(D-156) Screening the Mechanical Performance of Fluorescent Proteins in Living Cells

     Mechanical forces play an integral role in numerous cell-level physiological and pathophysiological events [1]. The process of mechanotransduction, or the conversion of mechanical forces into biochemical signals, allows cells to sense and respond to mechanical forces.  This occurs by specific proteins transmitting forces which induce deformations that alter protein function, such as the unfolding of a domain to expose a cryptic binding site [2]. The main tools for studying the molecular loads on proteins inside cells are Forster resonance energy transfer (FRET)-based molecular tension sensors (MTS). Made from two fluorescent proteins (FPs) linked by an extensible domain, tension-sensing modules are integrated into existing proteins to visualize the pN-level loads felt by those proteins in cells. Prior work has assumed that the optical properties of the FPs are independent of the load on the sensor. However, recent work has shown that FPs can undergo reversible mechanical switching, whereby repeated cycles of mechanical loading can turn off and on their fluorescence [3]. FP mechanical switching could confound results when using MTS. On the other hand, FP mechanical switching also offers the opportunity to create a new class of MTS to directly measure force-dependent changes in protein function in cells. To better design MTS to realize both goals, a palette of FPs with known mechanical properties is required. This work sets out to quantify the mechanostability of a range of commonly used FPs in cellular loading conditions to gain an understanding of the design space currently available to biosensor engineers.

       To do so, candidate FPs were screened in the context of a previously developed synthetic actin binding tension sensor (ABDTS) and an unloaded control sensor (ABDTL). By observing the changes in FP stoichiometry, which quantifies the relative number of acceptor and donor FPs,and FRET efficiency obtained from sensitized emission-based FRET measurements and analyzing the results in the context of a mathematical model of FP mechanical switching, we characterized the mechanical stability of a range of FPs inside cells for the first time.

        So far, we have screened two MTS containing three unique FPs: mTFP1-mVenus and mTFP1-YPet. In mTFP1-mVenus, we see a decrease in FRET efficiency and an increase in stoichiometry in ABDTS compared to the unloaded control ABDTL. This suggests that there is significant acceptor mechanical switching and that mTFP1 is more mechanically stable than mVenus. In mTFP1-YPet, there is little difference in FRET efficiency and stoichiometry between ABDTS and the ABDTL. This suggests there is very little mechanical switching in either the donor or acceptor FP and that YPet is more mechanically stable than mVenus and has similar stability to mTFP1.

This project has shown that in the context of cellular loads in the actin cytoskeleton, the mTFP1-YPet tension sensor module has less FP mechanical switching while retaining similar optical properties to the commonly used mTFP1-mVenus tension sensor module. Therefore, the mTFP1-YPet FP pair is suited for making an improved MTS to measure calibrated molecular tensions in the absence of FP mechanical switching. On the other hand, the acceptor mechanical switching present in the mTFP1-mVenus tension sensor module provides a means to directly measure force-dependent changes in protein function and thus could be leveraged to probe the process of mechanotransduction more directly. This marks the first characterization of FP mechanical properties and opens the door for new MTS designs to advance our understanding of mechanotransduction.

Future work should focus on expanding our current FP library to include other commonly used FP’s to both better understand the mechanical sensitivity of preexisting MTS and expand the toolset for protein engineers. More MTS should be made that cover a wider range of mechanical stability to increase the resolution with which we can characterize force-dependent protein function. Additionally, molecular simulation work can be used to not only predict FP stability, but it can also assist in future FP design to create FPs with custom mechanical stabilities. Finally, as new MTS are made, they can be incorporated into mechanosensitive proteins and their mechanical switching can be used to better understand how previously measured forces within these proteins actually impact protein deformations that alter protein function.

1. Wolfenson, H., Yang, B., & Sheetz, M. P. (2019). Steps in Mechanotransduction Pathways that Control Cell Morphology. Annual review of physiology, 81, 585–605. https://doi.org/10.1146/annurev-physiol-021317-121245


2. Hoffman, B., Grashoff, C. & Schwartz, M. Dynamic molecular processes mediate cellular mechanotransduction. Nature 475, 316–323 (2011). https://doi.org/10.1038/nature10316


3. Ganim, Z., & Rief, M. (2017). Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding. Proceedings of the National Academy of Sciences of the United States of America, 114(42), 11052–11056.  https://doi.org/10.1073/pnas.1704937114