Figure 1

Formation of gap junctions and hemichannels with Cx26-WT or -D50N under the condition of 1.9 mM Ca²⁺ concentration. Left column: 2-dimensional images. Right column: 3-dimensional movies. (A) Gjb2 WT-transfected HeLa cells were co-stained for TGN46, a marker for the trans-Golgi network (red), and for Cx26-WT-FLAG proteins (blue). The cells with green signals (eGFP) are the transfected cells. Blue arrows indicate gap junction plaques that formed between adjacent Cx26 expressing cells. Cx26-WT proteins are not localized in the trans-Golgi network marked with anti-TGN46 antibodies (red), and Cx26-WT blue dots may represent hemichannels (blue arrowheads) in the cell membrane. (B) Gjb2 c.148 G > A-transfected HeLa cells were also co-stained for TGN46 (red) and Cx26-D50N-FLAG proteins (blue). Similarly to Cx26-WT, the mutant Cx26, Cx26-D50N, formed gap junctions at the cell-cell contact zones (blue arrows). Cx26-D50N is not observed in the trans-Golgi network and seems to form hemichannels in the cell periphery (blue arrowheads). (C) Gjb2 WT-transfected cells were co-labeled with rhodamine-conjugated WGA (red) and Cx26-WT-FLAG (blue). The transfected cells show green signals (eGFP). Blue arrows indicate gap junction plaques connecting adjacent Cx26-WT-expressing cells. Purple signals (purple arrowheads) represent the co-localization of Cx26-WT and WGA, which are putative hemichannels. (D) Gjb2 c.148 G > A-transfected cells were double-stained for rhodamine-labeled WGA (red) and Cx26-D50N-FLAG (blue). Gap junction formation (blue) is indicated with blue arrows. Purple signals (purple arrowheads) indicate the co-localization of Cx26-D50N and WGA, suggesting hemichannel formation in the cell surface. Scale bars: 20 μm.