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G Raposo

Publications and source records attributed to G Raposo.

At least 37 records · Page 2Linked to original sources
PubMed1999

Peptide loading in the endoplasmic reticulum accelerates trafficking of peptide:MHC class II complexes in B cells.

In a combination of biochemical and immunoelectron-microscopical approaches we studied intracellular trafficking and localization of the endoplasmic-reticulum (ER)-formed complexes of murine MHC class II molecule I-Ab and an antigenic peptide Ealpha52-68 covalently linked to its beta-chain. The association with the peptide in the ER leads to sharp acceleration of the intracellular trafficking of the complexes to the plasma membrane. Within the cells, Ealpha52-68:I-Ab complexes accumulate in the multivesicular MHC class II compartment (MIIC), but not in denser multilaminar or intermediate type MIICs. The changes in the trafficking of ER-formed complexes result solely from the presence of the tethered peptide, since wild-type class II molecules traffic similarly in bare lymphocyte syndrome cells and in wild-type antigen-presenting cells.

Animals↗
PubMed1999

Association of myosin I alpha with endosomes and lysosomes in mammalian cells.

Myosin Is, which constitute a ubiquitous monomeric subclass of myosins with actin-based motor properties, are associated with plasma membrane and intracellular vesicles. Myosin Is have been proposed as key players for membrane trafficking in endocytosis or exocytosis. In the present paper we provide biochemical and immunoelectron microscopic evidence indicating that a pool of myosin I alpha (MMIalpha) is associated with endosomes and lysosomes. We show that the overproduction of MMIalpha or the production of nonfunctional truncated MMIalpha affects the distribution of the endocytic compartments. We also show that truncated brush border myosin I proteins, myosin Is that share 78% homology with MMIalpha, promote the dissociation of MMIalpha from vesicular membranes derived from endocytic compartments. The analysis at the ultrastructural level of cells producing these brush border myosin I truncated proteins shows that the delivery of the fluid phase markers from endosomes to lysosomes is impaired. MMIalpha might therefore be involved in membrane trafficking occurring between endosomes and lysosomes.

Actins↗
PubMed1999

Early endosomes are required for major histocompatiblity complex class II transport to peptide-loading compartments.

Antigen presentation to CD4(+) T lymphocytes requires transport of newly synthesized major histocompatibility complex (MHC) class II molecules to the endocytic pathway, where peptide loading occurs. This step is mediated by a signal located in the cytoplasmic tail of the MHC class II-associated Ii chain, which directs the MHC class II-Ii complexes from the trans-Golgi network (TGN) to endosomes. The subcellular machinery responsible for the specific targeting of MHC class II molecules to the endocytic pathway, as well as the first compartments these molecules enter after exit from the TGN, remain unclear. We have designed an original experimental approach to selectively analyze this step of MHC class II transport. Newly synthesized MHC class II molecules were caused to accumulate in the Golgi apparatus and TGN by incubating the cells at 19 degrees C, and early endosomes were functionally inactivated by in vivo cross-linking of transferrin (Tf) receptor-containing endosomes using Tf-HRP complexes and the HRP-insoluble substrate diaminobenzidine. Inactivation of Tf-containing endosomes caused a marked delay in Ii chain degradation, peptide loading, and MHC class II transport to the cell surface. Thus, early endosomes appear to be required for delivery of MHC class II molecules to the endocytic pathway. Under cross-linking conditions, most alphabetaIi complexes accumulated in tubules and vesicles devoid of gamma-adaptin and/or mannose-6-phosphate receptor, suggesting an AP1-independent pathway for the delivery of newly synthesized MHC class II molecules from the TGN to endosomes.

3,3'-Diaminobenzidine↗
PubMed1998

Antigen-dependent and -independent Ca2+ responses triggered in T cells by dendritic cells compared with B cells.

Dendritic cells (DCs) are much more potent antigen (Ag)-presenting cells than resting B cells for the activation of naive T cells. The mechanisms underlying this difference have been analyzed under conditions where ex vivo DCs or B cells presented known numbers of specific Ag-major histocompatibility complex (MHC) complexes to naive CD4(+) T cells from T cell antigen receptor (TCR) transgenic mice. Several hundred Ag-MHC complexes presented by B cells were necessary to elicit the formation of a few T-B conjugates with small contact zones, and the resulting individual T cell Ca2+ responses were all-or-none. In contrast, Ag-specific T cell Ca2+ responses can be triggered by DCs bearing an average of 30 Ag-MHC complexes per cell. Formation of T-DC conjugates is Ag-independent, but in the presence of the Ag, the surface of the contact zone increases and so does the amplitude of the T cell Ca2+ responses. These results suggest that Ag is better recognized by T cells on DCs essentially because T-DC adhesion precedes Ag recognition, whereas T-B adhesion requires Ag recognition. Surprisingly, we also recorded small Ca2+ responses in T cells interacting with unpulsed DCs. Using DCs purified from MHC class II knockout mice, we provide evidence that this signal is mostly due to MHC-TCR interactions. Such an Ag-independent, MHC-triggered calcium response could be a survival signal that DCs but not B cells are able to deliver to naive T cells.

Animals↗
PubMed1998

Deficient peptide loading and MHC class II endosomal sorting in a human genetic immunodeficiency disease: the Chediak-Higashi syndrome.

The Chediak-Higashi syndrome (CHS) is a human recessive autosomal disease caused by mutations in a single gene encoding a protein of unknown function, called lysosomal-trafficking regulator. All cells in CHS patients bear enlarged lysosomes. In addition, T- and natural killer cell cytotoxicity is defective in these patients, causing severe immunodeficiencies. We have analyzed major histocompatibility complex class II functions and intracellular transport in Epstein Barr Virus-transformed B cells from CHS patients. Peptide loading onto major histocompatibility complex class II molecules and antigen presentation are strongly delayed these cells. A detailed electron microscopy analysis of endocytic compartments revealed that only lysosomal multilaminar compartments are enlarged (reaching 1-2 micron), whereas late multivesicular endosomes have normal size and morphology. In contrast to giant multilaminar compartments that bear most of the usual lysosomal markers in these cells (HLA-DR, HLA-DM, Lamp-1, CD63, etc.), multivesicular late endosomes displayed reduced levels of all these molecules, suggesting a defect in transport from the trans-Golgi network and/or early endosomes into late multivesicular endosomes. Further insight into a possible mechanism of this transport defect came from immunolocalizing the lysosomal trafficking regulator protein, as antibodies directed to a peptide from its COOH terminal domain decorated punctated structures partially aligned along microtubules. These results suggest that the product of the Lyst gene is required for sorting endosomal resident proteins into late multivesicular endosomes by a mechanism involving microtubules.

Amino Acid Sequence↗
PubMed1998

Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes.

Dendritic cells (DCs) are professional antigen presenting cells with the unique capacity to induce primary and secondary immune responses in vivo. Here, we show that DCs secrete antigen presenting vesicles, called exosomes, which express functional Major Histocompatibility Complex class I and class II, and T-cell costimulatory molecules. Tumor peptide-pulsed DC-derived exosomes prime specific cytotoxic T lymphocytes in vivo and eradicate or suppress growth of established murine tumors in a T cell-dependent manner. Exosome-based cell-free vaccines represent an alternative to DC adoptive therapy for suppressing tumor growth.

Adenocarcinoma↗
PubMed1998

A novel tetanus neurotoxin-insensitive vesicle-associated membrane protein in SNARE complexes of the apical plasma membrane of epithelial cells.

The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP-containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.

Amino Acid Sequence↗
PubMed1997

Ii chain controls the transport of major histocompatibility complex class II molecules to and from lysosomes.

Major histocompatibility complex class II molecules are synthesized as a nonameric complex consisting of three alpha beta dimers associated with a trimer of invariant (Ii) chains. After exiting the TGN, a targeting signal in the Ii chain cytoplasmic domain directs the complex to endosomes where Ii chain is proteolytically processed and removed, allowing class II molecules to bind antigenic peptides before reaching the cell surface. Ii chain dissociation and peptide binding are thought to occur in one or more postendosomal sites related either to endosomes (designated CIIV) or to lysosomes (designated MIIC). We now find that in addition to initially targeting alpha beta dimers to endosomes, Ii chain regulates the subsequent transport of class II molecules. Under normal conditions, murine A20 B cells transport all of their newly synthesized class II I-A(b) alpha beta dimers to the plasma membrane with little if any reaching lysosomal compartments. Inhibition of Ii processing by the cysteine/serine protease inhibitor leupeptin, however, blocked transport to the cell surface and caused a dramatic but selective accumulation of I-A(b) class II molecules in lysosomes. In leupeptin, I-A(b) dimers formed stable complexes with a 10-kD NH2-terminal Ii chain fragment (Ii-p10), normally a transient intermediate in Ii chain processing. Upon removal of leupeptin, Ii-p10 was degraded and released, I-A(b) dimers bound antigenic peptides, and the peptide-loaded dimers were transported slowly from lysosomes to the plasma membrane. Our results suggest that alterations in the rate or efficiency of Ii chain processing can alter the postendosomal sorting of class II molecules, resulting in the increased accumulation of alpha beta dimers in lysosome-like MIIC. Thus, simple differences in Ii chain processing may account for the highly variable amounts of class II found in lysosomal compartments of different cell types or at different developmental stages.

Animals↗
PubMed1997

Assembly of an abundant endogenous major histocompatibility complex class II/peptide complex in class II compartments.

To identify the intracellular site(s) of formation of an endogenous class II/peptide complex in a human B cell line, we employed kinetic pulse-chase labeling experiments followed by subcellular fractionation by Percoll density gradient centrifugation and immunogold labeling on ultrathin cryosections. For direct demonstration of assembly of such complexes, we used the monoclonal antibody YAe, which detects an endogenous complex of the mouse class II molecule I-Ab with a 17-amino acid peptide derived from the alpha chain of HLA-DR (DR alpha52-68). We show that in human B lymphocytes, these class II/peptide complexes assemble and transiently accumulate in major histocompatibility complex class II-enriched compartments before reaching the cell surface.

Animals↗
PubMed1997

Accumulation of major histocompatibility complex class II molecules in mast cell secretory granules and their release upon degranulation.

To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by their protein content and their accessibility to endocytic tracers. Type I granules display internal membrane vesicles and are accessed by exogenous molecules after a time lag of 20 min; type II granules are reached by the endocytic tracer later and possess a serotonin-rich electron-dense core surrounded by a multivesicular domain. In these type I and type II granules, MHC class II molecules, mannose-6-phosphate receptors and lysosomal membrane proteins (lamp1 and lamp2) localize to small intralumenal vesicles. These 60-80-nm vesicles are released along with inflammatory mediators during mast cell degranulation triggered by IgE-antigen complexes. These observations emphasize the intimate connection between the endocytic and secretory pathways in cells of the hematopoietic lineage which allows regulated secretion of the contents of secretory lysosomes, including membrane proteins associated with small vesicles.

Animals↗
PubMed1997

Association of Engrailed homeoproteins with vesicles presenting caveolae-like properties.

We report here that the homeoproteins Engrailed-1 and Engrailed-2 are present in specific non-nuclear subcellular compartments. Using electron microscopy, we observed that chick-Engrailed-2 expressed in COS-7 cells associates with membrane fractions that are characterized as caveolae. This characterization is based on morphological, biochemical and immunological criteria such as, in particular, the absence of clathrin coat and the presence of caveolin and cholera toxin-binding sites. These data are fully confirmed by subcellular fractionation experiments, which demonstrate that transfected chick-Engrailed-2 is present in low density membrane fractions that are resistant to Triton X-100, enriched in caveolin and solubilized by the addition of a cholesterol-binding detergent, a set of properties highly characteristic of caveolae. The association of Engrailed-2 with specific membrane fractions observed after transfection in COS-7 cells is also observed for endogenous Engrailed-1 and Engrailed-2 expressed at late embryonic stages in the cerebellum and posterior mesencephalon of the rodent. Indeed, the two proteins are present in membrane fractions that bear all the characteristics of microdomains or caveolae-like domains, i.e. Triton X-100 resistance, saponin solubilization, low density on sucrose gradients, enrichment in glycosphingolipid GM1, absence of transmembrane Neural Cell Adhesion Molecule, presence of the glypiated (GPI-anchored) glycoprotein F3/F11 and of the acylated growth-associated protein GAP-43. Finally we demonstrate that part of the membrane-associated Engrailed, either expressed in COS-7 cells or endogenously present in neural tissues, is not accessible to proteolytic enzymes unless the membranes have been permeabilized with detergent. This study suggests that, in addition to their well-known presence in the nucleus, Engrailed proteins are also associated with caveolae-like vesicles that are primarily transported anterogradely into the axon, and that they can get access to a compartment compatible with secretion.

Animals↗
PubMed1996

B lymphocytes secrete antigen-presenting vesicles.

Antigen-presenting cells contain a specialized late endocytic compartment, MIIC (major histocompatibility complex [MHC] class II-enriched compartment), that harbors newly synthesized MHC class II molecules in transit to the plasma membrane. MIICs have a limiting membrane enclosing characteristic internal membrane vesicles. Both the limiting membrane and the internal vesicles contain MHC class II. In this study on B lymphoblastoid cells, we demonstrate by immunoelectron microscopy that the limiting membrane of MIICs can fuse directly with the plasma membrane, resulting in release from the cells of internal MHC class II-containing vesicles. These secreted vesicles, named exosomes, were isolated from the cell culture media by differential centrifugation followed by flotation on sucrose density gradients. The overall surface protein composition of exosomes differed significantly from that of the plasma membrane. Exosome-bound MHC class II was in a compact, peptide-bound conformation. Metabolically labeled MHC class II was released into the extracellular medium with relatively slow kinetics, 10 +/- 4% in 24 h, indicating that direct fusion of MIICs with the plasma membrane is not the major pathway by which MHC class II reaches the plasma membrane. Exosomes derived from both human and murine B lymphocytes induced antigen-specific MHC class II-restricted T cell responses. These data suggest a role for exosomes in antigen presentation in vivo.

Animals↗
PubMed1996

Characterization of MHC Class II Compartments by Immunoelectron Microscopy

At present the best way to determine the precise intracellular localization of proteins, in a potentially semiquantitative way, is the combination of ultrathin cryosectioning and immunogold labeling. This paper focuses on the intracellular localization of MHC class II molecules, which are involved in the T helper response to exogenous antigens. Newly synthesized MHC class II heterodimers associate with invariant chain molecules, which in turn direct the MHC class II complex to the endocytotic route. Proteolytic digestion of the invariant chain frees MHC class II molecules so that they can bind antigenic peptides. Immunoelectron microscopy has been an important tool to identify the endocytotic compartments that are enriched in MHC class II and that are the potential sites of antigenic peptide binding. The methods that can be used to characterize MHC class II compartments (MIICs) in various antigen-presenting cells (APCs) are described in detail. In all APCs studies so far, MIICs are situated late in the endocytotic pathway and display lysosomal characteristics. Still, immunoelectron microscopy allows us to define subsets of MIICs, which can be distinguished by their morphology, accessibility to endocytotic tracers, and expression of invariant chain and HLA-DM. Different types of MIICs can be found that display internal vesicles (multivesicular), internal membrane sheets (multilaminar), or both. The multivesicular type of MIIC contains detectable invariant chain and is the primary site of antigen entry. The multilaminar MIIC is situated later in the endocytotic route and has lost most of the invariant chain antigenicity. These data suggest a sequential maturation of MIICs, which correlates with the degradation of invariant chain and the subsequent binding of antigenic peptides.

Journal Article↗
PubMed1995

Major histocompatibility complex class II compartments in human B lymphoblastoid cells are distinct from early endosomes.

In human B lymphoblastoid cell lines, the majority of major histocompatibility complex (MHC) class II heterodimers are located on the cell surface and in endocytic compartments, while invariant chain (Ii)-associated class II molecules represent biosynthetic intermediates which are present mostly in the endoplasmic reticulum and Golgi complex. To investigate the origin of the MHC class II-positive compartments and their relation to early endosomes, the intracellular distribution of MHC class II molecules and Ii in relation to endocytic tracers was studied in human lymphoblastoid B cells by immunoelectronmicroscopy on ultrathin cryosections. Cross-linking of surface immunoglobulins, followed by a brief period of internalization of the immune complexes, did not alter the intracellular distribution of MHC class II molecules. While early endosomes were abundantly labeled for the cross-linked immunoglobulins, < 1% of total MHC class II molecules were detectable in early endosomes. MHC class II- and Ii-positive structures associated with the trans-Golgi network can be reached by endocytosed bovine serum albumin (BSA)-gold conjugates after 30 min of internalization. Prolonged exposure to BSA-gold allowed visualization of later endocytic compartments, in which a progressive loss of Ii was observed: first the lumenal portion, and then the cytoplasmic portion of Ii escaped detection, culminating in the formation of MHC class II-positive compartments (MIIC) devoid of Ii. The loss of Ii also correlated with a transition from a multivesicular to a multilaminar, electron-dense MIIC. The intracellular compartments in which class II molecules reside (MIIC) are therefore a heterogeneous set of structures, part of the later aspects of the endocytic pathway.

Antigen-Presenting Cells↗
PubMed1995

How MHC class II molecules reach the endocytic pathway.

We have examined trafficking of major histocompatibility complex (MHC) class II molecules in human B cells exposed to concanamycin B, a highly specific inhibitor of the vacuolar H(+)-ATPases required for acidification of the vacuolar system and for early to late endosomal transport. Neutralization of vacuolar compartments prevents breakdown of the invariant chain (Ii) and blocks conversion of MHC class II molecules to peptide-loaded, SDS-stable alpha beta dimers. Ii remains associated with alpha beta and this complex accumulates internally, as ascertained biochemically and by morphological methods. In concanamycin B-treated cells, a slow increase (> 20-fold) in surface expression of Ii, mostly complexed with alpha beta, is detected. This surface-disposed fraction of alpha beta Ii is nevertheless a minor population that reaches the cell surface directly, or is routed via early endosomes as intermediary stations. In inhibitor-treated cells, the bulk of newly synthesized alpha beta Ii is no longer accessible to fluid phase endocytic markers. It is concluded that the majority of alpha beta Ii is targeted directly from the trans-Golgi network to the compartment for peptide loading, bypassing the cell surface and early endosomes en route to the endocytic pathway.

Anti-Bacterial Agents↗
PubMed1995

Misfolded major histocompatibility complex class I molecules accumulate in an expanded ER-Golgi intermediate compartment.

Misfolded membrane proteins are rapidly degraded, often shortly after their synthesis and insertion in the endoplasmic reticulum (ER), but the exact location and mechanisms of breakdown remain unclear. We have exploited the requirement of MHC class I molecules for peptide to achieve their correct conformation: peptide can be withheld by introducing a null mutation for the MHC-encoded peptide transporter, TAP. By withholding TAP-dependent peptides, the vast majority of newly synthesized class I molecules fails to leave the endoplasmic reticulum and is degraded. We used mice transgenic for HLA-B27 on a TAP1-deficient background to allow visualization by immunoelectron microscopy of misfolded HLA-B27 molecules in thymic epithelial cells. In such HLA transgenic animals, the TAP mutation can be considered a genetic switch that allows control over the extent of folding of the protein of interest, HLA-B27, while the rate of synthesis of the constituent subunits remains unaltered. In TAP1-deficient, HLA-B27 transgenic animals, HLA-B27 molecules fail to assemble correctly, and do not undergo carbohydrate modifications associated with the Golgi apparatus, such as conversion to Endoglycosidase H resistance, and acquisition of sialic acids. We show that such molecules accumulate in an expanded network of tubular and fenestrated membranes. This compartment has its counterpart in normal thymic epithelial cells, and is identified as an ER-Golgi intermediate. We detect the presence of ubiquitin and ubiquitin-conjugating enzymes in association with this compartment, suggesting a nonlysosomal mode of degradation of its contents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Thy-1 triggers mouse thymocyte apoptosis through a bcl-2-resistant mechanism.

Programmed cell death plays an important role during thymocyte development, since a vast majority (97%) of mouse cortical thymocytes die in thymus, whereas only 3% of these cells are rescued from cell death and positively selected. Although it seems well established that thymocyte fate depends upon appropriate surface-expressed T cell receptor, little is known about the molecular mechanism(s) responsible for the massive thymocyte elimination that occurs in the thymus. We report here that Thy-1 is capable of triggering mouse thymocyte death in vitro through a bcl-2-resistant mechanism. We have previously shown that Thy-1 is involved in mouse thymocyte adhesion to thymic stroma through interaction with an epithelial cell ligand. To examine the Thy-1 signaling function in thymocytes, we have mimicked its interaction with stromal cells by culturing mouse thymocytes onto tissue culture plates coated with monoclonal antibodies (mAb) directed at distinct Thy-1 epitope regions. mAb recognizing determinants in a defined Thy-1 structural domain, but not others, were found to induce marked thymocyte apoptosis as evidenced by morphological and biochemical data. Use of a quantitative DNA dot blot assay indicated that Thy-1-mediated thymocyte apoptosis was not blocked by RNA or protein synthesis inhibitors, EGTA, or by cyclosporin A, and differed, therefore, from "activation-driven cell death". Moreover, Thy-1(+)-transfected, but not wild-type AKR1 (Thy-1-d) thymoma cells underwent apoptosis after ligation with apoptosis-inducing, Thy-1-specific mAb. In contrast to thymocytes, the latter event was inhibitable by RNA and protein synthesis inhibitors, an indication that thymocytes, but not thymoma cells, contain the molecular components necessary for Thy-1-driven apoptosis. We further showed that Thy-1-triggered thymocyte death is a developmentally regulated process operative in fetal thymocytes from day 17 of gestation, but not in peripheral T cells. Indeed, the target of apoptosis by anti-Thy-1 was found to reside mainly within the CD4+8+3- and CD4+8+3lo double positive immature thymocyte subsets. Finally, it is of major interest that Thy-1-mediated apoptosis, which was found to be readily detectable in thymocytes from bcl-2-transgenic mice, represents a thus far unique experimental system for studying bcl-2-resistant thymocyte death mechanism(s).

Animals↗