Unit: Biology of Cell Interactions - CNRS URA 2582
Director: DAUTRY-VARSAT Alice
The research of this unit focuses on (i) the mechanisms of entry, signalisation and the intracellular fate of cytokine receptors (ii) the mechanisms of entry and development of intracellular bacteria, the Chlamydia, which are mainly responsible for pneumopathies, sexually transmitted diseases and blindness.
1. RECEPTOR DYNAMICS AND INTRACELLULAR TRAFFIC
(F. Gesbert, N. Sauvonnet)
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.
The research of this unit focuses on the mechanisms of entry, signalisation and the intracellular fate of cytokine receptors. Studies on the trafficking of the antigen receptor have been initiated in this unit by the group of Andrés Alcover. Results regarding this subject are in the report of the Unit of Lymphocyte Cell Biology, created in 2005 and directed by Andrés Alcover.
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 other internalization pathways exist.
The common cytokine receptor γc belongs to the type I cytokine receptor family. It is shared by interleukin (IL-) 2, 4, 7, 9, 15 and IL-21 receptors. 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 γc 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. Regulation by ubiquitination can be controled by a balance between the activity of ubiquitin ligases and ubiquitin hydrolases. We have shown that coexpression of γc and of the ubiquitin ligase c-Cbl results in an important decrease in γc expression. Conversly, expression of the ubiquitin hydrolase DUB-2 results in an increase in γc expression. We have also shown that DUB-2 is able to counteract c-Cbl effect on γc expression, in a dose dependent manner. Thus, we propose that the ubiquitin-ligase/ubiquitin-hydrolase c-Cbl/DUB-2 can regulate the expression of the common cytokine receptorγc.
2. Invasion OF HOST CELLS BY INTRACELLULAR BACTERIA, CHLAMYDIA
(A. Subtil, M. E. Balañá, G. Boncompain, C. Delevoye)
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.
Molecular mechanisms of Chlamydia entry
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.
Another small GTPase, Arf6, is known for its role in membrane trafficking and actin polymerization. We have shown that infection by C. caviae induces rapid and transient activation of Arf6, whose activity is required for entry. The downstream effector of Arf6, the phosphatidylinositol 4-phosphate 5-kinase, and its product, the phosphatidylinositol 4,5-biphosphate, accumulate at the entry sites and are necessary for bacteria entry. On the contrary, the activity of phospholipase D, another Arf6 effector, is not required for entry. Finally, our data suggest that during Chlamydia entry, Arf6 activity controls actine polymerization rather than membrane recruitment to the entry site.
Systematic search for type III secreted proteins
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 twenty-four new proteins which are candidate to be secreted by several species of Chlamydia 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. Our working model is that these proteins have co-evolved with the SNARE machinery for a role in membrane fusion.
Chlamydia pneumoniae and atherosclerosis : clinical studies
The pathological and clinical relevance of C. pneumoniae in atherosclerosis is still unknown. Reliable current diagnostic tests for endovascular bacteria require biopsies during atherectomy or bypass surgery and can only be performed at advanced stages of atherosclerosis. We developped a new diagnostic approach to detect endocoronary bacteria using balloons that have been inserted endoluminally to perform coronary angioplasty. DNA was extracted from this material and nested PCR was performed. We detected C. pneumoniae DNA in 40% of the balloons from patients with native atherosclerotic stenosis, but not in balloons from patients with restenosis. Moreover, we found that the presence of endovascular bacteria is correlated with the severity of coronary artery disease. Thus, we have developped a new tool to explore the link between atherosclerosis and infections at earlier stages of atherosclerosis than existing methods. It could help selecting patients for therapeutic trials.
Figure 1. Dynamics and intracellular traffic of membrane receptors. Membrane receptors at the cell surface can be internalised in intracellular compartment (endocytosis). Then, they can be recycled back to the plasma membrane (recycling) or sorted and degraded in lysosomes (degradation).
Figure 2. Working model for the link between endocytosis and actin cytoskeleton. A core complex composed of dynamin, cortactin, Arp2/3 complex and F-actin appears to be involved in both clathrin-dependent and -independent pathways. Cortactin is proposed to play a central role in connecting endocytosis to actin dynamics through dynamin. Syndapin, intersectin and mAbp1 are actin-dynamin interacting proteins specifically required in clathrin-coated pits and vesicles formation.
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, ubiquitination, intracellular traffic, cytokine receptor, interleukin 2, small GTPase, SNARE, Chlamydia, type III secretion