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  Director : Dautry-Varsat Alice (adautry@pasteur.fr)



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 in the external milieu. These interactions take place at the plasma membrane and may induce intracellular signals or even allow the entry of pathogens into the cell. 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. Dautry-Varsat, A. Alcover, V. Das, F. Gesbert, B. Nal, N. Sauvonnet)

Membrane receptors recognize their extracellular ligand and the ligand-receptor complexes are then endocytosed. Receptors and ligands are then sorted in intracellular membrane compartments. 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.

We have developped new tools to specifically and efficiently block clathrin-mediated endocytosis : dominant negative mutants of the protein Eps15. This has allowed to identify 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 could not be detected in clathrin-coated structures on the plasma membrane, unlike transferrin receptors which are markers of the clathrin-dependent endocytosis pathway. Instead, 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.

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 recent work lead to the identification of a short sequence located in the intracellular part of the IL2 receptor b chain (IL2Rb). This sequence is responsible for the specific targetting of IL-2Rb to the lysosomes. In addition, we observed that the IL2Rb chain is mono-ubquitinated. 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 IL2Rb ubiquitination prevents its sorting to lysosomes, and thus its degradation, while its endocytosis remains unmodified. Similar results were obtained by modifying the previously described sorting sequence in IL2Rb, to prevent the addition of ubiquitin. Our work is now focused on various aspects of the ubiquitin-dependent regulation of expression of the IL2Rb chain. In addition to the identification of the ubiquitination target residues, we are studying the proteins that associate to the ubiquitinated IL-2Rb during its trafficking from early endosomes to late endosomes/lysosomes.

T cell antigen receptor polarization at the "immunological synapse". Role of the actin cytoskeleton.

A key event of an immune response is the recognition by T lymphocytes of 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 clusters. This organized cell to cell contact has been termed the immunological synapse, by analogy with the neural 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 investigated the role of ezrin, a protein that links the plasma membrane with the actin cytoskeleton. We observed, by confocal microscopy, that ezrin accumulates together with fillamentous actin at the site of contact of T cells with antigen presenting cells, indicating that ezrin 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, as well as the accumulation of protein kinase C theta, a key signaling molecule for T cell activation, at the immunological synapse. Furthermore, 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, which helps molecular clustering at the immunological synapse and T cell activation.

2. Invasion BY INTRACELLULAR BACTERIA, CHLAMYDIA(A. Dautry-Varsat, A. Subtil, M. E. Balana, 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 analyse 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 have shown the involvement of cholesterol and sphingolipid-rich membrane microdomains (also named " lipid rafts ") in Chlamydia entry. 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 Chamydia 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 EB at the end of the cycle.

Keywords: Endocytosis, intracellular traffic, interleukin 2 receptor, T cell receptor, ezrin, immunological synapse, actin cytoskeleton, Chlamydia, bacterial type III secretion

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  Office staff Researchers Scientific trainees Other personnel
  GOISNARD, Christiane, (cgoisnar@pasteur.fr) ALCOVER Andrés, IP,aalcover@pasteur.fr

DAUTRY-VARSAT Alice, IP,adautry@pasteur.fr

GESBERT, Franck, IP,fgesbert@pasteur.fr

SUBTIL Agathe, CNRS,asubtil@pasteur.fr

BALANA, Maria-Eugenia, Post-doctoral fellow,mbalana@pasteur.fr

DAS Vincent, PhD student,vdas@pasteur.fr

DELEVOYE Cédric, PhD student,delevoye@pasteur.fr

NAL Béatrice, Post-doctoral fellow,bnal@pasteur.fr

SAUVONNET Nathalie, Post-doctoral fellow,nathsauv@pasteur.fr

WYPLOSZ Benjamin, MD PhD student (part time)bwyplosz@pasteur.fr

DUJEANCOURT, Annick, IP, Technician,adujean@pasteur.fr)

PERRINET, Stéphanie, CNRS, Technician,perrinet@pasteur.fr)

MALARDE, Valérie, IP, Technician (part-time),vmalarde@pasteur.fr)

GOISNARD, Christiane, IP, Secretary (part-time),cgoisnar@pasteur.fr

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