The coordination of cell growth during fission yeast mating requires Ras1-GTP hydrolysis

The spatial and temporal control of polarity is fundamental to the survival of all organisms. Cells define their polarity using highly conserved mechanisms that frequently rely upon the action of small GTPases, such as Ras and Cdc42. Schizosaccharomyces pombe is an ideal system with which to study the control of cell polarity since it grows from defined tips using Cdc42-mediated actin remodeling. Here we have investigated the importance of Ras1-GTPase activity for the coordination of polarized cell growth during fission yeast mating. Following pheromone stimulation, Ras1 regulates both a MAPK cascade and the activity of Cdc42 to enable uni-directional cell growth towards a potential mating partner. Like all GTPases, when bound to GTP, Ras1 adopts an active conformation returning to an inactive state upon GTP-hydrolysis, a process accelerated through interaction with negative regulators such as GAPs. Here we show that, at low levels of pheromone stimulation, loss of negative regulation of Ras1 increases signal transduction via the MAPK cascade. However, at the higher concentrations observed during mating, hyperactive Ras1 mutations promote cell death. We demonstrate that these cells die due to their failure to coordinate active Cdc42 into a single growth zone resulting in disorganized actin deposition and unsustainable elongation from multiple tips. These results provide a striking demonstration that the deactivation stage of Ras signaling is fundamentally important in modulating cell polarity

The Chemokine CXCL12 Is Essential for the Clearance of the Filaria Litomosoides sigmodontis in Resistant Mice

Litomosoides sigmodontis is a cause of filarial infection in rodents. Once infective larvae overcome the skin barrier, they enter the lymphatic system and then settle in the pleural cavity, causing soft tissue infection. The outcome of infection depends on the parasite's modulatory ability and also on the immune response of the infected host, which is influenced by its genetic background. The goal of this study was to determine whether host factors such as the chemokine axis CXCL12/CXCR4, which notably participates in the control of immune surveillance, can influence the outcome of the infection. We therefore set up comparative analyses of subcutaneous infection by L. sigmodontis in two inbred mouse strains with different outcomes: one susceptible strain (BALB/c) and one resistant strain (C57BL/6). We showed that rapid parasite clearance was associated with a L. sigmodontis-specific CXCL12-dependent cell response in C57BL/6 mice. CXCL12 was produced mainly by pleural mesothelial cells during infection. Conversely, the delayed parasite clearance in BALB/c mice was neither associated with an increase in CXCL12 levels nor with cell influx into the pleural cavity. Remarkably, interfering with the CXCL12/CXCR4 axis in both strains of mice delayed filarial development, as evidenced by the postponement of the fourth molting process. Furthermore, the in vitro growth of stage 4 filariae was favored by the addition of low amounts of CXCL12. The CXCL12/CXCR4 axis thus appears to have a dual effect on the L. sigmodontis life cycle: by acting as a host-cell restriction factor for infection, and as a growth factor for worms

A Flow Cytometry-Based FRET Assay to Identify and Analyse Protein-Protein Interactions in Living Cells

Försters resonance energy transfer (FRET) microscopy is widely used for the analysis of protein interactions in intact cells. However, FRET microscopy is technically challenging and does not allow assessing interactions in large cell numbers. To overcome these limitations we developed a flow cytometry-based FRET assay and analysed interactions of human and simian immunodeficiency virus (HIV and SIV) Nef and Vpu proteins with cellular factors, as well as HIV Rev multimer-formation.Amongst others, we characterize the interaction of Vpu with CD317 (also termed Bst-2 or tetherin), a host restriction factor that inhibits HIV release from infected cells and demonstrate that the direct binding of both is mediated by the Vpu membrane-spanning region. Furthermore, we adapted our assay to allow the identification of novel protein interaction partners in a high-throughput format.The presented combination of FRET and FACS offers the precious possibility to discover and define protein interactions in living cells and is expected to contribute to the identification of novel therapeutic targets for treatment of human diseases

Identification of an ERK inhibitor as a therapeutic drug against tau aggregation in a new cell-based assay

Formation of Tau aggregates is a common pathological feature of tauopathies and their accumulation directly correlates with cytotoxicity and neuronal degeneration. Great efforts have been made to understand Tau aggregation and to find therapeutics halting or reversing the process, however, progress has been slowed due to the lack of a suitable method for monitoring Tau aggregation. We developed a cell-based assay allowing to detect and quantify Tau aggregation in living cells. The system is based on the FRET biosensor CST able to monitor the molecular dynamic of Tau aggregation in different cellular conditions. We probed candidate compounds that could block Tau hyperphosphorylation. In particular, to foster the drug discovery process, we tested kinase inhibitors approved for the treatment of other diseases. We identified the ERK inhibitor PD-901 as a promising therapeutic molecule since it reduces and prevents Tau aggregation. This evidence establishes the CST cell-based aggregation assay as a reliable tool for drug discovery and suggests that PD-901 might be a promising compound to be tested for further preclinical studies on AD

Les neutraligands à l'assaut des chimiokines

International audienceEn s'inspirant des virus et parasites qui se prémunissent du système immunitaire de l'hôte grâce à des substances « neutralisantes » qui bloquent l'action des chimiokines, les chercheurs développent de nouveaux composés aux vertus antiinflammatoires et anticancéreuses porteuses d'espoirs

Neutralizing endogenous chemokines with small molecules : principles and potential therapeutic applications

International audienc

Hypoxia differentially regulated CXCR4 and CXCR7 signaling in colon cancer

International audienceBackground: HIF-1α and CXCR4/CXCL12 have crucial roles in the metastatic process of colorectal cancer. Our aim was to study the significance of targeting HIF-1α and the CXCR4/CXCL12 axis in colorectal cancer to prevent the dissemination process in vitro. Methods: We investigated CXCR4 and CXCR7 mRNA and protein expression in human colon carcinomas and the modulation of their expression by hypoxia and HIF-1α in colon cancer cell lines. The migration of tumor cells in a Boyden chamber was studied after CXCR4 inhibition with siRNA or the CXCR4/CXCL12 neutraligand, chalcone 4. Results: Analysis of a cohort of colon polyps and chromosome-unstable carcinomas showed that the expression of CXCR4 and CXCR7 was similar to that of the normal mucosa in the polyps and early-stage carcinomas but significantly increased in late stage carcinomas. Our data demonstrate that hypoxia strongly induced the expression of CXCR4 transcript and protein at the cell membrane, both regulated by HIF-1α, whereas CXCR7 expression was independent of hypoxia. After transient hypoxia, CXCR4 levels remained stable at the cell membrane up to 48 hours. Furthermore, reducing CXCR4 expression impaired CXCL12-induced Akt phosphorylation, whereas Erk activation remained unchanged. In contrast, reducing CXCR7 expression did not affect Akt nor Erk activation. In the presence of CXCR4 or CXCR7 siRNAs, a significant reduction in cell migration occurred (37% and 17%, respectively). Although irinotecan inhibited cell migration by 20% (p <0.001), the irinotecan and chalcone 4 combination further increased inhibition to 40% (p <0.001). Conclusion: We demonstrated, for the first time, that hypoxia upregulated CXCR4 but not CXCR7 expression in tumor cells and that the CXCR4 receptor protein level remains high at the cell membrane when the tumor cells return to normoxia for up to 48 hours. In addition we showed the interest to inhibit the CXCR4 signaling by inhibiting both the HIF-1α and CXCR4/CXCL12 pathway. CXCR4 seems to be a relevant target because it is continuously expressed and functional both in normoxic and hypoxic conditions in tumor cells

An Antedrug of the CXCL12 Neutraligand Blocks Experimental Allergic Asthma without Systemic Effect in Mice

International audienceThe chemokine receptor CXCR4 and its chemokine CXCL12 are involved in normal tissue patterning but also in tumor cell growth and survival as well as in the recruitment of immune and inflammatory cells, as successfully demonstrated using agents that block either CXCL12 or CXCR4. In order to achieve selectivity in drug action on the CXCR4/CXCL12 pair, in particular in the airways, drugs should be delivered as selectively as possible in the treated tissue and should not diffuse in the systemic circulation, where it may reach undesired organs. To this end, we used a previously unexploited Knoevenagel reaction to create a short lived drug, or soft drug, based on the CXCL12-neutralizing small molecule, chalcone 4, which blocks binding of CXCL12 to CXCR4. We show that the compound, carbonitrile-chalcone 4, blocks the recruitment of eosinophils to the airways in ovalbumin-sensitized and challenged mice in vivo when administered directly to the airways by the intranasal route, but not when administered systemically by the intraperitoneal route. We show that the lack of effect at a distant site is due to the rapid degradation of the molecule to inactive fragments. This approach allows selective action of the CXCL12 neutraligands although the target protein is widely distributed in the organism

Plasma membrane translocation of REDD1 governed by GPCRs contributes to mTORC1 activation

International audienceThe mTORC1 kinase promotes cell growth in response to growth factors by activation of receptor tyrosine kinase. It is regulated by the cellular energy level and the availability of nutrients. mTORC1 activity is also inhibited by cellular stresses through overexpression of REDD1 (regulated in development and DNA damage responses). We report the identification of REDD1 in a fluorescent live-imaging screen aimed at discovering new proteins implicated in G-protein-coupled receptor signaling, based on translocation criteria. Using a sensitive and quantitative plasma membrane localization assay based on bioluminescent resonance energy transfer, we further show that a panel of endogenously expressed GPCRs, through a Ca 2+ /calmodulin pathway, triggers plasma membrane translocation of REDD1 but not of its homolog REDD2. REDD1 and REDD2 share a conserved mTORC1-inhibitory motif characterized at the functional and structural level and differ most in their N-termini. We show that the N-terminus of REDD1 and its mTORC1-inhibitory motif participate in the GPCR-evoked dynamic interaction of REDD1 with the plasma membrane. We further identify REDD1 as a novel effector in GPCR signaling. We show that fast activation of mTORC1 by GPCRs correlates with fast and maximal translocation of REDD1 to the plasma membrane. Overexpression of functional REDD1 leads to a reduction of mTORC1 activation by GPCRs. By contrast, depletion of endogenous REDD1 protein unleashes mTORC1 activity. Thus, translocation to the plasma membrane appears to be an inactivation mechanism of REDD1 by GPCRs, which probably act by sequestering its functional mTORC1-inhibitory motif that is necessary for plasma membrane targeting