Background: 

Connexin 43 (Cx43) is one of the most widely expressed connexins in the body. Beside its channel forming capacity, Cx43 has also an important role as an adapter protein with binding sites for various proteins at its cytoplasmic C-terminal part. Since connexin expression is often altered during processes with enhanced cell migration such as wound healing, angiogenesis and arteriosclerosis, we analyzed whether Cx43 affects migration in a gap junction dependent or independent manner.

Methods: 

HeLa cells, which as wild type do not express any connexins, were stably transfected with cDNAs encoding either full length Cx43 (Cx43fl, aa 1-382) or a C-terminus truncated Cx43 mutant, defined here as the "N-terminal part" (Cx43NT-GFP; aa 1-258). Alternatively, a cDNA encoding only the cytoplasmic C-terminal tail was transfected (Cx43CT-GFP; aa 258-382). Both mutants were coupled with the green fluorescent protein (GFP). 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 localization 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 punctated expression of Cx43 at cell-cell-contact regions and also in the cytoplasm. To analyze cell coupling, the transfer of a gap junction permeable dye (Calcein-AM) between two differently stained HeLa populations, coincubated for 3 hours, was measured. FACS analysis (n = 4) indicated functional cell coupling between cells expressing Cx43fl (64 ± 16%) and to a lesser but still significant extent in HeLa-Cx43NT-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 for 24 hours using wound assay chambers (Ibidi) in which the cells migrate into a defined gap (n = 5). HeLa-Cx43CT-GFP cells showed significantly increased migration distances (accumulated distance 212 ± 16 mm) compared to HeLa cells expressing Cx43NT-GFP (accumulated distance 157 ± 17 mm). These results suggest that Cx43 enhances cell migration in a gap junction independent manner. In fact its C-terminal part seems to be essential for this function, which does not require membrane localization of the molecule.