Fig. 8.

Cx43 tubulin-binding domain is required for epicardial cell polarity. (A-D) E11.5 epicardial explants were transfected with plasmids expressing either wild-type Cx43 fused with GFP (A,B) or Cx43 with the putative tubulin-binding domain deleted (Cx43dT) and fused with GFP (C,D). Transfection of GFP alone was also carried out as another control. Transfected cells were identified by GFP expression (A,C). Staining with γ-tubulin antibody (B,D), shown by the red dot enclosed by the white arrow, delineated the orientation of the MTOC. (E) In Cx43 KO epicardial cells, the MTOC failed to align with the direction of cell migration, as indicated by a very high percentage of non-forward-facing MTOC. Quantitative motion analysis revealed a significant increase in the extension and retraction of cell protrusions and in roundness of the Cx43dT cells compared to the Cx43 transfected cells (P-value indicated in graph). Significant differences were also observed when Cx43dT was compared with the GFP transfected cells (roundness, P=0.0001; cell protrusions, P=0.0261; cell retractions, P=0.0255). Scale bar: 50 μm.