Unit: Biology of Cell Interactions - CNRS URA 2582
Director: DAUTRY-VARSAT Alice
The research of this unit is focused on the mechanisms of entry and intracellular fate of some key receptors of the immune system, the T lymphocyte antigen receptor and the receptors of a cytokine, the interleukin 2, and of intracellular bacteria, the Chlamydia. These bacteria are mainly responsible for pneumopathies, sexually transmitted diseases and blindness.
1. RECEPTOR DYNAMICS AND INTRACELLULAR TRAFFIC (A. Alcover, A. Dautry-Varsat, F. Gesbert, N. Sauvonnet, S. Charrin, V. Das, M. I. Thoulouze)
Membrane receptors bind their extracellular ligand and the ligand-receptor complexes are endocytosed. Receptors and ligands are then sorted in membrane compartments towards degradation or recycling. Endocytosis leads to changes in the expression, function and localisation of membrane components.
Receptor-mediated endocytosis through clathrin-coated pits and vesicles has been by far the most thoroughly investigated. Receptor-ligand complexes concentrate in coated pits which invaginate to form coated vesicles that bud from the plasma membrane and rapidly loose their coat. These vesicles then fuse with intracellular compartments named endosomes. However, in the recent years, it has appeared that there exist other internalization pathways.
The common cytokine receptorγc belongs to the type I cytokine receptor family. It is shared by interleukin (IL-) 2, 4, 7, 9, 15 and IL-21 receptors. Thus, the γc receptor plays a major role in lymphocyte proliferation and differentiation, leading when mutated to X-linked severe combined immunodeficiency. Having identified IL-2, an essential growth factor for lymphocytes, as the first physiological ligand entering by a new clathrin-independent mechanism, we studied the endocytic pathway of γc. This receptor is rapidly internalized and reaches early endosomes. We have shown that endocytosis of the common γc cytokine receptor is clathrin-independent by using a dominant-negative mutant of Eps15 or RNA interference to knock down clathrin heavy chain. This pathway requires the GTPase dynamin. In addition, it requires actin polymerization. To further characterize the function of dynamin in clathrin-independent endocytosis, in particular its connection with the actin cytoskeleton, we focused on dynamin binding proteins that interact with F-actin. We compared the involvement of these proteins in the clathrin-dependent and independent pathways. Thus, we observed that intersectin, syndapin and mAbp1, which are necessary for the uptake of transferrin (Tf), a marker of the clathrin route, are not required for γc receptor endocytosis. Strikingly, cortactin is needed for both γ c and Tf internalizations. These results reveal the ubiquitous action of cortactin in internalization processes and suggest its role as a linker between actin dynamics and clathrin-dependent and -independent endocytosis.
Sorting of intracellular receptors : a role for ubiquitin
After internalization, IL2 receptors reach endosomes where they are sorted and can be recycled back to the plasma membrane or addressed to late endosomes/lysosomes where they are degraded. We have shown that IL2 receptor β chains are mono-ubiquitinated, which is essential for this intracellular sorting. Ubiquitin is a small, very conserved, protein that has been recently shown to regulate the intracellular traffic to late endosomes/lysosomes. Inhibition of ubiquitination prevents IL2 receptor β sorting to lysosomes, and thus its degradation, while its endocytosis remains unmodified. Our work is now focused on various aspects of the ubiquitin-dependent regulation of expression of the IL2 receptor β chain and of γc chain. In addition to the identification of the ubiquitination target residues, we are studying the proteins involved in the ubiquitination of receptors, which control their trafficking from early endosomes to late endosomes/lysosomes and thus their overall expression.
T cell antigen receptor polarization at the immunological synapse. Role of the actin cytoskeleton and of intracellular vesicular trafficking.
A key event of an immune response is the recognition by T lymphocytes of peptide antigens displayed on the surface of antigen presenting cells. Soon upon antigen recognition, T cell antigen receptors (TCR), adhesion molecules, as well as signaling and cytoskeletal components, translocate to the contact site between the T cell and the antigen presenting cell and segregate into different clusters. This organized cellular junction, where communication between the T lymphocyte and the antigen presenting cell takes place, has been called the immunological synapse. We investigate the mechanisms that lead to the formation of the immunological synapse and result in T cell activation. In particular, we analyze the role of the actin cytoskeleton and of intracellular traffic in these processes.
We have shown that ezrin, a protein that links the membrane with the actin cytoskeleton transiently polarizes towards the antigen presenting cell (Figure 1). Moreover, we observed that over-expression of a truncated form of ezrin inhibited T cell receptor clustering at the immunological synapse, as well as later events of T cell activation, such as IL-2 gene activation. These data provide evidence for an important role of ezrin in T lymphocytes. By linking membrane and actin cytoskeleton components, ezrin may help molecular clustering at the immunological synapse and modulate T cell activation.
We also showed that intracellular vesicular trafficking is a key process in the formation of the immunological synapse. By using confocal microscopy, time-lapse digital imaging and quantitative image analysis, we showed that recycling endosomes can transport T cell receptors from other sites of the cell membrane to the immunological synapse, and that this transport is necessary for efficient T cell polarization at the immunological synapse (Figure 2). Moreover, we showed that SNARE proteins, that control the fusion between recycling vesicles and the plasma membrane, are required. These results show that intracellular vesicular transport is a key mechanism to transport T cell receptors and to promote their polarization at the immunological synapse.
2. Invasion OF HOST CELLS BY INTRACELLULAR BACTERIA, CHLAMYDIA (A. Dautry-Varsat, A. Subtil, M. E. Balañá, C. Delevoye, B. Wyplosz)
Chlamydiae are bacteria that proliferate only within eukaryotic host cells. The three species pathogenic to humans, Chlamydia trachomatis, Chlamydia psittaci and Chlamydia pneumoniae, cause a number of diseases, including trachoma, pelvic inflammatory disease, pneumonia and trachoma.
Primary infections are often minor or asymptomatic; the sequelae, blindness, sterility or ectopic pregnancy appear long after infection. Throughout their cycle in the host cell, chlamydiae remain in a membrane-bound compartment called an inclusion (Figure 3). At the end of the cycle, the host cell is lysed and infectious forms are produced. We analyze the mechanisms used by the bacteria to enter the cells, grow and modify them.
Since Chlamydia are strict intracellular parasites, their development depends on their capacity of being internalized by the host cell, in particular by epithelial cells, which are their main target (Figure 4). To that end Chlamydiae, like other pathogens, have evolved strategies that utilize the existing endocytic machineries and signaling pathways. Indeed, we have shown that within five minutes of infection by Chlamydia caviae GPIC strain, several events take place in the immediate vicinity of invasive bacteria: GM1-containing microdomains cluster, tyrosine-phosphorylated proteins accumulate, and intense actin polymerization occurs. We have also shown that actin polymerization is controlled by the small GTPases Cdc42 and Rac, which become activated upon infection.
During Chlamydia development cycle, the volume of the inclusions increases considerably, until they occupy a large portion of the cytoplasm. The inclusion membrane contains lipids that come from the host-cell. It also contains proteins produced by the bacteria which proliferate inside the inclusion. We have shown that Chlamydia use a secretion mechanism, which in other bacterial pathogens is involved in delivery of bacterial proteins within or through the membrane of eukaryotic host-cells. We have developed a systematic approach to find proteins secreted by Chlamydia by this type of secretion mechanism (named type III) into the host cell during infection. We have identified several candidate proteins and their characterization is underway. These proteins are very likely important in Chlamydia pathogenicity.
IncA is one of the proteins synthesized and secreted onto the inclusion membrane by the bacteria. IncA proteins from different species of Chlamydiae show little sequence similarity. We have shown that both C. trachomatis and C. caviae IncA are conserved. Both proteins associate with themselves to form multimers. When artificially expressed by the host cell, they localize to the endoplasmic reticulum. Strikingly, heterologous expression of IncA in the endoplasmic reticulum completely inhibits concomitant inclusion development. The development of a C. trachomatis strain that does not express IncA is not inhibited by artificial IncA expression, showing that the disruptive effect observed with the wild type strain requires direct interactions between IncA molecules at the inclusion membrane and on the endoplasmic reticulum. Finally, we modeled IncA tetramers in parallel four helix bundles based on the structure of the SNARE complex, a conserved structure involved in membrane fusion in eukaryotic cells and found that IncA tetramers were highly stable in this model. We propose that these proteins may have co-evolved with the SNARE machinery for a role in membrane fusion.
Figure 1. T lymphocyte (Tc) interacting with a cell presenting a bacterial superantigen (APC) observed by confocal microscopy. The actin cytoskeleton associated protein ezrin (red) and the T cell antigen receptor (green) are polarized towards the antigen presenting cell (immunofluorescence, right). On the left, the morphology of the two cells is seen by interferential contrast microscopy.
Figure 2. Polarized transport of T cell receptors towards the immune synapse via endosomal vesicles. Human T lymphocyte (Tc) encountering a cell presenting a bacterial superantigen (APC). T cell receptors present in endosomal vesicles were labeled with a fluorescent anti-T cell receptor antibody, which was previously internalized. The picture displays the merge between the flurescence image (green) and the interference contrast image (gray), and shows that T cell receptor-containing vesicles polarize and are in close apposition to the cell-cell contact zone where the immune synapse forms.
Figure 3. Scheme of the infectious cycle of Chlamydia. The whole cycle takes place in the host-cell, in 48 to 72 hours. The infectious form of Chlamydia (EB) differentiates once inside the cell in the proliferative form (RB), which multiply and differentiate to EBs at the end of the cycle.
Figure 4. Chlamydia at the surface of an epithelial cell, as observed by scanning electron microscopy. The bacteria are stained with antibodies coupled to gold particles (white dots on the picture). Source: M. E. Balañá, with M. C. Prévost and S. Giroux, (electron microscopy platform, Institut Pasteur).
Figure 5. Scheme of the interactions between bacteria and the epithelial host-cell. Attachment to the cells involves membrane microdomains. Remodelling of the actin cytoskeleton follows, and bacteria enter by a process resembling phagocytosis. Signal transduction is detected early after infection. Type III secretion is active soon after entry. From their localisation at the frontier between the host and the pathogens, Inc proteins (triangles) are attracting candidates to play a role in a variety of functions: escape from lysosomal degradation pathways, microtubule-dependent migration of the inclusion towards the centrosome, import of nutrients and of lipids from the host cell. Some bacterial proteins are secreted and reach the cell cytosol (stars).
Keywords: Endocytosis, intracellular traffic, cytokine receptor, interleukin 2, T cell receptor, ezrin, immunological synapse, actin cytoskeleton, SNARE, Chlamydia, bacterial type III secretion, cell biology, immunology