ABSTRACT
Molecular tension sensors measure piconewton forces experienced by individual proteins in the context of the cellular microenvironment. Current genetically-encoded tension sensors use FRET to report on extension of an elastic peptide encoded in a cellular protein of interest. Here we present the development and characterization of a new type of molecular tension sensor based on bioluminescence resonance energy transfer (BRET) which exhibits more desirable spectral properties and an enhanced dynamic range compared to other molecular tension sensors. Moreover, it avoids many disadvantages of FRET measurements in cells, including heating of the sample, autofluorescence, photobleaching, and corrections of direct acceptor excitation. We benchmark the sensor by inserting it into the canonical mechanosensing focal adhesion protein vinculin, observing highly resolved gradients of tensional changes across focal adhesions. We anticipate that the BRET-TS will expand the toolkit available to study mechanotransduction at a molecular level and allow potential extension to an in vivo context.
INTRODUCTION
The mechanical microenvironment of a cell guides cellular processes such as migration, differentiation, division, and signaling1, and is thus often altered in disease2,3. At a molecular level, mechanical stimuli alter conformations of mechanosensing proteins to communicate the stimulus to the cell interior and effect a downstream cellular response in a process called mechanotransduction4,5. Measuring tensions sensed by cellular proteins and how tensions are altered by cellular context will lead to better understanding of the molecular mechanisms used in mechanosensing in disease-related processes such as tumor migration.
Tools to measure tensions at a cell and tissue level such as traction force microscopy and atomic force microscopy are well-established. Tools to measure molecular level tensions have only emerged more recently to characterize the pN forces present across mechanosensitive proteins6 and have been used to measure tensions sensed by focal adhesions proteins such as vinculin, talin, and integrins, as well as cadherins7–10. Generally, genetically encodable molecular tension sensors contain an elastic peptide linker with well-characterized extension in response to applied force flanked by two fluorescent proteins (FP) allowing for measurement of tension across a protein typically utilizing Förster resonance energy transfer (FRET). The extent of linker stretching due to mechanical force can measured by FRET and subsequently be correlated to a quantifiable force. Until recently, the state of the art genetically-encoded FRET tension was TSMod, a distance dependent FRET-based sensor that can measure tensions in the 1-6 pN range7. The dynamic range of TSMod, however, is limited by the photophysical properties of the fluorescent protein pair, mTFP1 and venus(A206K)11. Recent studies reported improved FRET tension sensors by using more optimal fluorescent proteins, utilizing different linkers, and engineering FP termini to achieve closer distances that yielded higher FRET under no load12,13. A computational model has also been described to allow researchers to optimize force range or sensitivity based on experimental needs12. Nonetheless, these sensors are still constrained by the limitations of FRET, namely autofluorescence, direct acceptor excitation, photobleaching, and incompatibility for in vivo use.
We developed a genetically-encodable molecular tension sensor based on bioluminescence resonance energy transfer (BRET) that overcomes several limitations of FRET. BRET is a distance and orientation dependent phenomenon analogous to FRET but is initiated by a chemiluminescent reaction of a luciferase protein with its substrate instead of light excitation14. Upon luciferase excitation, non-radiative energy transfer can occur to excite a closely linked acceptor fluorescent protein that produces a distinct emission spectrum. Our BRET tension sensor, which we will refer to as BRET-TS, takes advantage of the uncharacteristically bright Nanoluciferase (NanoLuc), allowing in vitro and live cell detection of signal using standard plate readers and microscopes. The luminescent-fluorescent protein pair of NanoLuc15 and mNeonGreen16 has recently been shown to exhibit robust resonance energy transfer efficiency17. Here we report on using these proteins as a sensitive BRET donor-acceptor pair in a molecular tension sensor. Using BRET as a distance reporter in molecular tension sensors allows for more sensitive readouts due to the lack of autofluorescence and phototoxicity. BRET also offers the potential for use in vivo where light excitation is not able to penetrate into tissue. We demonstrate the utility of this tension sensor in vitro and in cell based assays, using vinculin as the model system to show an enhanced dynamic range compared to field standard FRET-based sensors.
RESULTS AND DISCUSSION
Sensor design and characterization
In the BRET-based molecular tension sensor (BRET-TS), the NanoLuc donor and mNeonGreen acceptor proteins flank a 40 amino acid flexible spider silk flagelliform domain (GPGGA)8 (Figure 1A). Spider silk exhibits predictable changes in length under tension18. Both proteins are terminally truncated to enhance the base BRET efficiency by bringing them in closer proximity17. We first expressed recombinant BRET-TS and the commonly used FRET-TSMod to compare their spectral properties7.
In the absence of tension, the unloaded BRET sensor boasts a robust ~60% BRET efficiency upon addition of NanoLuc’s chemiluminescent substrate, furimazine (Figure 1B). Moreover, the emission maxima of NanoLuc (460 nm) and mNeonGreen (517 nm) are spectrally separated by 57 nm. In contrast, the unloaded TSMod FRET tension sensor exhibits only 25% energy transfer with extensive spectral overlap between mTFP and Venus (Figure 1C)7. Recent iterations of FRET tension sensors using Clover and Ruby derived FPs display improved spectral separation but still only achieve unloaded energy transfer efficiencies of 35%.12
A BRET efficiency of 60% approaches energy transfer efficiencies observed for unloaded spider silk peptides flanked by organic fluorophores Cy3 and Cy57,26 used to calibrate the sensors, despite the usually detrimental contribution of increased distance that FPs contribute. This is likely due to the fact that in BRET-TS, the mNeonGreen acceptor has a higher quantum efficiency than the NanoLuc donor17, a trend that is reversed in the mTFP1 / venus and clover / ruby FRET pairs. The enhanced BRET efficiency at zero force translates to a larger dynamic range, as the sensor has the potential to detect a 60% change in energy transfer when force is applied, making it easier to detect higher forces and resolve the entire range of forces. The large spectral separation of the donor and acceptor is also desirable for ratiometric imaging, where spectral filters are used to separate emission from donor and acceptor.
Upon application of tension to the elastic peptide, we expect the spider silk linker to stretch, separating the protein pair and causing a decrease in the resonance energy transfer (RET) efficiency. Thus, we next aimed to ensure that BRET-TS exhibits the expected changes in RET with distance between the donor and acceptor. We initially characterized the distance dependence of BRET by inserting a series of rigid alpha helical linkers (HL) A(EAAAK)nA (Figure 2A) of known lengths between mNeonGreen and NanoLuc in the context of recombinant proteins expressed in E. Coli19. Expectedly, as the distance between the protein pair increases, a decrease in BRET is observed when measured in bulk using a standard plate reader (Figure 2B).
We next encoded the series of alpha helical linkers sandwiched between mNeonGreen and NanoLuc into a cell surface expressed protein to characterize RET as a function of distance in cells. Constructs were transiently transfected into HEK293T or U2OS cells, and BRET read out on a plate reader on via ratiometric imaging. Remarkably, the cellular BRET was easily detectable on a plate reader, underscoring the brightness and low background of NanoLuc (Figure S1). To detect BRET localization in live cells, furimazine was added to the media and the ratio of emission of mNeonGreen to NanoLuc was measured simultaneously or sequentially using an EM-CCD camera outfitted with spectral filters specific to NanoLuc and mNeonGreen emissions (Figure 2C). The pixel-by-pixel ratio of the two images was then calculated. Emission from NanoLuc alone as well as emission from the donor and acceptor connected by a short dipeptide “GF” linker were also measured for reference. Again, a drastic decrease in BRET is observed with increasing linker length (Figure 2D). Heterogeneity in the BRET signal, likely reflecting a diversity in mechanical microenvironments sensed by the cell-surface BRET-TS, is also apparent in the cell experiments that is masked in the in vitro bulk experiments.
To ensure the BRET observed was due to intramolecular rather than intermolecular effects, we co-transfected cell surface receptors containing either NanoLuc or mNeonGreen alone. We did not observe any BRET signal, consistent with a lack of intermolecular BRET in this context (Figure S2A). Similar results were obtained when inserting a 3C proteolytic cleavage site in the recombinant protein version. Cleavage by HRV 3C protease abolished BRET resulting in only the expected NanoLuc emission (Figure S2B).
To determine if the ratios derived from in vitro and in cell linker experiments were similar, we plotted the BRET efficiency as a function of distance. We first calculated histograms of ratiometric images from Figure 2B to determine average NeonGreen to NanoLuc ratios. To convert BRET ratios to BRET efficiencies, we first corrected for the Nanoluc/NeonGreen emission overlap and calculated BRET efficiency using spectral deconvolution or the ratio of intensities (Supplemental Methods).
We next calculated the Förster radius (R0) of mNeonGreen-NanoLuc to be 51 Å based on the experimentally determined spectral overlap (J=2.84*1015 nm4 M−1 cm−1) and using NanoLuc’s previously reported quantum yield20. This is the highest reported Förster radius of any NanoLuc-FP pair21. Using this R0 and our experimental BRET efficiencies, we then calculated R values for the helical linkers using standard FRET equations and plotted them against BRET efficiency (Figure 2E). The curves fit well to a sigmoidal function. A similar curve is obtained using the R values previously calculated for these linkers19 (Figure S3). The BRET efficiencies measured in vitro and in cells were remarkably similar for a given linker, and we observed no significant difference between the image splitter and filter wheel methods of ratiometric imaging.
Measuring tension across vinculin
Finally, we wanted to validate BRET-TS in a known mechanosensing protein. We chose the focal adhesion protein vinculin that has previously been shown to experience tensions on the order of 1 to 6 pN using molecular tension sensors7. This system provides a good opportunity to benchmark the dynamic range of BRET-TS against state-of-the-art FRET tension sensors. We inserted the BRET-TS into a previously described site within the protein after residue 883 (VinTS). We also created a force-insensitive control construct lacking the carboxyl terminal F-actin binding tail (VinTL) (Figure 3A). As this tension sensor is 84 amino acids smaller than TSMod, we did not repeat previously reported functional characterization of vinculin with the inserted sensor7.
Remarkably, luciferase signal from VinTS was clearly observable in a bulk plate reader assay in comparison to zero signal in untransfected cells (Figure S4). In contrast, direct excitation of mNeonGreen at 450nm resulted in the same signal in both untransfected and transfected cells due to cellular autofluorescence. For imaging of VinTS in focal adhesions, constructs were transfected in U2OS cells and plated sparsely on fibronectin. The cells were subsequently imaged upon addition of furimazine. We observed a gradient in tension across peripheral focal adhesions22 in VinTS that was not observed in the force-insensitive mutant VinTL (Figure 3B-H). Tension gradients were not observed in the original FRET-TS TSMod due to insufficient dynamic range7, but were recently detected in improved FRET-TS’s12. BRET efficiency is lowest at the cell periphery, which corresponds to higher forces. BRET efficiency gradually increases (force decreases) moving towards the nucleus (Figure 3C). This gradient is abolished in the actin binding mutant lacking the carboxy-terminus of vinculin and in point mutants of the actin or talin binding domains (Figure 3H and S5)23. BRET-TS provides better resolution in highlighting the gradient across the focal adhesion compared to previous studies; we observe an average 31% change in BRET across peripheral focal adhesions, compared to 5% and 15% BRET gradients reported for TSMod and improved TSMods, respectively7,12. VinTS has already been used to dissect molecular mechanisms of collagen matrix formation in stem cells24 and measure the effect of osteocytes on migration potential of breast cancer cells25. The enhanced dynamic range of VinTS will allow further studies of focal adhesion tensions in cellular processes.
CONCLUSIONS
We developed a genetically-encodable BRET-based tension sensor that is smaller in size, offers better spectral separation, and a larger dynamic range for detecting pN forces over FRET based molecular tension sensors due to the desirable photophysical properties of the mNeonGreen-NanoLuc pair17. Moreover, BRET offers other key improvements over FRET-based sensors such as lack of photo toxicity, greatly simplified image processing and increased signal-to-noise due to the use of bioluminescence as the energy donor. Though the enhanced dynamic range and potential for in vivo imaging of BRET-TS will be advantageous for most applications of molecular tension sensors, BRET-TS would not be suitable for measuring tensions related to cellular processes that happen in <10 seconds due to the longer exposure times required. Moreover, BRET-TS is not compatible with optical sectioning strategies utilizing laser scanning, such as confocal microscopy, and would necessitate the use of computational optical sectioning.
Further optimization of the force regime can be accomplished through substitution of the flexible spider silk flagelliform linker with other recently described molecular spring domains13,26. Furthermore, we believe this sensor will be aptly suited for live animal in vivo measurement of molecular scale tensions. Finally, this BRET pair should be widely applicable to use in other biosensors.
Author Contributions
E.J.A., K.J.T. and W.R.G. designed experiments, collected data, and analyzed data. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Funding Sources
This research was supported by an NIH NIGMS R35 GM119483 grant, an UMN ACS IRG-118198, and pilot funds from NIH U54 CA210190 grant. E.J.A. received salary support from a Biotechnology Training Grant NIH T32GM008347 and 3M Graduate Fellowship. W.R.G. is a Pew Biomedical Scholar.
Supplemental sequences
ACKNOWLEDGMENT
We would like to thank the Physical Sciences of Oncology Center at UMN for resources and support. Thanks to Fluorescence Innovations as well as Thomas Pengo of the University Imaging Centers for support and helpful discussions. Thanks to Brenton Hoffman for helpful discussions. We would also like to thank Hideki Aihara and Nick Levinson for use of their plate readers.
ABBREVIATIONS
- BRET
- Bioluminescence resonance energy transfer
- FRET
- Förster resonance energy transfer
- FP
- fluorescent protein
- TS
- tension sensor
- RET
- resonance energy transfer
- HL
- helical linker
- FA
- focal adhesion
- RLU
- relative light units
- Nluc
- NanoLuc