Overall, these results show that FRET signal measured by molecular tension probes is determined by a combination of sensor architecture and measurement approach. This work provides a controlled comparison and a reference for selecting & interpreting tension sensors in future mechanobiology studies.
We finally examined the effect of fluorophore orientation using circular permutation. Orientation changed the FRET readout in a strongly module-dependent manner, demonstrating that changes in signal cannot be interpreted solely on the basis of donor–acceptor distance.
At the sub-FA level, this translated into clear differences in spatial readout. Vinculin tension increased from proximal to distal regions of peripheral adhesions and exceeded ~10-15 pN, as revealed by CC-S2. Best spatial gradient, once again, resolved by binary-response sensors FL and CC-S2.
Largest differences emerged when comparing mechanical sensor modules. Among the six modules tested, the binary-response FL and CC-S2 showed the clearest separation between loaded and unloaded states and the broadest FRET distributions, indicating a higher capacity to resolve vinculin tension.
We then compared different donor–acceptor pairs within the same sensor architecture. Clover–mScarlet-I produced the highest unloaded FRET, whereas Clover–mRuby2 resulted in very low apparent FRET values and large spread in data, possibly linked to the inefficient maturation of mRuby2.
However, when the same constructs were analyzed using FLIM versus sensitized-emission FRET, the measured efficiencies shifted, and importantly, the magnitude of that shift depended on the construct. This shows that even the unloaded reference state is not universal.
We first compared multiple no-tension unloaded constructs, including tailless control, actin-binding mutant, C-terminal control and cytosol-localized module. All showed higher FRET than VinTS, confirming that the sensor reports force-induced changes across FAs.
To address this, we avoided cross-study comparisons and kept the sample preparation, imaging parameters and analysis pipeline identical, and varied only the sensor design. This allowed us to directly isolate how individual design parameters influence the measured FRET readout.
Part of the difficulty is that there is no single “standard” tension sensor. Different designs use different fluorophores, linkers, force regimes, sensitivities...Some respond gradually, others almost like a switch. Even imaging modality varies (intensity vs FLIM), and results are not comparable.
Despite the strong interest in this technology, much of the literature still comes from a small number of groups with long-standing expertise, raising an important question of how transferable across different labs this technique is, and how can we make it easier for those who want to implement it.
FRET-based tension sensors are one of the few approaches that allow to reveal pN-scale forces across specific proteins in living cells. They've been applied across many systems, but when you start comparing results across studies, numbers often do not align, even in similar biological conditions.
🔬New preprint out!
In this study, we systematically compare FRET-based tension sensors to understand how sensor design and measurement modality shape the FRET readout, and how this impacts the resolution of molecular tension measurements in cells.
Enjoy the read!
www.biorxiv.org/content/10.6...
Check out the latest development for DNA-PAINT technology, from none other than the leading Jungmann(s). Such an exciting field!
Our latest work on directional DNA hairpin 🧬 unzipping and mechanical anisotropy is out! Same sequence, different mechanical properties! By sequence design we can turn a classic two state folder into a soft, compliant molecule 🧸 that visit a stable transient ensemble! Have fun with the box!📦
Mark your calendars! Mechanobiology event of the year is around the corner. Can’t wait to return to beautiful Barcelona and reconnect with my old colleagues from IBEC.
Garcia Lab did it again! Such a fantastic read if you are interested in FAs mechanics and biology. Congratulations to @garcialabgt.bsky.social
Still buzzing after the @cellmech2025.bsky.social conference in Leuven!
It was great to connect with so many experts in mechanobiology and to be part of this close, dynamic community. We enjoyed the inspiring talks and left with plenty of new ideas.💡
Already looking forward to the next edition!
Interested in how cells respond when squeezed? Check the review paper just published by Laura Faure, @valeriaventurini.bsky.social and myself on this. Really thorough and clear revision by Laura and Valeria!
Always an honor to hear @johannaivaska.bsky.social share her insights, this time on how composite ECM components influence cell behavior, and how fibronectin-dense regions affect (or not) YAP/Oct-1 localization. An inspiring talk from an inspiring scientist. @cellmech2025.bsky.social.
Great experience presenting my work at @cellmech2025.bsky.social, where I shared how TFM and FRET sensors reveal the relationship between cellular tractions and focal adhesion tension. Sacrificed the food and drinks, but the (rather intense) discussions made up for it 💪🏻
Exciting week ahead! @cellmech2025.bsky.social is looking truly incredible and we are happy to be here as @rochalab.bsky.social. Let’s share some quality science! 🔬
How do #stemcells integrate information to coordinate fate decisions? Delighted to finally see our work showing how growth factors regulate the mechano-osmotic state of the #nucleus and #chromatin to control #pluripotency exit out! www.nature.com/articles/s41... see 🧵 👇
Very happy to see our preprint on how the mechanosensitive ion channel Piezo1 controls intercellular junction maturation by balancing cortex and membrane tension now published in @jcellsci.bsky.social Congratulations to Ahsan & team! journals.biologists.com/jcs/article/...
Huge respect for this technically wild study from the Oakes lab👏
Combining laser ablation, TFM, vinculin tension probes, optogenetics and FRAP, they show that the recruitment of zyxin & paxillin, but not of vinculin and its tension, correlate with local tension changes. Interesting and challenging!
How to turn a layer of fibroblasts into a tulip 🌷?
Check out our new pre-print on shape-programmable living surfaces.
Led by @pauguillamat.bsky.social
www.biorxiv.org/content/10.1...
Thanks Isaac, it’s very nice of you! It’s almost there :))
Congratulations! Such a clever and simple approach. Would definitely like to give it a try!
One major drawback of DNA force sensors is their super short lifetime—making them rather unsuitable for most bio studies. Huge congrats (and thanks) to isaacli.bsky.social’s team for solving one more problem for the field! Looks like it’s time to image some integrin forces 😎
Can we break past the usual limits of #SuperResolution #Microscopy—achieving deep-imaging, high-res, large FOV imaging without the trade-offs?
🧬🔬 YES: with #DNA_PAINT + Spinning Disk Confocal + Optical Photon Reassignment (#SDC-OPR)!
📄 www.nature.com/articles/s41... @natcomms.nature.com!
🧵👇
