Cécile Lagaudrière-gesbert - Academia.edu (original) (raw)

Papers by Cécile Lagaudrière-gesbert

Research paper thumbnail of Summary Rho GTPases link cytoskeletal rearrangements and activation processes induced via the tetraspanin CD 82 in T lymphocytes

Research paper thumbnail of CD9, CD63, CD81, and CD82 are components of a surface tetraspan network connected to HLA-DR and VLA integrins

European Journal of Immunology, 1996

ABSTRACT CD9, CD63, CD81, and CD82 are glycoproteins of unknown function which belong to the tetr... more ABSTRACT CD9, CD63, CD81, and CD82 are glycoproteins of unknown function which belong to the tetraspan superfamily. These molecules have short cytoplasmic sequences, four transmembrane domains and two unequal extracellular regions. Here, we show that these molecules are associated with each other on cell surface and with other glycoproteins such as very late antigen (VLA) integrins and HLA-DR antigens. Moreover, the VLA integrins and HLA-DR antigens were also found to be associated. The interactions of these molecules were analyzed by transfection experiments. It is demonstrated that overexpression of CD9 antigen in Raji cells leads to a lower efficiency of precipitation of CD81 and CD82, suggesting a direct interaction between these molecules. In these cells, the co-precipitation of CD81 and CD82 was not modified, suggesting that these tetraspans did not compete for association. However, in COS-7 cells, transfection of both CD81 and CD82 led to a marked reduction of the number of CD9/CD81 or CD9/CD82 complexes compared to single-transfected cells, and this was associated with the appearance of CD81/CD82 complexes. Therefore, in this cellular system, CD9 competes with CD81 and CD82 for association with the other tetraspan proteins. Finally, the tetraspans do not compete for the association with integrins or HLA-DR. Indeed, when CD9 was expressed in Raji cells, it was incorporated into the pre-existing complexes of these molecules with CD81 and CD82. These data suggest the existence of a tetraspan network which, by connecting several molecules, may organize the positioning of cell surface proteins and play a role in signal transduction, cell adhesion, and motility.

Research paper thumbnail of Functional Analysis of Four Tetraspans, CD9, CD53, CD81, and CD82, Suggests a Common Role in Costimulation, Cell Adhesion, and Migration: Only CD9 Upregulates HB-EGF Activity

Cellular Immunology, 1997

Molecules of the tetraspan superfamily are engaged in multimolecular complexes containing other p... more Molecules of the tetraspan superfamily are engaged in multimolecular complexes containing other proteins such as beta 1 integrins and MHC antigens. Although their functions are not clear, they have been suggested to play a role in cell adhesion and migration, signal transduction, and costimulation. We have in this paper directly compared the functional properties of four tetraspans, CD9, CD53, CD81, and CD82. mAbs to any of these molecules were able to deliver a costimulatory signal for CD3-mediated activation of the T cell line Jurkat. CD82 mAbs were the most efficient in triggering this effect. Moreover, engagement of CD9, CD81, and CD82 induced the homotypic aggregation of the megakaryocytic cell line HEL, and inhibited the migration of this cell line. Similar results were obtained with the preB cell line NALM-6 using the CD9 and CD81 mAbs. The CD81 mAb 5A6 produced the strongest effects. Therefore, the tetraspans are recognized by mAbs which produce similar effects on the same cell lines. This is consistent with the tetraspans being included in large molecular complexes and possibly forming a tetraspan network (the tetraspan web). We also demonstrate that the tetraspans are likely to keep specific functional properties inside this network. Indeed, we have demonstrated that the human CD9 is able, like the monkey molecule, to upregulate the activity of the transmembrane precursor of heparin-binding EGF as a receptor for the diphtheria toxin when cotransfected in murine LM cells. Neither CD81, nor CD82 had such activity. By using chimeric CD9/CD81 molecules we demonstrate that this activity requires the second half of CD9, which contains the large extracellular loop, the fourth transmembrane region, and the last short cytoplasmic domain.

Research paper thumbnail of Negri bodies are viral factories with properties of liquid organelles

Nature Communications, 2017

Replication of Mononegavirales occurs in viral factories which form inclusions in the host-cell c... more Replication of Mononegavirales occurs in viral factories which form inclusions in the host-cell cytoplasm. For rabies virus, those inclusions are called Negri bodies (NBs). We report that NBs have characteristics similar to those of liquid organelles: they are spherical, they fuse to form larger structures, and they disappear upon hypotonic shock. Their liquid phase is confirmed by FRAP experiments. Live-cell imaging indicates that viral nucleocapsids are ejected from NBs and transported along microtubules to form either new virions or secondary viral factories. Coexpression of rabies virus N and P proteins results in cytoplasmic inclusions recapitulating NBs properties. This minimal system reveals that an intrinsically disordered domain and the dimerization domain of P are essential for Negri bodies-like structures formation. We suggest that formation of liquid viral factories by phase separation is common among Mononegavirales and allows specific recruitment and concentration of viral proteins but also the escape to cellular antiviral response.

Research paper thumbnail of Negri bodies and other virus membrane-less replication compartments

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

Research paper thumbnail of Functional Analysis of Four Tetraspans, CD9, CD53, CD81, and CD82, Suggests a Common Role in Costimulation, Cell Adhesion, and Migration: Only CD9 Upregulates HB-EGF Activity

Cellular Immunology, 1997

Molecules of the tetraspan superfamily are engaged in multimolecular complexes containing other p... more Molecules of the tetraspan superfamily are engaged in multimolecular complexes containing other proteins such as beta 1 integrins and MHC antigens. Although their functions are not clear, they have been suggested to play a role in cell adhesion and migration, signal transduction, and costimulation. We have in this paper directly compared the functional properties of four tetraspans, CD9, CD53, CD81, and CD82. mAbs to any of these molecules were able to deliver a costimulatory signal for CD3-mediated activation of the T cell line Jurkat. CD82 mAbs were the most efficient in triggering this effect. Moreover, engagement of CD9, CD81, and CD82 induced the homotypic aggregation of the megakaryocytic cell line HEL, and inhibited the migration of this cell line. Similar results were obtained with the preB cell line NALM-6 using the CD9 and CD81 mAbs. The CD81 mAb 5A6 produced the strongest effects. Therefore, the tetraspans are recognized by mAbs which produce similar effects on the same cell lines. This is consistent with the tetraspans being included in large molecular complexes and possibly forming a tetraspan network (the tetraspan web). We also demonstrate that the tetraspans are likely to keep specific functional properties inside this network. Indeed, we have demonstrated that the human CD9 is able, like the monkey molecule, to upregulate the activity of the transmembrane precursor of heparin-binding EGF as a receptor for the diphtheria toxin when cotransfected in murine LM cells. Neither CD81, nor CD82 had such activity. By using chimeric CD9/CD81 molecules we demonstrate that this activity requires the second half of CD9, which contains the large extracellular loop, the fourth transmembrane region, and the last short cytoplasmic domain.

Research paper thumbnail of Naive T Lymphocytes Duration of the Interactions with the Adhesion, Synapse Formation, and Dendritic Cell Maturation Controls

Research paper thumbnail of Rho GTPases link cytoskeletal rearrangements and activation processes induced via the tetraspanin CD82 in T lymphocytes

Journal of cell science, Jan 15, 2002

Activation of T lymphocytes requires the engagement of the T-cell receptor and costimulation mole... more Activation of T lymphocytes requires the engagement of the T-cell receptor and costimulation molecules through cell-to-cell contacts. The tetraspanin CD82 has previously been shown to act as a cytoskeleton-dependent costimulation molecule. We show here that CD82 engagement leads to the tyrosine phosphorylation and association of both the Rho GTPases guanosine exchange factor Vav1 and adapter protein SLP76, suggesting that Rho GTPases participate in CD82 signaling. Indeed, broad inactivation of all Rho GTPases, or a specific blockade of RhoA, Rac1 or Cdc42, inhibited the morphological changes linked to CD82 engagement but failed to modulate the inducible association of CD82 with the actin network. Rho GTPase inactivation, as well as actin depolymerization, reduced the ability of CD82 to phosphorylate Vav and SLP76 and to potentiate the phosphorylation of two early TcR signaling intermediates: the tyrosine kinases ZAP70 and membrane adapter LAT. Taken together, this suggests that an a...

Research paper thumbnail of CD82, tetra-span-transmembrane protein, is a regulated transducing molecule on U937 monocytic cell line

Journal of leukocyte biology, 1995

The mononuclear cell surface protein IA4, recently classified as CD82, was originally identified ... more The mononuclear cell surface protein IA4, recently classified as CD82, was originally identified in our laboratory by the IA4 monoclonal antibody (mAb), because of its high expression on three lymphoblastoid, LAK-susceptible, variant cell lines. We have characterized CD82 as a new activation/differentiation marker of mononuclear cells. This protein belongs to the new family of TST proteins (tetra spans transmembrane), which includes CD9, CD37, CD53, CD63, and CD81 (TAPA-1). Here we demonstrate that cross-linking of IA4 mAbs induces an increase of intracellular free calcium in U937 cells and tyrosine phosphorylation of various proteins. Our data indicate that the intracellular calcium increase is initiated by a phospholipase C (PLC)-induced PtdIns(1,4,5)P3 second messenger followed by a more stable change, linked to extracellular calcium entry. This transducing signal was dependent on dual engagement of both CD82 and Fc receptors. Surface cross-linking of CD82 together with Fc recept...

Research paper thumbnail of CD82, member of the tetra-span-transmembrane protein family, is a costimulatory protein for T cell activation

Journal of immunology (Baltimore, Md. : 1950), 1995

It is now well documented that full activation of T cells requires a two-signal triggering that c... more It is now well documented that full activation of T cells requires a two-signal triggering that can be mimicked, in the absence of accessory cells, by co-immobilization of mAbs directed to stimulatory/accessory molecules (CD2, CD3, CD28, adhesion molecules, etc.). In this report, we describe that engagement of CD82 can delivery such a costimulatory signal for full activation of the human T cell line Jurkat, leading to strong IL-2 production and cell differentiation. The CD82 Ag, which belongs to the new tetra-span-transmembrane family (CD9, CD37, CD53, CD63, and CD81 (TAPA-1)), has been identified originally in our laboratory for its enhanced expression on three LAK-susceptible cell lines, and has been characterized as an activation/differentiation marker of mononuclear cells. Jurkat cells, stimulated in vitro by co-immobilization of anti-CD82 and anti-CD3 mAbs, produced high levels of IL-2, became strongly adherent to plastic dishes, and developed dendritic processes. These morphol...

Research paper thumbnail of Focal Adhesion Kinase Is Involved in Rabies Virus Infection through Its Interaction with Viral Phosphoprotein P

Journal of Virology, 2014

The rabies virus (RABV) phosphoprotein P is a multifunctional protein: it plays an essential 33 r... more The rabies virus (RABV) phosphoprotein P is a multifunctional protein: it plays an essential 33 role in viral transcription and replication, and in addition RABV P has been identified as an 34 interferon antagonist. Here, a yeast two hybrid screen revealed that RABV P interacts with the 35 focal adhesion kinase (FAK). The binding involved the domain 106-131 corresponding to the 36 dimerization domain of P and the C-ter domain of FAK containing the proline rich domains 37 PRR2 and PRR3. The P-FAK interaction was confirmed in infected cells by co-38 immunoprecipitation and co-localization of FAK with P in Negri bodies. By alanine 39 scanning, we identified a single mutation in the P protein that abolishes this interaction. The 40 mutant virus containing a substitution of Arg to Ala in position 109 (PR109A) in P, which 41 did not interact with FAK, is affected at a post-transcriptional step involving protein synthesis 42 and viral RNA replication. Furthermore, FAK depletion inhibited viral protein expression in 43 infected cells. This provides the first evidence of an interaction of RAV with FAK which 44 positively regulates infection.

Research paper thumbnail of Proteolysis and antigen presentation by MHC class II molecules

Advances in Immunology, 2002

ABSTRACT Proteolysis is the primary mechanism used by all cells not only to dispose of unwanted p... more ABSTRACT Proteolysis is the primary mechanism used by all cells not only to dispose of unwanted proteins but also to regulate protein function and maintain cellular homeostasis. Proteases that reside in the endocytic pathway are the principal actors of terminal protein degradation. The proteases contained in the endocytic pathway are classified into four major groups based on the active-site amino acid used by the enzyme to hydrolyze amide bonds of proteins: cysteine, aspartyl, serine, and metalloproteases. The presentation of peptide antigens by major histocompatibility complex (MHC) class II molecules is strictly dependent on the action of proteases. Class II molecules scour the endocytic pathway for antigenic peptides to bind and present at the cell surface for recognition by CD4+ T cells. The specialized cell types that support antigen presentation by class II molecules are commonly referred to as professional antigen presenting cells (APCs), which include bone marrow-derived B lymphocytes, dendritic cells (DCs), and macrophages. In addition, the expression of certain endocytic proteases is regulated either at the level of gene transcription or enzyme maturation and their activity is controlled by the presence of endogenous protease inhibitors.

Research paper thumbnail of Interaction of CD82 Tetraspanin Proteins with HTLV-1 Envelope Glycoproteins Inhibits Cell-to-Cell Fusion and Virus Transmission

Virology, 2000

The entry of retroviruses into their target cell involves interactions between the virus envelope... more The entry of retroviruses into their target cell involves interactions between the virus envelope glycoproteins and their cellular receptors, as well as accessory ligand-receptor interactions involving adhesion molecules that can also participate in fusion. We have studied the contribution of CD82 proteins to the transmission of the human T-cell leukemia virus type 1 (HTLV-1), which is greatly dependent on cell-to-cell contacts. CD82 proteins belong to a class of cell surface molecules, the tetraspanins, that can act as molecular facilitators in cellular adhesion processes. The coexpression of CD82 proteins with HTLV-1 envelope glycoproteins resulted in marked inhibition of syncytium formation, whereas CD82 proteins had no effect on syncytium formation induced by human immunodeficiency virus type 1 (HIV-1) envelope proteins. The presence of CD82 proteins also inhibited cell-to-cell transmission of HTLV-1. Coimmunoprecipitation and cocapping experiments showed that CD82 associates with HTLV-1 envelope glycoproteins, both within the cell and at the cell surface. Finally, whereas the intracellular maturation of HTLV-1 glycoproteins was not modified by the presence of CD82 proteins, HTLV-1 protein coproduction delayed the intracellular maturation of CD82 proteins. There thus seems to be a reciprocal interaction between virus and cell proteins, and the cellular proteins involved in adhesion modulate retrovirus transmission both positively, as shown in other systems, and negatively, as shown here.

Research paper thumbnail of Uncoating ATPase Hsc70 is recruited by invariant chain and controls the size of endocytic compartments

Proceedings of the National Academy of Sciences, 2002

Targeting of class II major histocompatibility complex molecules to endocytic compartments is med... more Targeting of class II major histocompatibility complex molecules to endocytic compartments is mediated by their association with the invariant chain (Ii). Although the identity of certain sorting signals located in Ii's cytoplasmic tail is known, proteins that interact with Ii's cytoplasmic tail in living cells remain to be identified. Synthesis of a biotinylated trimeric Ii cytoplasmic tail allowed the retrieval of two proteins that interact with this domain. We identify one of them as the 70-kDa heat-shock cognate protein (hsc70), the uncoating ATPase of clathrin-coated vesicles, and the other as its mitochondrial homologue, the glucose-regulated protein grp75. Expression of Ii in COS cells results in the formation of large endocytic compartments. We observe extensive colocalization of hsc70 with Ii in these macrosomes. Expression of a dominantnegative (K71M) green fluorescent protein-tagged version of hsc70 counteracted the ability of Ii to modify the endocytic pathway, demonstrating an interaction in vivo of Ii with hsc70 as part of the machinery responsible for macrosome formation.

Research paper thumbnail of Dendritic Cell Maturation Controls Adhesion, Synapse Formation, and the Duration of the Interactions with Naive T Lymphocytes

The Journal of Immunology, 2004

The initiation of adaptive immune responses requires the direct interaction of dendritic cells (D... more The initiation of adaptive immune responses requires the direct interaction of dendritic cells (DCs) with naive T lymphocytes. It is well established that the maturation state of DCs has a critical impact on the outcome of the response. We show here that mature DCs form stable conjugates with naive T cells and induce the formation of organized immune synapses. Immature DCs, in contrast, form few stable conjugates with no organized immune synapses. A dynamic analysis revealed that mature DCs can form long-lasting interactions with naive T cells, even in the absence of Ag. Immature DCs, in contrast, established only short intermittent contacts, suggesting that the premature termination of the interaction prevents the formation of organized immune synapses and full T cell activation.

Research paper thumbnail of Analysis of Protease Activity in Live Antigen-presenting Cells Shows Regulation of the Phagosomal Proteolytic Contents During Dendritic Cell Activation

Journal of Experimental Medicine, 2002

Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagoso... more Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagosomes in live antigen-presenting cells. We find that an ingested particle sequentially encounters distinct protease activities during phagosomal maturation. Incorporation of active proteases into the phagosome of the macrophage cell line J774 indicates that phagosome maturation involves progressive fusion with early and late endocytic compartments. In contrast, phagosome biogenesis in bone marrow-derived dendritic cells (DCs) and macrophages preferentially involves endocytic compartments enriched in cathepsin S. Kinetics of phagosomal maturation is faster in macrophages than in DCs. Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide. This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation. Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.

Research paper thumbnail of Structure of the Tetraspanin Main Extracellular Domain. A PARTIALLY CONSERVED FOLD WITH A STRUCTURALLY VARIABLE DOMAIN INSERTION

Journal of Biological Chemistry, 2001

The tetraspanin family of membrane glycoproteins is involved in the regulation of cellular develo... more The tetraspanin family of membrane glycoproteins is involved in the regulation of cellular development, proliferation, activation, and mobility. We have attempted to predict the structural features of the large extracellular domain of tetraspanins (EC2), which is very important in determining their functional specificity. The tetraspanin EC2 is composed of two subdomains: a conserved three-helix subdomain and a variable secondary structure subdomain inserted within the conserved subdomain. The occurrence of key disulphide bridges and other invariant residues leads to a conserved relative topology of both subdomains and also suggests a structural classification of tetraspanins. Using the CD81 EC2 structure as a template, the structures of two other EC2s were predicted by homology modeling and indicate a conserved shape, in which the variable subdomain is located at one side of the structure. The conserved and variable subdomains might contain sites that correspond, respectively, to common and specific interactions of tetraspanins. The tetraspanin EC2 seems to correspond to a new scheme of fold conservation/variability among proteins, namely the insertion of a structurally variable subdomain within an otherwise conserved fold.

Research paper thumbnail of The engagement of CD82 is a costimulatory signal inducing cytokines production by normal resting or in vitro differentiated T lymphocytes

Research paper thumbnail of Signaling through CD82 and other tetraspanins induces sustained morphological changes involved in T cell activation

Research paper thumbnail of Signaling through the tetraspanin CD82 triggers its association with the cytoskeleton leading to sustained morphological changes and T cell activation

European Journal of Immunology, 1998

In this report, we provide new evidence of a crosstalk between T cell activation and adhesion pro... more In this report, we provide new evidence of a crosstalk between T cell activation and adhesion processes through a functional cytokeleton. We show that CD82 signaling induces longlasting adhesion, spreading and development of membrane extensions, involving actin polymerization. Addition of various co-stimuli (phorbol 12-myristate 13-acetate or monoclonal antibodies to CD3 or CD2) increases the CD82-induced morphological alterations and, reciprocally, CD82 engagement synergizes with these stimuli to induce T cell activation as indicated by both primary tyrosine phosphorylation and IL-2 production. Different kinases are involved in both processes. CD82 co-signaling involves src kinases including p56 lck. On the other hand, the CD82-induced alterations of cell morphology are negatively regulated by cAMP-dependent kinases independently of activation of src kinases. Simultaneously with cytoskeletal rearrangements, we observed an inducible association of CD82 with the cytoskeletal matrix. In addition, the potentiating and stabilizing effects induced by CD82 crosslinking on tyrosine phosphorylation were abolished by cytoskeleton-disrupting agents. These results suggest that the actin polymerization triggered by CD82, through its ability to associate with the cytoskeletal matrix, is the primary step involved in the CD82 induced costimulatory activity. Our data provide further evidence for a direct role of the actin cytoskeleton as a major component for sustained signal transduction in T cells and suggest that tetraspanins could be "membrane organizers" connecting both surface and intracellular molecules.

Research paper thumbnail of Summary Rho GTPases link cytoskeletal rearrangements and activation processes induced via the tetraspanin CD 82 in T lymphocytes

Research paper thumbnail of CD9, CD63, CD81, and CD82 are components of a surface tetraspan network connected to HLA-DR and VLA integrins

European Journal of Immunology, 1996

ABSTRACT CD9, CD63, CD81, and CD82 are glycoproteins of unknown function which belong to the tetr... more ABSTRACT CD9, CD63, CD81, and CD82 are glycoproteins of unknown function which belong to the tetraspan superfamily. These molecules have short cytoplasmic sequences, four transmembrane domains and two unequal extracellular regions. Here, we show that these molecules are associated with each other on cell surface and with other glycoproteins such as very late antigen (VLA) integrins and HLA-DR antigens. Moreover, the VLA integrins and HLA-DR antigens were also found to be associated. The interactions of these molecules were analyzed by transfection experiments. It is demonstrated that overexpression of CD9 antigen in Raji cells leads to a lower efficiency of precipitation of CD81 and CD82, suggesting a direct interaction between these molecules. In these cells, the co-precipitation of CD81 and CD82 was not modified, suggesting that these tetraspans did not compete for association. However, in COS-7 cells, transfection of both CD81 and CD82 led to a marked reduction of the number of CD9/CD81 or CD9/CD82 complexes compared to single-transfected cells, and this was associated with the appearance of CD81/CD82 complexes. Therefore, in this cellular system, CD9 competes with CD81 and CD82 for association with the other tetraspan proteins. Finally, the tetraspans do not compete for the association with integrins or HLA-DR. Indeed, when CD9 was expressed in Raji cells, it was incorporated into the pre-existing complexes of these molecules with CD81 and CD82. These data suggest the existence of a tetraspan network which, by connecting several molecules, may organize the positioning of cell surface proteins and play a role in signal transduction, cell adhesion, and motility.

Research paper thumbnail of Functional Analysis of Four Tetraspans, CD9, CD53, CD81, and CD82, Suggests a Common Role in Costimulation, Cell Adhesion, and Migration: Only CD9 Upregulates HB-EGF Activity

Cellular Immunology, 1997

Molecules of the tetraspan superfamily are engaged in multimolecular complexes containing other p... more Molecules of the tetraspan superfamily are engaged in multimolecular complexes containing other proteins such as beta 1 integrins and MHC antigens. Although their functions are not clear, they have been suggested to play a role in cell adhesion and migration, signal transduction, and costimulation. We have in this paper directly compared the functional properties of four tetraspans, CD9, CD53, CD81, and CD82. mAbs to any of these molecules were able to deliver a costimulatory signal for CD3-mediated activation of the T cell line Jurkat. CD82 mAbs were the most efficient in triggering this effect. Moreover, engagement of CD9, CD81, and CD82 induced the homotypic aggregation of the megakaryocytic cell line HEL, and inhibited the migration of this cell line. Similar results were obtained with the preB cell line NALM-6 using the CD9 and CD81 mAbs. The CD81 mAb 5A6 produced the strongest effects. Therefore, the tetraspans are recognized by mAbs which produce similar effects on the same cell lines. This is consistent with the tetraspans being included in large molecular complexes and possibly forming a tetraspan network (the tetraspan web). We also demonstrate that the tetraspans are likely to keep specific functional properties inside this network. Indeed, we have demonstrated that the human CD9 is able, like the monkey molecule, to upregulate the activity of the transmembrane precursor of heparin-binding EGF as a receptor for the diphtheria toxin when cotransfected in murine LM cells. Neither CD81, nor CD82 had such activity. By using chimeric CD9/CD81 molecules we demonstrate that this activity requires the second half of CD9, which contains the large extracellular loop, the fourth transmembrane region, and the last short cytoplasmic domain.

Research paper thumbnail of Negri bodies are viral factories with properties of liquid organelles

Nature Communications, 2017

Replication of Mononegavirales occurs in viral factories which form inclusions in the host-cell c... more Replication of Mononegavirales occurs in viral factories which form inclusions in the host-cell cytoplasm. For rabies virus, those inclusions are called Negri bodies (NBs). We report that NBs have characteristics similar to those of liquid organelles: they are spherical, they fuse to form larger structures, and they disappear upon hypotonic shock. Their liquid phase is confirmed by FRAP experiments. Live-cell imaging indicates that viral nucleocapsids are ejected from NBs and transported along microtubules to form either new virions or secondary viral factories. Coexpression of rabies virus N and P proteins results in cytoplasmic inclusions recapitulating NBs properties. This minimal system reveals that an intrinsically disordered domain and the dimerization domain of P are essential for Negri bodies-like structures formation. We suggest that formation of liquid viral factories by phase separation is common among Mononegavirales and allows specific recruitment and concentration of viral proteins but also the escape to cellular antiviral response.

Research paper thumbnail of Negri bodies and other virus membrane-less replication compartments

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

Research paper thumbnail of Functional Analysis of Four Tetraspans, CD9, CD53, CD81, and CD82, Suggests a Common Role in Costimulation, Cell Adhesion, and Migration: Only CD9 Upregulates HB-EGF Activity

Cellular Immunology, 1997

Molecules of the tetraspan superfamily are engaged in multimolecular complexes containing other p... more Molecules of the tetraspan superfamily are engaged in multimolecular complexes containing other proteins such as beta 1 integrins and MHC antigens. Although their functions are not clear, they have been suggested to play a role in cell adhesion and migration, signal transduction, and costimulation. We have in this paper directly compared the functional properties of four tetraspans, CD9, CD53, CD81, and CD82. mAbs to any of these molecules were able to deliver a costimulatory signal for CD3-mediated activation of the T cell line Jurkat. CD82 mAbs were the most efficient in triggering this effect. Moreover, engagement of CD9, CD81, and CD82 induced the homotypic aggregation of the megakaryocytic cell line HEL, and inhibited the migration of this cell line. Similar results were obtained with the preB cell line NALM-6 using the CD9 and CD81 mAbs. The CD81 mAb 5A6 produced the strongest effects. Therefore, the tetraspans are recognized by mAbs which produce similar effects on the same cell lines. This is consistent with the tetraspans being included in large molecular complexes and possibly forming a tetraspan network (the tetraspan web). We also demonstrate that the tetraspans are likely to keep specific functional properties inside this network. Indeed, we have demonstrated that the human CD9 is able, like the monkey molecule, to upregulate the activity of the transmembrane precursor of heparin-binding EGF as a receptor for the diphtheria toxin when cotransfected in murine LM cells. Neither CD81, nor CD82 had such activity. By using chimeric CD9/CD81 molecules we demonstrate that this activity requires the second half of CD9, which contains the large extracellular loop, the fourth transmembrane region, and the last short cytoplasmic domain.

Research paper thumbnail of Naive T Lymphocytes Duration of the Interactions with the Adhesion, Synapse Formation, and Dendritic Cell Maturation Controls

Research paper thumbnail of Rho GTPases link cytoskeletal rearrangements and activation processes induced via the tetraspanin CD82 in T lymphocytes

Journal of cell science, Jan 15, 2002

Activation of T lymphocytes requires the engagement of the T-cell receptor and costimulation mole... more Activation of T lymphocytes requires the engagement of the T-cell receptor and costimulation molecules through cell-to-cell contacts. The tetraspanin CD82 has previously been shown to act as a cytoskeleton-dependent costimulation molecule. We show here that CD82 engagement leads to the tyrosine phosphorylation and association of both the Rho GTPases guanosine exchange factor Vav1 and adapter protein SLP76, suggesting that Rho GTPases participate in CD82 signaling. Indeed, broad inactivation of all Rho GTPases, or a specific blockade of RhoA, Rac1 or Cdc42, inhibited the morphological changes linked to CD82 engagement but failed to modulate the inducible association of CD82 with the actin network. Rho GTPase inactivation, as well as actin depolymerization, reduced the ability of CD82 to phosphorylate Vav and SLP76 and to potentiate the phosphorylation of two early TcR signaling intermediates: the tyrosine kinases ZAP70 and membrane adapter LAT. Taken together, this suggests that an a...

Research paper thumbnail of CD82, tetra-span-transmembrane protein, is a regulated transducing molecule on U937 monocytic cell line

Journal of leukocyte biology, 1995

The mononuclear cell surface protein IA4, recently classified as CD82, was originally identified ... more The mononuclear cell surface protein IA4, recently classified as CD82, was originally identified in our laboratory by the IA4 monoclonal antibody (mAb), because of its high expression on three lymphoblastoid, LAK-susceptible, variant cell lines. We have characterized CD82 as a new activation/differentiation marker of mononuclear cells. This protein belongs to the new family of TST proteins (tetra spans transmembrane), which includes CD9, CD37, CD53, CD63, and CD81 (TAPA-1). Here we demonstrate that cross-linking of IA4 mAbs induces an increase of intracellular free calcium in U937 cells and tyrosine phosphorylation of various proteins. Our data indicate that the intracellular calcium increase is initiated by a phospholipase C (PLC)-induced PtdIns(1,4,5)P3 second messenger followed by a more stable change, linked to extracellular calcium entry. This transducing signal was dependent on dual engagement of both CD82 and Fc receptors. Surface cross-linking of CD82 together with Fc recept...

Research paper thumbnail of CD82, member of the tetra-span-transmembrane protein family, is a costimulatory protein for T cell activation

Journal of immunology (Baltimore, Md. : 1950), 1995

It is now well documented that full activation of T cells requires a two-signal triggering that c... more It is now well documented that full activation of T cells requires a two-signal triggering that can be mimicked, in the absence of accessory cells, by co-immobilization of mAbs directed to stimulatory/accessory molecules (CD2, CD3, CD28, adhesion molecules, etc.). In this report, we describe that engagement of CD82 can delivery such a costimulatory signal for full activation of the human T cell line Jurkat, leading to strong IL-2 production and cell differentiation. The CD82 Ag, which belongs to the new tetra-span-transmembrane family (CD9, CD37, CD53, CD63, and CD81 (TAPA-1)), has been identified originally in our laboratory for its enhanced expression on three LAK-susceptible cell lines, and has been characterized as an activation/differentiation marker of mononuclear cells. Jurkat cells, stimulated in vitro by co-immobilization of anti-CD82 and anti-CD3 mAbs, produced high levels of IL-2, became strongly adherent to plastic dishes, and developed dendritic processes. These morphol...

Research paper thumbnail of Focal Adhesion Kinase Is Involved in Rabies Virus Infection through Its Interaction with Viral Phosphoprotein P

Journal of Virology, 2014

The rabies virus (RABV) phosphoprotein P is a multifunctional protein: it plays an essential 33 r... more The rabies virus (RABV) phosphoprotein P is a multifunctional protein: it plays an essential 33 role in viral transcription and replication, and in addition RABV P has been identified as an 34 interferon antagonist. Here, a yeast two hybrid screen revealed that RABV P interacts with the 35 focal adhesion kinase (FAK). The binding involved the domain 106-131 corresponding to the 36 dimerization domain of P and the C-ter domain of FAK containing the proline rich domains 37 PRR2 and PRR3. The P-FAK interaction was confirmed in infected cells by co-38 immunoprecipitation and co-localization of FAK with P in Negri bodies. By alanine 39 scanning, we identified a single mutation in the P protein that abolishes this interaction. The 40 mutant virus containing a substitution of Arg to Ala in position 109 (PR109A) in P, which 41 did not interact with FAK, is affected at a post-transcriptional step involving protein synthesis 42 and viral RNA replication. Furthermore, FAK depletion inhibited viral protein expression in 43 infected cells. This provides the first evidence of an interaction of RAV with FAK which 44 positively regulates infection.

Research paper thumbnail of Proteolysis and antigen presentation by MHC class II molecules

Advances in Immunology, 2002

ABSTRACT Proteolysis is the primary mechanism used by all cells not only to dispose of unwanted p... more ABSTRACT Proteolysis is the primary mechanism used by all cells not only to dispose of unwanted proteins but also to regulate protein function and maintain cellular homeostasis. Proteases that reside in the endocytic pathway are the principal actors of terminal protein degradation. The proteases contained in the endocytic pathway are classified into four major groups based on the active-site amino acid used by the enzyme to hydrolyze amide bonds of proteins: cysteine, aspartyl, serine, and metalloproteases. The presentation of peptide antigens by major histocompatibility complex (MHC) class II molecules is strictly dependent on the action of proteases. Class II molecules scour the endocytic pathway for antigenic peptides to bind and present at the cell surface for recognition by CD4+ T cells. The specialized cell types that support antigen presentation by class II molecules are commonly referred to as professional antigen presenting cells (APCs), which include bone marrow-derived B lymphocytes, dendritic cells (DCs), and macrophages. In addition, the expression of certain endocytic proteases is regulated either at the level of gene transcription or enzyme maturation and their activity is controlled by the presence of endogenous protease inhibitors.

Research paper thumbnail of Interaction of CD82 Tetraspanin Proteins with HTLV-1 Envelope Glycoproteins Inhibits Cell-to-Cell Fusion and Virus Transmission

Virology, 2000

The entry of retroviruses into their target cell involves interactions between the virus envelope... more The entry of retroviruses into their target cell involves interactions between the virus envelope glycoproteins and their cellular receptors, as well as accessory ligand-receptor interactions involving adhesion molecules that can also participate in fusion. We have studied the contribution of CD82 proteins to the transmission of the human T-cell leukemia virus type 1 (HTLV-1), which is greatly dependent on cell-to-cell contacts. CD82 proteins belong to a class of cell surface molecules, the tetraspanins, that can act as molecular facilitators in cellular adhesion processes. The coexpression of CD82 proteins with HTLV-1 envelope glycoproteins resulted in marked inhibition of syncytium formation, whereas CD82 proteins had no effect on syncytium formation induced by human immunodeficiency virus type 1 (HIV-1) envelope proteins. The presence of CD82 proteins also inhibited cell-to-cell transmission of HTLV-1. Coimmunoprecipitation and cocapping experiments showed that CD82 associates with HTLV-1 envelope glycoproteins, both within the cell and at the cell surface. Finally, whereas the intracellular maturation of HTLV-1 glycoproteins was not modified by the presence of CD82 proteins, HTLV-1 protein coproduction delayed the intracellular maturation of CD82 proteins. There thus seems to be a reciprocal interaction between virus and cell proteins, and the cellular proteins involved in adhesion modulate retrovirus transmission both positively, as shown in other systems, and negatively, as shown here.

Research paper thumbnail of Uncoating ATPase Hsc70 is recruited by invariant chain and controls the size of endocytic compartments

Proceedings of the National Academy of Sciences, 2002

Targeting of class II major histocompatibility complex molecules to endocytic compartments is med... more Targeting of class II major histocompatibility complex molecules to endocytic compartments is mediated by their association with the invariant chain (Ii). Although the identity of certain sorting signals located in Ii's cytoplasmic tail is known, proteins that interact with Ii's cytoplasmic tail in living cells remain to be identified. Synthesis of a biotinylated trimeric Ii cytoplasmic tail allowed the retrieval of two proteins that interact with this domain. We identify one of them as the 70-kDa heat-shock cognate protein (hsc70), the uncoating ATPase of clathrin-coated vesicles, and the other as its mitochondrial homologue, the glucose-regulated protein grp75. Expression of Ii in COS cells results in the formation of large endocytic compartments. We observe extensive colocalization of hsc70 with Ii in these macrosomes. Expression of a dominantnegative (K71M) green fluorescent protein-tagged version of hsc70 counteracted the ability of Ii to modify the endocytic pathway, demonstrating an interaction in vivo of Ii with hsc70 as part of the machinery responsible for macrosome formation.

Research paper thumbnail of Dendritic Cell Maturation Controls Adhesion, Synapse Formation, and the Duration of the Interactions with Naive T Lymphocytes

The Journal of Immunology, 2004

The initiation of adaptive immune responses requires the direct interaction of dendritic cells (D... more The initiation of adaptive immune responses requires the direct interaction of dendritic cells (DCs) with naive T lymphocytes. It is well established that the maturation state of DCs has a critical impact on the outcome of the response. We show here that mature DCs form stable conjugates with naive T cells and induce the formation of organized immune synapses. Immature DCs, in contrast, form few stable conjugates with no organized immune synapses. A dynamic analysis revealed that mature DCs can form long-lasting interactions with naive T cells, even in the absence of Ag. Immature DCs, in contrast, established only short intermittent contacts, suggesting that the premature termination of the interaction prevents the formation of organized immune synapses and full T cell activation.

Research paper thumbnail of Analysis of Protease Activity in Live Antigen-presenting Cells Shows Regulation of the Phagosomal Proteolytic Contents During Dendritic Cell Activation

Journal of Experimental Medicine, 2002

Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagoso... more Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagosomes in live antigen-presenting cells. We find that an ingested particle sequentially encounters distinct protease activities during phagosomal maturation. Incorporation of active proteases into the phagosome of the macrophage cell line J774 indicates that phagosome maturation involves progressive fusion with early and late endocytic compartments. In contrast, phagosome biogenesis in bone marrow-derived dendritic cells (DCs) and macrophages preferentially involves endocytic compartments enriched in cathepsin S. Kinetics of phagosomal maturation is faster in macrophages than in DCs. Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide. This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation. Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.

Research paper thumbnail of Structure of the Tetraspanin Main Extracellular Domain. A PARTIALLY CONSERVED FOLD WITH A STRUCTURALLY VARIABLE DOMAIN INSERTION

Journal of Biological Chemistry, 2001

The tetraspanin family of membrane glycoproteins is involved in the regulation of cellular develo... more The tetraspanin family of membrane glycoproteins is involved in the regulation of cellular development, proliferation, activation, and mobility. We have attempted to predict the structural features of the large extracellular domain of tetraspanins (EC2), which is very important in determining their functional specificity. The tetraspanin EC2 is composed of two subdomains: a conserved three-helix subdomain and a variable secondary structure subdomain inserted within the conserved subdomain. The occurrence of key disulphide bridges and other invariant residues leads to a conserved relative topology of both subdomains and also suggests a structural classification of tetraspanins. Using the CD81 EC2 structure as a template, the structures of two other EC2s were predicted by homology modeling and indicate a conserved shape, in which the variable subdomain is located at one side of the structure. The conserved and variable subdomains might contain sites that correspond, respectively, to common and specific interactions of tetraspanins. The tetraspanin EC2 seems to correspond to a new scheme of fold conservation/variability among proteins, namely the insertion of a structurally variable subdomain within an otherwise conserved fold.

Research paper thumbnail of The engagement of CD82 is a costimulatory signal inducing cytokines production by normal resting or in vitro differentiated T lymphocytes

Research paper thumbnail of Signaling through CD82 and other tetraspanins induces sustained morphological changes involved in T cell activation

Research paper thumbnail of Signaling through the tetraspanin CD82 triggers its association with the cytoskeleton leading to sustained morphological changes and T cell activation

European Journal of Immunology, 1998

In this report, we provide new evidence of a crosstalk between T cell activation and adhesion pro... more In this report, we provide new evidence of a crosstalk between T cell activation and adhesion processes through a functional cytokeleton. We show that CD82 signaling induces longlasting adhesion, spreading and development of membrane extensions, involving actin polymerization. Addition of various co-stimuli (phorbol 12-myristate 13-acetate or monoclonal antibodies to CD3 or CD2) increases the CD82-induced morphological alterations and, reciprocally, CD82 engagement synergizes with these stimuli to induce T cell activation as indicated by both primary tyrosine phosphorylation and IL-2 production. Different kinases are involved in both processes. CD82 co-signaling involves src kinases including p56 lck. On the other hand, the CD82-induced alterations of cell morphology are negatively regulated by cAMP-dependent kinases independently of activation of src kinases. Simultaneously with cytoskeletal rearrangements, we observed an inducible association of CD82 with the cytoskeletal matrix. In addition, the potentiating and stabilizing effects induced by CD82 crosslinking on tyrosine phosphorylation were abolished by cytoskeleton-disrupting agents. These results suggest that the actin polymerization triggered by CD82, through its ability to associate with the cytoskeletal matrix, is the primary step involved in the CD82 induced costimulatory activity. Our data provide further evidence for a direct role of the actin cytoskeleton as a major component for sustained signal transduction in T cells and suggest that tetraspanins could be "membrane organizers" connecting both surface and intracellular molecules.