Figure 2.

Both the PBM and GBA motif of DAPLE are required to promote apical cell constriction. (A) Quantification of the relative apical area of DAPLE-transfected cells compared with neighboring, untransfected cells shows that hDAPLE ΔPBM and hDAPLE GBA* mutants fail to promote apical constriction in MDCK (left) or EpH4 (right) cells. Results are presented as box-and-whiskers plots (error bars indicate minimum to maximum range) of n = 4–9 independent experiments per condition. *, P < 0.05; **, P < 0.01 using the Mann–Whitney U test. (B) Confocal fluorescence microscopy pictures of MDCK cells sparsely expressing the indicated MYC-hDAPLE constructs and costained for MYC (magenta) and ZO-1 (green) show that hDAPLE GBA* mutant is enriched at apical junctions like WT, while hDAPLE ΔPBM mutant is not. Scale bars, 10 µm. (C) Diagram depicting the different functions of the PBM and the GBA motif in DAPLE-induced apical cell constriction. A possible effector pathway to promote apical cell constriction though G protein activation is shown on the right, along with the treatments used in D to test it (green). (D) Box-and-whiskers plots (error bars indicate minimum to maximum range) for the quantification of relative apical area show that DAPLE-mediated apical constriction requires the activity of myosin (inhibited by blebbistatin), ROCK (inhibited by Y-27632), free Gβγ (inhibited by Gallein, but not its inactive analogue, fluorescein) and p114RhoGEF (inhibited by siRNA). n = 4 independent experiments per condition. *, P < 0.05 using the Mann–Whitney U test. The immunoblot (IB) on the bottom right shows the reduction of p114RhoGEF expression upon siRNA treatments.