Mechanical forces play an undisputed elementary role in the interactions between cells and the surrounding extracellular matrix (ECM) . Not only are these forces essential for the cells migratory behavior, they also influence proliferation (including tumor growth) and differentiation [2–4]. These forces are transferred across focal adhesions (FAs) which connect ECM and cell skeleton through patches of activated integrin proteins. Since the origin of exerted cellular forces lies in these FAs, they are the ideal starting point for characterization of mechanotransduction pathways. Consequently, studying how the properties of the ECM affect the cellular forces is key in understanding how cells connect to their environment and alter their behavior appropriately. While the scientific field of cellular mechanosensation has been studied for years, recent developments in imaging techniques and force sensor development enable us to dig deeper.
To investigate the forces applied in FAs, the well established TSMod force sensor is used. This sensor consists of two fluorescent proteins separated by an elastic linker. This elastic linker keeps both chromophores close to establish a high Förster Resonance Energy Transfer (FRET) state. This sensor is incorporated in the Vinculin protein, sandwiched by its head (Vh) and tail (Vt) domain. Only when a force is applies to the FA complex, the fluorescent proteins separate resulting in a lower FRET signal. Two types of sensor were used, the tension sensitive (TS) and a tail-less mutant (TL, positive control). FRET efficiencies are calculated by measuring accurate donor lifetimes, which lowers when FRET towards acceptor (Venus) quenches the donor (mTFP1)