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Tension-tuned receptors for synthetic mechanotransduction and intercellular force detection

Abstract

Cells interpret mechanical stimuli from their environments and neighbors, but the ability to engineer customized mechanosensing capabilities has remained a synthetic and mechanobiology challenge. Here we introduce tension-tuned synthetic Notch (SynNotch) receptors to convert extracellular and intercellular forces into specifiable gene expression changes. By elevating the tension requirements of SynNotch activation, in combination with structure-guided mutagenesis, we designed a set of receptors with mechanical sensitivities spanning the physiologically relevant picoNewton range. Cells expressing these receptors can distinguish between varying tensile forces and respond by enacting customizable transcriptional programs. We applied these tools to design a decision-making circuit, through which fibroblasts differentiate into myoblasts upon stimulation with distinct tension magnitudes. We also characterize cell-generated forces transmitted between cells during Notch signaling. Overall, this work provides insight into how mechanically induced changes in protein structure can be used to transduce physical forces into biochemical signals. The system should facilitate the further programming and dissection of force-related phenomena in biological systems.

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Fig. 1: Design of customized mechanosensation.
Fig. 2: Tuning the tensile strength of sNRR domains.
Fig. 3: Band-pass filtering in mechanogenetic circuits.
Fig. 4: Intercellular mechanotransduction via cell-generated, ubiquitination-dependent endocytic forces.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

D.C.S. and A.M.M. were supported through Graduate Research Fellowship awards from the National Science Foundation. J.C.T was supported through a Cross-Disciplinary Fellowship awarded through BUnano (Boston University Nanotechnology Innovation Center). D.C.S and J.C.T. received support through the Boston University training program in Quantitative Biology and Physiology (QBP, NIH grant T32GM008764). D.C.S. was a recipient of a Kilachand Fellowship through the Multicellular Design Program (Boston University). Support for this work was provided through a seed grant from the Center for Multiscale & Translational Mechanobiology (Boston University) and through NIH research grants R35 GM128859 (to J.T.N.) and R01 HL147585 (to J.T.N.). Additional support was provided through the Reidy Family Career Development Professorship at Boston University (to J.T.N.). The schematics in Fig. 4 were created using biorender.com.

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D.C.S, J.C.T., A.M.M. and J.T.N. designed and performed experiments and analyzed the data. D.C.S. and J.T.N. wrote the manuscript. All authors edited the manuscript.

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Correspondence to John T. Ngo.

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J.T.N. and D.C.S. are inventors on an issued patent (U.S. Patent, 10,858,443) held by the Trustees of Boston University. J.C.T. and A.M.M. declare no competing interests.

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Supplementary Video 1

Time-lapse imaging of biotin-FITC induced trans-cellular activation. HEK293-FT reporter cells (UAS-H2B-mCherry) expressing anti-FITC receptors with the indicated mechanosensitive domains were cocultured with HEK293-FT antibiotin-TMD-DLL1 sender cells. trans-cellular coupling was induced by treatment with 2 nM biotin-FITC. Cells were imaged live for 24 h. Fluorescence emissions from T2A-BFP (blue, to identify receiver cells) and H2B-mCherry (red, to monitor reporter activity) were captured every 1.5 h. Sender cells were not fluorescently marked (that is, lacked BFP). Top row: merged images from detection of T2A-BFP (blue), H2B-mCherry (red) and transmitted light. Bottom row: emission from H2B-mCherry detection shown in grayscale.

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Sloas, D.C., Tran, J.C., Marzilli, A.M. et al. Tension-tuned receptors for synthetic mechanotransduction and intercellular force detection. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-022-01638-y

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