Figure 2
Formation of gap junctions and hemichannels with Cx26-G12R or Cx26-G45E under the condition of high extracellular Ca2+ concentration (3.8 mM Ca2+). Left column, 2-dimensional images. Right column, 3-dimensional movies. (A) The Gjb2 c.34 G > C-transfected HeLa cells were co-stained for the trans-Golgi network marker TGN46 (red) and for Cx26-G12R-FLAG proteins (blue). The cells with green signals (eGFP) are transfected cells. Blue arrows indicate the gap junction plaques between adjacent Cx26-G12R expressing cells. Cx26-G12R proteins are not localized in the trans-Golgi network stained with anti-TGN46 antibodies (red), and Cx26-G12R blue dots (blue arrowheads) are putative hemichannels in the cell surface. (B) The Gjb2 c.134 G > A-transfected HeLa cells were co-stained for TGN46 (red) and Cx26-G45E-FLAG proteins (blue). Cx26-G45E also formed gap junctions at the cell-cell contact zones (blue arrows). Blue dots of Cx26-G45E (blue arrowheads) are not seen in the trans-Golgi network (red). (C) Gjb2 c.34 G > C-transfected cells were co-labeled with rhodamine-conjugated WGA (red) and Cx26-G12R-FLAG (blue). The transfected cells show green signals (eGFP). Small gap junction plaques (blue arrows) are seen between adjacent Cx26-G12R-expressing cells. Purple signals (purple arrowheads) represent the co-localization of Cx26-G12R and WGA, suggesting the formation of hemichannels in the cell surface. (D) The Gjb2 c.134 G > A-transfected cells were double-stained for rhodamine-labeled WGA (red) and Cx26-G45E-FLAG (blue). Gap junction formation is not apparent. Purple signals (purple arrowheads) indicate the co-localization of Cx26-G45E and WGA, which are putative hemichannels in the cell surface. Scale bars: 20 μm.
