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A reversible shearing DNA probe for visualizing mechanically strong receptors in living cells

Nature Cell Biology volume 23, pages 642–651 (2021)Cite this article

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Abstract

In the last decade, DNA-based tension sensors have made significant contributions to the study of the importance of mechanical forces in many biological systems. Albeit successful, one shortcoming of these techniques is their inability to reversibly measure receptor forces in a higher regime (that is, >20 pN), which limits our understanding of the molecular details of mechanochemical transduction in living cells. Here, we developed a reversible shearing DNA-based tension probe (RSDTP) for probing molecular piconewton-scale forces between 4 and 60 pN transmitted by cells. Using these probes, we can easily distinguish the differences in force-bearing integrins without perturbing adhesion biology and reveal that a strong force-bearing integrin cluster can serve as a ‘mechanical pivot’ to maintain focal adhesion architecture and facilitate its maturation. The benefits of the RSDTP include a high dynamic range, reversibility and single-molecule sensitivity, all of which will facilitate a better understanding of the molecular mechanisms of mechanobiology.

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Fig. 1: Design and characterization of the RSDTPs.
Fig. 2: Revealing the magnitude and spatial dynamics of integrin forces in living cells with RSDTPs.
Fig. 3: Imaging of different levels of integrin force in real time with multiplexed RSDTPs.
Fig. 4: Mechanically strong integrins maintain the FA architecture.
Fig. 5: Drug treatment shows that the activity of myosin II dominates strong integrin forces.
Fig. 6: Characterization of actin-polymerization-driven integrin forces.

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Data availability

The raw image data generated and analysed that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

The behaviour of FAs in cells was analysed using the Focal Adhesion Analysis Server (FAAS): a web tool for analysing FA dynamics (https://faas.bme.unc.edu).

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (21775115, 32071305, 31670760, 11704286 and 11674403), the start-up funding from Wuhan University for financial support, and the Fundamental Research Funds for the Central Universities (2042018kf02). We thank V. Ma for comments on the manuscript and for helpful discussion.

Author information

Authors and Affiliations

  1. The Institute for Advanced Studies, Wuhan University, Wuhan, China

    Hongyun Li, Yuru Hu, Pengxiang Liu, Feng Sun, Wei Chen, Xinghua Zhang, Wenxu Wang, Liang Wang, Piyu Wu & Zheng Liu

  2. College of Life Sciences, State Key Laboratory of Virology, Wuhan University, Wuhan, China

    Chen Zhang & Xinghua Zhang

  3. School of Physics, Sun Yat-sen University, Guangzhou, China

    Jie Ma

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Contributions

Z.L. and H.L. conceived and initiated the project. H.L. designed and performed all the experiments with the help of Y.H., F.S., P.L., W.C., J.M., W.W., L.W. and P.W. C.Z and X.Z. performed the SMMT experiments. Z.L. supervised the project and wrote the manuscript.

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Correspondence to Zheng Liu.

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Peer review information Nature Cell Biology thanks Khalid Salaita and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Synthesis scheme and characterizations of RSDTPs.

a, The structures and oligonucleotide sequences of 17-pN, 45-pN and 56-pN RSDTPs. b, Detailed chemical schemes for the synthesis of RSDTPs. c, Representative HPLC traces of DNA strand I’ modified with a Cy3B dye (denoted by dashed rectangle frame.) (d) Analysis of assay products by 10% denaturing polyacrylamide gel electrophoresis, lane 1: strand I’-Cy3B; lane 2: strand I’-Cy3B-c(RGDfK); lane 3: extra-long single-stranded DNA (elssDNA, highlighted by a black arrow). Note that, the weaker band above is a complex formed by a small amount of elssDNA and the template strand. The image is the representative of at least three independent biological replicates. e, Representative electrospray ionization mass spectrums of elssDNA.

Source data

Extended Data Fig. 2 Schematic diagrams of the composition of the hairpin tension probes for calibration and experimental method of single molecule magnetic tweezer experiments.

a, The geometries and compositions of the force probes used for calibration. All probes, combined with a complementary strand (blue) at their 3′end, were ligated to the biotin modified dsDNA handle. For the 56-pN probe, the sequence highlighted by a rectangular is only used to extend the entire length of probe without significantly affecting the mechanical properties. b, Schematic depiction of the single-molecule magnetic tweezer experiments. The probes are immobilized to the streptavidin-coated glass surface through their DNA handles. To unfold the probe, pulling forces were applied to a streptavidin-coated magnetic bead by permanent magnets. c, Mechanical stability measurements for 56-pN reversible shearing DNA probe. The results show that the 56-pN probe does not unfold even if probe was exposed to a lower level of forces (30–44 pN) for more than 10 minutes, and the unfolding event is only observed when applied force increases above 50 pN and lasted for more than ten seconds.

Extended Data Fig. 3 Characterization of AuNPs and RSDTPs functionalized surfaces preparation.

a-b, Representative TEM images of AuNPs (a) and corresponding size distribution profiles (b). c, UV-VIS spectra of 5 nm AuNPs. d, Stepwise procedures for preparing RSDTPs functionalized surfaces (see online methods). e, The representative AFM image showing the spatial distribution of the AuNPs immobilized on a glass coverslip.

Source data

Extended Data Fig. 4 Determination of DNA probe density on Au nanoparticles surface.

To calibrate the surface density of the DNA tension sensor, we used the method described by Wang and Ha et al.23 to plot the relationship between the fluorescence grayscale units of the sample (mean intensity) and the DNA probe density. First, we incubated aqueous solutions containing 25 nM, 12.5 nM, and 6.25 nM Cy3B-unfolded DNA probe (a scrambled DNA hairpin probe annealing with a complementary strand, the structure was shown in a) on the AuNPs-modified coverslips for 30 minutes, then washed off the unbinding probes and measured the average fluorescence intensity of glass as a function of the incubation concertation of the Cy3B-unfolded DNA probe (b). There is a linear relationship between the mean fluorescence intensity of the surface and the DNA incubation concentration within this concentration range (c). By extrapolation, incubation of 0.05 nM Cy3B-unfolded DNA probe on the surface should correspond to mean fluorescence intensity of 14.4. we also found a molecular density of 0.149 molecules/μm2 on the surface (0.05 nM) using a single-molecule TIRF image (b). Taken together, 96.6 units in mean fluorescence intensity on the Cy3B-unfolded DNA probe surface is equivalent to 1 molecule/ μm2. Given that the average QE of the Cy3B-folded DNA probe is 98.3% (Fig. 1 in main text), the 1.6 units mean fluorescence intensity on the RSDTP-Cy3B coated surface should be equivalent to 1 molecule/ μm2. We could also plot a calibration curve between intensity and molecular density for RSDTP (Cy3B) coated surface, as shown in d. This calibration curve allows us to determine RSDTP (Cy3B) surface density by measuring the average intensity of a coverslip. For example, the 45-pN RSDTP coated surface’s molecular density was estimated to be 671 molecules/μm2 (e).

Source data

Extended Data Fig. 5 Determination of fluorescence quenching efficiency (QE) of RSDTPs on a coverslip.

a, Estimate the distance between the AuNP surface and fluorophores on mechanically unfolding RSDTPs with different rupture forces by assuming a contour length of 0.44 nm per nt. b, Plots of the quenching efficiency as a function of distance from fluorophore to AuNPs with diameters of 5 nm and 10 nm based on the NSET model (Supplementary Note 2). The quenching efficiency (QE)-distance simulation results suggested that the effect of 5 nm AuNP on the Cy3B fluorophore beyond a distance of 20 nm is negligible. c, Schematic diagram of measuring the fluorescence quenching efficiency of RSDTPs on a solid surface. To mimic the folded and unfolded states of different RSDTPs, we hybridize complementary DNA strands (preheated to 95 °C) with different length to a scrambled hairpin probe (sequences shown in e). The QE values are calculated by measuring the fluorescence intensities before and after the addition of complementary strands. d, An example of QE measurement. The QE value of 17-pN RSDTP was calculated as 97.3% by measuring the readout noise of EMCCD, surface fluorescence intensities before and after addition of complementary strands. e, Table listing the sequences of the DNA used in this assay.

Extended Data Fig. 6 Control experiments confirmed the reliability and reversibility of the tension probes.

a, Comparison of DIC images for cells plated on sensors with and without RGD peptide. No cell attachment was found on surface coated with sensors lacking RGD peptide, suggesting that tension signal is specially generated through RGD-integrin interactions. b, Representative DIC images of NIH 3T3 cells seeded on tension probes and fibronectin-coated coverslips at different time points. c, A 45-pN RSDTP that lacks the quencher oligonucleotide was used to further validate the stability and reversibility of tension probes. The images show that the fluorescent background of the surface that lacks the quencher oligonucleotide is three times higher than a surface with the quencher strand DNA. Despite the high fluorescence background of these surfaces, we can still detect a tension signal during cell spreading on these surfaces, because Au NP plays a second quencher here. After treating cells with Cyto D, we found that the tension signals disappeared immediately and didn’t leave any dark features, which are often used to determine the stability of tension probes on the surface18. The fluorescence profiles along the yellow lines in the images are also shown in the right panel.

Extended Data Fig. 7 Further confirmation of the force gradient within FA and the relationship between FA longevity and mechanically hotspots.

a, Representative 17-pN (Atto647N), 56-pN (Cy3B) and the overlay tension images for a single NIH/3T3 cells chosen from 2 independent replicates spreading on a coverslip encoded with multiplexed RSDTPs. b, Representative 56-pN tension image along with GFP-paxillin of a 3T3 cell chosen from 4 independent replicates. Contour of GFP-paxillin was overlaid with tension signal. c, Zoomed-in time-lapse images from yellow-boxed region in b showing adhesion structures bearing weak forces undergo a rapid disassembly process. d, Longevity of weak and strong adhesions. The box represents 25th and 75th percentiles, the median is denoted by the middle horizontal line with mean value is labeled above each box and whiskers represent 1.5-fold the interquartile range. n = 49 for weak adhesions (structures without 56-pN signal) and n = 34 for strong adhesions (structures contain 56-pN signal) in the cell from b. Two-tailed, Student’s t-test was used to measure statistical significance, and P-values are shown on the graph.

Source data

Extended Data Fig. 8 Testing the reliability of photocleavable RSDTPs.

a, Representative RICM and tension images before and after UV illumination (time=30 s, UV-light is operating with a periodic illumination, power density = 1.3 × 10-4 μW/μm2) for cells seeding on different photocleavable RSDTPs coated surfaces. After cleavage of the loop of probes, the cell exhibited a rapid contraction on 17-pN photocleavable RSDTP and slow contraction on 56-pN photocleavable RSDTP, suggested that the RSDTP have been switched to a TGT-like probe. The images were representative of at least 2 independent replicates. b, FA dynamics of GFP-Paxillin expressing 3T3 cells seeded on 56-pN RSDTP, 56-pN TGT and 56-pN photocleavable RSDTP coated surface in response to UV illumination. Same illumination condition as in a were used here. No significant effect on cells seeded on 56-pN RSDTPs and 56-pN TGT probe coated surfaces were observed. The images were representative of at least 3 independent replicates.

Extended Data Fig. 9 Images of myosin-independent integrin force.

a, Tension images of blebbistatin pre-treated cells adhering to RSDTPs with different rupture forces. NIH 3T3 cells were pretreated with 10 μM blebbistatin for 30 minutes and then seeded on the coverslips. The images were representative of 3 independent replicates. b, Representative 45-pN tension images of a Y-27632 pretreated NIH 3T3 cell chosen from 2 independent replicates before and after adding Cytochalasin D. c, Co-localization of the integrin tension signal with GFP-Paxillin and GFP-Actin in 3T3 cells treated with Y27632 on 45-pN RSDTP coated surface. Images of RICM (gray), GFP (green), tension (red) and overlay channels were shown. Line scan analysis plot of the region highlighted with dashed white arrows in the overlay images shows the co-localization of the tension with GFP-paxillin and GFP-actin. The images were representative of 3 independent replicates. d, Representative 33-pN tension images of a Y-27632 pretreated NIH 3T3 cell chosen from 3 independent biological replicates before and after adding 100 mM sucrose. The right panel shows the change of the thin edge tension signal before and after hypertonic treatment, n=9 cells.

Source data

Extended Data Fig. 10 Single-molecule subunit counting of the force-bearing integrins at thin edges of Y27632 pretreated NIH 3T3 cells.

a, Left, a representative tension image (45-pN RSDTP) of a single living cell treated with Y27632 showing punctate fluorescent points. Right, representative intensity traces, under continuous excitation sufficient to cause bleaching of the fluorophores, from four clusters denoted by yellow arrows in the tension image. Below, histogram and gaussian fit of fluorescence intensities for the bleaching steps in the traces (n = 39 steps from cell 1). b, Additional data were performed on two fixed cells, and similar results were obtained. (n = 38 steps for cell 2; n = 36 steps for cell 3).

Source data

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Supplementary Notes 1 and 2.

Reporting Summary

Supplementary Video 1

Time-lapse video showing RICM and tension imaging of NIH-3T3 cells spreading on the coverslips functionalized by RSDTPs with different rupture forces (top, 17 pN; middle, 45 pN; bottom, 56 pN). The left panel represents the RICM channel, the middle panel represents the integrin tension reported as fluorescent signals by the RSDTPs, and the right channel represents the overlay of RICM and tension channels. Images were acquired for a duration of 80 min at 5-min intervals.

Supplementary Video 2

Time-lapse video showing the dynamics of FAs (marked by GFP–paxillin) in NIH-3T3 cells spreading on 56-pN TGTs, 56-pN RSDTPs, mixed-probe surface (2% 56-pN RSDTPs and 98% 56-pN TGTs) or fibronectin-functionalized surfaces. Images were acquired for a duration of 60 min at 1-min intervals.

Supplementary Video 3

Time-lapse video showing the changes in FA dynamics and 56-pN tension signals before and after UV illumination when a NIH-3T3 cell expressing GFP–paxillin spread on a mixed-probe surface (2% 56-pN photocleavable RSDTPs and 98% 56-pN non-fluorescent TGTs). Images were acquired every 2 min, UV illumination condition: 365 nm, 0.25 Hz frequency, 1.3 × 10–4 μW μm–2.

Supplementary Video 4

Time-lapse video showing the dynamics of the 17-pN (Cy3B) and 56-pN (Atto647N) tension signals along with the cytoskeleton during the recovery of myosin activity. NIH-3T3 cells were pretreated with 20 μM Y-27632 before seeding on a multiplexed tension probe surface. Y-27632 was removed by washing with culture medium without drugs twice, and video was recorded at t = 0 when the fresh medium was added. Images were acquired for a duration of 80 min at 5-min intervals.

Supplementary Video 5

Time-lapse video showing GFP–actin (inverted contrast) and tension map (red) of a Y-27632-pretreated NIH-3T3 cell spreading on a 45-pN RSDTP functionalized surface.

Supplementary Tables

Supplementary Tables 1 and 2.

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Li, H., Zhang, C., Hu, Y. et al. A reversible shearing DNA probe for visualizing mechanically strong receptors in living cells. Nat Cell Biol 23, 642–651 (2021). https://doi.org/10.1038/s41556-021-00691-0

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