Fig. 3. The disruption of AJs by distinct mechanisms leads to a strong activation of Rap1. (A) Serum-starved epithelial FRT cell monolayers were either left untreated and lysed (Ctrl), or incubated in serum-free DMEM supplemented with 4 mM EGTA for 30 minutes at 37°C to disrupt E-cadherin-mediated cell-cell contacts. Treated cells were then either lysed (EGTA) or allowed to reform adherens junctions by further incubation with fresh medium containing 1.8 mM Ca²⁺ for 45 minutes at 37°C before lysis (EGTA+Ca²⁺). Cell lysates were then processed for measurement of active Rap1 levels by the pull-down assay. Notice that the disruption of adherens junctions by EGTA treatment led to a strong activation of Rap1, while the amounts of active Rap1 returned to the basal level as E-cadherin-mediated cell-cell adhesion was re-established by Ca²⁺ restoration. Relative levels of total Rap1 in whole cell lysates are showed in the lower blot as control. (B) In parallel experiments, cells were subjected to the above calcium switch procedure while plated onto glass coverslips, and further processed for immunofluorescence microscopy analysis. The staining of cortical actin with FITC-conjugated phalloidin confirms the effectiveness of the calcium switch procedure. (C) Serum-starved epithelial FRT cell monolayers were either left untreated (–) or treated with 5 µM PAO for the indicated times at 37°C to disrupt cell-cell contacts. Cells were then processed for measurement of active Rap1 levels by the pull-down assay. Notice that PAO treatment clearly affected the GTP-loading of Rap1 (GTP-Rap1) within 30 minutes, while total Rap1 levels did not change. (D) FRT cells plated onto glass coverslips were either left untreated (Ctrl) or treated with 5 µM PAO for 30 minutes (PAO), and processed for immunofluorescence microscopy analysis. Notice the effectiveness of PAO treatment in disrupting cell-cell adhesion, as detected by staining F-actin with FITC-conjugated phalloidin, and the now fibroblast-like morphology of the treated cells. (E) Serum-starved epithelial FRT cell monolayers were subjected to the calcium switch procedure, and further processed for measurement of the active levels of both Rap1 and Rac1 through specific pull-down assays. Notice that the changes in Rap1 activity that occurred during the calcium switch were closely paralleled by inverse changes in Rac1 activity.