Figure 2. Microtubule organizing center, Golgi apparatus, and microtubules fail to reorient in Cx43α1 KO MEFs, which also display a reduction in stabilized microtubules.

(A–D). The polarity of Cx43 wildtype and KO MEFs (n = 75 and 67 cells respectively) at the wound edge were examined by immunostaining with a GM130 antibody (A, B) to delineate the Golgi apparatus, and with a γ-tubulin antibody to delineate the microtubule organizing center (C, D). In wildtype cells (A,B), the Golgi and the MTOC were usually forward facing relative to the direction of wound closure (white arrow), but in KO MEFs (C,D), the position of the Golgi and MTOC appear to be randomized. (E,F). Orientation of the MTOC/Golgi was scored with each cell divided into one 120 degree sector facing the lead edge, and a second sector comprising the remainder 240 degrees (E). Cells with MTOCs located in the sector facing the lead edge were considered oriented and scored 1, cells with MTOCs positioned outside of this were considered not oriented and scored 0. Compilation of such scoring showed a significant reduction in the reorientation of the MTOC in Cx43 KO MEFs when compared to the wildtype MEFs, and this reduction was not statistically different when compared with cells with non-orientated MTOC when cultured in the absence of serum (F). These observations suggest KO MEFs have a defect in reorientation of the MTOC at the wound edge. (G–L) Immunostaining with an α-tubulin antibody showed microtubules align with the direction of wound closure in wildtype MEFs, but not in KO MEFs (G vs J). Much of the microtubules in wildtype MEFs were also immunostained by a Glu-tubulin antibody (H), while little or no Glu-tubulin staining was observed in the KO MEFs (K). Data presented as mean ± SEM. All scale bars represent 25 µm.