpha also induced nitric oxide production through activation of endothelial nitric oxide synthase (eNOS) present in the same membrane compartment. The dual activation of superoxide and nitric oxide-generating systems provided a spatially favorable environment for nitration of tyrosine-containing proteins localized to rafts. Perturbation of membrane raft structural integrity with cholesterol-sequestering compounds caused the delocalization of NADPH oxidase subunits and eNOS from the rafts and inhibited TNF-alpha-induced ROS production and protein tyrosine nitration. Together, these data provide evidence that membrane rafts and caveolae play a role in the spatial regulation of NADPH oxidase and subsequent ROS/reactive nitrogen species in endothelial cells.     

Spatiotemporal analysis of differential Akt regulation in plasma membrane microdomains

As a central kinase in the phosphatidylinositol 3-kinase pathway, Akt has been the subject of extensive research; yet, spatiotemporal regulation of Akt in different membrane microdomains remains largely unknown. To examine dynamic Akt activity in membrane microdomains in living cells, we developed a specific and sensitive fluorescence resonance energy transfer-based Akt activity reporter, AktAR, through systematic testing of different substrates and fluorescent proteins. Targeted AktAR reported higher Akt activity with faster activation kinetics within lipid rafts compared with nonraft regions of plasma membrane. Disruption of rafts attenuated platelet-derived growth factor (PDGF)-stimulated Akt activity in rafts without affecting that in nonraft regions. However, in insulin-like growth factor-1 (IGF)-1 stimulation, Akt signaling in nonraft regions is dependent on that in raft regions. As a result, cholesterol depletion diminishes Akt activity in both regions. Thus, Akt activities are differentially regulated in different membrane microdomains, and the overall activity of this oncogenic pathway is dependent on raft function. Given the increased abundance of lipid rafts in some cancer cells, the distinct Akt-activating characteristics of PDGF and IGF-1, in terms of both effectiveness and raft dependence, demonstrate the capabilities of different growth factor signaling pathways to transduce differential oncogenic signals across plasma membrane.     

Mycobacterium tuberculosis lipoprotein-induced association of TLR2 with protein kinase C zeta in lipid rafts contributes to reactive oxygen species-dependent inflammatory signalling in macrophages

Membrane lipid rafts are enriched in cholesterol and play an important role as signalling platforms. However, the roles of lipid rafts and associated signalling molecules in the innate immune responses to mycobacteria remain unknown. Here we show that stimulation with Mycobacterium tuberculosis 19 kDa lipoprotein, a TLR2/1 agonist, results in translocation of TLR2 to lipid rafts, coalescence of lipid rafts and production of reactive oxygen species (ROS) that drive pro-inflammatory responses. Disruption of lipid raft organization markedly reduced lipoprotein-induced ROS and inflammatory responses. Remarkably, the atypical protein kinase C (PKC) ζ was specifically recruited into detergent-resistant membrane fractions and associated with TLR2. PKCζ activity was critical for lipoprotein-dependent ROS generation, raft coalescence and the pro-inflammatory responses by macrophages. Moreover, lipid raft organization was required for 19 kDa mediated PKCζ activation. These results demonstrate that TLR2 trafficking and raft coalescence play an essential role for the initiation of lipoprotein-induced innate immune responses via TLR2 and ROS signalling. In addition, PKCζ targets to lipid rafts and may act as a critical adaptor molecule to regulate lipid raft dynamics during TLR2 signalling.     

Lipolysis products from triglyceride-rich lipoproteins increase endothelial permeability, perturb zonula occludens-1 and F-actin, and induce apoptosis

Products generated from lipoprotein lipase-mediated hydrolysis of triglyceride-rich lipoproteins (TGRL) are reported to increase endothelial layer permeability. We hypothesize that these increases in permeability result from the active rearrangement and dissolution of the junctional barrier in human aortic endothelial cells, as well as induction of the apoptotic cascade. Human aortic endothelial cells were treated with TGRL lipolysis products generated from coincubation of human TGRL plus lipoprotein lipase. Measurement of transendothelial electrical resistance demonstrated a time-dependent decrease in endothelial barrier function in response to TGRL lipolysis products. Immunofluorescent localization of zonula occludens-1 (ZO-1) showed radial rearrangement along cell borders after 1.5 h of treatment with lipolysis products. A concurrent redistribution of F-actin from the cell body to the cell margins was observed via rhodamine phalloidin staining. Immunofluorescent imaging for occludin and vascular endothelial cadherin showed that these proteins relocalize as well, although these changes are less prominent than for ZO-1. Western analysis of cells exposed to lipolysis products for 3 h revealed the fragmentation of ZO-1, a reduction in occludin, and no change of vascular endothelial cadherin. Lipolysis products also increased caspase-3 activity and induced nuclear fragmentation. Treatments did not cause oncosis in cells at any point during the incubation. These results demonstrate that TGRL lipolysis products play an important role in the regulation of endothelial permeability, the organization of the actin cytoskeleton, the localization and expression of junctional proteins, especially ZO-1, and the induction of apoptosis.     

The death-inducing signalling complex is recruited to lipid rafts in Fas-induced apoptosis

Membrane microdomains known as lipid rafts have been shown recently to be involved in Fas signalling and apoptosis in T and B cell lines. Here, we have investigated further the role of lipid rafts in Fas-induced apoptosis in non-transformed human CD4 T cells. We show that Fas-induced apoptosis in CD4 T cells was inhibited by the lipid raft disrupter methyl-beta-cyclodextrin. When lipid rafts were isolated from control and Fas ligand treated cells, we found that a small proportion of Fas was present in the raft fraction in untreated cells and that this was greatly increased upon Fas ligation. The other components of the Death Inducing Signalling Complex (DISC), FADD, and procaspase 8, were also present at higher levels in the raft fraction isolated from Fas ligand treated cells. We conclude that formation of the DISC occurs in lipid rafts and that these membrane microdomains are required for efficient Fas signalling and apoptosis.     

A novel approach to examining compositional heterogeneity of detergent-resistant lipid rafts

Lipid rafts play an important role in cell signalling, cell adhesion and other cellular functions. Compositional heterogeneity of lipid rafts provides one mechanism of how lipid rafts provide the spatial and temporal regulation of cell signalling and cell adhesion. The constitutive presence of some signalling receptors/molecules and accumulation of others in the lipid raft allows them to interact with each other and thereby facilitate relay of signals from the plasma membrane to the cell interior. Devising a method that can analyze these lipid microdomains for the presence of signalling receptors/molecules on an individual raft basis is required to address the issue of lipid raft heterogeneity. SDS-PAGE analysis, currently used for analyses of detergent-resistant lipid rafts, does not address this question. We have designed a cell-free assay that captures detergent-resistant lipid rafts with an antibody against a raft-resident molecule and detects the presence of another lipid raft molecule. Our results suggest that detergent-resistant lipid rafts, also known as detergent-resistant membranes, are heterogeneous populations on an immortalized mouse T-cell plasma membrane with respect to antigen receptor/signalling complex and other signalling/adhesion proteins. This cell-free assay provides a simple and quick way to examine the simultaneous presence of two proteins in the lipid rafts and has the potential to estimate trafficking of molecules in and out of the lipid microdomains during cell signalling on a single detergent-resistant lipid raft basis.     

MHC class II molecules traffic into lipid rafts during intracellular transport

There have been many studies demonstrating that a portion of MHC class II molecules reside in detergent-insoluble membrane domains (commonly referred to as lipid rafts). We have proposed that the function of raft association is to concentrate specific MHC class II-peptide complexes in plasma membrane microdomains that can facilitate efficient T cell activation. We now show that MHC class II becomes lipid raft associated before binding antigenic peptides. Using pulse-chase radiolabeling techniques, we find that newly synthesized MHC class II and MHC class II-invariant chain complexes initially reside in a detergent-soluble membrane fraction and acquire detergent insolubility as they traffic to lysosomal Ag processing compartments. Monensin, an inhibitor of protein transport through the Golgi apparatus, blocks association of newly synthesized MHC class II with lipid rafts. Treatment of cells with leupeptin, which inhibits invariant chain degradation, leads to the accumulation of MHC class II in lipid rafts within the lysosome-like Ag-processing compartments. Raft fractionation of lysosomal membranes confirmed the presence of MHC class II in detergent-insoluble microdomains in Ag-processing compartments. These findings indicate that newly synthesized MHC class II complexes are directed to detergent-insoluble lipid raft microdomains before peptide loading, a process that may facilitate the loading of similar peptides on MHC class II complexes in these microdomains.     

Candida albicans Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses

Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-β-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated β-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.     

Cold Induces Micro- and Nano-Scale Reorganization of Lipid Raft Markers at Mounds of T-Cell Membrane Fluctuations

Whether and how cold causes changes in cell-membrane or lipid rafts remain poorly characterized. Using the NSOM/QD and confocal imaging systems, we found that cold caused microscale redistribution of lipid raft markers, GM1 for lipid and CD59 for protein, from the peripheral part of microdomains to the central part on Jurkat T cells, and that cold also induced the nanoscale size-enlargement (1/3- to 2/3-fold) of the nanoclusters of lipid raft markers and even the colocalization of GM1 and CD59 nanoclusters. These findings indicate cold-induced lateral rearrangement/coalescence of raft-related membrane heterogeneity. The cold-induced re-distribution of lipid raft markers under a nearly-natural condition provide clues for their alternations, and help to propose a model in which raft lipids associate themselves or interact with protein components to generate functional membrane heterogeneity in response to stimulus. The data also underscore the possible cold-induced artifacts in early-described cold-related experiments and the detergent-resistance-based analyses of lipid rafts at 4°C, and provide a biophysical explanation for recently-reported cold-induced activation of signaling pathways in T cells. Importantly, our fluorescence-topographic NSOM imaging demonstrated that GM1/CD59 raft markers distributed and re-distributed at mounds but not depressions of T-cell membrane fluctuations. Such mound-top distribution of lipid raft markers or lipid rafts provides spatial advantage for lipid rafts or contact molecules interacting readily with neighboring cells or free molecules.     

Lipid rafts serve as signaling platforms for Tie2 receptor tyrosine kinase in vascular endothelial cells

The Tie2 receptor tyrosine kinase plays a pivotal role in vascular and hematopoietic development. The major intracellular signaling systems activated by Tie2 in response to Angiopoietin-1 (Ang1) include the Akt and Erk1/2 pathways. Here, we investigated the role of cholesterol-rich plasma membrane microdomains (lipid rafts) in Tie2 regulation. Tie2 could not be detected in the lipid raft fraction of human umbilical vein endothelial cells (HUVECs) unless they were first stimulated with Ang1. After stimulation, a minor fraction of Tie2 associated tightly with the lipid rafts. Treatment of HUVECs with the lipid raft disrupting agent methyl-β-cyclodextrin selectively inhibited Ang1-induced Akt phosphorylation, but not Erk1/2 phosphorylation. It has been reported that inhibition of FoxO activity is an important mechanism for Ang1-stimulated Tie2-mediated endothelial function. Consistent with this, we found that phosphorylation of FoxO mediated by Tie2 activation was attenuated by lipid raft disruption. Therefore, we propose that lipid rafts serve as signaling platforms for Tie2 receptor tyrosine kinase in vascular endothelial cells, especially for the Akt pathway.     

Gag induces the coalescence of clustered lipid rafts and tetraspanin-enriched microdomains at HIV-1 assembly sites on the plasma membrane

The HIV-1 structural protein Gag associates with two types of plasma membrane microdomains, lipid rafts and tetraspanin-enriched microdomains (TEMs), both of which have been proposed to be platforms for HIV-1 assembly. However, a variety of studies have demonstrated that lipid rafts and TEMs are distinct microdomains in the absence of HIV-1 infection. To measure the impact of Gag on microdomain behaviors, we took advantage of two assays: an antibody-mediated copatching assay and a Förster resonance energy transfer (FRET) assay that measures the clustering of microdomain markers in live cells without antibody-mediated patching. We found that lipid rafts and TEMs copatched and clustered to a greater extent in the presence of membrane-bound Gag in both assays, suggesting that Gag induces the coalescence of lipid rafts and TEMs. Substitutions in membrane binding motifs of Gag revealed that, while Gag membrane binding is necessary to induce coalescence of lipid rafts and TEMs, either acylation of Gag or binding of phosphatidylinositol-(4,5)-bisphosphate is sufficient. Finally, a Gag derivative that is defective in inducing membrane curvature appeared less able to induce lipid raft and TEM coalescence. A higher-resolution analysis of assembly sites by correlative fluorescence and scanning electron microscopy showed that coalescence of clustered lipid rafts and TEMs occurs predominately at completed cell surface virus-like particles, whereas a transmembrane raft marker protein appeared to associate with punctate Gag fluorescence even in the absence of cell surface particles. Together, these results suggest that different membrane microdomain components are recruited in a stepwise manner during assembly.     

Ternary SNARE complexes are enriched in lipid rafts during mast cell exocytosis

Lipid rafts are membrane microdomains rich in cholesterol and glycosphingolipids that have been implicated in the regulation of intracellular protein trafficking. During exocytosis, a class of proteins termed SNAREs mediate secretory granule-plasma membrane fusion. To investigate the role of lipid rafts in secretory granule exocytosis, we examined the raft association of SNARE proteins and SNARE complexes in rat basophilic leukemia (RBL) mast cells. The SNARE protein SNAP-23 co-localized with a lipid raft marker and was present in detergent-insoluble lipid raft microdomains in RBL cells. By contrast, only small amounts (<20%) of the plasma membrane SNARE syntaxin 4 or the granule-associated SNARE vesicle-associated membrane protein (VAMP)-2 were present in these microdomains. Despite this, essentially all syntaxin 4 and most of VAMP-2 in these rafts were present in SNARE complexes containing SNAP-23, while essentially none of these complexes were present in nonraft membranes. Whereas SNAP-23 is membrane anchored by palmitoylation, the association of the transmembrane protein syntaxin 4 with lipid rafts was because of its binding to SNAP-23. After stimulating mast cells exocytosis, the amount of syntaxin 4 and VAMP-2 present in rafts increased twofold, and these proteins were now present in raft-associated phospho-SNAP-23/syntaxin 4/VAMP-2 complexes, revealing differential association of SNARE fusion complexes during the process of regulated exocytosis.     

大鼠肝再生过程中肝细胞质膜微区差异蛋白质鉴定与分析

动物肝是具有极强再生能力的器官,研究并阐明肝再生的机制可为肝移植等与肝损伤相关的疾病治疗提供理论依据.质膜包括“脂筏(lipid rafts)”和“质膜微囊(caveolae)”的微区,具有参与胞吞胞饮、信号转导、运输胆固醇等重要功能.肝再生过程中,肝质膜微区脂筏蛋白质受到内部调控的影响会发生改变. 捕获脂筏微区信号蛋白分布的变化,对于理解和阐明肝再生过程中信号通路途径有重要意义.本研究应用成熟的大鼠2/3肝切除模型结合蔗糖密度梯度离心法,提取假手术组与肝再生组大鼠肝细胞质膜,并进一步纯化获得质膜微区蛋白质.通过SDS-PAGE分离以及ESI-Q-TOF质谱鉴定,对获得的质膜微区蛋白质进行差异分析. 结果显示,有30个微区蛋白质差异表达,其中13个上调、17个下调.生物信息学分析表明,所鉴定到的蛋白质主要参与细胞增殖、程序性死亡、细胞凋亡等调控,同时涉及到与肝再生密切相关的血管生成等信号通路.本文为质膜微区蛋白质的研究提供了方法上的参考以及相关基础数据,为后续临床肝再生的研究奠定了一定的基础.     

Direct visualization of Ras proteins in spatially distinct cell surface microdomains

Localization of signaling complexes to specific microdomains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains, including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent microdomain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins.     

Role of lipid rafts in membrane delivery of renal epithelial Na+-K+-ATPase, thick ascending limb

Lipid rafts are cholesterol- and shingolipid-enriched membrane microdomains implicated in membrane signaling and trafficking. To assess renal epithelial raft functions through the characterization of their associated membrane proteins, we have isolated lipid rafts from rat kidney by sucrose gradient fractionation after detergent treatment. The low-density fraction was enriched in cholesterol, sphingolipid, and flotillin-1 known as lipid raft markers. Based on proteomic analysis of the low-density fraction, the protein with the highest significance score was the alpha-subunit of Na(+)-K(+)-ATPase (NKA), whose raft association was validated by simultaneous immunoblotting. The beta-subunit of NKA was copurified from the low-density fraction. To test the role of lipid rafts in sorting and membrane delivery of renal-transporting epithelia, we have chosen to study thick ascending limb (TAL) epithelium for its high NKA activity and the property to be stimulated by antidiuretic hormone (ADH). Cultured rabbit TAL cells were studied. Cholesterol depletion and detergent extraction at warmth caused a shift of NKA to the higher-density fractions. Comparative preparations from blood monocytes revealed the absence of NKA from rafts in these nonpolarized cells. Short-term exposure of rabbit TAL cells to ADH (1 h) caused translocation and enhanced raft association of NKA via cAMP activation. Preceding cholesterol depletion prevented this effect. TAL-specific, glycosylphosphatidylinositol-anchored Tamm Horsfall protein was copurified with NKA in the same raft fraction, suggesting functional interference between these products. These results may have functional implications regarding the turnover, trafficking, and regulated surface expression of NKA as the major basolateral ion transporter of TAL.     

Nitric oxide inhibition of adenylyl cyclase type 6 activity is dependent upon lipid rafts and caveolin signaling complexes

Several cell types, including cardiac myocytes and vascular endothelial cells, produce nitric oxide (NO) via both constitutive and inducible isoforms of NO synthase. NO attenuates cardiac contractility and contributes to contractile dysfunction in heart failure, although the precise molecular mechanisms for these effects are poorly defined. Adenylyl cyclase (AC) isoforms type 5 and 6, which are preferentially expressed in cardiac myocytes, may be inhibited via a direct nitrosylation by NO. Because endothelial NO synthase (eNOS and NOS3), beta-adrenergic (betaAR) receptors, and AC6 all can localize in lipid raft/caveolin-rich microdomains, we sought to understand the role of lipid rafts in organizing components of betaAR-G(s)-AC signal transduction together with eNOS. Using neonatal rat cardiac myocytes, we found that disruption of lipid rafts with beta-cyclodextrin inhibited forskolin-stimulated AC activity and cAMP production, eliminated caveolin-3-eNOS interaction, and increased NO production. betaAR- and G(s)-mediated activation of AC activity were inhibited by beta-cyclodextrin treatment, but prostanoid receptor-stimulated AC activity, which appears to occur outside caveolin-rich microdomains, was unaffected unless eNOS was overexpressed and lipid rafts were disrupted. An NO donor, SNAP, inhibited basal and forskolin-stimulated cAMP production in both native cardiac myocytes and cardiac myocytes and pulmonary artery endothelial cells engineered to overexpress AC6. These effects of SNAP were independent of guanylyl cyclase activity and were mimicked by overexpression of eNOS. The juxtaposition of eNOS with betaAR and AC types 5 and 6 results in selective regulation of betaAR by eNOS activity in lipid raft domains over other G(s)-coupled receptors localized in nonraft domains. Thus co-localization of multiple signaling components in lipid rafts provides key spatial regulation of AC activity.     

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.     

Rafts--the current picture

Although evidences that cell membrane contains microdomains are accumulating, the exact properties, diversity and levels of organization of small lipid patches built mainly of cholesterol and sphingomyelin, termed rafts, remain to be elucidated. Our understanding of the cell membrane is increasing with each new raft feature discovered. Nowadays rafts are suggested to act as sites of cell signaling events, to be a part of protein sorting machinery but also they are used by several pathogens as gates into the cells. It is still unclear how rafts are connected to the membrane skeleton and cytoskeleton and with how many different types of rafts are we actually dealing with. This review summarizes some of the most recent discoveries trying to make a view of the complex raft properties.