Inhibition of gap junction and adherens junction assembly by connexin and A-CAM antibodies.

Abstract

Abstract. We examined the roles of the extracellular domains of a gap junction protein and a cell adhesion molecule in gap junction and adherens junction formation by altering cell interactions with antibody Fab fragments. Using immunoblotting and immunocytochemistry we demonstrated that Novikoff cells contained the gap junction protein, cormexin43 (Cx43), and the cell adhesion molecule, A-CAM (N-cadherin). Cells were dissociated in EDTA, allowed to recover, and reaggregated for 60 min in media containing Fab fragments prepared from a number of antibodies. We observed no cell-cell dye transfer 4 min after microinjection in 90% of the cell pairs treated with Fab fragments of antibodies for the first or second extracellular domain of Cx43, the second extracellular domain of connexin32 (Cx32) or A-CAM. Cell-cell dye transfer was detected within 30 s in cell pairs treated with control Fab fragments (pre-immune serum, antibodies to the rat major histocompatibility complex or the amino or carboxyl termini of Cx43). We observed no gap junctions by freeze-fracture EM and no adherens junctions by thin section EM between cells treated with the Fab fragments that blocked cell-cell dye transfer. Gap junctions were found on •50% of the cells in control samples using freeze-fracture EM. We demonstrated with reaggregated Novikoff cells that: (a) functional interactions of the extracellular domains of the connexins were necessary for the formation of gap junction channels; (b) cell interactions mediated by A-CAM were required for gap junction assembly; and (c) Fab fragments of antibodies for A-CAM or connexin extracellular domains blocked adherens junction formation.

Wounding alters epidermal connexin expression and gap junction-mediated intercellular communication.

“…Based on the observation that presumptive migratory cells became uncoupled, we postulate that keratinocyte activation may be partly initiated by the loss of connexin expression and a concomitant decrease in GJIC. The molecular mechanisms by which GJIC affect activation are not known although the modulation of cell-cell adhesion subsequent to the inhibition of GJIC could be involved (Meyer et al, 1992;Paul et al, 1995). The second aspect of wound repair that correlated with altered GJIC is the restoration of normal epidermal growth.…”

The Second PDZ Domain of Zonula Occludens-1 Is Dispensable for Targeting to Connexin43 Gap Junctions

“…Alternatively, the presence of low levels of ZO-1 at the edge of Cx43-GFP plaques could result indirectly via macromolecular associations between GJs and other cell-cell junctions that incorporate ZO-1. Adherens junctions are logical candidates in this respect as they are known to play a pivotal role in GJ stability (Hertig et al 1996;Jongen et al 1991;Li et al 2005;Meyer et al 1992;Musil et al 1990;Wei et al 2005) and cadherin-associated catenin complexes have been shown to bind both Cx43 (Ai et al 2000;Wei et al 2005) and ZO-1 (Itoh et al 1997).…”

Connexin 43 is critical to maintain the homeostasis of the blood–testis barrier via its effects on tight junction reassembly

“…The formation of both the AJ and the GJ can be blocked with the use of antibodies against either N-cadherin or Cx43 (26), and Cx43 can be transported directly to existing AJ sites through microtubules (27). Cx43 has also been reported to promote endothelial attachment of leukocytes induced by cytokines or tumor cells during metastasis when it was overexpressed (28)(29)(30).…”