Stress fiber organization regulated by MLCK and Rho-kinase in cultured human fibroblasts

To understand the roles of Rho-kinase and myosin light chain kinase (MLCK) for the contraction and organization of stress fibers, we treated cultured human foreskin fibroblasts with several MLCK, Rho-kinase, or calmodulin inh ibitors and analyzed F-actin organization in the cells. Some cells were tra nsfected with green fluorescent protein (GFP)-labeled actin, and the effect s of inhibitors were also studied in these living cells. The Rho-kinase inh ibitors Y-27632 and HA1077 caused disassembly of stress fibers and focal ad hesions in the central portion of the cell within 1 h. However, stress fibe rs located in the periphery of the cell were not severely affected by the R ho-kinase inhibitors. When these cells were washed with fresh medium, the c entral stress fibers and focal adhesions gradually reformed, and within 3 h the cells were completely recovered. ML-7 and KT5926 are specific MLCK inh ibitors and caused disruption and/or shortening of peripheral stress fibers , leaving the central fibers relatively intact even though their number was reduced. The calmodulin inhibitors W-5 and W-7 gave essentially the same r esults as the MLCK inhibitors. The MLCK and calmodulin inhibitors, but not the Rho-kinase inhibitors, caused cells to lose the spread morphology, indi cating that the peripheral fibers play a major role in keeping the flattene d state of the cell. When stress fiber models were reactivated, the periphe ral fibers contracted before the central fibers. Thus our study shows that there are at least two different stress fiber systems in the cell. The cent ral stress fiber system is dependent more on the activity of Rho-kinase tha n on that of MLCK, while the peripheral stress fiber system depends on MLCK .