Cellular responses to mechanical perturbation are vital to cell physiology. In 
particular, migrating cells have been shown to sense substrate stiffness and 
alter cell morphology and speed. Zyxin is a focal adhesion protein that responds 
to external mechanical forces; however, the mechanisms of zyxin recruitment at 
force-bearing sites are unknown. Using force-sensing microfabricated substrates, 
we simultaneously measured traction force and zyxin recruitment at force-bearing 
sites. GFP-tagged zyxin accumulates at force-bearing sites at the leading edge, 
but not at the trailing edge, of migrating epithelial cells. Zyxin recruitment 
at force-bearing sites depends on Rho-kinase and myosin II activation, 
suggesting that zyxin responds not only to the externally applied force, as 
previously shown, but also to the internally generated actin-myosin force. Zyxin 
in turn recruits vasodilator-stimulated phosphoprotein, a regulator of actin 
assembly, to force-bearing sites. To dissect the domains of zyxin that are 
essential for this unique force-dependent accumulation, we generated two zyxin 
truncation mutants: one lacking the LIM domain (ΔLIM) and one containing only 
the LIM domain with all three LIM motifs (LIM). GFP-tagged ΔLIM does not 
localize to the force-bearing sites, but GFP-tagged zyxin LIM-domain is 
sufficient for the recruitment to and dynamics at force-bearing focal adhesions. 
Furthermore, one or two LIM motifs are not sufficient for force-dependent 
accumulation, suggesting that all three LIM motifs are required. Therefore, the 
LIM domain of zyxin recruits zyxin to force-bearing sites at the leading edge of 
migrating cells.