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SummaryHIV-1-infected lymphocytes improperly respond to T cell antigen receptor (TCR) stimulation. To document this phenomenon, we studied the capacity of HIV-1-infected lymphocytes to form immunological synapses. We show here that HIV-1-infected T cells poorly conjugated with antigen-presenting cells, and when they formed conjugates, the synapses were abnormal. TCR and Lck accumulated in the recycling endosomal compartment, and their clustering at the synapse was severely reduced. These phenomena were, to a large extent, caused by Nef, a viral protein affecting intracellular trafficking and signaling pathways. Concomitantly, in HIV-infected cells, tyrosine phosphorylation at the synapse and the patterns of tyrosine phosphorylated proteins were disturbed in a Nef-dependent manner. These findings underscore the importance of Lck and TCR endosomal trafficking in synapse formation and early T cell signaling. Alteration of endocytic and signaling networks at the immunological synapse likely impacts the function and fate of HIV-1-infected cells.

International audience; Endocytosis is critical for many cellular functions. We show that endocytosis of the common gammac cytokine receptor is clathrin independent by using a dominant-negative mutant of Eps15 or RNA interference to knock down clathrin heavy chain. This pathway is synaptojanin independent and requires the GTPase dynamin. In addition, this process 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 gammac receptor endocytosis. Strikingly, cortactin is needed for both gammac 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.

The developmental cycle of Chlamydiaceae occurs in a membrane compartment called an inclusion. IncA is a member of a family of proteins synthesized and secreted onto the inclusion membrane by bacteria. IncA proteins from different species of Chlamydiaceae show little sequence similarity. We report that the biochemical properties of Chlamydia trachomatis and Chlamydia caviae are conserved. Both proteins self-associate 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. Using truncated forms of IncA from C. caviae, we show that expression of the C-terminal cytoplasmic domain of the protein at the surface of the endoplasmic reticulum is sufficient to disrupt the bacterial developmental cycle. On the other hand, 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 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. Both C. trachomatis and C. caviae IncA tetramers were highly stable in this model. In conclusion, we show that the property of IncA proteins to assemble into multimeric structures is conserved between chlamydial species, and we propose that these proteins may have co-evolved with the SNARE machinery for a role in membrane fusion.

Bacteria of the Chlamydiales order are very successful intracellular organisms that grow in human and animal cells, and even in amoebae. They fulfill several essential functions to enter their host cells, establish an intracellular environment favorable for their multiplication and exit the host cell. They multiply in a unique organelle called the inclusion, which is isolated from the endocytic but not the exocytic pathway. A combination of host cell factors and of proteins secreted by the bacteria, from within the inclusion, contribute to the establishment and development of this inclusion. Here we review recent data on the entry mechanisms and maturation of the inclusion.

The mechanism by which T cell antigen receptors (TCR) accumulate at the immunological synapse has not been fully elucidated. Since TCRs are continuously internalized and recycled back to the cell surface, we investigated the role of polarized recycling in TCR targeting to the immunological synapse. We show here that the recycling endosomal compartment of T cells encountering activatory antigen-presenting cells (APCs) polarizes towards the T cell-APC contact site. Moreover, TCRs in transit through recycling endosomes are targeted to the immunological synapse. Inhibition of T cell polarity, constitutive TCR endocytosis, or recycling reduces TCR accumulation at the immunological synapse. Conversely, increasing the amount of TCRs in recycling endosomes before synapse formation enhanced their accumulation. Finally, we show that exocytic t-SNAREs from T cells cluster at the APC contact site and that tetanus toxin inhibits TCR accumulation at the immunological synapse, indicating that vesicle fusion mediated by SNARE complexes is involved in TCR targeting to the immunological synapse.

Problem: The aim of the present study was to define the expression of CXCR4 and CCR5 on non-cultured non-stimulated primary human trophoblast cells (TCs) immediately after their immunopurification. Method of study: We have evaluated by flow cytometric analysis and immunofluorescence, highly purified primary TCs prepared from first trimester (8.2 ± 0.3 weeks, n = 15) and term (Caesarean section, n = 10) placentae for the cell surface and intracellular expression of CXCR4 and CCR5. Results: There was a high level of individual variability for CXCR4 and CCR5 expression between trophoblast batches. In first trimester and term placentae TCs, we found a greater number of TCs preparations expressing intracellular CXCR4 than CCR5 (P

Upon antigen recognition, T cells undergo substantial membrane and cytoskeletal rearrangements that lead to the formation of the immunological synapse and are necessary for subsequent T-cell activation. However, little is known about how membrane and cytoskeletal molecules interact during these processes. Here we discuss the involvement of the membrane-microfilament linker ezrin. We propose that ezrin is a component of the cytoskeleton-mediated architecture of the immunological synapse that plays a role in T-cell receptor clustering, protein kinase C theta translocation and intracellular signaling.

Infection with an obligate intracellular bacterium, theChlamydia trachomatislymphogranuloma venereum (LGV/L2) strain or the guinea pig inclusion conjunctivitis serovar ofChlamydia psittaci, leads to apoptosis of host cells. The apoptosis is not affected by a broad-spectrum caspase inhibitor, and caspase-3 is not activated in infected cells, suggesting that apoptosis mediated by these two strains ofChlamydiais independent of known caspases. Overexpression of the proapoptotic Bcl-2 family member, Bax, was previously shown to induce caspase-independent apoptosis, and we find that Bax is activated and translocates from the cytosol to the mitochondria inC. psittaci-infected cells.C. psittaci-induced apoptosis is inhibited in host cells overexpressing Bax inhibitor-1 and is inhibited through overexpression of Bcl-2, which blocks both caspase-dependent and -independent apoptosis. As Bax and mitochondria are ideally located to sense stress-related metabolic changes emanating from the interior of an infected cell, it is likely that Bax-dependent apoptosis may also be observed in cells infected with other intracellular pathogens.

Down-regulation of cell surface growth factor receptors plays a key role in the tight control of cellular responses. Recent reports suggest that the ubiquitin system, in addition to participating in degradation by the proteasome of cytosolic and nuclear proteins, might also be involved in the down-regulation of various membrane receptors. We have previously characterized a signal in the cytosolic part of the interleukin 2 receptor beta chain (IL2Rbeta) responsible for its targeting to late endosomes/lysosomes. In this report, the role of the ubiquitin/proteasome system on the intracellular fate of IL2Rbeta was investigated. Inactivation of the cellular ubiquitination machinery in ts20 cells, which express a thermolabile ubiquitin-activating enzyme E1, leads to a significant decrease in the degradation rate of IL2Rbeta, with little effect on its internalization. In addition, we show that a fraction of IL2Rbeta can be monoubiquitinated. Furthermore, mutation of the lysine residues of the cytosolic region of a chimeric receptor carrying the IL2Rbeta targeting signal resulted in a decreased degradation rate. When cells expressing IL2Rbeta were treated either by proteasome or lysosome inhibitors, a significant decrease in receptor degradation was observed. Our data show that ubiquitination is required for the sorting of IL2Rbeta toward degradation. They also indicate that impairment of proteasome function might more generally affect intracellular routing.

Clathrin-dependent endocytosis has long been presented as the only efficient mechanism by which transmembrane receptors are internalized. We selectively blocked this process using dominant-negative mutants of Eps15 and showed that clathrin-mediated endocytosis of transferrin was inhibited, while endocytosis of interleukin 2 (IL2) receptors proceeded normally. Ultrastructural and biochemical experiments showed that clathrin-independent endocytosis of IL2 receptors exists constitutively in lymphocytes and is coupled to their association with detergent-resistant membrane domains. Finally, clathrin-independent endocytosis requires dynamin and is specifically regulated by Rho family GTPases. These results define novel properties of receptor-mediated endocytosis and establish that the IL2 receptor is efficiently internalized through this clathrin-independent pathway.

Chlamydia spp. are strictly intracellular pathogens that grow inside a vacuole, called an inclusion. They possess genes encoding proteins homologous to components of type III secretion machineries, which, in other bacterial pathogens, are involved in delivery of bacterial proteins within or through the membrane of eukaryotic host cells. Inc proteins are chlamydial proteins that are associated with the inclusion membrane and are characterized by the presence of a large hydrophobic domain in their amino acid sequence. To investigate whether Inc proteins and other proteins exhibiting a similar hydropathic profile might be secreted by a type III system, we used a heterologous secretion system. Chimeras were constructed by fusing the N-terminal part of these proteins with a reporter, the Cya protein of Bordetella pertussis, and these were expressed in various strains of Shigella flexneri. We demonstrate that these hybrid proteins are secreted by the type III secretion system of S. flexneri, thereby providing evidence that IncA, IncB and IncC are secreted by a type III mechanism in chlamydiae. Moreover, we show that three other proteins from Chlamydia pneumoniae, all of which have in common the presence of a large hydrophobic domain, are also secreted by S. flexneri type III secretion machinery.