Rho-associated kinase-dependent contraction of stress fibres and the organization of focal adhesions

Abstract

Stress fibres and associated focal adhesions in cells constitute a contractile apparatus that regulates cell motility and contraction. Rho-kinase, an effector molecule of small GTPases, regulates non-muscle cell motility and contractility. Rho-kinase mediates the contraction of stress fibres in a Ca(2+)-independent manner, and is responsible for slower and more finely tuned contraction of stress fibres than that regulated by myosin light chain kinase activity in living cells. The specific inhibition of the Rho-kinase activity causes cells to not only lose their stress fibres and focal adhesions, but also to appear to lose their cytoplasmic tension. Activated Rho-kinase is also involved in the organization of newly formed stress fibres and focal adhesions in living cells.

Figure 1.

Effects of Rho-kinase (Y-27632) and MLCK (ML-7) inhibitors on peripheral and central stress fibres of human foreskin fibroblasts. The cells in (a–c) are doubly stained with rhodamine-labelled phalloidin for F-actin staining (red) and anti-vinculum for focal adhesion staining (green). Yellow staining indicates the overlap of the two colours, where F-actin and vinculin co-localize. (a) Untreated control; (b) cell treated with Y-27632 (10 µM) for 1 h; (c) cell treated with ML-7 (25 µM) for 1 h. (b) Cell treated with Y-27632 showed a reduced number of central stress fibres; however, peripheral stress fibres remained unchanged. (c) Peripheral stress fibres were significantly reduced after the treatment with ML-7, but not central stress fibres. All photographs are taken under confocal laser-scanning microscopy. Scale bar, 20 µm.

Figure 2.

Schematic of the two regulatory actin–myosin contractile systems in non-muscle cells. P-MLC induced cell contraction. P-MLC, phosphorylated MLC; MP, myosin phosphatase; Cat, catalytic subunit of myosin phosphatase; MBS, myosin-binding subunit of myosin phosphatase; P-MBS, phosphorylated MBS.