Cells are frequently subject to mechanical forces. Keratinocytes in the skin are exposed to forces from shear, pressure or stretch, endothelial cells in the blood vessels are subject to the shear forces of the blood flow; cells in our lungs feel forces from inflation, cells in the heart the rhythmic contractions of the heartbeat, and bone cells must bear forces from our weight. Of course, cells also generate their own intracellular forces as is obvious during cell division or cell migration.
Over the last years it has become apparent, that the ability of cells to sense these forces and to translate them into biochemical information is crucial for a wide range of biological processes, and also has important implications for diseases such as arteriosclerosis, osteoporosis or cancer. However, the molecular mechanisms that underlie cells’ mechanosensitivity are largely elusive.
Limitations of currently available technologies
One reason for our limited understanding of mechanotransduction is our inability to measure mechanical forces inside cells. Currently used methods such as traction force microscopy reliably report cellular traction forces but lack sub-cellular resolution; atomic force microscopy, on the other hand, allows highly sensitive force measurements across single proteins but its application to living cells is limited.
We have developed a light-microscopy-based technique that allows measurement of pico-Newton (pN) forces across distinct molecules in living cells. The method is based on a physical effect called 'Förster resonance energy transfer' (FRET) which can occur between two adjacent fluorophores (Fig.1a).
We used the fact that FRET is highly distance-dependent and inserted an extensible linker between the two fluorophores. The peptide-linker has been derived from the spider silk protein flagelliform, which can be stretched by a few pN. Thus, forces across the tension sensor will stretch the linker, separate the fluorophores and reduce FRET (Fig.1b, Grashoff et al., Nature, 2010).
Insertion of this biosensor into proteins of interest allows visualization of tension across distinct molecules in living cells. To measure forces in cell-matrix adhesions we generated a vinculin tension sensor. Our experiments revealed that vinculin is indeed exposed to mechanical forces in cell adhesions and is subject to an interesting spatio-temporal regulation: while vinculin is subject to high forces in protruding areas of migrating cells it bears no significant tension in retracting parts of the cell (Fig.1c, Grashoff et al., Nature, 2010). This is the first example of an intracellular force measurement across a distinct molecule in living cells.
In the future, this technology should allow us to investigate molecular mechanisms of force transduction in great detail and to develop a more comprehensive understanding of cellular mechanotransduction.
Grashoff C et al. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature. 2010, 466:263-266. »
Doyle AD and Yamada KM. Cell biology: Sensing tension. Nature. 2010, 466:192-193. »
Wrighton KH. Sensing and controlling protein dynamics. Nat Rev Mol Cell Biol. 2010, 11:680-681. »