Rho‐kinase dependent organization of stress fibers and focal adhesions in cultured fibroblasts

The activation of Rho‐kinase is known to modulate the organization of the actin‐based cytoskeletal systems, including the formation of stress fibers and focal adhesions. Rho‐kinase likely plays a more crucial and complex role in the organization of actin‐based cytoskeletal systems than in that of myosin light chain kinase (MLCK). In order to understand the role of Rho‐kinase in the organization of stress fibers and focal adhesions, we treated cultured fibroblasts with a Rho‐kinase inhibitor and analyzed the stress fiber and focal adhesion organization under conventional fluorescence microscopy and replica electron microscopy. Some of the cells were transfected with GFP‐labeled paxillin, actin or α‐actinin, and the effects of the inhibitor were monitored in the living cells. The Rho‐kinase inhibitor caused disassembly of the stress fibers and focal adhesions in the central portion of the cell within 1 h. However, the stress fibers and focal adhesions located in the cell periphery were not as severely affected by the Rho‐kinase inhibitor. The time‐lapse video recording revealed that when these cells were washed with a fresh medium in order to remove the Rho‐kinase inhibitor, the stress fibers and focal adhesions located in the center of the cell gradually reorganized and, within 1.5–2 h, the cells completely recovered. This observation strongly suggests that the activation of Rho‐kinase plays an important role in the organization of the central stress fibers and focal adhesions.