Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates

Mechanical forces play a major role in the regulation of cell adhesion and cytoskeletal organization. In order to explore the molecular mechanism unde rlying this regulation, we have investigated the relationship between local force applied by the cell to the substrate and the assembly of focal adhes ions. A novel approach was developed for real-time, high-resolution measure ments of forces applied by cells at single adhesion sites. This method comb ines micropatterning of elastomer substrates and fluorescence imaging of fo cal adhesions in live cells expressing GFP-tagged vinculin, Local forces ar e correlated with the orientation, total fluorescence intensity and area of the focal adhesions, indicating a constant stress of 5.5 +/- 2 nN mum-2. T he dynamics of the force-dependent modulation of focal adhesions were chara cterized by blocking actomyosin contractility and were found to be on a tim e scale of seconds. The results put clear constraints on the possible molec ular mechanisms for the mechanosensory response of focal adhesions to appli ed force.