A Novel Role for p115RhoGEF in Regulation of Epithelial Plasticity
Figure 5
Overexpression of p115RhoGEF leads to activation of RhoA and catalytic activity of p115RhoGEF is required for its regulation of epithelial plasticity.
A) Control and p115-OE MCF7 cells and cells were transfected with a FRET-based RhoA biosensor. Cells were imaged for CFP and FRET and FRET/CFP ratio representative of RhoA activity was calculated. Warmer colors correspond to high RhoA activity. High RhoA activity was seen at cell-cell junctions and within the cell body in cells that overexpressed p115RhoGEF as compared to control MCF7 cells. B) Total RhoA activity was quantitated by calculating FRET intensity per unit area of the cell from a total of 30 cells (n = 3). Cells that overexpressed p115RhoGEF showed a significant increase in total RhoA activity as compared to control cells (mean ± SE). C) Junctional RhoA activity was quantitated by calculating FRET intensity per unit length of the junction from a total of 30 cells (n = 3). Cells that overexpressed p115RhoGEF showed a significant increase in junctional RhoA as compared to control cells (mean ± SE). D) Control and p115-OE MDA-MB-231 cells were transfected with the FRET-based RhoA biosensor. High RhoA activity was seen around the cell periphery and within the entire cell body of p115-OE MDA-MB-231 cells as compared to control cells that showed RhoA activation at protrusions and in areas in the rear of the cell. E) Total RhoA activity was quantitated by calculating FRET intensity per unit area of the cell from 25 cells (n = 3). p115-OE MDA-MB-231 cells showed a significant increase in total RhoA activity as compared to control cells (mean ± SE). F) MCF7 cells overexpressing a catalytically inactive p115RhoGEFDH mutant (GEF mutant p115) showed no change in junctional E-cadherin staining intensity as compared to cells that overexpressed wild type p115RhoGEF G) Quantitation of junctional staining intensity of E-cadherin in cells that overexpressed p115RhoGEFDH mutant did not show a significant difference as compared to control cells (average ± S.E.). 150 cell-cell junctions were analyzed from 3 separate experiments. H) Representative immunoblots showing expression of p115RhoGEFDH mutant in MCF7. I) Actin staining of p115RhoGEFDH mutant–expressing MDA-MB -231 cells did not show the change in morphology seen upon overexpression of wild type p115RhoGEF. J) Quantitation of change in morphology in terms of circularity of the cells reveal no significant difference between control cells and cells that overexpressed p115RhoGEFDH mutant (mean ± SE). 75 cells were analyzed from 3 separate experiments. K) Representative immunoblots showing overexpression of p115RhoGEFDH mutant as compared to endogenous p115RhoGEF in control cells.