| Gap junctions are conglomerations of intercellular channels that are composed of a family of related but distinct proteins called connexin(Cx)s. It is as yet unknown how the assembly of Cxs into gap junctions is initiated upon cell-cell contact. E-cadherin-mediated cell-cell adhesion has been implicated as a prerequisite for the assembly of Cxs into gap junctions, however, the molecular mechanisms have remained elusive. We investigated whether the assembly of Cx43 and Cx32 into gap junctions was contingent upon E-cadherin mediated cell-cell adhesion. Using E-cadherin and Cx null cells, and by introducing Cx43 and Cx32, either alone or in combination with E-cadherin, we found that E-cadherin-mediated cell-cell adhesion was neither essential nor sufficient to initiate the assembly of Cxs into gap junctions de novo, and facilitated the growth of only preformed gap junctions composed of Cx43. We demonstrated that the carboxyl terminal (CT) domain of Cx43 played a critical role in the de novo assembly of gap junctions and their growth. Our findings suggest that direct or indirect interaction of Cx43 with the actin cytoskeleton via ZO-1 may play an important role in the de novo assembly of Cx43 into gap junctions and/or in their stability. We also demonstrated that cell polarization initiated the assembly of Cx32 into gap junctions and augmented the assembly of Cx43 independent of E-cadherin expression, and the CT domains of Cxs played critical roles in their polarization dependent assembly. Overall, these findings suggest that the assembly of Cxs into gap junctions at the site of cell-cell contact is governed by a complex interplay of E-cadherin, the carboxy-termini of Cxs and the actin cytoskeleton. |
