Search

International audience

International audience; The development of a tetO/TetR system in the fungus Neurospora crassa is described. The system includes (i) a synthetic gene encoding a TetR variant fused to GFP, and (ii) a standard tetO array integrated homologously, as a proof of principle, near the his-3 gene. The localization of TetR-GFP at the tetO array (observed by fluorescence microscopy) can be disrupted by the application of tetracycline. The full-length array is stable during vegetative growth, but it triggers strong repeat-induced point mutation (RIP) by the RID-dependent as well as the DIM-2-dependent pathways during the sexual phase. Thus, both RIP pathways must be inactivated to allow the faithful inheritance of the unmodified construct. In summary, this study introduces a new molecular tool into Neurospora research, and suggests that the standard tetO array can self-engage in recombination-independent homologous pairing.

Human leishmaniasis is a public health problem worldwide for which the development of a vaccine remains a challenge. T cell-mediated immune responses are crucial for protection. Peptide vaccines based on the identification of immunodominant T cell epitopes able to induce T cell specific immune responses constitute a promising strategy. Here, we report the identification of human leukocyte antigen class-I (HLA-I) and -II (HLA-II)-restricted multi-epitope peptides from Leishmania proteins that we have previously described as vaccine candidates. Promastigote Surface Antigen (PSA), LmlRAB (L. major large RAB GTPase) and Histone (H2B) were screened, in silico, for T cell epitopes. 6 HLA-I and 5 HLA-II-restricted multi-epitope peptides, able to bind to the most frequent HLA molecules, were designed and used as pools to stimulate PBMCs from individuals with healed cutaneous leishmaniasis. IFN-γ, IL-10, TNF-α and granzyme B (GrB) production was evaluated by ELISA/CBA. The frequency of IFN-γ-producing T cells was quantified by ELISpot. T cells secreting cytokines and memory T cells were analyzed by flow cytometry. 16 of 25 peptide pools containing HLA-I, HLA-II or HLA-I and -II peptides were able to induce specific and significant IFN-γ levels. No IL-10 was detected. 6 peptide pools were selected among those inducing the highest IFN-γ levels for further characterization. 3/6 pools were able to induce a significant increase of the percentages of CD4+IFN-γ+, CD8+IFN-γ+ and CD4+GrB+ T cells. The same pools also induced a significant increase of the percentages of bifunctional IFN-γ+/TNF-α+CD4+ and/or central memory T cells. We identified highly promiscuous HLA-I and -II restricted epitope combinations from H2B, PSA and LmlRAB proteins that stimulate both CD4+ and CD8+ T cell responses in recovered individuals. These multi-epitope peptides could be used as potential components of a polytope vaccine for human leishmaniasis., Author summary The control of leishmaniasis, a neglected tropical disease of public health importance, caused by protozoan parasites of the genus Leishmania, mainly relies on chemotherapy, which is highly toxic. Currently, there is no vaccine against human leishmaniasis. Peptide-based vaccines consisting of T cell epitopes identified within proteins of interest by epitope predictive algorithms are a promising strategy for vaccine development. Here, we identified multi-epitope peptides composed of HLA-I and -II-restricted epitopes, using immunoinformatic tools, within Leishmania proteins previously described as potential vaccine candidates. We showed that multi-epitope peptides used as pools were able to activate IFN-γ producing CD4+ as well as CD8+ T cells, both required for parasite elimination. In addition, granzyme B-producing CD4+ T cells, bifunctional CD4+ IFN-γ+/TNF-α+ and/or TNF-α+/IL-2+ T cells as well as CD4+ and CD8+ central memory T cells, all involved in Leishmania infection control, were significantly increased in response to multi-epitope peptide stimulation. As far as we know, no study has described the detection of both CD4+ and CD8+ T cell populations in response to stimulation by both HLA-I and II-restricted peptides in humans. The immunogenic HLA-I and -II-restricted multi-epitope peptides identified in this study could constitute potential vaccine candidates against human leishmaniasis.

International audience; Background information: Eukaryotic cilia and flagella are sophisticated organelles composed of several hundreds of proteins that need to be incorporated at the right time and the right place during assembly.Results: Two methods were used to investigate this process in the model protist Trypanosoma brucei: inducible expression of epitope-tagged labelled proteins and fluorescence recovery after photobleaching of fluorescent fusion proteins. This revealed that skeletal components of the radial spokes (RSP3), the central pair (PF16) and the outer dynein arms (DNAI1) are incorporated at the distal end of the growing flagellum. They display low or even no visible turnover in mature flagella, a finding further confirmed by monitoring a heavy chain of the outer dynein arm. In contrast, the membrane-associated protein arginine kinase 3 (AK3) showed rapid turnover in both growing and mature flagella, without particular polarity and independently of intraflagellar transport.Conclusion: These results demonstrate different modes of incorporation for structural and membrane-associated proteins in flagella.Significance: The existence of two distinct modes for incorporation of proteins in growing flagella suggests the existence of different targeting machineries. Moreover, the absence of turnover of structural elements supports the view that the length of the mature flagellum in trypanosomes is not modified after assembly.

International audience; Von Willebrand factor (VWF) modulates factor VIII (FVIII) clearance and the anti-FVIII immune response. Despite the high affinity that defines the FVIII/VWF interaction, association/dissociation kinetics dictates 2% to 5% FVIII being present as free protein. To avoid free FVIII when studying the FVIII-VWF complex in vivo, we designed a FVIII-nanobody fusion protein, with the nanobody part being directed against VWF. This fusion protein, designated FVIII-KB013bv, had a 25-fold higher affinity compared with B-domainless FVIII (BDD-FVIII) for VWF. In vitro analysis revealed full cofactor activity in 1-stage clotting and chromogenic assays (activity/antigen ratio 1.0 ± 0.3 and 1.1 ± 0.3, respectively). In vivo, FVIII-013bv displayed a twofold increased mean residence time compared with BDD-FVIII (3.0 hours vs 1.6 hours). In a tail clip–bleeding assay performed 24 hours after FVIII infusion, blood loss was significantly reduced in mice receiving FVIII-KB013bv vs BDD-FVIII (15 ± 7 μL vs 194 ± 146 μL; P = .0043). Unexpectedly, when examining anti-FVIII antibody formation in FVIII-deficient mice, the immune-response toward FVIII-KB013bv was significantly reduced compared with BDD-FVIII (1/8 vs 14/16 mice produced anti-FVIII antibodies after treatment with FVIII-KB013bv and BDD-FVIII, respectively). Our data show that a stabilized interaction between FVIII and VWF is associated with a prolonged survival of FVIII and a reduced immune response against FVIII.

International audience; In the field of live viral vaccines production, there is an unmet need for in vitro tests complying a 3R approach (Refine, Replace and Reduce the use of animal experimentation) to replace the post-licensing safety tests currently assayed in animals. Here, we performed a pilot study evaluating whether virulence of rabies virus, RABV, can be forecast by an in vitro test of neurite outgrowth. The rationale to use neurite outgrowth as a read-out for this test is based on the salient property of the cytoplasmic domain of the G-protein (Cyto-G) of virulent RABV strains - not of attenuated RABV strains - to stimulate neurite outgrowth in vitro. We observed that neurite elongation triggered by the Cyto-Gs encoded by different RABV field isolates correlate with the distinct virulence scores obtained in a mouse model of experimental rabies. Our results cast the idea that it could be feasible to predict RABV virulence by testing the in vitro property of a RABV strain to promote neurite outgrowth without the use of animal experimentation.

International audience; Experimental determination of membrane protein topology can be achieved using various techniques. Here we present the pho-lac dual reporter system, a simple, convenient, and reliable tool to analyze the topology of membrane proteins in vivo. The system is based on the use of two topological markers with complementary properties, the Escherichia coli β-galactosidase LacZ, which is active in the cytoplasm, and the E. coli alkaline phosphatase PhoA, which is active in the bacterial periplasm. Specifically, in this pho-lac gene system, the reporter molecule is a chimera composed of the mature PhoA that is in frame with the β-galactosidase α-peptide, LacZα. Hence, when targeted to the periplasm, the PhoA-LacZα dual reporter displays high alkaline phosphatase activity but no β-galactosidase activity. Conversely, when located in the cytoplasm, PhoA-LacZα has no phosphatase activity but exhibits high β-galactosidase activity in E. coli cells expressing the ω fragment of LacZ, LacZω (via the α-complementation phenomenon). The dual nature of the PhoA- LacZα reporter allows a simple way to normalize both enzymatic activities to obtain readily interpretable information about the subcellular location of the fusion site between the membrane protein under study and the reporter. In addition, the PhoA-LacZα reporter permits utilization of dual-indicator agar plates to easily discriminate between colonies bearing cytoplasmic fusions, periplasmic fusions, or out-of-frame fusions. In total, the phoA-lacZα fusion reporter approach is a straightforward and rather inexpensive method of characterizing the topology of membrane proteins in vivo.

Signaling through Toll-like receptors (TLRs), the main receptors in innate immunity, is essential for the defense of mucosal surfaces. It was previously shown that systemic TLR5 stimulation by bacterial flagellin induces an immediate, transient interleukin-22 (IL-22)-dependent antimicrobial response to bacterial or viral infections of the mucosa. This process was dependent on the activation of type 3 innate lymphoid cells (ILCs). The objective of the present study was to analyze the effects of flagellin treatment in a murine model of oral infection with Yersinia pseudotuberculosis (an invasive, Gram-negative, enteropathogenic bacterium that targets the small intestine). We found that systemic administration of flagellin significantly increased the survival rate after intestinal infection (but not systemic infection) by Y. pseudotuberculosis . This protection was associated with a low bacterial count in the gut and the spleen. In contrast, no protection was afforded by administration of the TLR4 agonist lipopolysaccharide, suggesting the presence of a flagellin-specific effect. Lastly, we found that TLR5- and MyD88-mediated signaling was required for the protective effects of flagellin, whereas neither lymphoid cells nor IL-22 was involved.

International audience; The bacterial two-hybrid (BACTH, for "Bacterial Adenylate Cyclase-Based Two-Hybrid") system is a simple and fast genetic approach to detecting and characterizing protein-protein interactions in vivo. This system is based on the interaction-mediated reconstitution of a cyclic adenosine monophosphate (cAMP) signaling cascade in Escherichia coli. As BACTH uses a diffusible cAMP messenger molecule, the physical association between the two interacting chimeric proteins can be spatially separated from the transcription activation readout, and therefore it is possible to analyze protein-protein interactions that occur either in the cytosol or at the inner membrane level as well as those that involve DNA-binding proteins. Moreover, proteins of bacterial origin can be studied in an environment similar (or identical) to their native one. The BACTH system may thus permit a simultaneous functional analysis of proteins of interest-provided the hybrid proteins retain their activity and their association state. This chapter describes the principle of the BACTH genetic system and the general procedures to study protein-protein interactions in vivo in E. coli.

In vivo single molecule tracking has recently developed into a powerful technique for measuring and understanding the transient interactions of transcription factors (TF) with their chromatin response elements. However, this method still lacks a solid foundation for distinguishing between specific and non-specific interactions. To address this issue, we took advantage of the power of molecular genetics of yeast. Yeast TF Ace1p has only five specific sites in the genome and thus serves as a benchmark to distinguish specific from non-specific binding. Here, we show that the estimated residence time of the short-residence molecules is essentially the same for Hht1p, Ace1p and Hsf1p, equaling 0.12–0.32 s. These three DNA-binding proteins are very different in their structure, function and intracellular concentration. This suggests that (i) short-residence molecules are bound to DNA non-specifically, and (ii) that non-specific binding shares common characteristics between vastly different DNA-bound proteins and thus may have a common underlying mechanism. We develop new and robust procedure for evaluation of adverse effects of labeling, and new quantitative analysis procedures that significantly improve residence time measurements by accounting for fluorophore blinking. Our results provide a framework for the reliable performance and analysis of single molecule TF experiments in yeast.

International audience; Translation is an essential step in gene expression. In this study, we used an improved SunTag system to label nascent proteins and image translation of single messenger ribonucleoproteins (mRNPs) in human cells. Using a dedicated reporter RNA, we observe that translation of single mRNPs stochastically turns on and off while they diffuse through the cytoplasm. We further measure a ribosome density of 1.3 per kilobase and an elongation rate of 13-18 amino acids per second. Tagging the endogenous POLR2A gene revealed similar elongation rates and ribosomal densities and that nearly all messenger RNAs (mRNAs) are engaged in translation. Remarkably, tagging of the heavy chain of dynein 1 (DYNC1H1) shows this mRNA accumulates in foci containing three to seven RNA molecules. These foci are translation sites and thus represent specialized translation factories. We also observe that DYNC1H1 polysomes are actively transported by motors, which may deliver the mature protein at appropriate cellular locations. The SunTag should be broadly applicable to study translational regulation in live single cells.

International audience; Dengue virus (DENV) is a mosquito-borne flavivirus responsible for life-threatening dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). The viral replication machinery containing the core non-structural protein 5 (NS5) is implicated in severe dengue symptoms but molecular details remain obscure. To date, studies seeking to catalogue and characterize interaction networks between viral NS5 and host proteins have been limited to the yeast two-hybrid system, computational prediction and co-immunoprecipitation (IP) of ectopically expressed NS5. However, these traditional approaches do not reproduce a natural course of infection in which a number of DENV NS proteins colocalize and tightly associate during the replication process. Here, we demonstrate the development of a recombinant DENV that harbours a TAP tag in NS5 to study host-virus interactions in vivo. We show that our engineered DENV was infective in several human cell lines and that the tags were stable over multiple viral passages, suggesting negligible structural and functional disturbance of NS5. We further provide proof-of-concept for the use of rationally tagged virus by revealing a high confidence NS5 interaction network in human hepatic cells. Our analysis uncovered previously unrecognized hnRNP complexes and several low-abundance fatty acid metabolism genes, which have been implicated in the viral life cycle. This study sets a new standard for investigation of host-flavivirus interactions.

International audience; Recombinant antibody single-chain variable fragments (scFv) are difficult to purify homogeneously from a protein complex mixture. The most effective, specific and fastest method of purification is an affinity chromatography on Protein L (PpL) matrix. This protein is a multi-domain bacterial surface protein that is able to interact with conformational patterns on kappa light chains. It mainly recognizes amino acid residues located at the VL FR1 and some residues in the variable and constant (CL) domain. Not all kappa chains are recognized, however, and the lack of CL can reduce the interaction. From a scFv composed of IGKV10-94 according to IMGT®, it is possible, with several mutations, to transfer the motif from the IGKV12-46 naturally recognized by the PpL, and, with the single mutation T8P, to confer PpL recognition with a higher affinity. A second mutation S24R greatly improves the affinity, in particular by modifying the dissociation rate (kd). The equilibrium dissociation constant (KD) was measured at 7.2 10(-11) M by surface plasmon resonance. It was possible to confer PpL recognition to all kappa chains. This protein interaction can be modulated according to the characteristics of scFv (e.g., stability) and their use with conjugated PpL. This work could be extrapolated to recombinant monoclonal antibodies, and offers an alternative for protein A purification and detection.

International audience; Isolating endogenous SUMOylated proteins is a challenging task due to the high reversibility of this posttranslational modifi cation. We have shown that SUMO traps are useful tools for the enrichment and isolation of proteins modifi ed by SUMO in vitro and in vivo. To characterize the affi nity and specifi city of different SUMO chains for these traps, that are based on SUMO-interacting motifs, we have used realtime surface plasmon resonance (SPR), which allows a label-free analysis of protein/protein interactions. Here, a protocol to determine the affi nities of multivalent SUMO traps for polySUMO chains or mono-SUMO molecules by SPR is presented.

International audience; An important modification of thrombolytic agents is resistance to plasminogen activator inhibitor-1 (PAI-1). In previous studies, a new truncated PAI-1-resistant variant was developed based on deletion of the first three domains in t-PA and the substitution of KHRR 128-131 amino acids with AAAA in the truncated t-PA. The novel variant expressed in a static culture system of Chinese Hamster Ovary (CHO) DG44 cells exhibited a higher resistance to PAI-1 when compared with the full-length commercial drug; Actylase. In the present study, the truncatedmutant protein was expressed in CHO DG44 cells in 50 ml orbital shaking bioreactors. The final yield of the truncatedmutant in the culture was 752 IU/ml, representing a 63% increase compared with the static culture system. Therefore, these results suggest that using the combined features of a transient and stable expression system is feasible for the production of novel recombinant proteins in the quantities needed for preclinical studies.

Beyond the Greatwall Protein phosphorylation and dephosphorylation provide a central mechanism that controls the eukaryotic cell division cycle and entry of cells into mitosis. A form of protein phosphatase 2A (PP2A) has an important role inhibiting phosphorylation-dependent activation of cyclin-dependent kinase 1 (CDK1) itself and also dephosphorylating substrates of the active CDK1 that promote mitosis. PP2A activity is inhibited when another protein kinase, known as Greatwall, is activated (see the Perspective by Virshup and Kaldis ). Mochida et al. (p. 1670 ) and Gharbi-Ayachi et al. (p. 1673 ) searched for substrates of Greatwall that might participate in the cell cycle regulatory machinery. When phosphorylated by Greatwall, a pair of small related proteins, Arpp19 and α-endosulfine, inhibited activity of PP2A. These effects were critical for regulation of mitosis in Xenopus egg extracts and in human cancer cells. Greatwall itself is phosphorylated and activated by CDK1—thus, apparently contributing to a feed-forward loop that contributes to the switchlike commitment of cells to mitosis.

International audience; The Drosophila I-R type of hybrid dysgenesis is a sterility syndrome (SF sterility) associated with the mobilization of the I retrotransposon in female germ cells. SF sterility results from a maternal-effect embryonic lethality whose origin has remained unclear since its discovery about 40 years ago. Here, we show that meiotic divisions in SF oocytes are catastrophic and systematically fail to produce a functional female pronucleus at fertilization. As a consequence, most embryos from SF females rapidly arrest their development with aneuploid or damaged nuclei, whereas others develop as non-viable, androgenetic haploid embryos. Finally, we show that, in contrast to mutants affecting the biogenesis of piRNAs, SF egg chambers do not accumulate persistent DNA double-strand breaks, suggesting that I-element activity might perturb the functional organization of meiotic chromosomes without triggering an early DNA damage response.

International audience; Mineralocorticoid receptor (MR) plays a critical role in brain function. However, the regulatory mechanisms controlling neuronal MR expression that constitutes a key element of the hormonal response are currently unknown. Two alternative P1 and P2 promoters drive human MR gene transcription. To examine promoter activities and their regulation during neuronal differentiation and in mature neurons, we generated stably transfected recombinant murine embryonic stem cell (ES) lines, namely P1-GFP and P2-GFP, in which each promoter drove the expression of the reporter gene green fluorescent protein (GFP). An optimized protocol, using embryoid bodies and retinoic acid, permitted us to obtain a reproducible neuronal differentiation as revealed by the decrease in phosphatase alkaline activity, the concomitant appearance of morphological changes (neurites), and the increase in the expression of neuronal markers (nestin, beta-tubulin III, and microtubule-associated protein-2) as demonstrated by immunocytochemistry and quantitative PCR. Using these cell-based models, we showed that MR expression increased by 5-fold during neuronal differentiation, MR being preferentially if not exclusively expressed in mature neurons. Although the P2 promoter was always weaker than the P1 promoter during neuronal differentiation, their activities increased by 7- and 5-fold, respectively, and correlated with MR expression. Finally, although progesterone and dexamethasone were ineffective, aldosterone stimulated both P1 and P2 activity and MR expression, an effect that was abrogated by knockdown of MR by small interfering RNA. In conclusion, we provide evidence for a tight transcriptional control of MR expression during neuronal differentiation. Given the neuroprotective and antiapoptotic role proposed for MR, the neuronal differentiation of ES cell lines opens potential therapeutic perspectives in neurological and psychiatric diseases.

International audience; Sjögren's syndrome (SS) or autoimmune epithelitis is characterized by focal lymphocytic infiltrates surrounding the tubular epithelium of exocrine glands and by overactivity of the B-cell population. Although T cells were long considered the main effectors in SS, recent findings indicating a key role for B cells have prompted studies of treatments designed to deplete the B-cell population. Among molecules that can be targeted to achieve B-cell depletion, CD20 and CD22 are surface antigens expressed specifically by B lymphocytes; and the cytokine B-cell-activating factor belonging to the TNF family (BAFF) is a TNF receptor ligand involved in B-cell differentiation, survival, and activation. The aim of this review is to discuss the clinical outcomes of SS patients treated with B-cell depletion.

Item does not contain fulltext The Drosophila Toll-signaling pathway controls the systemic antifungal host response. Gram-negative binding protein 3 (GNBP3), a member of the beta-glucan recognition protein family senses fungal infections and activates this pathway. A second detection system perceives the activity of proteolytic fungal virulence factors and redundantly activates Toll. GNBP3(hades) mutant flies succumb more rapidly to Candida albicans and to entomopathogenic fungal infections than WT flies, despite normal triggering of the Toll pathway via the virulence detection system. These observations suggest that GNBP3 triggers antifungal defenses that are not dependent on activation of the Toll pathway. Here, we show that GNBP3 agglutinates fungal cells. Furthermore, it can activate melanization in a Toll-independent manner. Melanization is likely to be an essential defense against some fungal infections given that the entomopathogenic fungus Beauveria bassiana inhibits the activity of the main melanization enzymes, the phenol oxidases. Finally, we show that GNBP3 assembles "attack complexes", which comprise phenoloxidase and the necrotic serpin. We propose that Drosophila GNBP3 targets fungi immediately at the inception of the infection by bringing effector molecules in direct contact with the invading microorganisms. 01 mei 2010

International audience; A series of retinoids designed to interfere with the repositioning of H12 have been synthesized to identify novel RARgamma antagonists based on the structure of known RARgamma agonists. The transcriptional activities of the novel ligands were revealed by cell-based reporting assays, using engineered cells containg RAR subtype-selective fusions of the RAR ligand-binding domains with the yeast GAL4 activator DNA-binding domain and the cognate luciferase reporter gene. Whereas none of the ligands exhibited features of a selective RARgamma antagonist, some of them are endowed with interesting activities. In particular 24a acts as a pan-RAR agonist that induces at high concentration a higher transactivation potential on RARalpha than TTNPB and synergizes at low concentration with TTNPB-bound RARalpha but not RARbeta or RARgamma. Similarly, 24c synergizes with TTNPB-bound RARgamma and exhibits RARalpha,beta antagonist activity. Compounds 24b and 25b are strong RARalpha,beta-selective antagonists without agonist or antagonist activities for RARgamma. Compounds 24b and 24c display weak RXR antagonist activity. In addition several pan-antagonists and partial agonist/antagonists have been defined.

International audience; Heparan sulfate proteoglycan receptor syndecan-1 interacts with the carboxyl-terminal LG4/5 domain in laminin 332 (alpha3LG4/5) and participates in cell adhesion and spreading. To dissect the function of syndecan-1 in these processes, we made use of a cell adhesion model in which syndecan-1 exclusively interacts with a recombinantly expressed alpha3LG4/5 fragment. Plating HT1080 cells on this fragment induces the formation of actin-containing protrusive structures in an integrin-independent manner. Here we show that syndecan-1-mediated formation of membrane protrusions requires dephosphorylation of tyrosine residues in syndecan-1. Accordingly, inhibition of phosphatases with orthovanadate decreases cell adhesion to the alpha3LG4/5 fragment. We demonstrate that the PDZ-containing protein syntenin-1, known to connect cytoskeletal proteins, binds to syndecan-1 in cells plated on the alpha3LG4/5 fragment and participates in the formation of membrane protrusions. We further show that syntenin-1 recruitment depends on the dephosphorylation of Tyr-309 located within syndecan-1 PDZ binding domain EFYA. We propose that tyrosine dephosphorylation of syndecan-1 may regulate its association with cytoskeleton components.

The YODA ( YDA ) mitogen-activated protein kinase pathway promotes elongation of the Arabidopsis zygote and development of its basal daughter cell into the extra-embryonic suspensor. Here, we show that the interleukin-1 receptor–associated kinase (IRAK)/Pelle-like kinase gene SHORT SUSPENSOR ( SSP ) regulates this pathway through a previously unknown parent-of-origin effect. SSP transcripts are produced in mature pollen but do not appear to be translated. Instead, they are delivered via the sperm cells to the zygote and the endosperm, where SSP protein transiently accumulates. Ectopic expression of SSP protein in the leaf epidermis is sufficient to activate YDA -dependent signaling. We propose that SSP protein produced from paternal transcripts upon fertilization triggers zygotic YDA activity, providing an essential temporal cue for the regulation of the asymmetric first division.

Multiple cell types arise from cells in the dermomyotome of the somite that express Pax3 and Pax7, and myogenesis is regulated by Notch signaling. The asymmetric cell fate determinant Numb is thought to promote differentiation of skeletal muscle and other lineages by negatively regulating Notch signaling. We used transgenesis to overexpress Numb spatiotemporally in Pax3+/Pax7+ somitic stem and progenitor cells in mouse embryos using a spatiotemporally regulated enhancer element from the Myf5 locus that can target muscle progenitor cells prior to cell commitment. Molecular analyses as well as examination of dermal and skeletal muscle cell fates in vivo show that although Numb is thought to be associated with muscle differentiation, unexpectedly the common stem/progenitor pool size for these lineages is increased in Numb-transgenic embryos. Prospective isolation of the relevant transgenic cells and analysis by quantitative reverse-transcription polymerase chain reaction demonstrated that, in this context, canonical Notch targets are not significantly downregulated. These findings were corroborated using a Notch reporter mouse during the formation of somites and prior to lineage segregation. Thus, we propose that Numb can regulate the self-renewal of dermal and muscle progenitors during a lineage progression. Disclosure of potential conflicts of interest is found at the end of this article.

International audience; Spinal muscular atrophy (SMA) is caused by reduced levels of the survival of motor neuron (SMN) protein. Although the SMN complex is essential for assembly of spliceosomal U small nuclear RNPs, it is still not understood why reduced levels of the SMN protein specifically cause motor neuron degeneration. SMN was recently proposed to have specific functions in mRNA transport and translation regulation in neuronal processes. The defective protein in Fragile X mental retardation syndrome (FMRP) also plays a role in transport of mRNPs and in their translation. Therefore, we examined possible relationships of SMN with FMRP. We observed granules containing both transiently expressed red fluorescent protein(RFP)-tagged SMN and green fluorescent protein(GFP)-tagged FMRP in cell bodies and processes of rat primary neurons of hypothalamus in culture. By immunoprecipitation experiments, we detected an association of FMRP with the SMN complex in human neuroblastoma SH-SY5Y cells and in murine motor neuron MN-1 cells. Then, by in vitro experiments, we demonstrated that the SMN protein is essential for this association. We showed that the COOH-terminal region of FMRP, as well as the conserved YG box and the region encoded by exon 7 of SMN, are required for the interaction. Our findings suggest a link between the SMN complex and FMRP in neuronal cells.

International audience; Transcriptional activators, several different coactivators, and general transcription factors are necessary to access specific loci in the dense chromatin structure to allow precise initiation of RNA polymerase II (Pol II) transcription. Histone acetyltransferase (HAT) complexes were implicated in loosening the chromatin around promoters and thus in gene activation. Here we demonstrate that the 2 MDa GCN5 HAT-containing metazoan TFTC/STAGA complexes contain a histone H2A and H2B deubiquitinase activity. We have identified three additional subunits of TFTC/STAGA (ATXN7L3, USP22, and ENY2) that form the deubiquitination module. Importantly, we found that this module is an enhancer of position effect variegation in Drosophila. Furthermore, we demonstrate that ATXN7L3, USP22, and ENY2 are required as cofactors for the full transcriptional activity by nuclear receptors. Thus, the deubiquitinase activity of the TFTC/STAGA HAT complex is necessary to counteract heterochromatin silencing and acts as a positive cofactor for activation by nuclear receptors in vivo.

The previous characterization and structural analyses of Sfi1p, a Saccharomyces cerevisiae centrin-binding protein essential for spindle pole body duplication, have suggested molecular models to account for centrin-mediated, Ca 2+ -dependent contractility processes (S. Li, A. M. Sandercock, P. Conduit, C. V. Robinson, R. L. Williams, and J. V. Kilmartin, J. Cell Biol. 173:867-877, 2006). Such processes can be analyzed by using Paramecium tetraurelia , which harbors a large Ca 2+ -dependent contractile cytoskeletal network, the infraciliary lattice (ICL). Previous biochemical and genetic studies have shown that the ICL is composed of diverse centrin isoforms and a high-molecular-mass centrin-associated protein, whose reduced size in the démaillé (dem1) mutant correlates with defective organization of the ICL. Using sequences derived from the high-molecular-mass protein to probe the Paramecium genome sequence, we characterized the Pt CenBP1 gene, which encodes a 460-kDa protein. PtCenBP1p displays six almost perfect repeats of ca. 427 amino acids (aa) and harbors 89 potential centrin-binding sites with the consensus motif LLX 11 F/LX 2 WK/R, similar to the centrin-binding sites of ScSfi1p. The smaller (260-kDa) protein encoded by the dem1 mutant Pt CenBP1 allele comprises only two repeats of 427 aa and 46 centrin-binding sites. By using RNA interference and green fluorescent protein fusion experiments, we showed that PtCenBP1p forms the backbone of the ICL and plays an essential role in its assembly and contractility. This study provides the first in vivo demonstration of the role of Sfi1p-like proteins in centrin-mediated Ca 2+ -dependent contractile processes.

International audience; Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.

A chimeric protein West Nile virus (WNV) vaccine capable of delivering both innate and adaptive immune signals was designed by fusing a modified version of bacterial flagellin (STF2 Delta ) to the EIII domain of the WNV envelope protein. This fusion protein stimulated interleukin-8 production in a Toll-like receptor (TLR)-5-dependent fashion, confirming appropriate in vitro TLR5 bioactivity, and also retained critical WNV-E-specific conformation-dependent neutralizing epitopes as measured by enzyme-linked immunosorbent assay. When administered without adjuvant to C3H/HeN mice, the fusion protein elicited a strong WNV-E-specific immunoglobulin G antibody response that neutralized viral infectivity and conferred protection against a lethal WNV challenge. This potent EIII-specific immune response requires a direct linkage of EIII to STF2 Delta , given that a simple mixture of the 2 components failed to induce an antibody response or to provide protection against virus challenge. The presence of a functional TLR5 gene in vivo is also required--TLR5-deficient mice elicited only a minimal antigen-specific response. These results confirm that vaccines designed to coordinately regulate the innate and adaptive immune responses can induce protective immune responses without the need for potentially toxic adjuvants. They also support the further development of an effective WNV vaccine and novel monovalent and multivalent vaccines for related flaviviruses.

Polyglutamylases are enzymes that form polyglutamate side chains of variable lengths on proteins. Polyglutamylation of tubulin is believed to regulate interactions of microtubules (MTs) with MT-associated proteins and molecular motors. Subpopulations of MTs are differentially polyglutamylated, yet only one modifying enzyme has been discovered in mammals. In an attempt to better understand the heterogeneous appearance of tubulin polyglutamylation, we searched for additional enzymes and report here the identification of six mammalian polyglutamylases. Each of them has a characteristic mode of catalysis and generates distinct patterns of modification on MTs, which can be further diversified by cooperation of multiple enzymes. Polyglutamylases are restricted to confined tissues and subtypes of MTs by differential expression and localization. In conclusion, we propose a multienzyme mechanism of polyglutamylation that can explain how the diversity of polyglutamylation on selected types of MTs is controlled at the molecular level.

We studied Tcirg1 gene expression on RANKL-induced osteoclastic differentiation of the mouse model RAW264.7 cells. We identified a mechanism involving PARP-1 inhibition release and JunD/Fra-2 binding, which is responsible for Tcirg1 gene upregulation. Introduction: The Tcirg1 gene encodes the a3 isoform of the V-ATPase a subunit, which plays a critical role in the resorption activity of the osteoclast. Using serial deletion constructs of the Tcirg1 gene promoter, we performed a transcriptional study to identify factor(s) involved in the regulation of the RANKL-induced gene expression. Materials and Methods: The promoter activity of serial-deletion fragments of the Tcirg1 gene promoter was monitored throughout the RAW264.7 cells differentiation process. We next performed sequence analysis, EMSA, UV cross-linking, qPCR, and gel supershift experiments to identify the factor(s) interacting with the promoter. Results: A deletion of the −1297−1244 region led to the disappearance of the RANKL-induced promoter activity. EMSA experiments showed the binding of two factors that undergo differential binding on RANKL treatment. Supershift experiments led us to identify the dimer JunD/Fra-2 as the binding activity associated with the −1297/−1268 Tcirg1 gene promoter sequence in response to RANKL. Moreover, we observed poly(ADP-ribose) polymerase-1 (PARP-1) binding to an adjacent site (−1270/−1256), and this interaction was disrupted after RANKL treatment. Conclusions: We provide data that identify junD proto-oncogene (JunD) and Fos-related antigen 2 (Fra-2) as the activator protein-1 (AP-1) factors responsible for the RANKL-induced upregulation of the mouse Tcirg1 gene expression. Moreover, we identified another binding site for PARP-1 that might account for the repression of Tcirg1 gene expression in pre-osteoclastic cells.

G protein-coupled receptors transmit extracellular signals into the cells by activating heterotrimeric G proteins, a process that is often followed by receptor desensitization. Monitoring such a process in real time and in living cells will help better understand how G protein activation occurs. Energy transfer-based approaches [fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET)] were recently shown to be powerful methods to monitor the G protein-coupled receptors (GPCRs)-G protein association in living cells. Here, we used a BRET technique to monitor the coupling between the protease-activated receptor 1 (PAR1) and Galpha(i1) protein. A specific constitutive BRET signal can be measured between nonactivated PAR1 and the Galpha(i1) protein expressed at a physiological level. This signal is insensitive to pertussis toxin (PTX) and probably reflects the preassembly of these two proteins. The BRET signal rapidly increases upon receptor activation in a PTX-sensitive manner. The BRET signal then returns to the basal level after few minutes. The desensitization of the BRET signal is concomitant with beta-arrestin-1 recruitment to the receptor, consistent with the known rapid desensitization of PARs. The agonist-induced BRET increase was dependent on the insertion site of fluorophores in proteins. Taken together, our results show that BRET between GPCRs and Galpha proteins can be used to monitor the receptor activation in real time and in living cells. Our data also revealed that PAR1 can be part of a preassembled complex with Galpha(i1) protein, resulting either from a direct interaction between these partners or from their colocalization in specific microdomains, and that receptor activation probably results in rearrangements within such complexes.

The scaffold attachment factor SAFB1 and its recently discovered homologue SAFB2 might provide an important link between pre-mRNA splicing, intracellular signalling and transcription. Using novel mono-specific antisera, we found endogenous SAFB2 protein has a different spatial distribution from SAFB1 within the nucleus where it is found in much larger nuclear complexes (up to 670 kDa in size), and a distinct pattern of expression in adult human testis. By contrast, SAFB1 protein predominantly exists either as smaller complexes or as a monomeric protein. Our results suggest stable core complexes containing components comprised of SAFB1, SAFB2 and the RNA binding proteins Sam68 and hnRNPG exist in parallel with free SAFB1 protein. We found that SAFB2 protein, like SAFB1, acts as a negative regulator of a tra2β variable exon. Despite showing an involvement in splicing, we detected no stable interaction between SAFB proteins and SR or SR-related splicing regulators, although these were also found in stable higher molecular mass complexes. Each of the detected alternative splicing regulator complexes exists independently of intact nucleic acids, suggesting they might be pre-assembled and recruited to nascent transcripts as modules to facilitate alternative splicing, and/or they represent nuclear storage compartments from which active proteins are recruited.

The respiratory syncytial virus (RSV) phosphoprotein (P) is a major polymerase co-factor that interacts with both the large polymerase fragment (L) and the nucleoprotein (N). The N-binding domain of RSV P has been investigated by co-expression of RSV P and N proteins in Escherichia coli. Pull-down assays performed with a series of truncated forms of P fused to glutathione S-transferase (GST) revealed that the region comprising the last nine C-terminal amino acid residues of P (233-DNDLSLEDF-241) is sufficient for efficient binding to N. Site-directed mutagenesis shows that the last four residues of this peptide are crucial for binding and must be present at the end of a flexible C-terminal tail. The presence of the P oligomerization domain (residues 100–160) was an important stabilizing factor for the interaction. The tetrameric full-length P fused to GST was able to pull down both helical and ring structures, whereas a monomeric C-terminal fragment of P (residues 161–241) fused to GST pulled down exclusively RNA–N rings. Electron-microscopy analysis of the purified rings showed the presence of two types of complex: undecamers (11N) and decamers (10N). Mass-spectrometry analysis of the RNA extracted from rings after RNase A treatment showed two peaks of 22 900 and 24 820 Da, corresponding to a mean RNA length of 67 and 73 bases, respectively. These results suggest strongly that each N subunit contacts 6 nt, with an extra three or four bases further protected from nuclease digestion by the ring structure at both the 5′ and 3′ ends.

International audience; CDC25 dual-specificity phosphatases play a central role in cell cycle control through the activation of Cyclin-Dependent Kinases (CDKs). Expression during mitosis of a stabilized CDC25B mutant (CDC25B-DDA), which cannot interact with the F-box protein βTrCP for proteasome-dependent degradation, causes mitotic defects and chromosome segregation errors in mammalian cells. We found, using the same CDC25B mutant, that stabilization and failure to degrade CDC25B during mitosis lead to the appearance of multipolar spindle cells resulting from a fragmentation of pericentriolar material (PCM) and abolish mitotic Plk1-dependent phosphorylation of Kizuna (Kiz), which is essential for the function of Kiz in maintaining spindle pole integrity. Thus, in mitosis Kiz is a new substrate of CDC25B whose dephosphorylation following CDC25B stabilization leads to the formation of multipolar spindles. Furthermore, endogenous Kiz and CDC25B interact only in mitosis, suggesting that Kiz phosphorylation depends on a balance between CDC25B and Plk1 activities. Our data identify a novel mitotic substrate of CDC25B phosphatase that plays a key role in mitosis control.

LeIF, a Leishmania protein similar to the eukaryotic initiation factor eIF4A, which is a prototype of the DEAD box protein family, was originally described as a Th1-type natural adjuvant and as an antigen that induces an IL12-mediated Th1 response in the peripheral blood mononuclear cells of leishmaniasis patients. This study aims to characterize this protein by comparative biochemical and genetic analysis with eIF4A in order to assess its potential as a target for drug development. We show that a His-tagged, recombinant, LeIF protein of Leishmania infantum, which was purified from Escherichia coli, is both an RNA-dependent ATPase and an ATP-dependent RNA helicase in vitro, as described previously for other members of the DEAD box helicase protein family. In vivo experiments show that the LeIF gene cannot complement the deletion of the essential TIF1 and TIF2 genes in the yeast Saccharomyces cerevisiae that encode eIF4A. In contrast, expression of LeIF inhibits yeast growth when endogenous eIF4A is expressed off only one of its two encoding genes. Furthermore, in vitro binding assays show that LeIF interacts with yeast eIF4G. These results show an unproductive interaction of LeIF with translation initiation factors in yeast. Furthermore, the 25 amino terminal residues were shown to enhance the ability of LeIF to interfere with the translation machinery in yeast.

Indirubin, an isomer of indigo, is a reported inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3 (GSK-3) as well as an agonist of the aryl hydrocarbon receptor (AhR). Indirubin is the active ingredient of a traditional Chinese medicinal recipe used against chronic myelocytic leukemia. Numerous indirubin analogs have been synthesized to optimize this promising kinase inhibitor scaffold. We report here on the cellular effects of 7-bromoindirubin-3'-oxime (7BIO). In contrast to its 5-bromo- and 6-bromo- isomers, and to indirubin-3'-oxime, 7BIO has only a marginal inhibitory activity towards CDKs and GSK-3. Unexpectedly, 7BIO triggers a rapid cell death process distinct from apoptosis. 7-Bromoindirubin-3'-oxime induces the appearance of large pycnotic nuclei, without classical features of apoptosis such as chromatin condensation and nuclear fragmentation. 7-Bromoindirubin-3'-oxime-induced cell death is not accompanied by cytochrome c release neither by any measurable effector caspase activation. Furthermore, the death process is not altered either by the presence of Q-VD-OPh, a broad-spectrum caspase inhibitor, or the overexpression of Bcl-2 and Bcl-XL proteins. Neither AhR nor p53 is required during 7BIO-induced cell death. Thus, in contrast to previously described indirubins, 7BIO triggers the activation of non-apoptotic cell death, possibly through necroptosis or autophagy. Although their molecular targets remain to be identified, 7-substituted indirubins may constitute a new class of potential antitumor compounds that would retain their activity in cells refractory to apoptosis.

Human intestinal cell differentiation is mediated by signaling pathways that remain largely undefined. We and others have shown that cell migration and differentiation along the crypt-villus axis is associated with temporal and spatial modulations of the repertoire, as well as with the function of integrins and E-cadherins and their substrates. Cross-talk between integrin and cadherin signaling was previously described and seems to coordinate this differentiation process. Here, we report that engagement of α6 and, to a lesser extent, α3 integrin subunits after HT-29 cell adhesion on laminin 5 increases the expression of E-cadherin, which then organizes into nascent adherens junctions. We further identify that phosphoinositide 3-kinase (PI 3-kinase) activation plays a key role in this cross-talk. Indeed, integrin-dependent adhesion on laminin 5 stimulates PI 3-kinase activity. Immunofluorescence and immunoprecipitation experiments revealed that activated PI 3-kinase is recruited at cell-cell contacts. Using LY294002, an inhibitor of PI 3-kinase activity, we found that this activation is essential for E-cadherin connection with the cytoskeleton and for biogenesis of adherens junctions. Finally, we demonstrated that PI 3-kinase could signal through Rac1b activation to control adherens junction assembly. Our results provide a mechanistic insight into integrin-cadherin cross-talk and identify a novel role for PI 3-kinase in the establishment of adherens junctions.

International audience; Protein transduction domains (PTDs) are promising tools for transducing presynthesized polypeptides across the plasma membrane. However, the development and optimization of PTDs are hampered by many technical problems and artifacts resulting notably from the tight binding of PTDs to the cell surface and the difficulty in discriminating, through imagery analyses, truly cytosolic from cytoplasmic vesicular compartments. To circumvent these problems, we have developed an unambiguous enzymatic assay of the cytosolic uptake of PTD-driven proteins, based on the processing by ubiquitin-specific C-terminal proteases (DUBs). This method, coupled with fluorometry and fluorescence microscopy, shows that the TAT PTD derived from human immunodeficiency virus type 1 is rapidly taken up by cells but fails to reach their cytosol, except when dendritic cells, which are known to take up circulating antigens for cross-presentation, are used. In addition to its usefulness in assessing cytosolic uptake, DUB processing of PTD-linked proteins can ensure the intracellular release of cargo proteins, which might prove helpful for MHC-I-based vaccination or intracellular delivery of biologically active polypeptides.

Cellular signal transduction proceeds through a complex network of molecular interactions and enzymatic activities. The timing of these molecular events is critical for the propagation of a signal and the generation of a specific cellular response. To define the timing of signalling events, we introduce the combination of high-resolution confocal microscopy with the application of small-molecule inhibitors at various stages of signal transduction in T cells. Inhibitors of Src-family tyrosine kinases and actin dynamics were employed to dissect the role of the lymphocyte-specific tyrosine kinase Lck in the formation and maintenance of T cell receptor/CD3-dependent contacts. Anti-CD3epsilon-coated coverslips served as a highly defined stimulus. The kinetics of the recruitment of the yellow fluorescent protein-tagged signalling protein ZAP-70 were detected by high-resolution confocal microscopy. The analysis revealed that at 5 min after receptor engagement, Lck activity was required for maintenance of contacts. In contrast, after 20 min of receptor engagement, the contacts were Lck-independent. The relevance of the timing of inhibitor application provides a pharmacological concept for the maturation of T cell-substrate contacts.

The climax of amphibian metamorphosis is marked by thyroid hormone-dependent tadpole tail resorption, implicating apoptosis of multiple cell types, including epidermal cells, fibroblasts, nerve cells, and muscles. The molecular cascades leading to and coordinating the death of different cell types are not fully elucidated. It is known that the mitochondrial pathway, and in particular the Bax and XR11 genes, regulates the balance between apoptosis and survival in muscle. However, the down-stream factors modulated by changes in mitochondrial permeability have not been studied in a functional context. To investigate further the mitochondrial-dependent pathway, we analyzed the regulation and the role of caspase 9 in Xenopus tadpoles. We report that caspase 9 mRNA is expressed in the tail before metamorphosis and increases before and during climax. Similarly, at the protein level, the production of active forms of caspase 9 increases in muscle tissue as metamorphosis progresses. To assess the functional role of caspase 9, we designed a dominant-negative protein. Overexpression of this dominant-negative abrogates both Bax-induced cell death in vitro and muscle apoptosis in vivo during natural metamorphosis. These findings consolidate a model of metamorphic muscle death that directly implicates the mitochondrial pathway and the apoptosome.

International audience; MCAK is a member of the kinesin-13 family of microtubule (MT)-depolymerizing kinesins. We show that the potent MT depolymerizer MCAK tracks (treadmills) with the tips of polymerizing MTs in living cells. Tip tracking of MCAK is inhibited by phosphorylation and is dependent on the extreme COOH-terminal tail of MCAK. Tip tracking is not essential for MCAK's MT-depolymerizing activity. We propose that tip tracking is a mechanism by which MCAK is preferentially localized to regions of the cell that modulate the plus ends of MTs.

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.

While the biology of nitrogen-fixing root nodules has been extensively studied, little is known about the evolutionary events that predisposed legume plants to form symbiosis with rhizobia. We have studied the presence and the expression of two pectic gene families in Medicago, polygalacturonases (PGs) and pectin methyl esterases (PMEs) during the early steps of the Sinorhizobium meliloti-Medicago interaction and compared them with related pollen-specific genes. First, we have compared the expression of MsPG3, a PG gene specifically expressed during the symbiotic interaction, with the expression of MsPG11, a highly homologous pollen-specific gene, using promoter-gus fusions in transgenic M. truncatula and tobacco plants. These results demonstrated that the symbiotic promoter functions as a pollen-specific promoter in the non-legume host. Second, we have identified the presence of a gene family of at least eight differentially expressed PMEs in Medicago. One subfamily is represented by one symbiotic gene (MtPER) and two pollen-expressed genes (MtPEF1 and MtPEF2) that are clustered in the M. truncatula genome. The promoter-gus studies presented in this work and the homology between plant PGs, together with the analysis of the PME locus structure and MtPER expression studies, suggest that the symbiotic MsPG3 and MtPER could have as ancestors pollen-expressed genes involved in polar tip growth processes during pollen tube elongation. Moreover, they could have been recruited after gene duplication in the symbiotic interaction to facilitate polar tip growth during infection thread formation.

The CB1 cannabinoid receptor (CB1R) displays a significant level of ligand-independent (i.e. constitutive) activity, either when heterologously expressed in nonneuronal cells or in neurons where CB1Rs are endogenous. The present study investigates the consequences of constitutive activity on the intracellular trafficking of CB1R. When transfected in HEK-293 cells, CB1R is present at the plasma membrane, but a substantial proportion ( approximately 85%) of receptors is localized in intracellular vesicles. Detailed analysis of CB1-EGFP expressed in HEK-293 cells shows that the intracellular CB1R population is mostly of endocytic origin and that treatment with inverse agonist AM281 traps CB1R at the plasma membrane through a monensin-sensitive recycling pathway. Co-transfection with dominant positive or dominant negative mutants of the small GTPases Rab5 and Rab4, but not Rab11, profoundly modifies the steady-state and ligand-induced intracellular distribution of CB1R, indicating that constitutive endocytosis is Rab5-dependent, whereas constitutive recycling is mediated by Rab4. In conclusion, our results indicate that, due to its natural constitutive activity, CB1R permanently and constitutively cycles between plasma membrane and endosomes, leading to a predominantly intracellular localization at steady state.

International audience; Histone H3 lysine 9 methylation is associated with long-term transcriptional repression through recruitment of heterochromatin protein 1 (HP1) proteins. These proteins are believed to promote the formation of dense chromatin structures interfering with DNA accessibility. During the G2 phase of the cell cycle, HP1 proteins are delocalized from foci of pericentromeric heterochromatin, while a wave of H3 serine 10 phosphorylation is initiated within these regions. Here, we show that in vivo phosphorylation of serine 10 in G2 can occur on histone tails methylated on lysine 9. Unexpectedly, this modification favours rather than prevents HP1 binding to chromatin. Dissociation of HP1 from the methylated histone H3 tails is observed only after a third modification by acetylation of lysine 14, which occurs in prophase. We propose that phosphoacetylation of histone H3 could be a general mechanism allowing the cell to overcome HP1-mediated transcriptional repression.

During the past few years several new interacting partners for G protein-coupled receptors (GPCRs) have been discovered, suggesting that the activity of these receptors is more complex than previously anticipated. Recently, candidate G protein-coupled receptor associated sorting protein (GASP-1) has been identified as a novel interacting partner for the delta opioid receptor and has been proposed to determine the degradative fate of this receptor. We show here that GASP-1 associates in vitro with other opioid receptors and that the interaction domain in these receptors is restricted to a small portion of the carboxyl-terminal tail, corresponding to helix 8 in the three-dimensional structure of rhodopsin. In addition, we show that GASP-1 interacts with COOH-terminus of several other GPCRs from subfamilies A and B and that two conserved residues within the putative helix 8 of these receptors are critical for the interaction with GASP-1. In situ hybridization and northern blot analysis indicate that GASP-1 mRNA is mainly distributed throughout the central nervous system, consistent with a potential interaction with numerous GPCRs in vivo. Finally, we show that GASP-1 is a member of a novel family comprising at least 10 members, whose genes are clustered on chromosome X. Another member of the family, GASP-2, also interacts with the carboxyl-terminal tail of several GPCRs. Therefore, GASP proteins may represent an important protein family regulating GPCR physiology.

International audience; We examined the expression and function of a gene we previously cloned from its downregulation in a muscle atrophy model. The encoded protein was named myodulin because of sequence homologies with the cartilage-specific chondromodulin-I (ChM-I) protein, its restricted expression in skeletal muscle tissue, and its modulating properties on vascular endothelial cells described here. We investigated the expression of myodulin in muscle fibers and cultured muscle cells. Myodulin RNA messengers were found in muscle fibers and their tendon extensions. Overexpression of myodulin fused to a FLAG peptide showed evidence of a muscle cell surface protein. Myodulin functions were assessed from similarities with chondromodulin-I. Coculture experiments using C(2)C(12) mouse myoblasts or myotubes, which stably overexpress myodulin, with H5V mouse cardiac vascular endothelial cells revealed that myodulin had a very active role in the invasive action of endothelial cells, without any evidence of extracellular myodulin secretion. Our results suggest that myodulin may be a muscle angiogenic factor operating through direct cell-to-cell interactions. This role is consistent with the correlation between modulations in myodulin expression and modifications in muscle microvascularization associated with activity-dependent muscle mass variations.

During re-epithelialization of cutaneous wounds, keratinocytes recapitulate several aspects of the embryonic process of epithelial-mesenchymal transition (EMT), including migratory activity and reduced intercellular adhesion. The transcription factor Slug modulates EMT in the embryo and controls desmosome number in adult epithelial cells, therefore, we investigated Slug expression and function during cutaneous wound re-epithelialization. Slug expression was elevated in keratinocytes bordering cutaneous wounds in mice in vivo, in keratinocytes migrating from mouse skin explants ex vivo, and in human keratinocytes at wound margins in vitro. Expression of the related transcription factor Snail was not significantly modulated in keratinocytes during re-epithelialization in vitro. Epithelial cell outgrowth from skin explants of Slug knockout mice was severely compromised, indicating a critical role for Slug in epithelial keratinocyte migration. Overexpression of Slug in cultured human keratinocytes caused increased cell spreading and desmosomal disruption, both of which were most pronounced at wound margins. Furthermore, in vitro wound healing was markedly accelerated in keratinocytes that ectopically expressed Slug. Taken together, these findings suggest that Slug plays an important role during wound re-epithelialization in adult skin and indicate that Slug controls some aspects of epithleial cell behavior in adult tissues as well as during embryonic development.

The organogenesis of nitrogen-fixing nodules in legume plants is initiated in specific root cortical cells and regulated by long-distance signaling and carbon allocation. Here, we explore cell-to-cell communication processes that occur during nodule initiation in Medicago species and their functional relevance using a combination of fluorescent tracers, electron microscopy, and transgenic plants. Nodule initiation induced symplasmic continuity between the phloem and nodule initials. Macromolecules such as green fluorescent protein could traffic across short or long distances from the phloem into these primordial cells. The created symplasmic field was regulated throughout nodule development. Furthermore, Medicago truncatula transgenic plants expressing a viral movement protein showed increased nodulation. Hence, the establishment of this symplasmic field may be a critical element for the control of nodule organogenesis.