Cooperative cell-cell actin network remodeling to perform Gap junction endocytosis.

BACKGROUND: The endocytosis of Gap junction plaques (GJP) requires cytoskeletal forces to internalize such large membranous structures. Actin, which partners the connexin proteins constituting Gap junctions and is located close to Annular Gap Junctions (AGJ), could be actively involved in this physiological process. RESULTS: Electron Microscopy and Light Microscopy images, associated with time-lapse analysis and 3D reconstruction, used at high resolution and enhanced using ImageJ based software analysis, revealed that: i) actin cables, originating from Donor cells, insert on the edge of GJP and contribute to their invagination, giving rise to AGJ, whereas actin cables on the Acceptor cell side of the plaque are not modified; ii) actin cables from the Donor cell are continuous with the actin network present over the entire GJP surface. These actin cables fuse at a single point distant from the plaque, which then detaches itself from the membrane, condensing to form an actin mass during the final internalization process; iii) the Acceptor cell participates in the last step of the endocytic invagination process by forming an annular actin structure known as an actin ring. CONCLUSIONS: Together, these data suggest that the endocytosis of GJP is an example of a unique cooperative mechanism between the Donor (the traction of its actin cables) and the Acceptor cells (forming the actin ring).

RéSUMé: CONTEXTE: L'endocytose des plaques de jonctions communicantes ou jonctions gap (GJP) nécessite les forces du cytosquelette pour internaliser ces grandes structures membranaires. L'actine, partenaire des connexines, proteins constitutives des jonctions gap (Gj), localisée proche des jonctions gap annulaires (GJA), pourrait être impliquée dans ce processus physiologique. RéSULTATS: L' imagerie par microscopie optique et électronique, associées avec des analyses vidéo et des reconstructions en relief/3D, examinées à haute résolution et améliorées après traitement par des logiciels développés sous ImageJ, montrent que: i) des câbles d'actine, originaires des cellules donneuses, s'insèrent sur le bord des plaques jonctionnelles et facilitent leur invagination pour former les GJA tandis que les câbles d'actine des cellules receveuses ne sont pas modifies; ii) les câbles d'actine des cellules donneuses sont en continuité avec le réseau d'actine qui couvre la totalité de la surface de la plaque. De plus, ces câbles fusionnent en un point unique, à distance de la plaque, qui se détache de la région membranaire pour former une masse d'actine à la fin du processus d'endocytose; iii) la cellule receveuse participe à l'étape ultime du processus d'endocytose de la plaque en formant un anneau d'actine. CONCLUSIONS: L'ensemble de nos résultats montrent que l'endocytose des plaques jonctionnelles est un exemple de coopération unique entre la cellule donneuse (grâce à la traction des câbles d'actine) et la cellule receveuse (anneau d'actine).

Multiple and complex influences of connexins and pannexins on cell death.

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.

Physiological roles of connexins and pannexins in reproductive organs.

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.

New cellular mechanisms of gap junction degradation and recycling.

BACKGROUND INFORMATION: Connexins (Cxs), 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. Because of 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. RESULTS: 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 AGJ 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. CONCLUSIONS: 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 internalisation 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.

The RNA-binding protein Mex3b regulates the spatial organization of the Rap1 pathway.

The four related mammalian MEX-3 RNA-binding proteins are evolutionarily conserved molecules for which the in vivo functions have not yet been fully characterized. Here, we report that male mice deficient for the gene encoding Mex3b are subfertile. Seminiferous tubules of Mex3b-deficient mice are obstructed as a consequence of the disrupted phagocytic capacity of somatic Sertoli cells. In addition, both the formation and the integrity of the blood-testis barrier are compromised owing to mislocalization of N-cadherin and connexin 43 at the surface of Sertoli cells. We further establish that Mex3b acts to regulate the cortical level of activated Rap1, a small G protein controlling phagocytosis and cell-cell interaction, through the activation and transport of Rap1GAP. The active form of Rap1 (Rap1-GTP) is abnormally increased at the membrane cortex and chemically restoring Rap1-GTP to physiological levels rescues the phagocytic and adhesion abilities of Sertoli cells. Overall, these findings implicate Mex3b in the spatial organization of the Rap1 pathway that orchestrates Sertoli cell functions.

Connexin a check-point component of cell apoptosis in normal and physiopathological conditions.

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.

Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model.

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 12days. Exposure to low concentrations of chromium (10µg/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.

Connexin 43 a check-point component of cell proliferation implicated in a wide range of human testis diseases.

Gap junction channels link cytoplasms of adjacent cells. Connexins, their constitutive proteins, are essential in cell homeostasis and are implicated in numerous physiological processes. Spermatogenesis is a sophisticated model of germ cell proliferation, differentiation, survival, and apoptosis, in which a connexin isotype, connexin 43, plays a crucial role as evidenced by genomic approaches based on gene deletion. The balance between cell proliferation/differentiation/apoptosis is a prerequisite for maintaining levels of spermatozoa essential for fertility and for limiting anarchic cell proliferation, a major risk of testis tumor. The present review highlights the emerging role of connexins in testis pathogenesis, focusing specifically on two intimately interconnected human testicular diseases (azoospermia with impaired spermatogenesis and testicular germ cell tumors), whose incidence increased during the last decades. This work proposes connexin 43 as a potential cancer diagnostic and prognostic marker, as well as a promising therapeutic target for testicular diseases.

Molecular connexin partner remodeling orchestrates connexin traffic: from physiology to pathophysiology.

Connexins, through gap junctional intercellular communication, are known to regulate many physiological functions involved in developmental processes such as cell proliferation, differentiation, migration and apoptosis. Strikingly, alterations of connexin expression and trafficking are often, if not always, associated with human developmental diseases and carcinogenesis. In this respect, disrupted trafficking dynamics and aberrant intracytoplasmic localization of connexins are considered as typical features of functionality failure leading to the pathological state. Recent findings demonstrate that interactions of connexins with numerous protein partners, which take place throughout connexin trafficking, are essential for gap junction formation, membranous stabilization and degradation. In the present study, we give an overview of the physiological molecular machinery and of the specific interactions between connexins and their partners, which are involved in connexin trafficking, and we highlight their changes in pathological situations.

The large GTPase dynamin2: a new player in connexin 43 gap junction endocytosis, recycling and degradation.

Connexins (Cx) are key regulators of cell proliferation, differentiation and apoptosis. Cx trafficking and endocytosis need interactions with a large number of signaling and scaffolding proteins. We demonstrate herein that Cx43-GFP gap junction plaque endocytosis was blocked in cells transfected by the dominant-negative form of dynamin2 (Dyn2K44A) and by dynasore, an inhibitor of dynamin GTPase activity, which reduced the association between dynamin2 and Cx43. Our data also reveal that recruitment of the GTPase at the plasma membrane and its activation by c-Src are key events for Cx43 internalization. In addition they show that dynamin2 participated in internalization and degradation of the gap junction plaque but also in recycling of Cx43 to the plasma membrane through respectively Rab5/Rab7 and Rab11 pathways. These results demonstrate for the first time that dynamin2 is a new Cx partner and report an innovating mechanistic model by which dynamin2 may control Cx43 gap junction plaque invagination, endocytosis, recycling and degradation. These processes are magnified in response to carcinogen exposure underlining their potential importance during carcinogenesis.

Testicular connexin 43, a precocious molecular target for the effect of environmental toxicants on male fertility.

Many recent epidemiological, clinical and experimental findings support the hypothesis that environmental toxicants are responsible for the increasing male reproductive disorders (congenital malformations, declining sperm counts and testicular cancer) over the past 20 years. It has also been reported that exposure to these toxicants, during critical periods of development (fetal and neonatal), represents a more considerable risk for animals and humans than exposure during adulthood. However, the molecular targets for these chemicals have not been clearly identified. Recent studies showed that a family of transmembranous proteins, named connexins, regulates numerous physiological processes involved in testicular development and function, such as Sertoli and germ cell proliferation, differentiation, germ cell migration and apoptosis. In the testis, knockout strategy revealed that connexin 43, the predominant connexin in this organ, is essential for spermatogenesis. In addition, there is evidence that many environmental toxicants could alter testicular connexin 43 by dysregulation of numerous mechanisms controlling its function. In the present work, we propose first to give an overview of connexin expression and intercellular gap junction coupling in the developing fetal and neonatal testes. Second, we underline the impact of maternally chemical exposure on connexin 43 expression in the perinatal developing testis. Lastly, we attempt to link this precocious effect to male offspring fertility.

Major involvement of connexin 43 in seminiferous epithelial junction dynamics and male fertility.

In different epithelia, cell membranes contacting one another form intercellular junctional complexes including tight, adherens and gap junctions, which could mutually influence the expression of each other. We have here investigated the role of Cx43 in the control of adherens and tight junction proteins (N-cadherin, beta-catenin, occludin and ZO-1) by using conditional Sertoli cell knockout Cx43 (SCCx43KO(-/-)) transgenic mice and specific anti-Cx43 siRNA. Gap junction coupling and Cx43 levels were reduced in SCCx43KO(-/-) as compared to Wild-type testes. Ultrastructural analysis revealed disappearance of gap junctions, the presence of tight and adherens junctions and persistent integrity of the blood-testis barrier in SCCx43KO(-/-) testis. Occludin, N-cadherin and beta-catenin levels were enhanced in SCCx43KO(-/-) mice as compared to Wild-type animals whereas ZO-1 levels were reduced. Cx43 siRNA blocked gap junction functionality in Sertoli cells and altered tight and adherens protein levels. The Cx43 control of tight and adherens junctions appeared channel-dependent since gap junction blockers (glycyrrhetinic acid and oleamide) led to similar results. These data suggest that the control of spermatogenesis by Cx43 may be mediated through Sertoli cell Cx43 channels, which are required, not only in cell/cell communication between Sertoli and germ cells, but also in the regulation of other junctional proteins essential for the blood-testis barrier.

Physiological and physiopathological aspects of connexins and communicating gap junctions in spermatogenesis.

Spermatogenesis is a highly regulated process of germ cell proliferation and differentiation, starting from spermatogonia to spermatocytes and giving rise to spermatids, the future spermatozoa. In addition to endocrine regulation, testicular cell-cell interactions are essential for spermatogenesis. This precise control is mediated through paracrine/autocrine pathways, direct intercellular contacts and through intercellular communication channels, consisting of gap junctions and their constitutive proteins, the connexins. Gap junctions are localized between adjacent Leydig cells, between Sertoli cells and between Sertoli cells and specific germ cells. This review focuses on the distribution of connexins within the seminiferous epithelium, their participation in gap junction channel formation, the control of their expression and the physiological relevance of these junctions in both the Sertoli-Sertoli cell functional synchronization and the Sertoli-germ cell dialogue. In this review, we also discuss the potential implication of disrupted connexin in testis cancer, since impaired expression of connexin has been described as a typical feature of tumoral proliferation.

Differential time course of FSH/FSH receptor complex endocytosis within sertoli and germ cells during rat testis development.

Follicle-stimulating hormone (FSH) is required for initiation and maintenance of spermatogenesis, a dynamic process of cell proliferation and maturation. By using FSH-gold particles and pulse-chase experiments, we analyzed the kinetics of FSH endocytosis in Sertoli and germ cells during development. Ultrastructural time-dependent analysis demonstrates that FSH was first located on plasma membrane, before being accumulated within the endosomal compartment, in the early endosomes, identified by morphological criteria and Rab-5 colocalization. Thereafter, FSH-gold was routed to the degradation pathway. The FSH endocytosis kinetic was similar in Sertoli cells, spermatogonia and spermatocytes. However, quantitative analysis of gold particles revealed differences in the dynamic of FSH accumulation in the endosomes between immature and mature rats. This age-dependent kinetic of FSH endocytosis, mostly detectable by ultrastructural analysis associated with quantitative data, argues for a potential new regulatory mechanism of the FSH signalling pathway that could occur during maturation of testicular cells.

[European regulation REACH and the assessment of testicular toxicity]. / Analyse de la spermatogenèse ex vivo : application à l'analyse de la toxicité testiculaire.

Several studies suggest that exposure to environmental pollutants is partly responsible for testicular pathologies that have considerably increased over the last decades (cryptorchidism, hypospadias, cancer, decrease in the number of ejaculated spermatozoa). However, the cellular and molecular mechanisms involved in this reprotoxicity remain mostly unknown. One of the challenges of the european regulation REACH is to improve the knowledge on the chemical, toxic and ecotoxic properties of substances used in everyday life. As for the testicular toxicity, the few in vivo models used are not always the most appropriate for mechanistic studies. Our laboratory has developed and validated on a physiological point of view, coculture systems of germ cells in bicameral chambers, which reproduce a blood-testis barrier, allowing the determination of the mechanisms responsible for the toxicity of organic or mineral compounds on spermatogenesis, while reducing greatly the number of animals required.

Heteromeric connexin 43/connexin 33 complex endocytosis: a connexin phosphorylation independent mechanism.

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.

Contraceptive steroids from pharmaceutical waste perturbate junctional communication in Sertoli cells.

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 ERalpha and only express the cytoplasmic beta isoform. ERbeta 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 43 gap junction plaque endocytosis implies molecular remodelling of ZO-1 and c-Src partners.

Gap junctions, through their constitutive proteins, connexins (Cx), are involved in several processes including regulation of cellular proliferation, tissue differentiation, homeostasis and neoplasic transformation. Internalization of the gap junction plaque to form annular gap junction is a dynamic process, which present similarities with endocytosis, and participates in the control of gap junction coupling. Cx43 exhibits dynamic trafficking that needs sequential implication of a large number of protein partners. We have recently shown that ZO-1 localized in both sides of the gap junction plaque was restricted to one side during internalization. The dissociation between ZO-1 and Cx43 particularly occurred on the face where c-Src specifically associated with Cx43 and was abnormally accelerated in response to a carcinogen. In this addendum we summarize and further discuss these results.

Connexin 33 impairs gap junction functionality by accelerating connexin 43 gap junction plaque endocytosis.

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.

Three-dimensional analysis of connexin 43 gap junction in the ex vivo rat seminiferous tubules: short-term effects of hormonal effectors.

Cx43 gap junctions are essential for proliferation, differentiation, and apoptosis of germ cells during spermatogenesis. However, only few and indirect observations have been reported on the distribution of Cx43, the predominant Cx within the seminiferous tubules. In the present study, we developed an innovative method that allows visualization of the three- dimensional localization of Cx43 associated with gap junctions and their functionality in isolated spermatogenic stage-specific seminiferous tubules. Cx43 gap junctions were present between myoid cells, between Sertoli cells, and between Sertoli and germ cells. Cx43 levels and coupling were stage-dependent with higher values at stages VI-VIII of spermatogenesis and markedly reduced at stages IX-X. Short-term exposure of seminiferous tubule fragments at stages VI-VIII and of the 42GPA9 Sertoli cell line transfected with a Cx43-GFP vector, to FSH, cAMP, DHT, and 17beta-E(2) significantly altered Cx43 distribution as well as gap junction coupling. These observations highlight a nongenomic effect of these testicular effectors on Cx43 gap junction.