This study evaluates the influence of vinculin in closed conformation on the mechanical properties of cells. We demonstrate that MEFvin(-/-) cells transfected with the eGFP-vinculin mutant A50I (talin-binding-deficient-vinculin in a constitutively closed conformation) show 2-fold lower stiffness and focal adhesion density compared to MEFvin(+/+) and MEFRRescue cells. MEFA50I I cells are as stiff as MEFvin-/- cells with similar focal adhesion density. Further, 2D traction microscopy indicates that MEFA50I and MEFvin(-/-) cells generate 3- to 4-fold less strain energy than MEFvin(+/+) and MEFRescue cells. These results demonstrate that vinculin's mechano-coupling function is dependent on its conformational state. (C) 2011 Elsevier Inc. All rights reserved.