Abstract
Extracellular matrix (ECM) rigidity serves as a crucial mechanical cue impacting diverse biological processes. However, understanding the molecular mechanisms of rigidity sensing has been limited by the spatial resolution and force sensitivity of current cellular force measurement techniques. Here we developed a method to functionalize DNA tension probes on soft hydrogel surfaces in a controllable and reliable manner, enabling molecular tension fluorescence microscopy for rigidity sensing studies. Our findings showed that fibroblasts respond to substrate rigidity by recruiting more force-bearing integrins and modulating integrin sampling frequency of the ECM, rather than simply overloading the existing integrin–ligand bonds, to promote focal adhesion maturation. We also demonstrated that ECM rigidity positively regulates the pN force of T cell receptor–ligand bond and T cell receptor mechanical sampling frequency, promoting T cell activation. Thus, hydrogel-based molecular tension fluorescence microscopy implemented on a standard confocal microscope provides a simple and effective means to explore detailed molecular force information for rigidity-dependent biological processes.
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All imaging data have been uploaded on Figshare (https://doi.org/10.6084/m9.figshare.24008127). Source data are provided with this paper.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (32150016, 21775115, 32071305 and 31871356), the Fundamental Research Funds for the Central Universities (2042021kf0030) and Innovation Funds for Postdocs in Hubei Province.
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Z.L. conceived the project and supervised the research. W.W. and W.C., with the help of P.L., H.L., J.F. and F.S., designed and performed the hydrogel-based mTFM and TFM coupling experiments and analyzed the data. W.W., C.W. and Y.H. designed and performed the T cell and podosome experiments. C.Z. and X.Z. performed single-molecule magnetic tweezer experiments, and K.J. provided intellectual input for the cell knockout experiments. Z.L., W.W. and W.C. wrote the paper with the help of others.
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Nature Methods thanks Sanjeevi Sivasankar, Tejeshwar Rao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Madhura Mukhopadhyay, in collaboration with the Nature Methods team. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Synthesis schemes of molecular tension probes.
a, RSDTPs, b, TGTs and c, Hairpins.
Extended Data Fig. 2 Development of PA hydrogel substrates functionalized with both TFM and mTFM.
a, Immobilization of fluorescent nanobeads on the surfaces of prepared hydrogels for TFM. b, Introduction of lipo-acid groups into hydrogel network. c, Covalent attachment of DNA tension probes to hydrogel surfaces through Au NPs. d, Recipes of PA hydrogels with different stiffness (n = 3 samples) and, e, corresponding results of stiffness characterization using AFM (16 × 16 stiffness map within an 80 µm × 80 µm region of interest). The images are representative of three independent experimental results.
Extended Data Fig. 3 Calibration of RSDTPs attached to handles with different stiffness using magnetic tweezer.
a, Design and sequences of handles with different stiffness. b, Illustration of calibration setup using magnetic tweezer. c, Representative force-displacement calibration curves of 17 pN (left), 45 pN (middle), and 56 pN (right) RSDTPs. d, Distribution of unfolding forces of RSDTPs attached to handles with different stiffness (400 bp, n = 51, 46, 64 events for different RSDTPs. 4000 bp, n = 45, 121, 191 events for different RSDTPs. 13571 nt, n = 39, 45, 30 events for different RSDTPs).
Extended Data Fig. 4 Effects of CytoD on the mechanics of MEFs.
a, Representative timelapse confocal imaging of paxillin-GFP, 17pN-RSDTPs, TFM of cells spreading on 30 kPa PA hydrogel before and after treatment of cytochalasin D (20 μM), and corresponding plots of b, total intensity of mTFM and c, total traction forces measured by TFM (Control, n = 44 cells. CytoD, n = 41 cells). Data represent mean ± s.d. of three experiments. Two-tailed Student t-test is used to assess statistical significance.
Extended Data Fig. 5 The effect of gel deformation-induced aggregation of DNA tension probes on the validity of reported signals.
a, Representative paxillin-GFP, mTFM and bright field images of cells incubated on 3 kPa PA hydrogel coated with a control RSDTP that was hardly unfolded and normal 50 pN RSDTP, and corresponding quantification of b, spreading area and c, integrin tensions signals (n = 41, 20 cells for each condition). Data represent mean± s.d. of three experiments. Two-tailed Student’s t-test is used to assess statistical significance.
Extended Data Fig. 6 Characterization of the integrin tension distribution of multiple cell lines on soft (3 kPa) and hard (30 kPa) hydrogels.
Representative fluorescent images of paxillin-tagged a, C2C12, d, U2OS, g, U251, j, CHO, and m, HeLa cells on soft and hard hydrogel, and corresponding mTFM, simultaneously reported by mixed 17 pN-RSDTPs and 50 pN-RSDTPs at 37 °C (left), or 55 pN TGTs at room temperature 22 °C (right). The images are representative of three independent experimental results. FA area and the ratio of strongly loaded integrin to total loaded integrins of b, c, C2C12 (n = 25, 28 cells for each stiffness), e, f, U2OS (n = 31, 31 cells for each stiffness), h, i, U251 (n = 25, 34 cells for each stiffness), k, l, CHO (n = 18, 23 cells for each stiffness), and n, o, HeLa cells (n = 13, 28 cells for each stiffness) on soft and hard hydrogel. Data represent mean± s.d. of three independent experiments. Two-tailed Student’s t-test is used to assess statistical significance.
Extended Data Fig. 7 Effect of temperature on mechanical stability of RSDTPs and associated results of hydrogel-based mTFM.
a, Representative fluorescent images of paxillin-tagged MEFs on hydrogels (30 kPa) at 37 °C and room temperature (22 °C), and corresponding mTFM simultaneously reported by mixed 17 pN-RSDTPs and 50 pN-RSDTPs. Total intensity of mTFM per cell reported by b, 17 pN-RSDTPs, c, 50 pN-RSDTPs and d, their ratio (n = 56 cells). Data represent mean± s.d. of three independent experiments. Two-tailed Student’s t-test is used to assess statistical significance. The insensitivity of force ratio to the ambient temperature indicates that even after mechanical stability of RSDTPs was diminished by the increase of temperature from room temperature (22 °C) to 37 °C, the absolute mechanical stability difference between 17 pN RSDTP and 50 pN RSDTP remains significant enough to characterize the distribution of integrin tension.
Extended Data Fig. 8 The effect of substrate stiffness on integrin tension distribution of Vcl-KO MEF cells.
Representative eGFP-paxillin and tension maps of different RSDTPs of Vcl-KO MEF cells on a, 3 kPa hydrogel, b, 30 kPa hydrogel, imaged using confocal microscope, and c, glass substrates imaged using TIRF. The images are representative of three independent experimental results. d, Western blot results of constructed Vcl-KO MEF cells. The gel images are representative of three independent experimental results.
Extended Data Fig. 9 The reliability of locking strands in tracing molecular tension.
a. Sequences of 17pN-RSDTPs, paired locking strand, and unpaired locking strands. b, Representative paxillin-GFP and mTFM images of MEF cells plated on PA hydrogel with 17pN-RSDTPs in the absence of locking strands, after the addition of unpaired locking strands, and after addition of paired locking strands and c, corresponding plots of normalized accumulative intensity as a function of time (n = 6, 11, 9 cells for each condition). The images are representative of three independent experimental results. Data represent mean± s.d. of three independent experiments.
Extended Data Fig. 10 Efficiency of locking strands in memorizing mTFM.
a, Schematic diagram illustrating the flow of experiments. b, Representative Paxillin and mTFM images of MEFs seeded on 3 kPa and 30 kPa hydrogels functionalized with 50 pN RSDTPs before the addition of locking strands (left), after the addition of locking strands (middle), and after the treatment of Cytochalasin D (right), and corresponding c, plots of normalized accumulative intensity as a function of time (n = 6, 7 cells for each stiffness). The images are representative of three independent experimental results. Data represent mean± s.d. of three independent experiments. d, Total intensity of locked mTFM before and after the addition of CytoD and e, their ratios on 3 kPa and 30 kPa hydrogels (n = 6, 7 cells for each stiffness). Data represent mean± s.d. of three independent experiments. Two-tailed Student’s t-test is used to assess statistical significance.
Supplementary information
Supplementary Information
Supplementary Figs. 1–8.
Supplementary Video 1
Representative time-lapse confocal imaging of eGFP–paxillin, TFM, mTFM and bright field performed for MEF cells on 30 kPa hydrogels after the treatment of CytoD (20 µM). Images were captured for a duration of 14 min at 15 s intervals.
Supplementary Video 2
Representative time-lapse confocal imaging of eGFP–paxillin, TFM, mTFM and bright field conducted for MEF cells spreading and trypsinized on 30 kPa hydrogels. Images were acquired for a duration of 8 h 50 min at 10 min intervals.
Supplementary Video 3
Representative time-lapse confocal imaging of eGFP–LifeAct, TFM, mTFM and bright field of THP-1 cells on 30 kPa hydrogels. Images were acquired for a duration of 30 min at 30 s intervals.
Supplementary Video 4
Representative time-lapse confocal imaging of bright field and mTFM of naïve T cells seeded on 1 kPa hydrogels before and after the addition of locking strands. Images were acquired for a duration of 22 min at 60 s intervals.
Supplementary Video 5
Representative time-lapse confocal imaging of bright field and mTFM of naïve T cells seeded on 30 kPa hydrogels before and after the addition of locking strands. Images were acquired for a duration of 22 min at 60 s intervals.
Supplementary Data 1
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Wang, W., Chen, W., Wu, C. et al. Hydrogel-based molecular tension fluorescence microscopy for investigating receptor-mediated rigidity sensing. Nat Methods 20, 1780–1789 (2023). https://doi.org/10.1038/s41592-023-02037-0
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DOI: https://doi.org/10.1038/s41592-023-02037-0
