Fig. 2. The inhibition of Rap1 activity during cell adhesion does not require the integrity of the actin cytoskeleton, but is significantly influenced by cell density. (A) Confluent cells were harvested using 5 mM EDTA treatment, resuspended in serum-free medium in the presence (+) or absence (–) of 1 µM cytochalasin D (CytD), and either lysed (Su, suspension cells) or allowed to adhere for 1 hour at 37°C to FN-coated tissue culture dishes before measurement of active Rap1 (GTP-Rap1) by the pull-down assay. Western blotting of whole cell lysates was used to assess total levels of Rap1 (Total Rap1). Notice that the cell adhesion-dependent down-regulation of Rap1 activity did not require an intact actin cytoskeleton. (B) In parallel experiments, cells were seeded onto FN-coated glass coverslips either in the absence (Ctrl) or presence (CytD) of 1 µM CytD for 1 hour at 37°C to disrupt the actin cytoskeleton, and processed for immunofluorescence microscopy analysis to detect filamentous actin (F-actin). Notice the effectiveness of CytD treatment in disrupting the actin cytoskeleton. (C) Cells were harvested by trypsinization, extensively pipetted to generate a maximal dispersed cell suspension, replated at various densities (0.25x10⁵ to 1.5x10⁵ cells/cm²) in serum-free medium, and allowed to recover overnight at 37°C before measurement of Rap1 activity. Notice that the down-regulation of Rap1 activity during cell adhesion was clearly dependent on cell density.