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 and of intracellular bacteria, the Chlamydia. These bacteria are mainly responsible for pneumopathies, sexually transmitted diseases and blindness.

Cells communicate with their environment through membrane receptors, which recognize molecules and particles, the receptors' ligands, in the external milieu. These interactions take place at the plasma membrane and may induce intracellular signals and allow the entry in the cell of the receptors and their ligands. Our research is focused on the entry and intracellular traffic in eukaryotic cells of membrane receptors and intracellular bacteria, the Chlamydia. The receptors studied are expressed on lymphocytes and are essential for the immune response: the T lymphocyte antigen receptor and the receptors of a cytokine, the interleukin 2. Depending on the strain, the chlamydiae are the causative agents of sexually transmitted diseases, pulmonary infections and eye infections, and they may also be involved in atherosclerosis. We are currently characterizing their entry into and development within host cells.

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 then endocytosed. Receptors and ligands are then sorted in membrane compartments towards an intracellular location, degradation or recycling. Endocytosis leads to changes in the expression, function and localisation of membrane components.

Endocytosis pathways

Receptor mediated endocytosis allows cells to communicate with their environment via membrane receptors, which specifically bind macromolecules in the extracellular milieu. It is an essential process for cell homeostasy since it controls many functions including nutriment uptake, growth factor and hormone responses, antigen presentation and the entry of some pathogens.

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.

We have identified interleukin 2 (IL2), an essential growth factor for lymphocytes, as the first physiological ligand endocytosed by a new clathrin-independent mechanism. IL2 receptor endocytosis is rapid and efficient. IL2 receptors were found constituvely associated with membrane microdomains enriched in cholesterol and sphingolipids, named "rafts". The receptors are still in rafts when they reach endosomes. Finally, this new endocytosis pathway is specifically regulated by Rho family GTPases, while the GTPase dynamin is involved both in clathrin-dependent and independent endocytosis. Studies in progress aim at analysing the role of IL2 in the localisation of these receptors in membrane microdomains. They also aim at understanding the links between this new endocytic pathway and the signaling cascade induced by IL2 binding to its receptors. Finally, we are analyzing the role of the actin cytoskeleton in this endocytic pathway.

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 adressed to late endosomes/lysosomes where they are degraded. Our work lead to the identification of a short sequence located in the intracellular part of the IL2 receptor β chain (IL2Rβ). This sequence is responsible for the specific targeting of IL2Rβ to the lysosomes. In addition, we observed that ubquitination is essential for this intracellular sorting. Ubiquitin is a small, very conserved, protein whose function has been clearly established in the proteasome-dependent degradation of proteins and, more recently, has been shown to regulate the intracellular traffic to late endosomes/lysosomes. Inhibition of ubiquitination prevents IL2Rβsorting 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 IL2Rβ 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 by T lymphocytes, T cell antigen receptors (TCR), adhesion molecules, and 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 cell-to-cell contact, where communication between the T cell and the antigen presenting cell takes place, has been called the immunological synapse, by analogy with the neuronal synapse. Molecular organization at the immunological synapse is thought to be important to stabilize the T cell receptor signal transduction necessary for efficient T cell activation. 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 observed, by confocal microscopy, a strong reorganization of the T cell actin cytoskeleton at the site of contact with the antigen presenting cell. Thus, filamentous actin (F-actin) accumulates and transiently covers a large area of the contact site with the antigen presenting cell. Ezrin, a protein that links the membrane with the actin cytoskeleton also accumulates in the same area, indicating that it is involved in the reorganization of the actin cytoskeleton induced by antigen recognition (Figure 1). To investigate the functional importance of ezrin in T cell responses, we studied cells expressing a dominant negative mutant of ezrin. By confocal microscopy and quantitative image analysis, we observed that dominant negative ezrin inhibited T cell receptor clustering at the immunological synapse. In addition, this mutant inhibited the accumulation of protein kinase C (PKC)theta, a key signaling molecule for T cell activation that also translocates to the immunological synapse. Finally, we observed that dominant negative ezrin inhibits later events of T cell activation. These data provide evidence for a new role of ezrin in T lymphocytes. Ezrin may provide a link between membrane and actin cytoskeleton components that helps molecular clustering at the immunological synapse and T cell activation.

We also study the role of intracellular vesicular trafficking in the formation of the immunological synapse. By using confocal microscopy, time-lapse digital imaging and quantitative image analysis, we showed that the recycling endosomal compartment of the T lymphocyte polarizes towards the antigen presenting cell contact site upon antigen recognition. Endocytic vesicles are transported to the cell-cell contact site and recycle preferentially at the immunological synapse. This polarized vesicle transport facilitates the accumulation at the immunological synapse of membrane receptors that undergo cycles of endocytosis and recycling, like the T cell receptor. Consistently, perturbing T cell polarization, or the endocytic and recycling traffic of the T cell receptor, by pharmacological or biological approaches, results in reduced accumulation of T cell receptors at the immunological synapse. We propose that this intracellular transport mechanism facilitates the polarization of T cell receptors at the immunological synapse and is complementary of other mechanisms of receptor transport at the cell surface.

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 2). 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. Using dominant negative mutants of the protein Eps15 that we had prepared and characterized previously, we showed that Chlamydia entry does not involve clathrin-coated pits. Moreover, the entry of all three species of Chlamydia is inhibited by  cytochalasin D, an inhibitor of actin polymerization. This indicates that Chlamydia entry resembles more a process of phagocytosis, although it takes place in epithelial cells. We are currently studying the role of the actin cytoskeleton in this entry process.

We have shown the involvement in Chlamydia entry of membrane microdomains, enriched in cholesterol and sphingolipids (also named " lipid rafts "). Thus, Chlamydia are concentrated in these membrane microdomains  at the cell surface. If the formation of these microdomains is perturbed, for instance by modifying the plasma membrane cholesterol content, Chlamydia entry into host cells is inhibited. Furthermore, once internalized, the bacteria remain for several hours in these microdomains inside the inclusion. This could explain why Chlamydia inclusions lack classical endocytic markers. It could also explain certain properties of the inclusion, which seems to interact, via vesicular traffic, with the secretion pathway and not with the endocytic pathway.

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 are presently developping 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.


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. 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 3. 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 4. 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, interleukin 2 receptor, T cell receptor, ezrin, immunological synapse, actin cytoskeleton, Chlamydia, bacterial type III secretion

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