E-cadherin Differentially Regulates the Assembly of Connexin43 and Connexin32 into Gap Junctions in Human Squamous Carcinoma Cells

Abstract

It is as yet unknown how the assembly of connexins (Cx) into gap junctions (GJ) is initiated upon cell-cell contact. We investigated whether the trafficking and assembly of Cx43 and Cx32 into GJs were contingent upon cell-cell adhesion mediated by E-cadherin. We also examined the role of the carboxyl termini of these Cxs in initiating the formation of GJs. Using cadherin and Cx-null cells, and by introducing Cx43 and Cx32, either alone or in combination with E-cadherin, our studies demonstrated that E-cadherin-mediated cell-cell adhesion was neither essential nor sufficient to initiate GJ assembly de novo in A431D human squamous carcinoma cells. However, E-cadherin facilitated the growth and assembly of preformed GJs composed of Cx43, although the growth of cells on Transwell filters was required to initiate the assembly of Cx32. Our results also documented that the carboxyl termini of both Cxs were required in this cell type to initiate the formation of GJs de novo. Our findings also showed that GJ puncta composed of Cx43 co-localized extensively with ZO-1 and actin fibers at cell peripheries and that ZO-1 knockdown attenuated Cx43 assembly. These findings suggest that the assembly of Cx43 and Cx32 into GJs is differentially modulated by E-cadherin-mediated cell-cell adhesion and that direct or indirect cross-talk between carboxyl tails of Cxs and actin cytoskeleton via ZO-1 may regulate GJ assembly and growth.Gap junctions are conglomerations of intercellular channels that permit the direct exchange of ions, second messengers, and other molecules of less than 1500 Da between the cytoplasmic interiors of contiguous cells. The channels are composed of a family of proteins called connexins (Cx) 2 that are designated according to their molecular mass (1). The assembly of Cxs into GJs is a multistep process. The newly synthesized Cxs are cotranslationally inserted into the endoplasmic reticulum, which oligomerize to form hexamers called connexons. The connexons are delivered to the cell surface via Golgi and trans-Golgi network in small vesicles and dock with the connexons in the contiguous cell membranes to form intercellular channels. A GJ plaque is formed when several intercellular channels cluster (2, 3). Because of the short half-life of Cxs (2-5 h), the plaque is in a dynamic state and is thought to model and remodel constantly through recruitment of newly synthesized connexons to the periphery and through endocytosis of moribund plaque components from the center or through the endocytosis of the plaque in its entirety (4 -7). Although much has been learned about the intracellular transport of Cxs, the delivery of connexons to the cell surface, the formation of cell-to-cell channels, the spatio-temporal events and cellular factors required for the initial docking of connexons, the de novo formation of a nascent GJ plaque, its growth, and disassembly are poorly understood (2,8).The proximity between the plasma membranes of adjoining cells is, a priori, necessary for the formation of GJs, and cell-cell adhesi...

Characterization of the Structure and Intermolecular Interactions between the Connexin 32 Carboxyl-terminal Domain and the Protein Partners Synapse-associated Protein 97 and Calmodulin

“…Images of the scrape-loaded cells were captured using a CCD camera (Retiga 2000R, FAST 1394) with the aid of Volocity software (Improvision, Lexington, MA). The experiment was repeated 3 times and in each trial, 4 side by side images were captured and estimation of junction permeability was based on methods described previously where the number of fluorescent cells (containing Lucifer Yellow) were counted, excluding the cells containing dextran, which indicates the initially loaded cells (33,34). Data were analyzed statistically by analysis of variance with Tukey post hoc tests.…”

“…Scrape-loading Assay-Cells were scrape-loaded and immunostaining were performed as described in (33,34). Briefly, HeLa cells were seeded in six-well clusters at a density of 1.5 ϫ 10 6 /well and grew for 24 h prior and post-transfection.…”

The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells

“…Antibodies and Immunostaining-Rabbit polyclonal and mouse monoclonal antibodies against Cx32 and mouse anti-␤-catenin, and rabbit anti-␤-actin have been described previously (18,19,21). We also used rabbit polyclonal antibodies raised against the carboxyl tail (Sigma; C-3470) and the cytoplasmic loop of Cx32 (Sigma, C-3595).…”

“…In these studies we had fortuitously observed that the retrovirally expressed cytoplasmic tail-deleted Cx32 appeared to assemble into small gap junctions compared with those formed by the expression of the full-length Cx32 (18). Moreover, our previous study with cadherin-null human squamous carcinoma cells had also shown that the assembly of Cx32 into gap junctions was facilitated when cells acquired a partially polarized state and that the cytoplasmic tail of Cx32 (abbreviated as Cx32-CT) was required to initiate the formation of a gap junction plaque and/or its subsequent growth in these cells (19). These studies prompted us to explore the role of Cx32-CT in the assembly of gap junctions.…”

Connexins and Cadherin Cross-talk in the Pathogenesis of Prostate Cancer

“…To produce recombinant retrovirus for infection of target cells, amphotropic PTi67 cells were infected with the transiently produced recombinant retrovirus from EcoPack and selected in G418 (400 μg/ml). The recombinant retrovirus produced from the pooled polyclonal cultures of PTi67 cells was assayed for the virus titer by colony forming units as described [17][18][19][20][21][22].…”