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.
- Lemke SB, Weidemann T, Cost AL, Grashoff C, & Schnorrer F. A small proportion of Talin molecules transmit forces at developing muscle attachments in vivo. PLoS Biology Mar 27;17(3) (2019).
- Price AJ, Cost AL, Ungewiβ H, Waschke J, Dunn AR, & Grashoff C. Mechanical loading of desmosomes depends on the magnitude and orientation of external stress. Nat Commun 11; 9:5284 (2018).
- Freikamp, A., Mehlich, A., Klingner, C. & Grashoff, C. Investigating piconewton forces in cells by FRET-based molecular force microscopy. J Struct Biol 197, 37-42 (2017).
- Ringer, P., Weiβl, A., Cost, A.L., Freikamp, A., Sabass, B., Mehlich, A., Tramier, M., Rief, M. & Grashoff, C. Multiplexing molecular tension sensors reveals piconewton force gradient across talin-1. Nat Methods 14, 1090-1096 (2017).
- Freikamp, A., Cost, A.L. & Grashoff, C. The Piconewton Force Awakens: Quantifying Mechanics in Cells. Trends Cell Biol 26, 838-847 (2016).
- Austen, K., Ringer, P., Mehlich, A., Chrostek-Grashoff, A., Kluger, C., Klingner, C., Sabass, B., Zent, R., Rief, M. & Grashoff, C. Extracellular rigidity sensing by talin isoform-specific mechanical linkages. Nat Cell Biol 17, 1597-1606 (2015).
- Cost, A.L., Ringer, P., Chrostek-Grashoff, A. & Grashoff, C. How to Measure Molecular Forces in Cells: A Guide to Evaluating Genetically-Encoded FRET-Based Tension Sensors. Cell Mol Bioeng 8, 96-105 (2015).
- Austen, K., Kluger, C., Freikamp, A., Chrostek-Grashoff, A. & Grashoff, C. Generation and analysis of biosensors to measure mechanical forces within cells. Methods Mol Biol 1066, 169-184 (2013).
- Hoffman, B.D., Grashoff, C. & Schwartz, M.A. Dynamic molecular processes mediate cellular mechanotransduction. Nature 475, 316-323 (2011).
- Grashoff, C., Hoffman, B.D., Brenner, M.D., Zhou, R., Parsons, M., Yang, M.T., McLean, M.A., Sligar, S.G., Chen, C.S., Ha, T. & Schwartz, M.A. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature 466, 263-266 (2010).