Figure 5. Effect of Rho kinase inhibitor on growth factor-induced actin cytoskeletal organization and focal adhesion formation

Serum starved human lens epithelial cells were initially treated for 20 min with 5 μM Y-27632, a specific inhibitor of Rho kinase (the down stream effector of Rho GTPase), followed by treatment for 1 h with different growth factors. Pretreatment of cells with Y-27632 (right panel) abolished growth factor-mediated induction of stress fiber and focal adhesion formation, as compared to cells stimulated with growth factors alone (left panel). While growth factor treated cells appeared to undergo morphological changes and exhibited clear increases in actin stress fibers (indicated with arrows), cells pretreated with Y-27632 and stimulated with growth factors appear flat with a non-rigid morphology (B). Unlike other growth factors, cells pretreated with inhibitor, followed by EGF stimulation, exhibit a relatively high number of membrane ruffles, a hallmark of Rac GTPase activation and not affected by Y-27632 (A indicated with arrows). Images shown here indicate the double labeling of actin stress fibers (FITC staining) and focal adhesions (vinculin staining with TRITC conjugated-secondary antibody). While actin fibers were prominent, focal adhesions staining was weak in these images. However, direct viewing with confocal microscopy showed a definite positive staining for vinculin.