The ability to sense mechanical forces is vital to cell physiology. Yet, the molecular basis of mechano-signaling remains unclear. Previous studies have shown that zyxin, a focal adhesion protein, is recruited at force-bearing sites on the actin cytoskeleton and, therefore, identifying zyxin as a mechano-sensing protein candidate. Furthermore, zyxin accumulation at force-bearing sites requires the LIM domain located at the C-terminus of zyxin. The zyxin LIM domain consists of three LIM motifs, each containing two zinc-binding sites. Since individual LIM motifs do not accumulate at focal adhesions or force-bearing sites, we hypothesize that multiple zyxin LIM domains increase force sensitivity. Using a miniature force sensor and GFP-tagged LIM variants, we quantified the relationship between single, tandem dimer and trimer LIM protein localization and traction forces. While the presence of extra LIM domains affected VASP recruitment to focal adhesions, force sensitivity was not enhanced over the single LIM domain. Therefore, zyxin force sensitivity is optimal with a single LIM domain, while additional LIM domains fail to enhance force sensitivity. (C) 2012 Elsevier Inc. All rights reserved.