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 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.

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.

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.

Larval electroreceptors in the epidermis of mormyrid fish: II. The promormyromast.

Promormyromasts were found in the epidermis of the head of the larvae of five species of mormyrids bred in captivity. The promormyromast is a larval electroreceptor belonging to the specific lateral line system. In 12-day-old larvae this electroreceptor is characterized by a single sensory cell and two types of accessory cells. One type of accessory cell has dark cytoplasm, few microtubules, and contacts the sensory cell directly, whereas a second type has pale cytoplasm, many microtubules, and forms an outer layer not directly in contact with the sensory cell. This second type is referred to as a long pyriform accessory cell. This assembly of cells is situated below an intraepidermal cavity filled with acid polysaccharides. The bordering epidermal cells extend microvilli into the intraepidermal cavity. The apexes of the sensory cell, and of the two types of accessory cells, also open into the intraepidermal cavity but bear no microvilli. The promormyromast is innervated by an unmyelinated sensory nerve fiber passing through the basal membrane, which then splits into several branches between the accessory cells. These branches contact the periphery of the sensory cell with terminal boutons. At the site of each contact a ribbon-like structure surrounded by vesicles is present in the cytoplasm of the sensory cell. In older larvae of Campylomormyrus cassaicus, membrane foldings develop at the periphery of the pyriform accessory cells and accessory cell staining properties change just before transformation to become a mormyromast. The functional role of the promormyromast of the larval mormyrids is discussed.

Internalization of large double-membrane intercellular vesicles by a clathrin-dependent endocytic process.

Beyond its well-documented role in vesicle endocytosis, clathrin has also been implicated in the internalization of large particles such as viruses, pathogenic bacteria, and even latex beads. We have discovered an additional clathrin-dependent endocytic process that results in the internalization of large, double-membrane vesicles at lateral membranes of cells that are coupled by gap junctions (GJs). GJ channels bridge apposing cell membranes to mediate the direct transfer of electrical currents and signaling molecules from cell to cell. Here, we report that entire GJ plaques, clusters of GJ channels, can be internalized to form large, double-membrane vesicles previously termed annular gap junctions (AGJs). These internalized AGJ vesicles subdivide into smaller vesicles that are degraded by endo/lysosomal pathways. Mechanistic analyses revealed that clathrin-dependent endocytosis machinery-components, including clathrin itself, the alternative clathrin-adaptor Dab2, dynamin, myosin-VI, and actin are involved in the internalization, inward movement, and degradation of these large, intercellular double-membrane vesicles. These findings contribute to the understanding of clathrin's numerous emerging functions.

Comparative anatomy of the electrosensory lateral line lobe of mormyrids: the mystery of the missing map in the genus Stomatorhinus (family: Mormyridae).

Fish in the family Mormyridae produce weak electric organ discharges that are used in orientation and communication. The peripheral and central anatomy of the electrosensory system has been well studied in the species Gnathonemus petersii, but comparative studies in other species are scarce. Here we report on one genus of mormyrid that displays a remarkable change in the electrosensory lateral line lobe (ELL), the hypertrophied rhombencephalic structure that receives primary electroreceptor input. Although all other mormyrids studied have three distinct zones on each side of the ELL, fish of the genus Stomatorhinus exhibit only two. Therefore, the two-zone ELL is a unique derived characteristic shared by Stomatorhinus. We examined the cutaneous electroreceptors that project to the ELL in Stomatorhinus. All three types of electroreceptors previously described for G. petersii were present, but there was a significant change in one type, the mormyromast. Both mormyromast sensory cell types (A- and B-cells) are present, but the B-cell is not innervated in Stomatorhinus. We conclude that, although all cutaneous sensory cells are present, the missing B-cell afferents account for the loss of the dorsolateral zone of the ELL, and therefore the loss of an entire sensory map. Because mormyromasts are involved in electrolocation behavior, this anatomical difference is probably related to differences in electrolocation abilities. Stomatorhinus could prove to be an excellent system for linking evolutionary changes in behavior with modifications in their neural substrates.

Larval electroreceptors in the epidermis of mormyrid fish: I. Tuberous organs of type A and B.

Two types of larval electroreceptors, type A and B, are described in the epidermis of the head of larvae of three mormyrid species, Campylomormyrus cassaicus, Mormyrus rume proboscirostris and Pollimyrus isidori, bred in captivity. In each of these electroreceptor organs, a single sensory cell is found inside an intraepidermal cavity, sitting on a platform of accessory cells. The cavity is filled with microvilli originating both from the sensory cell and from the epidermal covering cells lining the intraepidermal cavity. These two types of tuberous larval electroreceptors differ in their distribution in the epidermis of the head, in the composition of their accessory cells, and by their innervation. The innervation found in type B organs is similar to that already described for electroreceptors of adult mormyrids. The sensorineural junction is composed of primary afferent terminal boutons, which contact the base of the sensory cell. Opposite each terminal bouton, a ribbon-like synaptic bar surrounded by vesicles is found in the cytoplasm of the sensory cell. In contrast, the base of the sensory cell in type A larval electroreceptors is not contacted by nervous terminal boutons, but instead forms closed appositions with specialized prolongations of accessory cells of the platform. The base of the sensory cell presents membrane evaginations, with hemispheric synaptic structures and few synaptic vesicles. These two types of electroreceptor organs degenerate at the time of the degeneration of the larval electric organ and the functional differentiation of the adult electric organ. The functional role of two tuberous electroreceptor types is examined.