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Results for “Membrane Microdomains”

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At least 19 recordsLinked to original sources

PubMed2005

Relationships between EGFR signaling-competent and endocytosis-competent membrane microdomains.

Membrane microdomains, the so-called lipid rafts, function as platforms to concentrate receptors and assemble the signal transduction machinery. Internalization, in most cases, is carried out by different specialized structures, the clathrin-coated pits. Here, we show that several endocytic proteins are efficiently recruited to morphologically identified plasma membrane lipid rafts, upon activation of the epidermal growth factor (EGF) receptor (EGFR), a receptor tyrosine kinase. Analysis of detergent-resistant membrane fractions revealed that the EGF-dependent association of endocytic proteins with rafts is as efficient as that of signaling effector molecules, such as Grb2 or Shc. Finally, the EGFR, but not the nonsignaling transferrin receptor, could be localized in nascent coated pits that almost invariably contained raft membranes. Thus, specialized membrane microdomains have the ability to assemble both the molecular machineries necessary for intracellular propagation of EGFR effector signals and for receptor internalization.

Cell Culture Techniques↗

PubMed2001

Caveolae/lipid rafts in fibroblast-like synoviocytes: ectopeptidase-rich membrane microdomains.

Membrane peptidases play important roles in cell activation, proliferation and communication. Human fibroblast-like synoviocytes express considerable amounts of aminopeptidase N/CD13, dipeptidyl peptidase IV/CD26, and neprilysin/CD10, transmembrane proteins previously proposed to be involved in the regulation of intra-articular levels of neuropeptides and chemotactic mediators as well as in adhesion and cell-cell interactions. Here, we report these peptidases in synoviocytes to be localized predominantly in glycolipid- and cholesterol-rich membrane microdomains known as 'rafts'. At the ultrastructural level, aminopeptidase N/CD13 and dipeptidyl peptidase IV/CD26 were found in caveolae, in particular in intracellular yet surface-connected vesicle-like structures and 'rosettes' made up of several caveolae. In addition, clusters of peptidases were seen at the cell surface in flat patches ranging in size from about 60 to 160 nm. Cholesterol depletion of synoviocytes by methyl-beta-cyclodextrin disrupted >90% of the caveolae and reduced the raft localization of aminopeptidase N/CD13 without affecting Ala-p-nitroanilide-cleaving activity of confluent cell cultures. In co-culture experiments with T-lymphocytes, cholesterol depletion of synoviocytes greatly reduced their capability to induce an early lymphocytic expression of aminopeptidase N/CD13. We propose caveolae/rafts to be peptidase-rich 'hot-spot' regions of the synoviocyte plasma membrane required for functional cell-cell interactions with lymphocytes. The peptidases may act in concert with other types of proteins such as receptors and signal transducers localized in these specialized membrane domains.

Blotting, Western↗

PubMed2002

Cholesterol-dependent gamma-secretase activity in buoyant cholesterol-rich membrane microdomains.

Buoyant membrane fractions containing presenilin 1 (PS1), an essential component of the gamma-secretase complex, and APP CTFbeta, a gamma-secretase substrate, can be isolated from cultured cells and brain by several different fractionation procedures that are compatible with in vitro gamma-secretase assays. Analysis of these gradients for amyloid beta protein (Abeta) and CTFgamma production indicated that gamma-secretase activity is predominantly localized in these buoyant membrane microdomains. Consistent with this localization, we find that gamma-secretase activity is cholesterol dependent. Depletion of membrane cholesterol completely inhibits gamma-secretase cleavage, which can be restored by cholesterol replacement. Thus, altering cholesterol levels may influence the development of Alzheimer's disease (AD) by influencing production and deposition of Abeta within cholesterol rich membrane microdomains.

Alzheimer Disease↗

PubMed2005

Proteomics of plasma membrane microdomains.

Plasma membrane microdomains represent subcompartments of the plasma membrane characterized by a specific lipid and protein composition. The recognition of microdomains in nearly all the eukaryotic membranes has accredited them with specialized functions in health and disease. Several proteomic studies have recently addressed the specific composition of plasma membrane microdomains, and will be reviewed in this paper. Peculiar information has been obtained, but a comprehensive view of the main protein classes required to define the microdomain proteome is still missing. The achievement of this information is slowed by the difficulties encountered in resolving and analyzing hydrophobic proteins, but it could help in understanding the overall function of plasma membrane microdomains and their involvement in human pathology.

Cell Membrane↗

PubMed2005

Developmental partitioning of myelin basic protein into membrane microdomains.

Specific membrane microdomains (including lipid rafts) exist in myelin but have not been fully characterized. Myelin basic protein (MBP) maintains the compactness of the myelin sheath and is highly posttranslationally modified. Thus, it has been suggested that MBP might also have other functions, e.g., in signal transduction. Here, the distribution of MBP and its modified forms was studied, spatially and temporally, by detailed characterization of membrane microdomains from developing and mature bovine myelin. Myelin membranes were extracted with three different detergents (Brij 96V, CHAPS, or Triton X-100) at 4 degrees C. The detergent-resistant membranes (DRMs), representing coalesced lipid rafts, were isolated as low-buoyant-density fractions on a sucrose density gradient. These myelin rafts were disrupted when cholesterol was depleted with methyl-beta-cyclodextrin. The use of CHAPS detergent led to enrichment of several myelin proteins, including phospho-Thr97-MBP, in the DRMs from mature myelin. Citrullinated and methylated MBP remained in "nonraft" microdomains. In contrast, the DRMs from early myelin were enriched in Golli-MBP, Fyn, Lyn, and CNP. The localization of various proteins in DRMs was further supported by the colocalization of these lipid raft components in cultured mouse oligodendrocytes. Thus, there is a developmental regulation of posttranslationally modified forms of MBP into specific membrane microdomains.

Animals↗

PubMed2006

Insulin resistance as a membrane microdomain disorder.

Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling, but their role in the pathogenesis of insulin resistance has not been investigated. Detergent-resistant membrane microdomains (DRMs), isolated in the low density fractions, are highly enriched in cholesterol, glycosphingolipids and various signaling molecules. TNFalpha induces insulin resistance in type 2 diabetes, but its mechanism of action is not fully understood. We have found a selective increase in the acidic glycosphingolipid ganglioside GM3 in 3T3-L1 adipocytes treated with TNFalpha, suggesting a specific function for GM3. We were able to extend these in vitro observations to living animals using obese Zucker fa/fa rats and ob/ob mice, in which the GM3 synthase mRNA levels in the white adipose tissues are significantly higher than in their lean controls. In the DRMs from TNFalpha-treated 3T3-L1 adipocytes, GM3 levels were doubled, compared to results in normal adipocytes. Additionally, insulin receptor (IR) accumulations in the DRMs were diminished, while caveolin and flotillin levels were unchanged. GM3 depletion was able to counteract the TNFalpha-induced inhibition of IR accumulation into DRMs. Together, these findings provide compelling evidence that in insulin resistance the insulin metabolic signaling defect can be attributed to a loss of IRs in the microdomains due to an accumulation of GM3.

3T3-L1 Cells↗

PubMed1999

Cholesterol-dependent localization of NAP-22 on a neuronal membrane microdomain (raft).

A membrane microdomain called raft has been under extensive study since the assembly of various signal-transducing molecules into this region has been envisaged. This domain is isolated as a low buoyant membrane fraction after the extraction with a nonionic detergent such as Triton X-100. The characteristic low density of this fraction is ascribed to the enrichment of several lipids including cholesterol. To clear the molecular mechanism of raft formation, several extraction methods were applied to solubilize raft components. Cholesterol extraction using methyl-beta-cyclodextrin was found to be effective to solubilize NAP-22, a neuron-enriched Ca(2+)-dependent calmodulin-binding protein as well as one of the main protein components of brain raft. Purified NAP-22 bound to the liposomes that were made from phosphatidylcholine and cholesterol. This binding was dependent on the amount of cholesterol in liposomes. Calmodulin inhibited this binding in a dose-dependent manner. These results suggest that the presence of a calcium-dependent regulatory mechanism works on the assembly of raft within the neuron.

Animals↗

PubMed2005

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

Pseudotypes of vesicular stomatitis virus with CD4 formed by clustering of membrane microdomains during budding.

Many plasma membrane components are organized into detergent-resistant membrane microdomains referred to as lipid rafts. However, there is much less information about the organization of membrane components into microdomains outside of lipid rafts. Furthermore, there are few approaches to determine whether different membrane components are colocalized in microdomains as small as lipid rafts. We have previously described a new method of determining the extent of organization of proteins into membrane microdomains by analyzing the distribution of pairwise distances between immunogold particles in immunoelectron micrographs. We used this method to analyze the microdomains involved in the incorporation of the T-cell antigen CD4 into the envelope of vesicular stomatitis virus (VSV). In cells infected with a recombinant virus that expresses CD4 from the viral genome, both CD4 and the VSV envelope glycoprotein (G protein) were found in detergent-soluble (nonraft) membrane fractions. However, analysis of the distribution of CD4 and G protein in plasma membranes by immunoelectron microscopy showed that both were organized into membrane microdomains of similar sizes, approximately 100 to 150 nm. In regions of plasma membrane outside of virus budding sites, CD4 and G protein were present in separate membrane microdomains, as shown by double-label immunoelectron microscopy data. However, virus budding occurred from membrane microdomains that contained both G protein and CD4, and extended to approximately 300 nm, indicating that VSV pseudotype formation with CD4 occurs by clustering of G protein- and CD4-containing microdomains.

Animals↗

PubMed1999

CD4 segregates into specific detergent-resistant T-cell membrane microdomains.

In T cells, glycolipids, glycoproteins attached to the membrane via a glycosylphosphatidylinositol (GPI) anchor, and Src-like tyrosine kinases are highly enriched in a membrane fraction resistant to solubilization by nonionic detergents. We have investigated the distribution of CD4 in T-cell membranes and found that approximately 10% of the CD4 co-receptor is associated with detergent-insoluble membrane microdomains, whilst the remaining 90% is in soluble membranes. Moreover, approximately 60% of the "insoluble CD4" is present in membrane microdomains containing GPI-anchored proteins and high glycolipid-dependent kinase activity, whereas the remaining 40% displays no association with GPI-anchored proteins and lacks glycolipid-associated kinase activity These results indicate that CD4 segregates at least into three different membrane microenvironments: 1) soluble membranes; 2) insoluble membrane microdomains containing GPI-anchored proteins; and 3) insoluble membrane microdomains devoid of GPI-anchored proteins. The level of CD4 in insoluble membranes was not modified upon triggering activation by T-cell receptor-crosslinking but detectable amounts of CD3 subunits were recruited into these specialized membranes under those conditions. The physical separation of CD4 into different membrane microenvironments raises the possibility of that some of the multiple functions of CD4 might segregate into distinct types of lipid microenvironment. The fact that components of T-cell receptor/CD3 complex were recruited into insoluble membranes upon stimulation is consistent with the CD4 present in this membrane fraction might participate in T-cell receptor-triggered activation events.

Animals↗

PubMed2004

TNFalpha-induced insulin resistance in adipocytes as a membrane microdomain disorder: involvement of ganglioside GM3.

Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling, but their role in the pathogenesis of insulin resistance has not been investigated. Detergent-resistant membrane microdomains (DRMs), isolated in the low-density fractions, are highly enriched in cholesterol, glycosphingolipids and various signaling molecules. Tumor necrosis factor alpha (TNFalpha) induces insulin resistance in type 2 diabetes, but its mechanism of action is not fully understood. In other studies we have found a selective increase in the acidic glycosphingolipid ganglioside GM3 in 3T3-L1 adipocytes treated with TNFalpha, suggesting a specific function for GM3. In the DRMs from TNFalpha-treated 3T3-L1 adipocytes, GM3 levels were doubled compared with results in normal adipocytes. Additionally, insulin receptor (IR) accumulations in the DRMs were diminished, whereas caveolin and flotillin levels were unchanged. Furthermore, insulin-dependent IR internalization and intracellular movement of the IR substrate 1(IRS-1) were both greatly suppressed in the treated cells, leading to an uncoupling of IR-IRS-1 signaling. GM3 depletion was able to counteract the TNFalpha-induced inhibitions of IR internalization and accumulation into DRMs. Together, these findings provide compelling evidence that in insulin resistance the insulin metabolic signaling defect can be attributed to a loss of IRs in the microdomains due to an accumulation of GM3.

Adipocytes↗

PubMed2004

Transmembrane adaptor proteins in membrane microdomains: important regulators of immunoreceptor signaling.

Membrane microdomains enriched in glycosphingolipids, cholesterol, glycosylphosphatidylinositol-anchored proteins and Src-family kinases (lipid rafts, GEMs) appear to play many important roles, especially in immunoreceptor signaling. Most transmembrane proteins are excluded from these specialized areas of membranes, notable exceptions being several palmitoylated proteins such as the T cell coreceptors CD4 and CD8, and several recently described transmembrane adaptor proteins, LAT, non-T cell activation linker (NTAL)/linker for activation of B cells (LAB), phosphoprotein associated with GEMs (PAG)/Csk-binding protein (Cbp) and LIME. All these molecules possess a very short N-terminal extracellular peptide (4-17 amino acids), transmembrane segment followed by a palmitoylation motif (CxxC) and cytoplasmic domain containing up to 10 tyrosine residues potentially phosphorylated by the Src- or Syk-family kinases. Tyrosine-phosphorylated transmembrane adaptors bind (directly via SH2 domains or indirectly) other signaling molecules such as several cytoplasmic adaptors and enzymes. LAT is indispensable for TCR signaling (and participates also at signal transduction initiated by some other receptors), NTAL/LAB appears to play a LAT-like role in signaling initiated by BCR and some Fc-receptors; PAG/Cbp cooperates with Csk, the cytoplasmic tyrosine kinase negatively regulating Src-family kinases. Additional transmembrane adaptors exist (TRIM, SIT, LAX) that are however not palmitoylated and therefore excluded from the lipid rafts; structurally and functionally, the zeta-chain family proteins tightly associated with immunoreceptors and activating NK-receptors may be also considered as transmembrane adaptors.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Organization of the vesicular stomatitis virus glycoprotein into membrane microdomains occurs independently of intracellular viral components.

The glycoprotein (G protein) of vesicular stomatitis virus (VSV) is primarily organized in plasma membranes of infected cells into membrane microdomains with diameters of 100 to 150 nm, with smaller amounts organized into microdomains of larger sizes. This organization has been observed in areas of the infected-cell plasma membrane that are outside of virus budding sites as well as in the envelopes of budding virions. These observations raise the question of whether the intracellular virion components play a role in organizing the G protein into membrane microdomains. Immunogold-labeling electron microscopy was used to analyze the distribution of the G protein in arbitrarily chosen areas of plasma membranes of transfected cells that expressed the G protein in the absence of other viral components. Similar to the results with virus-infected cells, the G protein was organized predominantly into membrane microdomains with diameters of approximately 100 to 150 nm. These results indicate that internal virion components are not required to concentrate the G protein into membrane microdomains with a density similar to that of virus envelopes. To determine if interactions between the G protein cytoplasmic domain and internal virion components were required to create a virus budding site, cells infected with recombinant VSVs encoding truncation mutations of the G protein cytoplasmic domain were analyzed by immunogold-labeling electron microscopy. Deletion of the cytoplasmic domain of the G protein did not alter its partitioning into the 100- to 150-nm microdomains, nor did it affect the incorporation of the G protein into virus envelopes. These data support a model for virus assembly in which the G protein has the inherent property of partitioning into membrane microdomains that then serve as the sites of assembly of internal virion components.

Animals↗

PubMed2000

Identification of low-density Triton X-100-insoluble plasma membrane microdomains in higher plants.

Low density Triton X-100-insoluble plasma membrane microdomains can be isolated from different mammalian cell types and are proposed to be involved in membrane trafficking, cell morphogenesis and signal transduction. Heterotrimeric G-proteins and their receptors are often associated with such domains, suggesting that these structures are involved in G-protein-coupled signaling. Here we report that detergent-insoluble plasma membrane microdomains also exist in higher plants and contain about 15% of membrane-bound heterotrimeric G-protein beta-subunit (Gbeta). Plasma membrane microdomains were isolated from tobacco leaves. They have low buoyant density relative to the surrounding plasma membrane, and are insoluble in Triton X-100 at 4 degrees C. Detergent-insoluble vesicles were examined by freeze-fracture electron microscopy. They have sizes in the range 100-400 nm, and often contain aggregated protein complexes. The majority of plasma membrane proteins cannot be detected in the Triton X-100-insoluble fraction, while few polypeptides are highly enriched. We identified six proteins with molecular masses of 22, 28, 35, 60, 67 and 94 kDa in detergent-insoluble fractions that are glycosylphosphatidylinositol (GPI)-anchored.

Cell Membrane↗

PubMed2003

A novel method for analysis of membrane microdomains: vesicular stomatitis virus glycoprotein microdomains change in size during infection, and those outside of budding sites resemble sites of virus budding.

Membrane proteins, including viral envelope glycoproteins, may be organized into areas of locally high concentration, commonly referred to as membrane microdomains. Some viruses bud from detergent-resistant microdomains referred to as lipid rafts. However, vesicular stomatitis virus (VSV) serves as a prototype for viruses that bud from areas of plasma membrane that are not detergent resistant. We developed a new analytical method for immunoelectron microscopy data to determine whether the VSV envelope glycoprotein (G protein) is organized into plasma membrane microdomains. This method was used to quantify the distribution of the G protein in microdomains in areas of plasma membrane that did not contain budding sites. These microdomains were compared to budding virus envelopes to address the question of whether G protein-containing microdomains were formed only at the sites of budding. At early times postinfection, most of the G protein was organized into membrane microdomains outside of virus budding sites that were approximately 100-150 nm, with smaller amounts distributed into larger microdomains. In contrast to early times postinfection, the increased level of G protein in the host plasma membrane at later times postinfection led to distribution of G protein among membrane microdomains of a wider variety of sizes, rather than a higher G protein concentration in the 100- to 150-nm microdomains. VSV budding occurred in G protein-containing microdomains with a range of sizes, some of which were smaller than the virus envelope. These microdomains extended in size to a maximum of 300-400 nm from the tip of the budding virion. The data support a model for virus assembly in which G protein organizes into membrane microdomains that resemble virus envelopes prior to formation of budding sites, and these microdomains serve as the sites of assembly of internal virion components.

Animals↗

PubMed2006

CDw78 defines MHC class II-peptide complexes that require Ii chain-dependent lysosomal trafficking, not localization to a specific tetraspanin membrane microdomain.

MHC class II molecules (MHC-II) associate with detergent-resistant membrane microdomains, termed lipid rafts, which affects the function of these molecules during Ag presentation to CD4+ T cells. Recently, it has been proposed that MHC-II also associates with another type of membrane microdomain, termed tetraspan microdomains. These microdomains are defined by association of molecules to a family of proteins that contain four-transmembrane regions, called tetraspanins. It has been suggested that MHC-II associated with tetraspanins are selectively identified by a mAb to a MHC-II determinant, CDw78. In this report, we have re-examined this issue of CDw78 expression and MHC-II-association with tetraspanins in human dendritic cells, a variety of human B cell lines, and MHC-II-expressing HeLa cells. We find no correlation between the expression of CDw78 and the expression of tetraspanins CD81, CD82, CD53, CD9, and CD37. Furthermore, we find that the relative amount of tetraspanins bound to CDw78-reactive MHC-II is indistinguishable from the amount bound to peptide-loaded MHC-II. We found that expression of CDw78 required coexpression of MHC-II together with its chaperone Ii chain. In addition, analysis of a panel of MHC-II-expressing B cell lines revealed that different alleles of HLA-DR express different amounts of CDw78 reactivity. We conclude that CDw78 defines a conformation of MHC-II bound to peptides that are acquired through trafficking to lysosomal Ag-processing compartments and not MHC-II-associated with tetraspanins.

Antigen Presentation↗

PubMed2001

Localization of neuronal growth-associated, microtubule-destabilizing factor SCG10 in brain-derived raft membrane microdomains.

Raft is a mobile membrane subdomain enriched in sphingolipid and cholesterol and also various signaling molecules. Previous observation suggested that brain-derived rafts contain tubulin but that rafts of non-neural origin do not. We hypothesized that SCG10, one of the neuronal growth-associated proteins (nGAPs), might be a neuron-specific molecule that anchors tubulin to neuronal rafts, and we explored biochemically its subcellular localization, interaction with tubulin, and effects on microtubule dynamics. In postnatal rat brain extracts, SCG10 was recovered mostly in membrane-associated fractions, and at least half was included in the raft fraction that was also enriched in GAP-43 and NAP-22. SCG10-enriched brain rafts also contained tubulin, and chemical cross-linking experiments revealed that SCG10 was closely associated with tubulin. In addition, SCG10 was able to inhibit polymerization of tubulin. These results indicate that SCG10 is a component of neuronal rafts as are other nGAPs, and suggest that SCG10 may be involved in signaling events in membranes for cytoskeletal reorganization around neuronal rafts.

Animals↗

PubMed2003

Constitutive localization of the gonadotropin-releasing hormone (GnRH) receptor to low density membrane microdomains is necessary for GnRH signaling to ERK.

Specialized membrane microdomains known as lipid rafts are thought to contribute to G-protein coupled receptor (GPCR) signaling by organizing receptors and their cognate signaling molecules into discrete membrane domains. To determine if the GnRHR, an unusual member of the GPCR superfamily, partitions into lipid rafts, homogenates of alpha T3-1 cells expressing endogenous GnRHR or Chinese hamster ovary cells expressing an epitope-tagged GnRHR were fractionated through a sucrose gradient. We found the GnRHR and c-raf kinase constitutively localized to low density fractions independent of hormone treatment. Partitioning of c-raf kinase into lipid rafts was also observed in whole mouse pituitary glands. Consistent with GnRH induced phosphorylation and activation of c-raf kinase, GnRH treatment led to a decrease in the apparent electrophoretic mobility of c-raf kinase that partitioned into lipid rafts compared with unstimulated cells. Cholesterol depletion of alpha T3-1 cells using methyl-beta-cyclodextrin disrupted GnRHR but not c-raf kinase association with rafts and shifted the receptor into higher density fractions. Cholesterol depletion also significantly attenuated GnRH but not phorbol ester-mediated activation of extracellular signal-related kinase (ERK) and c-fos gene induction. Raft localization and GnRHR signaling to ERK and c-Fos were rescued upon repletion of membrane cholesterol. Thus, the organization of the GnRHR into low density membrane microdomains appears critical in mediating GnRH induced intracellular signaling.

Animals↗