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International audience; Nonalcoholic fatty liver disease is a chronic liver disease which is associated with obesity and insulin resistance. We investigated the implication of REDD1 (Regulated in development and DNA damage response-1), a stress-induced protein in the development of hepatic steatosis. REDD1 expression was increased in the liver of obese mice and morbidly obese patients, and its expression correlated with hepatic steatosis and insulin resistance in obese patients. REDD1 deficiency protected mice from the development of hepatic steatosis induced by high-fat diet (HFD) without affecting body weight gain and glucose intolerance. This protection was associated with a decrease in the expression of lipogenic genes, SREBP1c, FASN, and SCD-1 in liver of HFD-fed REDD1-KO mice. Healthy mitochondria are crucial for the adequate control of lipid metabolism and failure to remove damaged mitochondria is correlated with liver steatosis. Expression of markers of autophagy and mitophagy, Beclin, LC3-II, Parkin, BNIP3L, was enhanced in liver of HFD-fed REDD1-KO mice. The number of mitochondria showing colocalization between LAMP2 and AIF was increased in liver of HFD-fed REDD1-KO mice. Moreover, mitochondria in liver of REDD1-KO mice were smaller than in WT. These results are correlated with an increase in PGC-1α and CPT-1 expression, involved in fatty acid oxidation. In conclusion, loss of REDD1 protects mice from the development of hepatic steatosis.

Extracellular matrix (ECM) elasticity is perceived by cells via focal adhesion structures, which transduce mechanical cues into chemical signalling to conform cell behavior. Although the contribution of ECM compliance to the control of cell migration or division is extensively studied, little is reported regarding infectious processes. We study this phenomenon with the extraintestinal Escherichia coli pathogen UTI89. We show that UTI89 takes advantage, via its CNF1 toxin, of integrin mechanoactivation to trigger its invasion into cells. We identify the HACE1 E3 ligase-interacting protein Optineurin (OPTN) as a protein regulated by ECM stiffness. Functional analysis establishes a role of OPTN in bacterial invasion and integrin mechanical coupling and for stimulation of HACE1 E3 ligase activity towards the Rac1 GTPase. Consistent with a role of OPTN in cell mechanics, OPTN knockdown cells display defective integrin-mediated traction force buildup, associated with limited cellular invasion by UTI89. Nevertheless, OPTN knockdown cells display strong mechanochemical adhesion signalling, enhanced Rac1 activation and increased cyclin D1 translation, together with enhanced cell proliferation independent of ECM stiffness. Together, our data ascribe a new function to OPTN in mechanobiology. Uropathogenic strains of Escherichia coli (UPEC) are a leading cause of urinary tract infections (UTIs) and invasion involves Rho GTPase members, notably Rac1, to drive actin cytoskeleton rearrangement leading to engulfment. Here, Petracchini et al. provide evidence of an ECM stiffnessmodulated role of Optineurin (OPTN), which regulates HACE1-dependant Rac1 activity and thus controls integrinmediated mechanotransduction and bacterial invasion.

Lymphatic collecting vessels and lymph nodes are inevitably embedded in adipose tissue. The physiological significance of this observation remains still not elucidated. However, obesity is characterized by impaired lymphatic function and increased vessel permeability. Inversely, lymphatic dysfunction induces obesity in mice, suggesting a significant interplay between lymphatic vessels and the adipose tissue. Therefore, understanding factors leading to lymphatic dysfunction might open new therapeutic windows to prevent obesity and associated comorbidities. The first step in this process requires a precise and detailed visualization of the lymphatic network in healthy and inflamed adipose tissue. Here, we describe a rapid, inexpensive, and efficient method that allows to label and analyze lymphatic and blood vessels. This approach takes advantage of the skin-draining brachial lymph node localization within the subcutaneous adipose tissue. The lymphatic arborization of this tissue can be revealed by injecting fluorochrome-conjugated lectins subcutaneously. Moreover, the in vivo labeling approach provides a way to evaluate lymphatic vessel density and functions. Coupled to blood vessel, adipocyte and immune cell staining, the protocol allows for high-resolution mapping of the subcutaneous adipose tissue by 3D imaging.

International audience; Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

The activation of adipose tissue macrophages (ATMs) in obesity is a key mediator of metabolic complications. Therefore, deciphering the cellular and molecular mechanisms initiating ATM activation could reveal regulatory pathways important to prevent obesity‐associated metabolic disorders. While stressed or dying adipocytes have been described as the source of factors activating ATMs, herein we discovered that endothelial cells directly communicate with a distinct population of ATMs to induce their transcriptional reprogramming in response to obesity in mice and humans. CD200R expressed by these perivascular ATMs regulated the acquisition of unique properties shaped by the obese adipose tissue vasculature. Systems biology and ATM-specific silencing in obese mice revealed a unique downstream signaling whereby CD200R act as a rheostat controlling multiple functions of ATMs. Together, these studies provide evidence for a CD200-CD200R axis triggering the reprogramming of ATMs by endothelial cells in obesity.

International audience; It has been shown that non-cytotoxic doses of Carbendazim (CBZ), a broad-spectrum benzimidazole fungicide, possess endocrine-disrupting (androgen-like) actions, ex vivo, on the pubertal rat seminiferous epithelium. Iprodione (IPR), a dicarboximide fungicide, is also known to be an endocrine-disrupter (anti-androgen). The effect of a mixture of these two pesticides was investigated in the validated rat seminiferous tubule culture model. Cultures were performed in the absence or presence of CBZ 50 nM or IPR 50 nM either alone or in mixture (Mix), over a 3-week period. Mix exerted a dramatic effect on two proteins (Connexin 43 and Claudin-11) of the blood-testis barrier and possessed similar effects to IPR on some germ cell populations. The presence of IPR together with CBZ (Mix) cancelled the effect of CBZ on the increase of the androgen-dependent TP1 and TP2 mRNAs and on the decrease of ER alpha, ER beta mRNAs. Nevertheless, CBZ alone or IPR alone or Mix induced toxicity on spermatogenesis resulting in a decrease of round spermatids (the precursors of spermatozoa). These results strongly suggest that, even at these low concentrations, the effects of IPR and of CBZ are not solely dependent on their respective anti-androgenic and androgen-like effects and should involve several mechanisms of action.

The endosomal system plays an essential role in cell homeostasis by controlling cellular signaling, nutrient sensing, cell polarity and cell migration. However, its place in the regulation of tissue, organ and whole body physiology is less well understood. Recent studies have revealed an important role for the endosomal system in regulating glucose and lipid homeostasis, with implications for metabolic disorders such as type 2 diabetes, hypercholesterolemia and non‐alcoholic fatty liver disease. By taking insights from in vitro studies of endocytosis and exploring their effects on metabolism, we can begin to connect the fields of endosomal transport and metabolic homeostasis. In this review, we explore current understanding of how the endosomal system influences the systemic regulation of glucose and lipid metabolism in mice and humans. We highlight exciting new insights that help translate findings from single cells to a wider physiological level and open up new directions for endosomal research.

Non-alcoholic fatty liver disease (NAFLD) represents a key feature of obesity-related type 2 diabetes with increasing prevalence worldwide. To our knowledge, no treatment options are available to date, paving the way for more severe liver damage, including cirrhosis and hepatocellular carcinoma. Here, we show an unexpected function for an intracellular trafficking regulator, the small Rab GTPase Rab24, in mitochondrial fission and activation, which has an immediate impact on hepatic and systemic energy homeostasis. RAB24 is highly upregulated in the livers of obese patients with NAFLD and positively correlates with increased body fat in humans. Liver-selective inhibition of Rab24 increases autophagic flux and mitochondrial connectivity, leading to a strong improvement in hepatic steatosis and a reduction in serum glucose and cholesterol levels in obese mice. Our study highlights a potential therapeutic application of trafficking regulators, such as RAB24, for NAFLD and establishes a conceptual functional connection between intracellular transport and systemic metabolic dysfunction. Non-alcoholic steatohepatitis (NASH) is characterized by lipid accumulation within hepatocytes and fibrosis. Seitz et al. show that the GTPase protein Rab24 is increased in the livers of people who are obese or have NASH.

Multiple myeloma (MM) evolves from a premalignant condition known as monoclonal gammopathy of undetermined significance (MGUS). However, the factors underlying the malignant transformation of plasmocytes in MM are not fully characterized. We report here that Eµ-directed expression of the antiapoptotic Bcl-B protein in mice drives an MM phenotype that reproduces accurately the human disease. Indeed, with age, Eµ-bcl-b transgenic mice develop the characteristic features of human MM, including bone malignant plasma cell infiltration, a monoclonal immunoglobulin peak, immunoglobulin deposit in renal tubules, and highly characteristic bone lytic lesions. In addition, the tumors are serially transplantable in irradiated wild-type mice, underlying the tumoral origin of the disease. Eµ-bcl-b plasmocytes show increased expression of a panel of genes known to be dysregulated in human MM pathogenesis. Treatment of Eµ-bcl-b mice with drugs currently used to treat patients such as melphalan and VELCADE efficiently kills malignant plasmocytes in vivo. Finally, we find that Bcl-B is overexpressed in plasmocytes from MM patients but neither in MGUS patients nor in healthy individuals, suggesting that Bcl-B may drive MM. These findings suggest that Bcl-B could be an important factor in MM disease and pinpoint Eµ-bcl-b mice as a pertinent model to validate new therapies in MM., Ligue nationale contre le cancer (France) (Equipe Labellisée Grant R08001AA), Fondation de France (Grant R08080AA), Fondation ARC pour la Recherche sur le Cancer (Grant PGA120140200777), Fondation ARC pour la Recherche sur le Cancer (Project 2015–2016), French Research National Agency (Grant ANR-11-LABX-0028-01)

Most delivery systems for small interfering RNA therapeutics depend on endocytosis and release from endo-lysosomal compartments. One approach to improve delivery is to identify small molecules enhancing these steps. It is unclear to what extent such enhancers can be universally applied to different delivery systems and cell types. Here, we performed a compound library screen on two well-established siRNA delivery systems, lipid nanoparticles and cholesterol conjugated-siRNAs. We identified fifty-one enhancers improving gene silencing 2-5 fold. Strikingly, most enhancers displayed specificity for one delivery system only. By a combination of quantitative fluorescence and electron microscopy we found that the enhancers substantially differed in their mechanism of action, increasing either endocytic uptake or release of siRNAs from endosomes. Furthermore, they acted either on the delivery system itself or the cell, by modulating the endocytic system via distinct mechanisms. Interestingly, several compounds displayed activity on different cell types. As proof of principle, we showed that one compound enhanced siRNA delivery in primary endothelial cells in vitro and in the endocardium in the mouse heart. This study suggests that a pharmacological approach can improve the delivery of siRNAs in a system-specific fashion, by exploiting distinct mechanisms and acting upon multiple cell types.

Reproductive organs are complex and well-structured tissues essential to perpetuate the species. In mammals, the male and female reproductive organs vary on their organization, morphology and function. Connectivity between cells in such tissues plays pivotal roles in organogenesis and tissue functions through the regulation of cellular proliferation, migration, differentiation and apoptosis. Connexins and pannexins can be seen as major regulators of these physiological processes. In the present review, we assembled several lines of evidence demonstrating that these two families of proteins are essential for male and female reproduction.

Connexins (Cx), the constitutive proteins of gap junctions, are key actors of many physiological processes. Therefore alterations of Cx expression and degradation lead to the development of physiopathological disorders. Due to the formation of a double membrane vesicle termed annular gap junction (AGJ), gap junction degradation is a unique physiological process for which many cellular aspects remain unclear. By using a combination of time-lapse fluorescence microscopy and high resolution transmission electron microscopy we evidenced new specific cellular events concerning gap junction degradation and recycling. Indeed, by time lapse video microscopy we demonstrated, for the first time to our knowledge, that an entire annular gap junction can be fully recycled back to the plasma membrane. Moreover, we dissected the degradative processes of gap junction by electron microscopy approaches. Interestingly, in addition to canonical autophagy and heterophagy pathways, previously described, we discovered that both pathways could sometimes intermingle. Strikingly, our results also highlighted a new lysosome-based autophagy pathway that could play a pivotal role in common autophagy degradation. The present investigation reveals that AGJ degradation is a more complex process that it was previously thought. First, a complete recycling of the gap junction plaque after its internalization could occur. Second, the degradation of this peculiar double membrane structure is possible through autophagy, heterophagy, hetero-autophagy or by lysosomal-based autophagy. Altogether, this work underlines novel aspects of gap junction degradation that could be extended to other cell biology processes. This article is protected by copyright. All rights reserved.

International audience; The endosomal protein-sorting machineries play vital roles in diverse physiologically important cellular processes. Much of the core membrane-sorting apparatus is conserved in evolution, such as retromer, which is involved in the recycling of a diverse set of cargoes via the retrograde trafficking route. Here, in an RNAi-based loss-of-function study, we identified that suppression of SNX12 leads to a severe blockage in CIM6PR (also known as IGF2R) transport and alters the morphology of the endocytic compartments. We demonstrate that SNX12 is involved in the early phase of CIM6PR transport, and mediates receptor recycling upstream of the other well-established SNX components of retromer. Ultra-structural analysis revealed that SNX12 resides on tubulo-vesicular structures, despite it lacking a BAR domain. Furthermore, we illustrate that SNX12 plays a key role in intraluminal vesicle formation and in the maturation of a subpopulation of early endosomes into late endosomes, thereby regulating selective endocytic transport of cargo for degradation. This study therefore provides evidence for the existence of early endosomal subpopulations that have differential roles in the sorting of the cargoes along endocytic degradative pathways.

Folliculogenesis requires communication between granulosa cells and oocytes, mediated by connexin-based gap junctions. Connexin 37 (Cx37)-deficient female mice are infertile. The present study assessed Cx37 deficiency in patients with primary ovarian insufficiency (POI). A candidate gene study was performed in patients and controls from the National Genotyping Center (Evry, France) including 58 Caucasian patients with idiopathic isolated POI and 142 Caucasian controls. Direct genomic sequencing of the coding regions of the GJA4 gene (encoding Cx37) was performed with the aim to identify a deleterious variant associated with POI and absent in ethnically matched controls. A single Cx37 variant absent in the control population was identified, namely a c.946G>A heterozygous substitution leading to a p.Gly316Ser variant that was present in two POI patients. This variant was absent in all Caucasian controls from various databases, and has been observed exclusively in African populations. This variant was identified to have a dominant negative effect in HeLa cells in vitro to alter connexon function (by 67.2±7.17%), as determined by Gap-fluorescence recovery after photobleaching. The alteration principally resulted from a decrease of cell surface connexons due to altered trafficking (by 47.73±8.59%). In marked contrast to this observation, a p.Pro258Ser variant frequent in all ethnic populations in databases had no functional effect in vitro. In conclusion, the present study reported on a Cx37 variant in two Caucasian POI patients, which was absent in control Caucasian populations, and which had a deleterious effect in vitro. It is therefore suggested that in the genetic context of the Caucasian population, this variant may contribute to POI.

International audience; Endocrine Disrupting Compounds (EDC) are found in many everyday products, like food packaging, food preservative or additive, pesticides residues, etc… Widely distributed throughout the environment and bioaccumulable in living organisms, persistent organic pollutants (POPs) are a specific class of EDC that accumulate in fat deposit. Some of them have been recognized as causing adverse effects on human’s health such as diabetes and cancer by mimicking hormone effects on metabolism. Cancer cells display high metabolic flexibility allowing them to grow in various cellular environments and favoring their proliferative and invasive capacities. Mitochondria are key players in this complex interplay as they produce ROS, generate energy and participate in nucleotide synthesis and in glutamine metabolism of cancer cells. Regarding the importance of hormones on prostate cancer risk and outcomes, we are developing multiple parameters in vitro assays conducted in a high-throughput screening format relevant for prostate cancer metabolism and aggressiveness. This screening method includes, inter alia a microscopy based analysis of mitochondria structure and function. We analyzed the effects of five EDCs (Aldrin, BDE28, TCDD, PCB153, PFOA) identified in the plasma of patients on two prostate cancer cell lines, 22RV1 (androgen-responsive) and DU145 (androgen-unresponsive). Each compound was tested in a dose dependent manner to determine its effects on total mitochondrial mass, mitochondrial membrane potential, ROS production, mitochondrial biogenesis and mitophagy. In addition, we performed an image based computational analysis of the mitochondrial network morphology and dynamics. This strategy allows us to extract some quantitative parameters on the mitochondrial network as fragmentation index, compactness, average volume, etc. When combined, morphological and functional parameters allow us to discriminate subtle perturbations of the mitochondrial structure-function induced by POPs in prostate cancer cells. We are confident that this multiparameter analysis strategy could represent a new perspective in identification and characterization of EDC based on their effects on cell metabolism (phenoscore) in order to estimate their potential risk on human health.

Cell death is a fundamental process for organogenesis, immunity and cell renewal. During the last decades a broad range of molecular tools were identified as important players for several different cell death pathways (apoptosis, pyroptosis, necrosis, autosis…). Aside from these direct regulators of cell death programs, several lines of evidence proposed connexins and pannexins as potent effectors of cell death. In the present review we discussed the potential roles played by connexins, pannexins and innexins in the different cell death programs at different scales from gap junction intercellular communication to protein-protein interactions. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Griseofulvin, a widely used antifungal antimitotic drug has been proposed as an anti-tumoral treatment by way of in vitro experiments. Recently, in vivo demonstration of griseofulvin efficacy against multiple myeloma in mice argues for its potential as therapeutics for cancer. Nevertheless, the molecular mechanisms by which griseofulvin disrupts cancerous cell progression are far from being understood. In the present study, we found that griseofulvin inhibits human germ cell tumor cell growth through activation of mitochondrial caspase pathway (caspase 9 and 3) leading to the activation of apoptosis rather than an alteration of cell proliferation. Strikingly, we demonstrated that griseofulvin triggered the expression level of connexin 43 (mRNA and protein), a well described tumor-suppressor gene, known to participate in apoptosis regulation. Consistently, together with microtubule instability, a mechanism classically associated with cell death in response to griseofulvin, we observed a disruption of connexin 43/tubulin association concomitant of an enhanced translocation of connexin 43, or an immunoreactive fragment of the protein, from the cytoplasm to the nucleus. Finally, by using siRNA approaches we demonstrated the requirement of connexin 43 in the apoptotic induction of griseofulvin on our tumor cell model. Altogether, these results described a new molecular mechanism connexin 43-dependent targeted by griseofulvin leading to apoptosis of human germ cell tumor cells.

Activation of the p53 pathway in adipose tissue contributes to insulin resistance associated with obesity. However, the mechanisms of p53 activation and the effect on adipocyte functions are still elusive. Here we found a higher level of DNA oxidation and a reduction in telomere length in adipose tissue of mice fed a high-fat diet and an increase in DNA damage and activation of the p53 pathway in adipocytes. Interestingly, hallmarks of chronic DNA damage are visible at the onset of obesity. Furthermore, injection of lean mice with doxorubicin, a DNA damage-inducing drug, increased the expression of chemokines in adipose tissue and promoted its infiltration by proinflammatory macrophages and neutrophils together with adipocyte insulin resistance. In vitro, DNA damage in adipocytes increased the expression of chemokines and triggered the production of chemotactic factors for macrophages and neutrophils. Insulin signaling and effect on glucose uptake and Glut4 translocation were decreased, and lipolysis was increased. These events were prevented by p53 inhibition, whereas its activation by nutlin-3 reproduced the DNA damage-induced adverse effects. This study reveals that DNA damage in obese adipocytes could trigger p53-dependent signals involved in alteration of adipocyte metabolism and secretory function leading to adipose tissue inflammation, adipocyte dysfunction, and insulin resistance.

Background NRD convertase, also termed Nardilysin, is a Zn++ metalloendopeptidase that specifically cleaves the N-terminus of arginine and lysine residues into dibasic moieties. Although this enzyme was found located within the testis, its function in male reproduction is largely unknown. In addition, the precise distribution of this enzyme within germ cells remains to be determined. Methods To answer these questions, we developed an immuno-gold electron microscopy analysis to detect Nardilysin at ultrastructural level in mice. In addition, we performed a quantitative analysis of these gold particles to statistically estimate the distribution of Nardilysin in the different subcellular compartments of differentiating late spermatids/spermatozoa. Results Expression of Nardilysin in wild-type mice was restricted to germ cells and markedly increased during the last steps of spermiogenesis. In elongated spermatids, we found the enzyme mainly localized in the cytoplasm, more precisely associated with two microtubular structures, the manchette and the axoneme. No labelling was detected over the membranous organelles of the spermatids. To test whether this localization is dependent of the functional microtubules organization of the flagella, we analysed the localization into a specific mouse mutant ebo/ebo (ébouriffé) known to be sterile due to an impairment of the final organization of the flagellum. In the ebo/ebo, the enzyme was still localized over the microtubules of the axoneme and over the isolated cytoplasmic microtubules doublets. Quantification of gold particles in wild-type and mutant flagella revealed the specific association of the enzyme within the microtubular area of the axoneme. Conclusions The strong and specific accumulation of Nardilysin in the manchette and axoneme suggests that the enzyme probably contributes either to the establishment of these specific microtubular structures and/or to their functional properties.

An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.

Résumé Les jonctions communicantes et leurs protéines constitutives, les connexines (Cxs), sont des constituants nécessaires à la cohésion tissulaire et reconnus comme suppresseurs de tumeurs. Le but de la présente revue est de faire le point sur l’organisation et le rôle des Cxs au sein du testicule et d’analyser leur expression en physiopathologie testiculaire. Organisées en structures hexamèriques formant un canal reliant directement les cytoplasmes des cellules adjacentes, les Cxs sont impliquées dans de nombreux processus physiologiques tels que la prolifération et la différenciation cellulaires. Le maintien d’une balance entre prolifération, différenciation et apoptose est un équilibre primordial évitant une prolifération cellulaire anarchique, risque de cancer. La spermatogenèse est un modèle sophistiqué de prolifération et de différenciation des cellules germinales dans lequel les Cxs jouent un rôle essentiel. Il est acquis qu’une altération de l’expression membranaire des Cxs est l’un des signes avant-coureurs de la cinétique tumorale germinale, et il a été suggéré que les toxiques environnementaux qui, dans leur grande majorité, affectent l’expression de ces protéines, puissent être impliqués dans le développement de cette pathologie. La recherche de molécules capables de freiner les effets délétères de toxiques carcinogènes sur les Cxs semble être à l’heure actuelle une voie intéressante ouvrant de nouvelles perspectives en santé humaine.

The role of gap junctions in proliferation, differentiation and apoptosis has been recently highlighted. Nevertheless, the molecular mechanisms that control these physiological events by acting on gap junction channels are still unknown. We have recently demonstrated that heteromeric gap junction plaques composed by Cx43 and Cx33 are unstable at the cell boundary and are rapidly internalized by endocytosis. In the present study, we analyze the phosphorylation status of Cx43 in homomeric (Cx43/Cx43) and heteromeric (Cx33/Cx43) complexes and their association with the tyrosine kinase c-Src. Our data show that c-Src interaction and P2 phosphorylation of Cx43, which are essential for homomeric Cx43 complex endocytosis, were altered in the heteromeric Cx33/Cx43 complex: lack of association between Cx33 and activated c-Src and disappearance of the P2 phosphorylated Cx43 isoform. The present findings demonstrate that the interaction of Cx33 with Cx43 within a same heteromeric complex may conduce to channel instability through alteration of the phosphorylation status of Cx43 independently of the control of the c-Src kinase. The data described here emphasize a new mechanism of Cx43 internalization Src kinase-independent.

The potential health impact of pharmaceutical waste is now a growing concern. Contraceptive steroids are prominent environmental contaminants and thus may act as endocrine disruptors. Numerous xenobiotics hamper Sertoli cells junctional communication which is known to participate in spermatogenesis control. This has been associated with male subfertility and testicular cancer. We investigated three contraceptive molecules found in the environment for their potential impact on Sertoli cells gap junction functionality: 17a-ethynylestradiol, medroxyprogesterone acetate and levonorgestrel. Four other non-steroid drugs also found in the environment were included in the study. Communication disruption was analyzed in vitro in murine seminiferous tubules and the 42GPA9 Sertoli cell line. Steroids modulated connexin43 trafficking and impaired junctional communication through rapid effects apparently acting on the cell membrane but not on Cx43 expression. The 4 non-steroid compounds showed no effect. Longer exposure to steroids increased gap junction impairment, which was associated in part with Na/K ATPase internalization. Estrogen receptors (ER) did not appear to be involved in gap junction disruption: Sertoli cells are devoid of ERα and only express the cytoplasmic β isoform. ERβ localization was not modified by either steroid. The threshold level was surprisingly low, around 10 −16 M. We conclude that steroidal pollutants disrupt Sertoli cells junctional communication in vitro at concentrations that can be found in the environment.

Connexin 33 (Cx33) is a testis-specific gap junction protein. We previously reported that Cx33 exerts dominant-negative effect on gap junction intercellular communication by sequestering Cx43 within early endosomes in Sertoli cells. However, the molecular mechanisms that drive this process are unknown. The present study analyzed: (i) the trafficking of Cx33 and Cx43 in wild-type Sertoli cells transfected with Cx33-DsRed2 and Cx43-green fluorescent protein vectors; (ii) the formation of heteromeric Cx33/Cx43 hemi-channels and their incorporation into gap junction plaques. Fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer and videomicroscopy studies demonstrated that Cx33 and Cx43 associated to form heteromeric oligomers that trafficked along microtubules to the plasma membrane. However, the plaques containing Cx33 were not functional. Immunoprecipitation experiments revealed that zonula occludens-1 (ZO-1), a scaffold protein proposed to secure Cx in gap junction plaques at the cell-cell boundary, associated with Cx33 in testis extracts. In cells expressing Cx33, Cx33 and ZO-1 specifically interacted with P(1) phosphorylated and P(0) unphosphorylated isoforms of Cx43, and the ZO-1 membranous signal level was reduced. It is suggested that alteration of Cx43/ZO-1 association by Cx33 could be one mechanism by which Cx33 exerts its dominant-negative effect on gap junction plaque.

A major side effect of the powerful immunosuppressive drug cyclosporine (CsA) is the development of a chronic nephrotoxicity whose mechanisms are not fully understood. Recent data suggest that tubular cells play a central role in the pathogenesis of chronic nephropathies. We have shown that CsA is responsible for endoplasmic reticulum (ER) stress in tubular cells. Autophagy has recently been described to be induced by ER stress and to alleviate its deleterious effects. In this study, we demonstrate that CsA induces autophagy in primary cultured human renal tubular cells through LC3II expression and autophagosomes visualization by electron microscopy. Autophagy is dependant on ER stress because various ER stress inducers activate autophagy, and salubrinal, an inhibitor of eIF2alpha dephosphorylation that protects cells against ER stress, inhibited LC3II expression. Furthermore, autophagy inhibition during CsA treatment with beclin1 siRNA significantly increases tubular cell death. Finally, immunohistochemical analysis of rat kidneys demonstrates a positive LC3 staining on injured tubular cells, suggesting that CsA induces autophagy in vivo. Taken together, these results demonstrate that CsA, through ER stress induction, activates autophagy as a protection against cell death.

Male infertility and cancers were recently linked, suggesting shared molecular bases. Because alteration of connexin functions were associated with male infertility and detected in many cancers, it is likely that connexins are one of the molecular machinery involved in the initiation of both pathologies. In addition, chemicals present within our environment can be at the origin of these two pathologies through their action on connexin expression and/or trafficking. However, taking advantage of this knowledge to develop strategies targeting connexins to cure male infertility and cancer still remains challenging.

Introduction L'activation de p53 dans l'adipocyte contribue au developpement de la resistance a l'insuline dans l'obesite murine. Cependant, les mecanismes responsables de son activation et l'impact sur les adipocytes restent meconnus. Materiels et Methodes Nous avons recherche la presence de dommages a l'ADN, principaux activateurs de p53, dans le tissu adipeux (TA) et les adipocytes de souris obeses sous regime riche en graisses, par quantification de l'oxydation de l'ADN, de la taille des telomeres, et des cassures de l'ADN. Nous avons etudie les consequences de ces dommages sur le TA in vivo en injectant des souris avec de la doxorubicine (drogue causant des dommages a l'ADN), in vitro sur des adipocytes murins et humains et l'implication de p53 grâce a des siARNs, un inhibiteur la pifithrine α et un activateur la nutlin-3a. Resultats Nous observons une augmentation de l'oxydation de l'ADN et une reduction de la taille des telomeres dans le TA des souris obeses. Dans l'adipocyte, nous montrons une augmentation des dommages a l'ADN et une activation de la voie p53. L'injection de doxorubycine a des souris augmente l'expression de chimiokines (CCL2, CCL7, CCL9, CXCL1) dans le TA et son infiltration par des macrophages pro-inflammatoires et des neutrophiles alors que les adipocytes deviennent resistants a l'insuline. In vitro, la doxorubicine stimule l'expression des memes chimiokines, et favorise l'attraction des macrophages. De plus, la doxorubicine inhibe la signalisation insulinique, le transport du glucose, la translocation de Glut4, et stimule la lipolyse. L'inhibition de p53 previent les effets des dommages a l'ADN alors que son activation par la nutlin-3 les mime. Conclusions Les dommages a l'ADN, retrouves dans les adipocytes de souris obeses, peuvent induire des signaux p53-dependants impliques dans l'alteration du metabolisme et des fonctions secretoires des adipocytes. Ces dommages a l'ADN conduiraient a une inflammation du TA, un dysfonctionnement des adipocytes, et au developpement de la resistance a l'insuline.

Objectifs Grâce a sa fonction energetique, la mitochondrie est un organite indispensable a la survie cellulaire, notamment pour la cellule cancereuse. L’objectif de nos travaux est de montrer que la mitochondrie constitue une cible des perturbateurs endocriniens (PE), pouvant expliquer en partie leurs effets biologiques observes dans les cancers hormono-dependants (sein, testicule, prostate). Materiel et methodes Analyse automatisee a haut debit de differents parametres mitochondriaux, en microscopie confocale et en cytometrie de flux, sur des lignees cellulaires humaines derivees de cancer hormono-dependants (sein : MCF7 ; prostate : 22RV1 et DU145 ; testicule : JKT-1), exposees a cinq familles de PE (organochlores, polybromes, PCB, perfluores, dioxines). Resultats Chaque lignee cellulaire a ete exposee a des doses croissantes de PE (de 10 −12 M a 10 −6 M) et nous avons pu mettre en evidence une augmentation de la production des radicaux libres aux plus faibles concentrations, correlee a une modification du potentiel de membrane des mitochondries et a une augmentation de la mitophagie, notamment pour le PCB153 et PFOA. Cet effet est d’autant plus marque dans les lignees cellulaires hormono-dependantes. Une analyse bioinformatique tridimensionnelle du reseau mitochondrial nous a egalement permis de mettre en evidence des modifications specifiques liees a une exposition aux PE, notamment une hyperfragmentation. Discussion La mise au point d’une analyse fonctionnelle et morphologique du reseau mitochondrial dans des cellules de cancers hormono-dependants nous permet donc de realiser une caracterisation multiparametrique des PE pouvant deboucher sur une methode de screening a haut debit utile aux industriels et agences reglementaires.

Rationnel La colonisation de souris axeniques avec un microbiote intestinal issu de souris obeses permet de transferer l’obesite et ses complications metaboliques. Ceci a permis de proposer le microbiote comme un facteur etiologique de l’obesite. Par contre, nous avons etudie la capacite du microbiote obese a induire l’obesite dans un modele de souris conventionnelles. Materiels et methodes Le contenu caecal de souris C57Bl/6 genetiquement (ob/ ob) ou nutritionnellement (HFD, High-Fat Diet) obeses a ete transplante chez des souris C57Bl/6 conventionnelles et non traitees aux antibiotiques. Les souris transplantees ont ete nourries avec un regime normal (NC, normal chow) pendant 3 semaines, puis avec un regime HFD pendant 6 semaines. Resultats Sous NC, les souris transplantees ont montre une augmentation de la tolerance a l’insuline et une diminution de la neoglucogenese hepatique comparativement aux souris controle non transplantees. De plus, ces ameliorations metaboliques ont ete retrouvees exacerbees lorsque que les souris transplantees ont ete nourries avec un regime HFD. Au niveau hepatique, nous avons observe une diminution de l’activite des enzymes clefs de la neoglucogenese (Glucose-6-phosphatase et Phosphoenolpyruvate Carboxyki-nase), associees a un changement d’expression des microRNAs impliques dans la sensibilite a l’insuline. Par ailleurs, nous avons observe une reduction de la masse grasse et de la taille des adipocytes. Au niveau intestinal (ileum) et du tissu adipeux mesenterique, nous avons observe l’augmentation d’expression de l’interferon-γ et la reduction de FoxP3. Les modifications metaboliques ci-dessus reportees se sont accompagnees a des profonds changements du microbiote intestinal. Conclusion Nous montrons pour la premiere fois un effet benefique du microbiote obese sur l’homeostasie glucidique, chez des souris conventionnelles non traitees aux antibiotiques. Nos resultats proposent une nouvelle vision de la dysbiose du microbiote intestinal. Cela pourrait permettre le developpement de strategies alternatives dans le domaine en forte expansion de la bacteriotherapie fecale. Declaration d’interet Les auteurs declarent ne pas avoir d’interet direct ou indirect (financier ou en nature) avec un organisme prive, industriel ou commercial en relation avec le sujet presente.

Gap junction protein connexins (Cxs) play essential roles in cell homeostasis, growth, differentiation and death. Therefore, Cx dysfunction has been associated with many diseases and with tumor development. Cxs control cell apoptosis through different molecular mechanisms. First, gap junction channels classically facilitate the influx and flux of apoptotic signals between adjacent cells and hemichannels between the intracellular and extracellular environments. Second, recent studies demonstrate that Cx proteins, independently from their functional role through channels or hemichannels and in conjunction with their intracytoplasmic localization, may act as signaling effectors able to activate the canonical mitochondrial apoptotic pathway. In the present review, we dissected both functions of Cx in apoptosis, providing new avenues for apoptosis-mediated cancer therapy.

Introduction Lors de l'obesite, la resistance a l'insuline est un element clef du developpement du diabete de type 2. Les lymphocytes residents du tissu adipeux (TA) ont recemment ete impliques dans la mise en place de cette resistance a l'insuline mais les mecanismes moleculaires restent inconnus. Nous avons observe que l'expression de la petite GTPase Rab4b est diminuee dans les lymphocytes T residents du tissu adipeux chez des patients obeses et diabetiques et des souris obeses (db/db et sous regime riche en lipides). Materiels et Methodes Pour comprendre le role de cette diminution d'expression de Rab4b dans les lymphocytes T dans le controle du metabolisme gluco-lipidique, nous avons genere un modele de souris invalidees pour Rab4b specifiquement dans les lymphocytes T et avons caracterise leurs defauts metaboliques et immunitaires. Resultats L'invalidation de Rab4b dans les lymphocytes T entraine une intolerance au glucose et une resistance a l'insuline sous regime normal, avec une augmentation des taux circulants d'acides gras non esterifies et l'accumulation de triglycerides dans le foie et les muscles, ceci sans modifier leur poids par rapport aux souris controles. L'absence de Rab4b altere la differenciation in vitro des lymphocytes T naifs en favorisant la differenciation en lymphocytes Th17 au depend des lymphocytes T regulateurs (Treg). In vivo, on observe une diminution des Treg et une augmentation des Th17 dans le TA qui s'accompagne d'une augmentation de secretion IL-6 et IL-17. Nous montrons que ce changement local du statut inflammatoire empeche l'expansion du TA (diminution du nombre d'adipocytes et augmentation de leur taille) en inhibant la differenciation adipocytaires. S'en suit une accumulation de macrophages pro-inflammatoires dans le TA et une resistance a l'insuline des adipocytes. Conclusions En conclusion, lors de l'obesite, la reduction de Rab4b dans les lymphocytes T pourrait etre un element important de la mise en place de la resistance a l'insuline de part son impact sur la fonction des lymphocytes T et leur dialogue avec les cellules du lignage adipocytaire.

International audience; Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durand's validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities. Since this metal is also known to affect cellular junctions, we investigated, in the present study, its potential influence on the Sertoli cell barrier and on junctional proteins present at this level such as connexin 43, claudin-11 and N-cadherin. Cultured seminiferous tubules in bicameral chambers expressed the three junctional proteins and ZO-1 for at least 12 days. Exposure to low concentrations of chromium (10 mu g/l) increased the trans-epithelial resistance without major changes of claudin-11 and N-cadherin expressions but strongly delocalized the gap junction protein connexin 43 from the membrane to the cytoplasm of Sertoli cells. The possibility that the hexavalent chromium-induced alteration of connexin 43 indirectly mediates the effect of the toxic metal on the blood testis barrier dynamic is postulated. (c) 2013 Elsevier Inc. All rights reserved.

RAB4A,B,C (CCV, EE, RE)Endocytic recycling ZFYVE20- VPS45, RABEP1, RUFY1, GRIPAP1, AKAP10, RAB11FIP1, CD2APRAB5A,B,C EE fusion, EE biogenesis EEA1, ZFYVE20- VPS45, APPL1/2, ANKFY1, RABEP1, RABEP2, PIK3R4, PIK3CB, OCRL, INPP5B, INPP4A, F8A1, VPS8 (yeast)RAB13 (EE, TJ) RE- to- PM transport, GLUT4 traffi ckingMICALL2, MICALL1RAB21 (EE) Integrin endocytosis, cytokinesisN.D.RAB22A EE- to- Golgi transport TBC1D2B, EEA1RAB31/RAB22B (EE, TGN, PG)TGN- to- EE transport, PG maturationTBC1D2B, OCRLRAB23 (PM, EE) PG- LY fusion, Hedgehog signaling, ciliogenesisN.D.RAB35 (PM, CCV, EE)Endocytic recycling, cytokine-sis, actin dynamicsOCRL, ACAP2, FSCN1RAB3A,B,C,D (SG, SV)Regulated exocytosis (SV cycle)RPH3A, RPH3AL, RIMS1/2, RAB3IP, SYN1, SYTL4/5RAB6A,B,C (Golgi)Golgi- to- PM, Golgi- to-endosome, intra- Golgi and ER- to- Golgi transport, cytokinesisGOLGB1, GCC2, OCRL, KIF20A, MYH9, MYH10, DENND5A, BICD1, CCDC64, TMF1, APBA3, VPS52RAB8A,B (RE, G4V)TGN- to- PM transport, GLUT4 and E- cadherin traffi cking, ciliogenesis, AJ assemblyOPTN, RPH3A, MYO5B, SYTL1, MICALL2, ODF2RAB10 (TGN, G4V)TGN- to- PM transport, GLUT4 traffi cking, PG maturationMICALL1, MYO5A/B/C, RIMS1RAB11A,B (TGN, RE)Endocytic recycling, RE- to-PM transport, cytokinesis, ciliogenesis, autophagyRAB11FIP1/FIP2/FIP3/FIP4/FIP5, EXOC6, RAB3IP, ZFYVE27*, MYO5B, TBC1D14RAB25 (RAB11C) (RE)RE- to- PM transport, integrin recyclingRAB11FIP1/FIP2/FIP3/FIP4/FIP5, RAB3IP, MYO5BRAB14 (EE, TGN) Golgi- to- EE transport KIF16B, ZFYVE20, RUFY1RAB15 (EE, RE) Endocytic recycling RABIF*, REP15RAB17 (RE, ML) Endocytic recycling, ciliogen-esis, melanosome traffi ckingN.D.RAB26 (SG) Regulated exocytosis RIMS1RAB33B (Golgi, ER- Golgi and intra- Golgi transport, autophagyGOLGA2, ATG16L1, RABEP1RAB37 (Golgi, SG)Mast cell degranulation, Wnt signaling, TNF- alpha secretionRIMS1, UNC13BRAB39A, B (Golgi, PG)Phagosomal acidifi cation, IL-1 secretionCASP1RAB7A,B (LE, LY, EE- to- LE and LE- to- Golgi transport, LY biogenesisRILP, VPS35 (retromer), VPS41 (HOPS complex), PIK3R4, FYCO1RAB9A,B (LE) Endosome- to- TGN transport PLIN3, GCC2, RHOBTB3, RABEPK, SGSM2, HPS4 (BLOC- 3 complex)RAB12 (RE, LY) Transport to LY N.D.RAB27A (ML, SG) Dynamics of LRO and SG RPH3A , RPH3AL MYO5AMLPH, SYTL1- 5, UNC13D, MYRIP, CORO1CRAB32 (TV, ML, Mito, APG)Autophagy, distribution of mitochondria, traffi cking of melanogenic enzyme to MLPRKAR2A, ANKRD7RAB34 (Golgi, PM, PG)Macropinocytosis, LY positioningRILP, UNC13BRAB36 (Golgi) Spatial distribution of LE and LY RILPRAB38 (TV, ML) Transport of tyrosinase to immature MLANKRD7RAB24 (APG?) - GABARAP