Force and focal adhesion assembly: A close relationship studied using elastic micro-patterned substrates Nathalie Q. Balaban, Ulrich S. Schwarz, Daniel Riveline, Polina Goichberg, Gila Tzur, Ilana Sabanay, Diana Mahalu, Sam Safran, Alexander Bershadsky, Lia Addadi, and Benjamin Geiger Nature Cell Biology 3: 466 - 472 (2001) Mechanical forces play a major role in the regulation of cell adhesion and cytoskeletal organization. In order to explore the molecular mechanism underlying this regulation, we have investigated the relationship between local force applied by the cell to the substrate and the assembly of the respective focal adhesions. For this purpose, a novel approach was developed for real-time and high-resolution measurements of forces applied by cells at single adhesion sites. The method combines micro-patterning of elastomer substrates and fluorescence imaging of focal adhesions in live cells expressing GFP-tagged vinculin. The force applied by the cell at individual focal adhesions is calculated, based on the deformations of the regular pattern and the simultaneous identification of near-by adhesion sites. These measurements indicate that local forces are correlated with the orientation, total fluorescence intensity and area of the focal adhesions. The linear dependence between force and area suggests that a constant stress of 5.5 +/- 2 nN/micron^2 is applied at the various focal adhesions of a cell. The dynamics of the force-dependent modulation of focal adhesions was characterized by blocking acto-myosin contractility, and the disruption was found to be on a time scale of seconds. The results put clear constraints on the possible molecular mechanisms for the mechanosensory response of focal adhesions to applied force.