Figure 6. Dominant Cx26 mutants inhibit dye transfer of cells co-expressing Cx30.

These are digital fluorescence images of confluent, bulk-selected HeLa cells that stably express wild type Cx30, or co-express Cx30 and an “empty vector” (vector/Cx30), as well as Cx26 (Cx30/Cx26) or the indicated Cx26 mutant. The cells were incubated in 0.1% LY (A) or 2% NB (B) for 5 min, and imaged either directly (LY) or following fixation (NB; visualized with TRITC-conjugated avidin) ~15 min after being wounded with a scalpel blade. In panel A, note that the wounded cells picked up LY in all cases, and, in contrast to cells expressing Cx30 alone, cells expressing Cx26 alone showed extensive transfer of LY to neighboring cells. None of the cells co-expressing a Cx26 mutant showed transfer of LY to neighboring cells except for cells co-expressing D179N, but this was less than in cells co-expressing wild type Cx26. Scale bar: 50 μm. In panel B, note that NB transferred extensively in cells expressing Cx30 alone, or co-expressing Cx30 and wild type Cx26, R143Q, or D179N, less extensively in cells co-expressing Cx30 and W44S, G59A, R75Q or R184Q, and not at all in cells co-expressing Cx30 and W44C, R75W, or C202F. Scale bar: 50 μm. In panel C, quantitative analysis of intercellular NB transfer after scrape-loading is shown. The columns represent the mean distance of NB transfer from the scrape line to the point where the fluorescence intensity dropped to 1.5x the background intensity. For each cell line, this was measured by acquiring at least 8 images from each of two to three different plates of cells. The error bars indicate ± s.e.m. Triple asterisks (***) above the error bars denote a significance level of p < 0.0001 when compared to vector/Cx30 expressing cells, Triple asterisks (***) below the error bars denote a significance level of p < 0.0001 when compared to Cx26/Cx30 expressing cells.