Isolation and in vitro contraction of stress fibers

Publisher Summary Isolated stress fibers are useful for studying their properties and functions, but they are also useful as a nonmuscle contraction model system for investigating the regulatory mechanism for the actomyosin-based contractility in nonmuscle cells. This chapter discusses the isolation of stress fibers en masse from cultured cells. It also describes the way to observe contraction of stress fibers. The isolation procedure is simple and provides stress fibers that are pure enough for biochemical as well as structural and other studies. The contraction of stress fibers is best observed by using those still attached to the substrate surface. The contraction is an actomyosin-based, ATP-driven contraction. Rho kinase is involved in stress fiber contraction. Both RhoA and Rho kinase are present in isolated stress fibers. The chapter explains that the stress fiber model can contract in the absence of Ca 2+ and that this contraction is inhibited by a Rho kinase inhibitor. In this case, the myosin regulatory light chain is phosphorylated, not by myosin light chain kinase, but by Rho kinase. The studies presented in the chapter indicate that stress fiber contraction is regulated by two independent systems: one by the Ca 2+ - dependent myosin light chain kinase system and the other by the Ca 2+ - independent Rho kinase system.