We are interested in the molecular processes underlying mechanical force transduction in cells. To investigate these processes, we developed a microscopy technique that allows the quantification of mechanical forces with piconewton-sensitivity in living cells. The method is based upon Fӧrster resonance energy transfer (FRET)–based biosensors, in which two fluorophores are connected by a mechanosensitive linker peptide that elongates in response to mechanical tension. Peptide extension leads to a decrease in FRET efficiency, which can be quantified with advanced microscopy methods such as fluorescence lifetime microscopy (FLIM). To allow quantitative measurements, we calibrated our probes using single-molecule force spectroscopy to determine the sensors’ force thresholds, their folding characteristics, and loading-rate sensitivities. In total, we generated four different tension sensor modules with force sensitivities at 1-6 piconewton (pN), 3-5 pN, 6-8 pN and 9-11 pN. By genetically inserting these individual biosensors into proteins of interest, mechanical tension across the respective target protein can be quantified. We applied our methods to study molecular forces across cell adhesion proteins like vinculin, talin and desmoplakin. Independent research labs adapted our method to investigate processes of force transduction in other subcellular structures like the cortical actin network, cell-cell junctions, and the glycocalxy. In summary, our technique can be utilized to investigate subcellular mechanical processes in living cells with piconewton sensitivity and in a spatio-temporally resolved manner.
- Kanoldt, V., Fischer, L., and Grashoff, C. (2019). Unforgettable force - crosstalk and memory of mechanosensitive structures. Biological chemistry 400, 687-698.
- Lemke, S.B., Weidemann, T., Cost, A.-L., Grashoff, C., and Schnorrer, F. (2019). A small proportion of Talin molecules transmit forces at developing muscle attachments in vivo. PLOS BIOLOGY 17.
- Price, A.J., Cost, A.-L., Ungewiß, H., Waschke, J., Dunn, A.R., and Grashoff, C. (2018). Mechanical loading of desmosomes depends on the magnitude and orientation of external stress. Nature Communications 9.
- Ringer, P., Weißl, A., Cost, A.-L., Freikamp, A., Sabass, B., Mehlich, A., Tramier, M., Rief, M., and Grashoff, C. (2017). Multiplexing molecular tension sensors reveals piconewton force gradient across talin-1. Nature methods 14, 1090-1096.
- Austen, K., Ringer, P., Mehlich, A., Chrostek-Grashoff, A., Kluger, C., Klingner, C., Sabass, B., Zent, R., Rief, M., and Grashoff, C. (2015). Extracellular rigidity sensing by talin isoform–specific mechanical linkages. Nature cell biology 17, 1597-1606.
- Cost, A.-L., Ringer, P., Chrostek-Grashoff, A., and Grashoff, C. (2014). How to Measure Molecular Forces in Cells: A Guide to Evaluating Genetically-Encoded FRET-Based Tension Sensors. Cellular and Molecular Bioengineering 8, 96-105.
- Austen, K., Kluger, C., Freikamp, A., Chrostek-Grashoff, A., and Grashoff, C. (2013). Generation and analysis of biosensors to measure mechanical forces within cells. Methods in molecular biology 1066, 169-184.
- Hoffman, B.D., Grashoff, C., and Schwartz, M.A. (2011). Dynamic molecular processes mediate cellular mechanotransduction. Nature 475, 316-323.
- Patla, I., Volberg, T., Elad, N., Hirschfeld-Warneken, V., Grashoff, C., Fässler, R., Spatz, J.P., Geiger, B., and Medalia, O. (2010). Dissecting the molecular architecture of integrin adhesion sites by cryo-electron tomography. Nature cell biology 12, 909-915.
- Grashoff, C., Hoffman, B.D., Brenner, M.D., Zhou, R., Parsons, M., Yang, M.T., McLean, M.A., Sligar, S.G., Chen, C.S., and Ha, T., et al. (2010). Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature 466, 263-266.