Genetically encoded force sensors for measuring mechanical forces in proteins

Abstract

There are three sources of free energy for cells: chemical potential, electrical potential and mechanical potential. There is little known about the last one since there have not been simple ways to measure stress in proteins in cells. We have now developed genetically encoded force sensors to assess the stress in fibrous proteins in living cells. These FRET based fluorescence sensors can be read out at video rates and provide real time maps of the stress distribution in cells, tissues and animals. The sensors can be inserted into specific proteins and in general do not disturb the normal function or anatomy. The original sensors used mutant GFPs linked by elastic linkers. These sensors provide a linear output with applied stress but the response is linear in strain. To improve contrast and dynamic range we have now developed a new class of sensors that are smaller making them less invasive, and have much higher intrinsic sensitivity since force modulates the angle between the donor and acceptor much more than the distance between them. Known as cpstFRET, the probe shows improved biocompatibility, wider dynamic range and higher sensitivity. 

Acknowledgements

This work was supported by a grant from US National Institutes of Health and The Children's Fund of Buffalo.

Figures and Tables

Figure 1 Cartoon diagrams of genetically encoded force sensors where the letter A in the fluorophore represents the acceptor Venus, the letter D represents the donor Cerulean. The arrows represent energy transfer from donor to acceptor. (A) stFRET; (B) sstFRET. (C) TSMod, mTFP1 is the donor and Venus is the acceptor. (D) PriSSM, using wild-type GFP and circularly permutated GFP for PRIM dimers.

Figure 2 Diagrams of inserting the force probes into host proteins. (A) Actinin-stFRET forming anti-parallel dimers. (B) Spectrin-stFRET exists as a hetero-dimer with α and β subunits. (C) Vinculin-TSMod associating at focal adhesions as monomers. (D) A protein construct of PriSSM with myosin motors on the ends.

Figure 3 A new and smaller FRET force sensor (cpstFRET) with higher sensitivity and wider dynamic range. (A) shows applied force twisting the fluorophores out of alignment and lower FRET. (B) shows cpstFRET incorporated into a spectrin α subunit that is part of a heterodimer.