SummaryAlthough gap junction plaque assembly has been extensively studied, mechanisms involved in plaque disassembly are not well understood. Disassembly involves an internalization process in which annular gap junction vesicles are formed. These vesicles undergo fission, but the molecular machinery needed for these fissions has not been described. The mechanoenzyme dynamin has been previously demonstrated to play a role in gap junction plaque internalization. To investigate the role of dynamin in annular gap junction vesicle fission, immunocytochemical, time-lapse and transmission electron microscopy were used to analyze SW-13 adrenocortical cells in culture. Dynamin was demonstrated to colocalize with gap junction plaques and vesicles. Dynamin inhibition, by siRNA knockdown or treatment with the dynamin GTPase inhibitor dynasore, increased the number and size of gap junction 'buds' suspended from the gap junction plaques. Buds, in control populations, were frequently released to form annular gap junction vesicles. In dynamin-inhibited populations, the buds were larger and infrequently released and thus fewer annular gap junction vesicles were formed. In addition, the number of annular gap junction vesicle fissions per hour was reduced in the dynamin-inhibited populations. We believe this to be the first report addressing the details of annular gap junction vesicle fissions and demonstrating a role of dynamin in this process. This information is crucial for elucidating the relationship between gap junctions, membrane regulation and cell behavior.
Gap junctions are composed of connexin (Cx) proteins and mediate intercellular communication required for many developmental and physiological processes. Here we describe the isolation and characterization of Cx48.5, a zebrafish connexin with the highest sequence identity to mammalian Cx46. Expression analysis showed that Cx48.5 is expressed in the adult and embryonic lens and heart, adult testis, and transiently in the embryonic otic vesicles. Injection of Cx48.5 cRNA into Xenopus oocytes elicited intercellular electrical coupling with voltage sensitivity similar to mammalian Cx46. In single oocytes, Cx48.5 also induced large outward currents on depolarization, consistent with gap-junctional hemichannels. Disruption of Cx48.5 expression in embryos with antisense morpholino oligos (morpholinos) revealed that Cx48.5 has an essential role in the maintenance of lens homeostasis. The morpholino-treated embryos also developed small lenses and eyes as well as severe cardiovascular abnormalities.Gap junctions are clusters of intercellular channels that allow the propagation of small metabolites, signaling molecules, and electrical impulses between adjacent cells. The protein units that make up the gap junction channels in vertebrates are called connexins and are coded for by a multigene family with 20 or more members in mammals (1). Gap junctions play vital roles in the functions of virtually all organs, including the lens and the heart. The evidence to date suggests that distinct sets of connexins may play a role in the development and function of organs like the lens and the heart. Three connexins are expressed in the mammalian lens. Connexin 43 is expressed in the undifferentiated lens epithelium, whereas Cx46 1 and Cx50 are typically considered lens-specific and are predominantly expressed in the lens fiber cells (for review, see Ref.2). Both Cx46 and Cx50 knock-out mice developed cataracts (3-5), and the Cx50 knock-out mice also developed small lenses and small eyes.Four connexins are abundantly expressed in the mammalian heart: Cx43, Cx40, Cx45, and Cx37. Connexin 43 is expressed in the atrial and ventricular working myocardium, Cx40 is found in the atrial working myocardium and the conducting bundle branches and Purkinje fibers, whereas Cx45 is restricted to the atrioventricular conduction system, and Cx37 is restricted to endothelial and endocardial cells (for reviews, see Refs. 6 -8). Mouse knock-out experiments have demonstrated that Cx43, Cx40, and Cx45 are all essential for cardiac development and function. For instance, connexin 43 knock-out mice died neonatally from pulmonary outflow tract obstruction (9). Connexin 40-deficient mice on the other hand exhibited slowed cardiac conduction and a partial atrioventricular block (10, 11). Last, Cx45 knock-out mice died by embryonic day 10 displaying the effects of a conduction block, endocardial cushion defects, and abnormalities of vascular development (12, 13). The Cx37 knock-out mice had no apparent cardiovascular abnormalities (14). Although the expression a...
It is becoming clear that in addition to gap junctions, playing a role in cell-cell communication, gap junction proteins, connexins, located in cytoplasmic-compartments may have other important functions. Mitochondrial connexin 43 (Cx43) is increased after ischemic preconditioning and has been suggested to play a protective role in the heart. How Cx43 traffics to the mitochondria and the interactions of mitochondria with other Cx43-containing structures are unknown. In this study, immunocytochemical, super-resolution and transmission electron microscopy were used to detect cytoplasmic Cx43-containing structure and to demonstrate their interactions with other cytoplasmic organelles. The most prominent cytoplasmic Cx43-containing structures, annular gap junctions, were demonstrated to form intimate associations with lysosomes as well as with mitochondria. Surprisingly, the frequency of associations between mitochondria and annular gap junctions was greater than that between lysosomes and annular gap junctions. The benefits of annular gap junction/mitochondrial associations are not known. However, it is tempting to suggest that the contact between annular gap junction vesicles and mitochondria facilitates Cx43 deliver to the mitochondria. Furthermore, it points to the need for investigating trafficking of Cx43 to cytoplasmic compartments and annular gap junction as more than only a vesicle destined for degradation.
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