Treatment of cultured cells with inhibitors of actomyosin contractility induces 
rapid deterioration of stress fibers, and disassembly of the associated focal 
adhesions (FAs). In this study, we show that treatment with the Rho kinase 
inhibitor Y-27632, which blocks actomyosin contractility, induces disarray in 
the FA-associated actin bundles, followed by the differential dissociation of 
eight FA components from the adhesion sites. Live-cell microscopy indicated that 
the drug triggers rapid dissociation of VASP and zyxin from FAs (τ values of 7-8 
min), followed by talin, paxillin and ILK (τ ~16 min), and then by FAK, vinculin 
and kindlin-2 (τ = 25-28 min). Examination of the molecular kinetics of the 
various FA constituents, using Fluorescence Recovery After Photobleaching 
(FRAP), in the absence of or following short-term treatment with the drug, 
revealed major changes in the kon and koff values of the different proteins 
tested, which are in close agreement with their differential dissociation rates 
from the adhesion sites. These findings indicate that mechanical, 
actomyosin-generated forces differentially regulate the molecular kinetics of 
individual FA-associated molecules, and thereby modulate FA composition and 
stability.