Glycolipid-enriched Membrane Domains Research Articles

Distinct Origin of Claudin7 in Early Tumor Endosomes Affects Exosome Assembly.

Microvesicles are the body's most powerful intercellular communication system and cancer-initiating cell microvesicles (CIC-TEX) reprogram Non-CIC towards fortified malignancy. Claudin7, a CIC-biomarker in gastrointestinal tumors, is recovered in TEX. Recent evidence suggesting individual cells delivering distinct microvesicles became of particular interest for claudin7, which is part of tight junctions (TJ) and glycolipid-enriched membrane domains (GEM), GEM-located claudin7 is palmitoylated. This offered the unique possibility of exploring the contribution of a CIC marker and its origin from distinct membrane domains on CIC-TEX biogenesis and activities. Proteome and miRNA analysis of wild-type, claudin7-knockdown and a rescue with claudin7 harboring a mutated palmitoylation site (mP) of a rat pancreatic and a human colon cancer line uncovered significant, only partly overlapping contributions of palmitoylated and non-palmitoylated claudin7 to TEX composition. Palmitoylated claudin7 facilitates GEM-integrated plasma membrane and associated signaling molecule recruitment; non-palmitoylated claudin7 supports recruitment of trafficking components, proteins engaged in fatty acid metabolism and TJ proteins into TEX. Claudin7mP also assists TEX recovery of selected miRNA. Thus, distinctly located claudin7 affects CIC-TEX composition and TJ-derived cld7 might play a unique role in equipping CIC-TEX with transporters and lipid metabolism-regulating molecules, awareness of distinct TEX populations being crucial facing therapeutic translation.

Pancreatic cancer stem cell markers and exosomes - the incentive push

Pancreatic cancer (PaCa) has the highest death rate and incidence is increasing. Poor prognosis is due to late diagnosis and early metastatic spread, which is ascribed to a minor population of so called cancer stem cells (CSC) within the mass of the primary tumor. CSC are defined by biological features, which they share with adult stem cells like longevity, rare cell division, the capacity for self renewal, differentiation, drug resistance and the requirement for a niche. CSC can also be identified by sets of markers, which for pancreatic CSC (Pa-CSC) include CD44v6, c-Met, Tspan8, alpha6beta4, CXCR4, CD133, EpCAM and claudin7. The functional relevance of CSC markers is still disputed. We hypothesize that Pa-CSC markers play a decisive role in tumor progression. This is fostered by the location in glycolipid-enriched membrane domains, which function as signaling platform and support connectivity of the individual Pa-CSC markers. Outside-in signaling supports apoptosis resistance, stem cell gene expression and tumor suppressor gene repression as well as miRNA transcription and silencing. Pa-CSC markers also contribute to motility and invasiveness. By ligand binding host cells are triggered towards creating a milieu supporting Pa-CSC maintenance. Furthermore, CSC markers contribute to the generation, loading and delivery of exosomes, whereby CSC gain the capacity for a cell-cell contact independent crosstalk with the host and neighboring non-CSC. This allows Pa-CSC exosomes (TEX) to reprogram neighboring non-CSC towards epithelial mesenchymal transition and to stimulate host cells towards preparing a niche for metastasizing tumor cells. Finally, TEX communicate with the matrix to support tumor cell motility, invasion and homing. We will discuss the possibility that CSC markers are the initial trigger for these processes and what is the special contribution of CSC-TEX.

The importance of claudin-7 palmitoylation on membrane subdomain localization and metastasis-promoting activities.

BackgroundClaudin-7 (cld7), a tight junction (TJ) component, is also found basolaterally and in the cytoplasm. Basolaterally located cld7 is enriched in glycolipid-enriched membrane domains (GEM), where it associates with EpCAM (EpC). The conditions driving cld7 out of TJ into GEM, which is associated with a striking change in function, were not defined. Thus, we asked whether cld7 serines or palmitoylation affect cld7 location and protein, particularly EpCAM, associations.ResultsHEK cells were transfected with EpCAM and wild type cld7 or cld7, where serine phopsphorylation or the palmitoylation sites (AA184, AA186) (cld7mPalm) were mutated. Exchange of individual serine phosphorylation sites did not significantly affect the GEM localization and the EpCAM association. Instead, cld7mPalm was poorly recruited into GEM. This has consequences on migration and invasiveness as palmitoylated cld7 facilitates integrin and EpCAM recruitment, associates with cytoskeletal linker proteins and cooperates with MMP14, CD147 and TACE, which support motility, matrix degradation and EpCAM cleavage. On the other hand, only cld7mPalm associates with TJ proteins.ConclusionCld7 palmitoylation prohibits TJ integration and fosters GEM recruitment. Via associated molecules, palmitoylated cld7 supports motility and invasion.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-015-0105-y) contains supplementary material, which is available to authorized users.

EpCAM‐associated claudin‐7 supports lymphatic spread and drug resistance in rat pancreatic cancer

Pancreatic cancer has a dismal prognosis because of early metastatic spread, a suggested feature of cancer-initiating cells (CIC). To control for a functional contribution of the pancreatic CIC-marker EpCAM, we explored metastasis formation by a stable EpCAM-knockdown (ASML-EpC(kd)) of the rat pancreatic adenocarcinoma line BSp73ASML (ASML(wt)). As EpCAM associates with claudin-7, an ASML-claudin-7-knockdown (ASML-cld7(kd)) was included to differentiate between EpC- and EpC-cld7-mediated effects. The metastatic capacity of ASML-EpC(kd) and more pronounced ASML-cld7(kd) cells is strikingly reduced. EpC-associated cld7 interferes with EpC-mediated cell-cell adhesion and supports migration. This requires cld7 phosphorylation and formation of an EpC-cld7-tetraspanin-alpha6beta4 complex in glycolipid-enriched membrane domains (GEM), where cld7 associates via the tetraspanin-alpha6beta4 complex with phosphorylated ezrin. The association of cld7 with alpha6beta4 and cytoskeleton strongly stimulates tumor cell migration. However, EpC does not actively contribute. Instead, GEM-located cld7 associates with presenilin-2, which facilitates EpC cleavage and thereby tumor cell proliferation. Finally, the EpC-cld7 complex promotes drug resistance. Both EpC and cld7 support MAPK and JNK activation, such that in ASML-EpC(kd) and ASML-cld7(kd) cells an undue expansion of proapoptotic molecules is observed. Only cld7 promotes activation of the PI3K/Akt pathway by a strong downregulation of Pten. Accordingly, cisplatin treatment prolongs the survival time of ASML-cld7(kd)-bearing rats. Taken together, cld7 supports tumorigenic features of EpC by provoking EpC cleavage and thereby its cotranscription factor activity. On the other hand, only cld7 is directly engaged in motility and apoptosis resistance. Thus, at least in concern of migrating CIC, it is cld7 that acts as a CIC biomarker.

Translocation of Glycosylphosphatidylinositol-Anchored Proteins from Plasma Membrane Microdomains to Lipid Droplets in Rat Adipocytes Is Induced by Palmitate, H2O2, and the Sulfonylurea Drug Glimepiride

Inhibition of lipolysis by palmitate, H(2)O(2), and the antidiabetic sulfonylurea drug, glimepiride, in rat adipocytes has been shown previously to rely on the concerted degradation of cAMP by the glycosylphosphatidylinositol (GPI)-anchored phosphodiesterase Gce1 and 5'-nucleotidase CD73, which both gain access to the lipid droplets (LDs). The present report demonstrates the translocation of Gce1 and CD73, harboring the intact GPI anchor, from detergent-insoluble glycolipid-enriched plasma membrane domains (DIGs) to the LDs in response to palmitate, H(2)O(2), and glimepiride by analysis of their steady-state distribution using photoaffinity labeling and activity determination as well as of their redistribution after pulse or equilibrium metabolic labeling. We were surprised to find that palmitate, H(2)O(2), and glimepiride induced the activation of the GPI-specific phospholipase C (GPI-PLC) at DIGs of rat adipocytes, leading to anchorless Gce1 and CD73. Inhibition of the GPI-PLC or the presence of nonhydrolyzable substrate analogs of Gce1 and CD73 interfered with the palmitate-, H(2)O(2)-, and glimepiride-induced 1) lipolytic cleavage of Gce1 and CD73, 2) translocation of their GPI-anchored versions from DIGs to LDs, 3) up-regulation of cAMP degradation, and 4) inhibition of lipolysis. These data suggest a novel insulin-independent antilipolytic mechanism in rat adipocytes, which relies on the palmitate-, H(2)O(2)-, and glimepiride-induced and GPI-PLC-dependent translocation of (c)AMP-degrading GPI-anchored proteins from the adipocyte plasma membrane to LDs. The findings may shed new light on the biogenesis and degradation of LDs in response to physiological and pharmacological stimuli.

The Multi‐PDZ domain protein MUPP1 as a lipid raft‐associated scaffolding protein controlling the acrosome reaction in mammalian spermatozoa

The success of acrosomal exocytosis, a complex process with a variety of interrelated steps, relies on the coordinated interaction of participating signaling molecules. Since scaffolding proteins are known to spatially organize sequential signaling pathways, we examined whether the Multi-PDZ domain protein MUPP1, recently identified in mammalian spermatozoa, is functionally active in controlling acrosomal secretion in mammalian sperm cells. To address this question, permeabilized mouse sperm were loaded with inhibitory antibodies against MUPP1 as well as with a photosensitive Ca(2+) chelator which allows a controlled release of acrosomal Ca(2+). The results revealed that MUPP1 controls initial tethering and docking of the acrosomal vesicle, whereas syntaxin 2, a t-SNARE protein also expressed in the acrosomal cap of mammalian spermatozoa, appears to take part in the final process of acrosomal fusion. Interestingly, using immunogold electron microscopy, it was found that MUPP1 is detectable in the region of the periacrosomal membrane. Furthermore, in isolated detergent-insoluble glycolipid-enriched membrane domains from epididymal spermatozoa, MUPP1 was found to show a striking association with the Triton X-100 insoluble membrane fraction, which did not change significantly upon sperm capacitation or partial chemical extraction of cholesterol. This evidence points to a role of MUPP1 as a membrane raft-associated molecular organizer, and suggests that mammalian spermatozoa may use a scaffolding protein and distinct membrane subdomains to spatially organize components involved in the process of acrosomal exocytosis.

Shiga toxin binding to globotriaosyl ceramide induces intracellular signals that mediate cytoskeleton remodeling in human renal carcinoma-derived cells.

Shiga toxin is a bacterial toxin consisting of A and B subunits. Generally, the essential cytotoxicity of the toxin is thought to be mediated by the A subunit, which possesses RNA cleavage activity and thus induces protein synthesis inhibition. We previously reported, however, that the binding of the Shiga toxin 1-B subunit to globotriaosyl ceramide, a functional receptor for Shiga toxin, induces intracellular signals in a manner that is dependent on glycolipid-enriched membrane domains, or lipid rafts. Although the precise role of this signaling mechanism is not known, here we report that Shiga-toxin-mediated intracellular signals induce cytoskeleton remodeling in ACHN cells derived from renal tubular epithelial carcinoma. Using confocal laser scanning microscopy, we observed that Shiga toxin 1-B treatment induces morphological changes in ACHN cells in a time-dependent manner. In addition, the morphological changes were accompanied by the redistribution of a number of proteins, including actin, ezrin, CD44, vimentin, cytokeratin, paxillin, FAK, and alpha- and gamma-tubulins, all of which are involved in cytoskeletal organization. The transient phosphorylation of ezrin and paxillin was also observed during the course of protein redistribution. Experiments using inhibitors for a variety of kinases suggested the involvement of lipid rafts, Src family protein kinase, PI 3-kinase, and RHO-associated kinase in Shiga toxin 1-B-induced ezrin phosphorylation. Shiga toxin 1-B-induced cytoskeletal remodeling should provide an in vitro model that can be used to increase our understanding of the pathogenesis of Shiga-toxin-mediated cell injury and the role of lipid-raft-mediated cell signaling in cytoskeletal remodeling.

Visualization of antigen presentation by actin-mediated targeting of glycolipid-enriched membrane domains to the immune synapse of B cell APCs.

Glycolipid-enriched membrane (GEM) domains, or lipid rafts, function in signaling in immune cells, but their properties during Ag presentation are less clear. To address this question, GEM domains were studied using fluorescence cell imaging of mouse CH27 B cells presenting Ag to D10 T cells. Our experiments showed that APCs were enriched with GEM domains in the immune synapse, and this occurred in an actin-dependent manner. This enrichment was specific to GEM domains, because a marker for non-GEM regions of the membrane was excluded from the immune synapse. Furthermore, fluorescence photobleaching experiments showed that protein in the immune synapse was dynamic and rapidly exchanged with that in other compartments of CH27 cells. To identify the signals for targeting GEM domains to the immune synapse in APCs, capping of the domains was measured in cells after cross-linking surface molecules. This showed that co-cross-linking CD48 with MHC class II was required for efficient capping and intracellular signaling. Capping of GEM domains by co-cross-linking CD48 and MHC class II occurred with co-capping of filamentous actin, and both domain capping and T cell-CH27 cell conjugation were inhibited by pretreating CH27 cells with latrunculin B. Furthermore, disruption of the actin cytoskeleton of the CH27 cells also inhibited formation of a mature immune synapse in those T cells that did conjugate to APCs. Thus, Ag presentation and efficient T cell stimulation occur by an actin-dependent targeting of GEM domains in the APC to the site of T cell engagement.

T cell glycolipid-enriched membrane domains are constitutively assembled as membrane patches that translocate to immune synapses.

In T cells, glycolipid-enriched membrane (GEM) domains, or lipid rafts, are assembled into immune synapses in response to Ag presentation. However, the properties of T cell GEM domains in the absence of stimulatory signals, such as their size and distribution in the plasma membrane, are less clear. To address this question, we used confocal microscopy to measure GEM domains in unstimulated T cells expressing a GEM-targeted green fluorescent protein molecule. Our experiments showed that the GEM domains were assembled into membrane patches that were micrometers in size, as evidenced by a specific enrichment of GEM-associated molecules and resistance of the patches to extraction by Triton X-100. However, treatment of cells with latrunculin B disrupted the patching of the GEM domains and their resistance to Triton X-100. Similarly, the patches were coenriched with F-actin, and actin occurred in the detergent-resistant GEM fraction of T cells. Live-cell imaging showed that the patches were mobile and underwent translocation in the plasma membrane to immune synapses in stimulated T cells. Targeting of GEM domains to immune synapses was found to be actin-dependent, and required phosphatidylinositol 3-kinase activity and myosin motor proteins. We conclude from our results that T cell GEM domains are constitutively assembled by the actin cytoskeleton into micrometer-sized membrane patches, and that GEM domains and the GEM-enriched patches can function as a vehicle for targeting molecules to immune synapses.

Differential effects of p-glycoprotein and multidrug resistance protein-1 on productive human immunodeficiency virus infection.

P-glycoprotein (P-gp) and multidrug-resistance protein-1 (MRP-1) are adenosine triphosphate-binding cassette proteins that may decrease intracellular concentrations of anti-human immunodeficiency virus (HIV) drugs. After HIV-1(IIIB) infection, HIV-1 protein and infectious virus production were decreased by at least 70-fold in CEM cells overexpressing P-gp but were increased by at least 50-fold in CEM cells overexpressing MRP-1, compared with control CEM cells. After transfection with the HIV-1(IIIB) genome, cells overexpressing P-gp and MRP-1 expressed similar amounts of HIV protein. Selective inhibitors of MRP-1 and P-gp partially reversed the effect of these transporters in a concentration-dependent manner. P-gp preferentially associated with glycolipid-enriched membrane (GEM) domains, which may be an important site for cellular binding and egress of HIV. In contrast, MRP-1 was not preferentially found in GEM domains. These results suggest that the inhibition of HIV productive infection by P-gp and augmentation by MRP-1 occur predominantly at a preintegration step but act through different mechanisms.

Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cgamma2 regulation in platelets.

The platelet collagen receptor glycoprotein VI (GPVI) and the fibrinogen receptor integrin alphaIIbbeta3 trigger intracellular signalling cascades involving the tyrosine kinase Syk, the adapter SLP-76 and phospholipase Cgamma2 (PLCgamma2). Similar pathways are activated downstream of immune receptors in lymphocytes, where they have been localized in part to glycolipid-enriched membrane domains (GEMs). Here we provide several lines of evidence that GPVI-mediated tyrosine phosphorylation of PLCgamma2 in platelets is dependent on GEM-organized signalling and utilizes the GEM resident adapter protein LAT (linker for activation of T cells). In sharp contrast, although fibrinogen binding to platelets stimulates alphaIIbbeta3-dependent activation of Syk and tyrosine phosphorylation of SLP-76 and PLCgamma2, it does not utilize GEMs to promote these responses or to support platelet aggregation. These results establish that GPVI and alphaIIbbeta3 trigger distinct patterns of receptor signalling in platelets, leading to tyrosine phosphorylation of PLCgamma2, and they highlight the role of GEMs in compartmentalizing signalling reactions involved in haemostasis.

Glycoprotein IIb-IIIa-dependent aggregation by glycoprotein Ibalpha is reinforced by a Src family kinase inhibitor (PP1)-sensitive signalling pathway.

It has been proposed that the receptor for von Willebrand factor (vWF), glycoprotein (GP)Ib-IX-V, signals through the same pathway as the collagen receptor, GPVI, namely via Src kinases, the Fc receptor (FcR) gamma-chain and Syk, leading to tyrosine phosphorylation of phospholipase Cgamma2 (PLCgamma2). The aim of the present study was to assess the functional significance of this pathway in platelet activation by GPIb-IX-V. In washed platelets, vWF/ristocetin and vWF/botrocetin stimulate weak tyrosine phosphorylation of the FcR gamma-chain, Syk and PLCgamma2, but not the adaptor LAT (linker for activation of T-cells), which is localized to glycolipid-enriched membrane domains. Increases in tyrosine phosphorylation were blocked by the Src family kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1). Under the same conditions, neither stimulus induced activation of PLCgamma2 nor functional responses, such as Ca(2+) elevation, secretion or GPIIb-IIIa-dependent aggregation. In contrast, in platelet-rich plasma (PRP), threshold concentrations of ristocetin or asialo-vWF stimulated GPIb-dependent biphasic aggregation, in which the second phase was blocked by PP1. Importantly, a significant component of the initial phase and the complete second phase of aggregation was blocked by GPIIb-IIIa receptor antagonists in PRP. Higher concentrations of ristocetin stimulated GPIIb-IIIa-independent agglutination in PRP. These results demonstrate that GPIb-IX-V initiates activation of GPIIb-IIIa in PRP through an undefined pathway that is reinforced by a PP1-sensitive pathway. In contrast, activation of GPIbalpha in washed platelets does not promote functional responses.

Glycoprotein IIb-IIIa-dependent aggregation by glycoprotein Ibα is reinforced by a Src family kinase inhibitor (PP1)-sensitive signalling pathway

It has been proposed that the receptor for von Willebrand factor (vWF), glycoprotein (GP)Ib-IX-V, signals through the same pathway as the collagen receptor, GPVI, namely via Src kinases, the Fc receptor (FcR) γ-chain and Syk, leading to tyrosine phosphorylation of phospholipase Cγ2 (PLCγ2). The aim of the present study was to assess the functional significance of this pathway in platelet activation by GPIb-IX-V. In washed platelets, vWF/ristocetin and vWF/botrocetin stimulate weak tyrosine phosphorylation of the FcR γ-chain, Syk and PLCγ2, but not the adaptor LAT (linker for activation of T-cells), which is localized to glycolipid-enriched membrane domains. Increases in tyrosine phosphorylation were blocked by the Src family kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1). Under the same conditions, neither stimulus induced activation of PLCγ2 nor functional responses, such as Ca2+ elevation, secretion or GPIIb-IIIa-dependent aggregation. In contrast, in platelet-rich plasma (PRP), threshold concentrations of ristocetin or asialo-vWF stimulated GPIb-dependent biphasic aggregation, in which the second phase was blocked by PP1. Importantly, a significant component of the initial phase and the complete second phase of aggregation was blocked by GPIIb-IIIa receptor antagonists in PRP. Higher concentrations of ristocetin stimulated GPIIb-IIIa-independent agglutination in PRP. These results demonstrate that GPIb-IX-V initiates activation of GPIIb-IIIa in PRP through an undefined pathway that is reinforced by a PP1-sensitive pathway. In contrast, activation of GPIbα in washed platelets does not promote functional responses.

The transmembrane adapter LAT plays a central role in immune receptor signalling.

The transmembrane adapter LAT (linker for activation of T cells) plays a central role in signalling by ITAM bearing receptors expressed on T cells, natural killer cells, mast cells and platelets. Receptor engagement leads to the phosphorylation of tyrosine residues present in the intracellular domain of LAT and formation of a multiprotein complex with other adapter molecules and enzymes including Grb2, Gads/SLP-76 and PLCgamma isoforms. These signalling events predominantly take place in glycolipid-enriched membrane domains. The constitutive presence of LAT in GEMs enables its function as the main scaffolding protein for the organization of GEM-localized signalling. The study of LAT-deficient mice and LAT-deficient cell lines further emphasizes the importance of LAT for these signalling cascades but also defines the existence of LAT-independent events downstream of the Syk-family kinase-ITAM complex.

Glycolipid-Enriched Membrane Domains Are Assembled into Membrane Patches by Associating with the Actin Cytoskeleton

Nonionic detergent lysates of cells contain a glycolipid-enriched membrane (GEM) fraction. It has been proposed that the GEM fraction represents poorly solubilized GEM microdomains, or lipid rafts. However, the properties of GEM domains in intact cells remain controversial. To study the properties of a GEM-associated protein using confocal microscopy, GFP was targeted to GEM domains using the N-terminal domain of p56lck (LckNT). Imaging of HeLa cells expressing LckNT-GFP showed that it was targeted to large actin-rich patches in the plasma membrane that contained up to a fivefold enrichment of protein. Double-labeling experiments showed that the patches were selectively enriched with other GEM-associated molecules. Furthermore, the patches were resistant to extraction by TX-100, and disrupting GEM domains by extracting cholesterol also disrupted colocalization of LckNT-GFP with F-actin. Analogous to the actin-rich patches in HeLa cells, LckNT-GFP colocalized with actin-rich membrane caps in stimulated T cells. Furthermore, disrupting the GEM-targeting signal of LckNT-GFP also inhibited its targeting to membrane caps. Altogether, these findings extend previous studies by showing that association of GEM domains with the actin cytoskeleton provides a mechanism for targeting signaling molecules to membrane patches and caps.

The Role of ITAM- and ITIM-coupled Receptors in Platelet Activation by Collagen

The major activation-inducing collagen receptor glycoprotein VI (GPVI) has been cloned within the last two years. It is a member of the Ig superfamily of proteins and is constitutively associated with the ITAM-bearing Fc receptor gamma-chain (FcR gamma-chain). GPVI signals through a pathway that involves several of the proteins used by Fc, B- and T-lymphocyte receptors and which takes place in glycolipid-enriched membrane domains in the plasma membrane known as GEMs. Responses to GPVI are regulated by PECAM-1 (CD31) and possibly other ITIM-bearing receptors. Despite a pivotal role for GPVI, there are important differences between signalling events to collagen and GPVI-specific ligands. This may reflect a role for co-receptors in the response to collagen.

Different initiation of pre-TCR and gammadeltaTCR signalling.

Lineage choice is of great interest in developmental biology. In the immune system, the alphabeta and gammadelta lineages of T lymphocytes diverge during the course of the beta-, gamma- and delta-chain rearrangement of T-cell receptor (TCR) genes that takes place within the same precursor cell and which results in the formation of the gammadeltaTCR or pre-TCR proteins. The pre-TCR consists of the TCRbeta chain covalently linked to the pre-TCRalpha protein, which is present in immature but not in mature T cells which instead express the TCRalpha chain. Animals deficient in pre-TCRalpha have few alphabeta lineage cells but an increased number of gammadelta T cells. These gammadelta T cells exhibit more extensive TCRbeta rearrangement than gammadelta T cells from wild-type mice. These observations are consistent with the idea that different signals emanating from the gammadeltaTCR and pre-TCR instruct lineage commitment. Here we show, by using confocal microscopy and biochemistry to analyse the initiation of signalling, that the pre-TCR but not the gammadeltaTCR colocalizes with the p56lck Src kinase into glycolipid-enriched membrane domains (rafts) apparently without any need for ligation. This results in the phosphorylation of CD3epsilon and Zap-70 signal transducing molecules. The results indicate clear differences between pre-TCR and gammadeltaTCR signalling.

Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts.

A number of recent studies have demonstrated the significance of detergent-insoluble, glycolipid-enriched membrane domains or lipid rafts, especially in regard to activation and signaling in T lymphocytes. These domains can be viewed as floating rafts composed of sphingolipids and cholesterol which sequester glycosylphosphatidylinositol (GPI)-linked proteins, such as Thy-1 and CD59. CD45, a 200-kDa transmembrane phosphatase protein, is excluded from these domains. We have found that human immunodeficiency virus type 1 (HIV-1) particles produced by infected T-cell lines acquire the GPI-linked proteins Thy-1 and CD59, as well as the ganglioside GM1, which is known to partition preferentially into lipid rafts. In contrast, despite its high expression on the cell surface, CD45 was poorly incorporated into virus particles. Confocal fluorescence microscopy revealed that HIV-1 proteins colocalized with Thy-1, CD59, GM1, and a lipid raft-specific fluorescent lipid, DiIC(16)(3), in uropods of infected Jurkat cells. CD45 did not colocalize with HIV-1 proteins and was excluded from uropods. Dot immunoassay of Triton X-100-extracted membrane fractions revealed that HIV-1 p17 matrix protein and gp41 were present in the detergent-resistant fractions and that [(3)H]myristic acid-labeled HIV Gag showed a nine-to-one enrichment in lipid rafts. We propose a model for the budding of HIV virions through lipid rafts whereby host cell cholesterol, sphingolipids, and GPI-linked proteins within these domains are incorporated into the viral envelope, perhaps as a result of preferential sorting of HIV Gag to lipid rafts.

The MAL Gene Is Expressed in Primary Mediastinal Large B-Cell Lymphoma

Primary mediastinal large B-cell lymphoma (PMBL) appears to be a distinct clinicopathologic entity among diffuse large B-cell lymphomas (DLBLs). To find molecular alterations associated with this disease, we compared the mRNAs expressed in 3 PMBLs and 3 peripheral DLBLs by differential display-reverse transcription (DDRT) and identified a mRNA specifically expressed in PMBLs. Sequence analysis showed that this mRNA is encoded by the MAL gene, the expression of which was shown to be restricted to the T-cell lineage during hematopoiesis. MAL gene expression was demonstrated by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) in 8 of 12 PMBLs. However, there was little or no MAL gene expression in 8 peripheral DLBLs. Immunohistochemical analysis evidenced expression of MAL protein in tumoral B cells restricted to the PMBL subtype. Finally, Southern blot studies did not demonstrate rearrangement of the MAL gene. Altogether, our results indicate that MAL expression is recurrent in PMBLs, providing further evidence that PMBL represents a distinct entity among DLBLs. Because MAL protein is located in detergent-insoluble glycolipid-enriched membrane (GEM) domains involved in lymphocyte signal transduction, abnormal expression of MAL protein in the B-lymphoid lineage may have significant implications in PMBL lymphomagenesis.

Translocation of tyrosine-phosphorylated TCRzeta chain to glycolipid-enriched membrane domains upon T cell activation.

Recent studies point to glycolipid-enriched membrane (GEM) microdomains as the critical sites for TCR-mediated signal transduction. However, whether the TCR complex is localized in the GEM domain is not well-defined. In the present study, we analyzed localization of the TCR-CD3 complex in the GEM domain by isolating the GEM fraction with sucrose density gradient centrifugation. Although 10% of TCRzeta chains was localized in the GEM fraction, most of the TCR complexes were excluded from the GEM before and after T cell activation, and the amount of TCRzeta in the GEM was not increased after activation. However, the tyrosine-phosphorylated form of TCRzeta was strongly concentrated in the GEM fraction upon TCR engagement. A kinetic study revealed that tyrosine phosphorylation of TCRzeta occurred initially in the Triton X-100-soluble membrane fraction followed by the accumulation of phosphorylated TCRzeta in the GEM. Thus, these results indicate that phosphorylated TCRzeta migrates into the GEM domains on T cell activation. We speculate that the GEM microdomains may function as a reservoir of activation signals from triggered TCR.