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.     

Cell membrane extensions, generated by mechanical constraint, are associated with a sustained lipid raft patching and an increased cell signaling

Platelet activation triggers an imbalance in plasma membrane phospholipids by a specific aminophospholipid outflux, resulting in filopodia formation. Similarly, the addition of a phospholipid excess in the outer leaflet of the plasma membrane induces cellular extensions and actin polymerization. The implication of membrane microdomains in sustaining these mechanical constraints remains, however, unknown and was investigated in human platelets and mouse fibroblasts. The disruption of lipid rafts by cholesterol depletion prevents actin polymerization and formation of cellular extensions. Phospholipid excess triggers raft patching underneath the cell extensions, recruitment of protein raft markers and increase of tyrosine phosphorylation of raft proteins. Using a mass spectrometric analysis of isolated platelet rafts, we identified tyrosine kinases and proteins implicated in the formation of cell membrane extensions, cell adhesion and motility. They are recruited to rafts in response to a mechanical constraint. Taken together, our results demonstrate that exogenous phospholipid addition causes a modulation of the lateral plasma membrane organization and an activation of the cell signaling triggering actin remodeling and the formation of cellular protrusions. Raft disruption abolishes these processes, demonstrating that their integrity is crucial for cell shape changes in response to a mechanical constraint on plasma membrane.     

Phosphotyrosine protein dynamics in cell membrane rafts of sphingosine-1-phosphate-stimulated human endothelium: Role in barrier enhancement

Sphingosine-1-phosphate (S1P), a lipid growth factor, is critical to the maintenance and enhancement of vascular barrier function via processes highly dependent upon cell membrane raft-mediated signaling events. Anti-phosphotyrosine 2 dimensional gel electrophoresis (2-DE) immunoblots confirmed that disruption of membrane raft formation (via methyl-β-cyclodextrin) inhibits S1P-induced protein tyrosine phosphorylation. To explore S1P-induced dynamic changes in membrane rafts, we used 2-D techniques to define proteins within detergent-resistant cell membrane rafts which are differentially expressed in S1P-challenged (1 μM, 5 min) human pulmonary artery endothelial cells (EC), with 57 protein spots exhibiting > 3-fold change. S1P induced the recruitment of over 20 cell membrane raft proteins exhibiting increasing levels of tyrosine phosphorylation including known barrier-regulatory proteins such as focal adhesion kinase (FAK), cortactin, p85α phosphatidylinositol 3-kinase (p85αPI3K), myosin light chain kinase (nmMLCK), filamin A/C, and the non-receptor tyrosine kinase, c-Abl. Reduced expression of either FAK, MLCK, cortactin, filamin A or filamin C by siRNA transfection significantly attenuated S1P-induced EC barrier enhancement. Furthermore, S1P induced cell membrane raft components, p-caveolin-1 and glycosphingolipid (GM1), to the plasma membrane and enhanced co-localization of membrane rafts with p-caveolin-1 and p-nmMLCK. These results suggest that S1P induces both the tyrosine phosphorylation and recruitment of key actin cytoskeletal proteins to membrane rafts, resulting in enhanced human EC barrier function.     

Lipid rafts-protein association and the regulation of protein activity

Lipid rafts are membrane microdomains enriched in saturated phospholipids, sphingolipids, and cholesterol. They have a varied but distinct protein composition and have been implicated in diverse cellular processes including polarized traffic, signal transduction, endo- and exo-cytoses, entrance of obligate intracellular pathogens, and generation of pathological forms of proteins associated with Alzheimer's and prion diseases. Raft proteins can be permanently or temporarily associated to lipid rafts. Here, we review recent advances on the biochemical and cell biological characterization of rafts, and on the emerging concept of the temporary residency of proteins in rafts as a regulatory mechanism of their biological activity.     

Dissecting the molecular function of reggie/flotillin proteins

Reggie-1/flotillin-2 and reggie-2/flotillin-1 are ubiquitously expressed, well-conserved proteins that are associated with membrane microdomains known as rafts. Studies from us and others have suggested a role in various cellular processes such as insulin signaling, T cell activation, membrane trafficking, phagocytosis, and epidermal growth factor receptor signaling. Recent findings also demonstrate that reggie-1 is associated with cell motility and transformation. However, the exact function of reggie proteins remains to be clarified. In this review, we will focus on some recent findings that have shed new light on the elusive molecular function of these highly interesting proteins. We will especially discuss the emerging role of reggie proteins in membrane receptor signaling and membrane trafficking, with emphasis on the regulation of the molecular function of reggies by post-translational modifications such as phosphorylation and lipid modifications.     

Protein and lipid trafficking induced in erythrocytes infected by malaria parasites

The human malaria parasite Plasmodium falciparum develops in a parasitophorous vacuolar membrane (PVM) within the mature red cell and extensively modifies structural and antigenic properties of this host cell. Recent studies shed significant new, mechanistic perspective on the underlying processes. There is finally, definitive evidence that despite the absence of endocytosis, transmembrane proteins in the host red cell membrane are imported in to the PVM. These are not major erythrocyte proteins but components that reside in detergent resistant membrane (DRM) rafts in red cell membrane and are detected in rafts in the PVM. Disruption of either erythrocyte or vacuolar rafts is detrimental to infection suggesting that raft proteins and lipids are essential for the parasitization of the red cell. On secretory export of parasite proteins: an ER secretory signal (SS) sequence is required for protein secretion to the PV. Proteins carrying an additional plastid targeting sequence (PTS) are also detected in the PV but subsequently delivered to the plastid organelle within the parasite, suggesting that the PTS may have a second function as an endocytic sorting signal. A distinct but yet undefined peptidic motif underlies protein transport across the PVM to the red cell (although all of the published data does not yet fit this model). Further multiple exported proteins transit through secretory 'cleft' structures, suggesting that clefts may be sorting compartments assembled by the parasite in the red cell.     

Biosynthesis, remodelling and functions of mammalian GPI-anchored proteins: recent progress

More than 100 mammalian proteins are post-translationally modified by glycosylphosphatidylinositol (GPI) at their C-termini and are anchored to the cell surface membrane via the lipid portion. GPI-anchored proteins (GPI-APs) have various functions, such as hydrolytic enzymes, receptors, adhesion molecules, complement regulatory proteins and other immunologically important proteins. GPI-anchored proteins are mainly associated with membrane microdomains or membrane rafts enriched in sphingolipids and cholesterol. It is thought that association with membrane rafts is important for GPI-APs in signal transduction and other functions. Here, we review recent progress in studies on biosynthesis, remodelling and functions of mammalian GPI-APs.     

脂筏在病毒感染中的作用

脂筏是细胞膜上富含鞘脂和胆固醇的微区结构,广泛分布于细胞的膜系统.脂筏中含有诸多信号分子和免疫受体,在细胞的生命活动中扮演非常重要的角色.更为重要的是,脂筏为细胞表面发生的蛋白质-蛋白质和蛋白质-脂类分子间的相互作用提供了平台.研究表明,很多病毒可以利用细胞膜表面的脂筏结构介导其侵入宿主细胞,一些病毒可以借助脂筏结构完成病毒颗粒的组装和出芽.本文将综述不同类型的病毒如SV40、HIV等借助脂筏完成入侵以及流感病毒等利用脂筏完成组装和出芽的证据及机理,并概述目前研究病毒与脂筏相互作用的方法及存在的问题.深入研究脂筏在病毒感染中的作用,将有助于对病毒与宿主细胞的相互作用的理解,从而可能发现新的、有效的对抗病毒的方法。     

Compartmentation of Fyn kinase with glycosylphosphatidylinositol-anchored molecules in oligodendrocytes facilitates kinase activation during myelination.

In many cell types, glycosylphosphatidylinositol (GPI)-anchored proteins are sequestered in detergent-resistant membrane rafts. These are plasma membrane microdomains enriched in glycosphingolipids and cholesterol and are suggested to be platforms for cell signaling. Concomitant with the synthesis of myelin glycosphingolipids, maturing oligodendrocytes progressively associate GPI-anchored proteins, including the adhesion molecules NCAM 120 and F3, in rafts. Here we show that these microdomains include Fyn and Lyn kinases. Both kinases are maximally active in myelin prepared from young animals, correlating with early stages of myelination. In the rafts, Fyn kinase is tightly associated with NCAM 120 and F3. In contrast, in oligodendrocyte progenitor cells lacking rafts or in raft-free membrane domains of more mature cells, F3 does not associate with Fyn. The addition of anti-F3 antibodies to oligodendrocytes results in stimulation of Fyn kinase specifically in rafts. Compartmentation of oligodendrocyte GPI-anchored proteins in rafts is thus a prerequisite for association with Fyn, permitting kinase activation. Interaction of oligodendrocyte F3 with axonal ligands such as L1 and ensuing kinase activation may play a crucial role in initiating myelination.     

Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins

Lipid rafts are microdomains present in the membrane of eukaryotic organisms and bacterial pathogens. They are characterized by having tightly packed lipids and a subset of specific proteins. Lipid rafts are associated with a variety of important biological processes including signaling and lateral sorting of proteins. To determine whether lipid rafts exist in the inner membrane of Borrelia burgdorferi, we separated the inner and outer membranes and analyzed the lipid constituents present in each membrane fraction. We found that both the inner and outer membranes have cholesterol and cholesterol glycolipids. Fluorescence anisotropy and FRET showed that lipids from both membranes can form rafts but have different abilities to do so. The analysis of the biochemically defined proteome of lipid rafts from the inner membrane revealed a diverse set of proteins, different from those associated with the outer membrane, with functions in protein trafficking, chemotaxis and signaling.     

The SPFH domain-containing proteins: more than lipid raft markers

Membrane microdomains with distinct lipid compositions, called lipid rafts, represent a potential mechanism for compartmentalizing cellular functions within the plane of biological membranes. SPFH domain-containing proteins are found in lipid raft microdomains in diverse cellular membranes. The functions of these proteins are just beginning to be elucidated. Recent advances in the understanding of structural features and their roles within lipid rafts include a potential function for SPFH proteins in the formation of membrane microdomains and lipid raft-associated processes, such as endocytosis and mechanosensation.     

Lipid rafts: cell surface platforms for T cell signaling

The Src family tyrosine kinase Lck is essential for T cell development and T cell receptor (TCR) signaling. Lck is post-translationally fatty acylated at its N-terminus conferring membrane targeting and concentration in plasma membrane lipid rafts, which are lipid-based organisational platforms. Confocal fluorescence microscopy shows that Lck colocalizes in rafts with GPI-linked proteins, the adaptor protein LAT and Ras, but not with non-raft membrane proteins including the protein tyrosine phosphatase CD45. The TCR also associates with lipid rafts and its cross-linking causes coaggregation of raft-associated proteins including Lck, but not of CD45. Cross-linking of either the TCR or rafts strongly induces specific tyrosine phosphorylation of the TCR in the rafts. Remarkably, raft patching alone induces signalling events analogous to TCR stimulation, with the same dependence on expression of key TCR signalling molecules. Our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalization of signaling proteins including Lck, LAT, and the TCR, while excluding CD45, thereby potentiating protein tyrosine phosphorylation and downstream signaling. We are currently testing this hypothesis as well as using imaging techniques such as fluorescence resonance energy transfer (FRET) microscopy to study the dynamics of proteins and lipids in lipid rafts in living cells undergoing signaling events. Recent data show that the key phosphoinositide PI(4,5)P2 is concentrated in T cell lipid rafts and that on stimulation of the cells it is rapidly converted to PI(3,4,5)P3 and diacylglycerol within rafts. Thus rafts are hotspots for both protein and lipid signalling pathways.     

Lipid raft heterogeneity in human peripheral blood T lymphoblasts: a mechanism for regulating the initiation of TCR signal transduction

Lateral mobility and spatial organization of proteins within the plasma membrane are likely to mediate the initial events coordinating T cell activation. Lipid rafts, distinct cholesterol/sphingolipid-rich membrane microdomains, provide a mechanism for this regulation by concentrating or excluding signaling proteins. We demonstrate in peripheral blood T cell lymphoblasts that immediate early phosphotyrosine signal transduction through the TCR complex is functionally dependent on a distinct population of lipid rafts. Specifically, cholesterol extraction destabilizes the membrane microdomains containing Lck, while the rafts containing the adapter protein linker for activation of T cells remain intact. Heterogeneity in the partitioning of these proteins in resting cells was confirmed by immunoelectron microscopy. After T cell activation, both Lck and the linker for activation of T cells colocalize to 50-100 nm microdomains in the plasma membrane, indicating that sequestration of these proteins into distinct lipid rafts may function to regulate the initiation of T cell signal transduction.     

MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration

Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and, similar to Rac1 KD cells, exhibited reduced cell spreading and migration. Results of rescue-of-function experiments by expression of MYADM or active Rac1L61 in cells knocked down for Rac1 or MYADM, respectively, are consistent with the idea that MYADM and Rac1 act on parallel pathways that lead to similar functional outcomes.     

Flotillins functionally organize the bacterial membrane

Proteins and lipids are heterogeneously distributed in biological membranes. The correct function of membrane proteins depends on spatiotemporal organization into defined membrane areas, called lipid domains or rafts. Lipid microdomains are therefore thought to assist compartmentalization of membranes. However, how lipid and protein assemblies are organized and whether proteins are actively involved in these processes remains poorly understood. We now have identified flotillins to be responsible for lateral segregation of defined membrane domains in the model organism Bacillus subtilis. We show that flotillins form large, dynamic assemblies that are able to influence membrane fluidity and prevent condensation of Laurdan stained membrane regions. Absence of flotillins in vivo leads to coalescence of distinct domains of high membrane order and, hence, loss of flotillins in the bacterial plasma‐membrane reduces membrane heterogeneity. We show that flotillins interact with various proteins involved in protein secretion, cell wall metabolism, transport and membrane‐related signalling processes. Importantly, maintenance of membrane heterogeneity is critical for vital cellular processes such as protein secretion.     

Exogenous Administration of Gangliosides Displaces GPI-anchored Proteins from Lipid Microdomains in Living Cells

Exogenous application of gangliosides to cells affects many cellular functions. We asked whether these effects could be attributed to the influence of gangliosides on the properties of sphingolipid-cholesterol microdomains on the plasma membrane, also termed rafts. The latter are envisaged as lateral assemblies of sphingolipids (including gangliosides), cholesterol, and a specific set of proteins. Rafts have been implicated in processes such as membrane trafficking, signal transduction, and cell adhesion. Recently, using a chemical cross-linking approach with Madin-Darby canine kidney (MDCK) cells permanently expressing a GPI-anchored form of growth hormone decay accelerating factor (GH-DAF) as a model system, we could show that GPI-anchored proteins are clustered in rafts in living cells. Moreover, this clustering was dependent on the level of cholesterol in the cell. Here we show that incubation of MDCK cells with gangliosides abolished subsequent chemical cross-linking of GH-DAF. Furthermore, insertion of gangliosides into the plasma membrane of MDCK GH-DAF cells renders GH-DAF soluble when subjected to extraction with Triton X-114 at 4 degrees C. Our data suggest that exogenous application of gangliosides displaces GPI-anchored proteins from sphingolipid-cholesterol microdomains in living cells.     

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.     

Proteomic analysis of detergent-resistant membranes from Candida albicans

Lipid rafts are membrane microdomains with a higher amount of saturated fatty acids and sterols than the rest of the membrane. They are more resistant to the action of non-anionic detergents, and are called, for this reason, detergent-resistant membranes (DRMs). Lipid rafts are involved in many cellular processes, like signaling, cytokinesis, response to environment, etc., and therefore must contain important proteins. We have obtained a fraction enriched in proteins from Candida albicans DRMs. The sample has been analyzed by SDS-PAGE and 29 proteins have been identified including markers for lipid rafts in Saccharomyces cerevisiae, like Pma1p and a glycosylphosphatidylinositol (GPI)-anchored protein belonging to the Phr family. Ecm33p, a GPI-anchored protein involved in cell wall biogenesis, has been found for the first time in lipid rafts. We have also identified proteins implicated in protein glycosylation, like the mannosyltransferases Mnn7p, Pmt2p and Mnt1p; proteins involved in lipid metabolism, like Erg11p and Scs7p; and heat shock proteins, like Ssa1p and Hsp90p. Most of the proteins identified are located in plasma, mitochondrial, Golgi or ER membranes, supporting the postulated existence of lipid-raft domains in all the membranes.     

脂筏的结构与功能

脂筏是膜脂双层内含有特殊脂质及蛋白质的微区.小窝是脂筏的一种类型,由胆固醇、鞘脂及蛋白质组成,以小窝蛋白为标记蛋白.脂筏的组分和结构特点有利于蛋白质之间相互作用和构象转化,可以参与信号转导和细胞蛋白质运转.一些感染性疾病、心血管疾病、肿瘤、肌营养不良症及朊病毒病等可能与脂筏功能紊乱有着密切的关系.     

Proteomic analysis of membrane rafts of melanoma cells identifies protein patterns characteristic of the tumor progression stage

The molecular mechanisms controlling the progression of melanoma from a localized tumor to an invasive and metastatic disease are poorly understood. In the attempt to start defining a functional protein profile of melanoma progression, we have analyzed by LC-MS/MS the proteins associated with detergent resistant membranes (DRMs), which are enriched in cholesterol/sphingolipids-containing membrane rafts, of melanoma cell lines derived from tumors at different stages of progression. Since membrane rafts are involved in several biological processes, including signal transduction and protein trafficking, we hypothesized that the association of proteins with rafts can be regulated during melanoma development and affect protein function and disease progression. We have identified a total of 177 proteins in the DRMs of the cell lines examined. Among these, we have found groups of proteins preferentially associated with DRMs of either less malignant radial growth phase/vertical growth phase (VGP) cells, or aggressive VGP and metastatic cells suggesting that melanoma cells with different degrees of malignancy have different DRM profiles. Moreover, some proteins were found in DRMs of only some cell lines despite being expressed at similar levels in all the cell lines examined, suggesting the existence of mechanisms controlling their association with DRMs. We expect that understanding the mechanisms regulating DRM targeting and the activity of the proteins differentially associated with DRMs in relation to cell malignancy will help identify new molecular determinants of melanoma progression.