Background: 

Cx43, widely expressed in most cells of the body has not only a role in forming gap junctions but may also function as part of a signalosome since it acts as an adapter protein binding other proteins to its cytoplasmic C-terminal end. Since it has been reported that expression of connexin 43 (Cx43) is altered during wound healing, a status being associated with enhanced cell migration, we analysed a potential role of Cx43 and its C-terminus in control of cell migration.

Methods: 

Both ends of connexin 43, which consists of four membrane spanning domains, the amino (NH₂) terminus and a long C-terminus are located in the cytoplasm. HeLa cells which as wild type do not express connexins, were stably transfected with cDNAs encoding either full length Cx43 (HeLa 43fl; AA 1–382) or Cx43 with the C-terminus truncated, named here as the "N-terminal part" (Cx43NT-GFP; AA 1–257). Alternatively, a cDNA encoding the cytoplasmic C-terminal tail only was transfected (Cx43CT-GFP; AA 258–382). All mutants were coupled with green fluorescent protein (GFP), respectively. HeLa cells expressing GFP only (HeLa-GFP) were used as additional controls.

Results: 

Confocal microscopy as performed in confluent HeLa cells showed cytosolic and mainly membranous localisation of Cx43NT-GFP, while Cx43CT-GFP and GFP alone were found to be expressed in the cytosol only. Likewise, immunofluorescence stainings of HeLa 43fl cells with Cx43 antibody showed Cx43 in the cytosol as well as punctated expression at cell-cell-contact regions. Cell coupling was analysed by the transfer of a gap junctional permeable dye (Calcein-AM) between two differently stained HeLa populations coincubated for 3 hours. Evaluation of gap junctional dye transfer by FACS analysis (n=4) indicated functional cell coupling between cells expressing HeLa 43fl (64 16%) and a slightly less but still significant coupling in HeLa 43NT-GFP cells (41 9%). In contrast, no coupling was observed in HeLa-GFP cells as well as in cells expressing Cx43CT-GFP. Cell migration was studied optically by time lapse microscopy (24 hrs) using Chemotaxis m-Slides (Ibidi) with 20% FCS as a migration stimulus (n=3). HeLa 43CT-GFP cells showed significantly increased migration distances compared to HeLa cells expressing Cx43NT-GFP.

These results suggest that Cx43 modulates cell migration in a gap junction independent manner. The C-terminal part of the Cx43 seems to be essential for this function, which does not require membrane localisation of the molecule.