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Cell stretching is amplified by active actin remodelling to deform and recruit proteins in mechanosensitive structures

Nature Cell Biology volume 22, pages 1011–1023 (2020)Cite this article

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Abstract

Detection and conversion of mechanical forces into biochemical signals controls cell functions during physiological and pathological processes. Mechanosensing is based on protein deformations and reorganizations, yet the molecular mechanisms are still unclear. Using a cell-stretching device compatible with super-resolution microscopy and single-protein tracking, we explored the nanoscale deformations and reorganizations of individual proteins inside mechanosensitive structures. We achieved super-resolution microscopy after live stretching on intermediate filaments, microtubules and integrin adhesions. Simultaneous single-protein tracking and stretching showed that while integrins followed the elastic deformation of the substrate, actin filaments and talin also displayed lagged and transient inelastic responses associated with active acto-myosin remodelling and talin deformations. Capturing acute reorganizations of single molecules during stretching showed that force-dependent vinculin recruitment is delayed and depends on the maturation of integrin adhesions. Thus, cells respond to external forces by amplifying transiently and locally cytoskeleton displacements, enabling protein deformation and recruitment in mechanosensitive structures.

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Fig. 1: Combining cell stretching with SRM.
Fig. 2: Combining cell stretching with live SRM or SPT.
Fig. 3: The acute mechanical response of β3-integrins to external forces is elastic.
Fig. 4: Inelastic mechanical response of talin to external forces correspond to talin unfolding.
Fig. 5: Transient and active remodelling of the acto-myosin network drives the inelastic response of actin.
Fig. 6: Small-scale stretching triggers zyxin reorganization but has no effect on vinculin recruitment in mature FAs.
Fig. 7: Small-scale stretching triggers vinculin recruitment in nascent adhesions without recruitment of additional talin.

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Data availability

All data 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

MATLAB codes for analysis of sptPALM trajectories and MSD computing are available from the corresponding author on reasonable request.

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Acknowledgements

We thank Z. Karatas, M. Fabre, R. Sterling and J. Carrere for technical assistance, T. Orré for helpful discussions, C. Poujol and S. Marais (Bordeaux Imaging Center) for technical help and F. Cordelières (Bordeaux Imaging Center) for support with the kymograph analysis (Kymo ToolBox). We thank J. B. Sibarita (IINS, Bordeaux, France) for support with the sptPALM analysis, V. Studer (IINS, Bordeaux, France) and P. O. Strale (Alvéole) for important discussions on microfabrication, M. Cabilic (IINS, Bordeaux, France) for the repositioning plugin developed in MetaMorph, T. Schlichthärle, L. Fischer and R. Jungmann (Max Planck Institute of Biochemistry, Munich, Germany) and C. Grashoff (Institute for Molecular Cell Biology, Münster, Germany) for providing materials, support and important discussions on DNA-PAINT, P. Kanchanawong (MBI, NUS, Singapore) for providing the talin1-22–tdEOS (talin-C), tdEOS-18–talin1 (talin-N) and mEos2-7–talinABS (THATCH) constructs. We acknowledge financial support from the French Ministry of Research and CNRS, ANR grant Integractome (to G.G.), ANR grant FastNano (to G.G.), ANR grant IntegrinNanoPlan (to G.G.), ANR Decav-Recav (to P.N.), ANR MecanoCav (to P.N.), Fondation pour la Recherche Médicale (to G.G.) and the Institut National du Cancer (to P.N.).

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Author notes

  1. These authors contributed equally: Sophie Massou, Filipe Nunes Vicente, Franziska Wetzel.

Authors and Affiliations

  1. Interdisciplinary Institute for Neuroscience, UMR 5297, Université de Bordeaux, Bordeaux, France

    Sophie Massou, Filipe Nunes Vicente, Amine Mehidi, Olivier Rossier & Grégory Giannone

  2. Interdisciplinary Institute for Neuroscience, UMR 5297, CNRS, Bordeaux, France

    Sophie Massou, Filipe Nunes Vicente, Amine Mehidi, Olivier Rossier & Grégory Giannone

  3. Laboratoire Photonique Numérique et Nanosciences, UMR 5298, Université de Bordeaux, Talence, France

    Franziska Wetzel, Dan Strehle & Pierre Nassoy

  4. Laboratoire Photonique Numérique et Nanosciences, UMR 5298, Institut d’Optique and CNRS, Talence, France

    Franziska Wetzel, Dan Strehle & Pierre Nassoy

  5. Cell Polarity, Migration and Cancer Unit, UMR 3691, Institut Pasteur Paris and CNRS, Paris, France

    Cecile Leduc

  6. Laboratoire Jean Perrin and Laboratoire de Physique Théorique de la Matière Condensée, CNRS - Sorbonne Université, Paris, France

    Raphaël Voituriez

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  1. Sophie Massou
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Contributions

G.G. conceived of and coordinated the project. G.G., P.N. and O.R. conceptualized the experiments and the stretching device. F.W., S.M., D.S. and F.N.V. developed and characterized the optical and mechanical properties of the stretching device. F.N.V. performed and analysed the experiments combining cell stretching and STED on microtubules and the actin cytoskeleton. F.N.V. and C.L. performed and analysed the experiments combining cell stretching and DNA-PAINT on vimentin intermediate filaments. F.N.V., S.M. and F.W. performed the experiments combining cell stretching and SRM/SPT on mature FAs. F.N.V. and G.G. analysed the experiments combining cell stretching and SRM/SPT on mature FAs. F.N.V. performed and analysed the experiments combining cell stretching and SRM/SPT on early nascent adhesions. A.M. developed analysis routines for kymographs. R.V. conceptualized the physical model. G.G. and P.N. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Grégory Giannone.

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Extended data

Extended Data Fig. 1 Mechanical properties of the stretchable substrate compatible with SRM and SPT.

a, Numerical simulation using COMSOL Multiphysics of 30 % (left), 60 % (middle) and 90 % (left) stretches for a 10 µm PDMS assembly. The first principal strain is color coded from 0 to 95 %, arrows display displacement fields (XY plane), scale factor 0.3. Scale bar, 1 mm. b, Numerical simulation of a 3 % stretch (left). The first principal strain is color coded from 0 to 4 %, arrows display displacement fields (XY plane), scale factor 0.03. Scale bar, 1 mm. Right, line plots of the first principle strain along the main axes of the stretching chamber. In both axes, the strain was in large parts nearly constant (for 0.5 mm < x,y < 2.5 mm). c, Images of fluorescent micropatterns (100 ×100 µm) on 10 µm PDMS assembly before (left) and after 30 % (middle left), 60 % (middle right) and 90 % (right) stretches. Scale bar, 50 µm. See Supplementary Video 2. d, Variation of micropatterns height (blue) and width (black) as function of time during successive trapeze-like patterns (30 %, 60 %, 90 %). Data are representative of 4 independent experiments (30 %) or 2 independent experiments (60 %, 90 %).

Extended Data Fig. 2 The stretchable elastic substrate enables to acquire SRM images of cellular structures deeper into the cells and live SRM images on cells that experience stretching.

a, Fluorescence image of vimentin-GFP in a vimentin Knock Out MEF on the 10 µm PDMS stretching device before (left) and after (right) 35% stretching (right direction) followed by rapid cell fixation. b, DNA-PAINT super-resolution images of vimentin after 35% stretching. Corresponding image of outlined area in right panel of a, highlighting the nucleus (left). Magnified section from outlined area in the left (right). Inset: Vimentin-GFP epifluorescence images. c, Fluorescence image of tubulin-GFP in a MEF on the 10 µm PDMS stretching device before (left) and after (right) 35% stretching (right direction) followed by rapid cell fixation. d, Confocal (left) and STED super-resolution images (right) of endogenous tubulin labeled with ATTO-647N after 35% stretching for the outlined area in the right panel of c displayed at a higher magnification. e, Low resolution confocal image of SiR-Tubulin in a MEF on the PDMS stretching device before (green) and after (magenta) 30 % stretching (left). Outlines represent cell contour. Live cell STED image of SiR-Tubulin after 30% stretching (right), corresponding to the outlined area in the left. Inset: SiR-Tubulin confocal image. f, Low resolution confocal image (left) and STED image (right) of SiR-Tubulin for the outlined area in the right panel of e displayed at a higher magnification. g, Low resolution confocal image of SiR-Actin in a MEF on the PDMS stretching device before (green) and after (magenta) 4 % stretching (left). Outlines represent cell contour. Live cell STED image of SiR-Actin after 4 % stretching, corresponding to the outlined area in the left panel (right). Inset: SiR-Actin confocal image. h, Low resolution confocal image (left) and STED image (right) of SiR-Actin for the outlined area in the right panel of g displayed at a higher magnification. Data are representative of 3 independent experiments (a-d, g, h) or 2 independent experiments (e, f).

Extended Data Fig. 3 The stretchable elastic substrate is compatible with single molecule localization microscopy.

a, Super‐resolution intensity images of purified mEos2 adsorbed on glass (left), 10 µm (middle) and 50 µm (right) PDMS assemblies. Scale bars, 500 nm. b, Spatial resolution (left) and density of mEos2 detection (right) for glass, 10 µm and 50 µm PDMS. Box plots: median (line) and mean (square) ± percentile (25%–75%). Density: mean ± s.e.m. Glass: n = 4472 point spread functions (PSFs). 10 µm: n = 3065 PSFs. 50 µm: n = 1864 PSFs. c, d, Super‐resolution intensity images of β3-integrin-mEos2 in a MEF obtained from a sptPALM sequence (50 Hz, 80 s) on 10 µm (c) or 50 µm (d) PDMS. Color-coded trajectories overlaid on GFP-Paxillin-labelled adhesion sites (greyscale) displaying free diffusion (magenta), confined diffusion (green) and immobilization (red) (right). Scale bars, 3 µm. e, Distributions of diffusion coefficient D of mEos2‐β3-integrin trajectories inside (left) and outside (right) FAs on 10 µm (light blue) or 50 µm (dark blue) PDMS. Immobilized proteins correspond to D < 0.017 µm2.s−1 (light blue line) for 10 µm PDMS and D < 0.041 µm2.s−1 (dark blue line) for 50 µm PDMS. f, Fraction of proteins undergoing free diffusion, confined diffusion or immobilization inside (left) and outside (right) FAs (mean ± s.e.m.). g, Cumulative distributions of trajectory lengths for 10 µm (light blue) and 50 µm (dark blue) PDMS. h, Densities of mEos2 trajectories inside (left) and outside (right) FAs for 10 µm (light blue) and 50 µm (dark blue) PDMS (mean ± s.e.m.). c, d, Data are representative of 2 independent experiments. e-h. Data pooled from 2 independent experiments: 10 µm, n = 5 cells (14720 trajectories); 50 µm, n = 5 cells (2028 trajectories). Where indicated, statistical significances were obtained using two-tailed Student’s t-tests or two-tailed Mann–Whitney rank sum tests. Statistical source data can be found at ED Source Data Fig. 3.

Source data

Extended Data Fig. 4 Elastic and inelastic mechanical response of talin-N to trapeze-like patterns.

a, Fluorescence image of paxillin-GFP in a MEF on the 10 µm PDMS stretching device before stretching (right direction) (top left). Projection of all tdEos-talin-N super‐resolution intensity images of a trapeze-like pattern time-lapse (stretching 2.2 %, 2 Hz, 40 s) (bottom left). Scale bar, 5 µm. Right, tdEos-talin-N kymographs generated from the trapeze-like pattern time-lapse (as shown in the left, dashed lines). Horizontal axis, space (500 nm); vertical axis, time (5 s). The magenta kymograph corresponds to the reference bead, and the green kymographs correspond to tdEos-talin-N. b, Displacement versus time plot for elastic responses of tdEos-talin-N (green lines) after normalization to the initial position of the reference bead (magenta line) (top). Corresponding mean displacement ± s.e.m. (bottom). c, Same as b for inelastic responses of tdEos-talin-N. b-c, Data pooled from 3 or 4 independent experiments. beads (n = 52 kymographs from 3 independent experiments); tdEos-talin-N (n = 98 kymographs pooled from 4 independent experiments). Source data can be found at ED Source Data Fig. 4.

Source data

Extended Data Fig. 5 Elastic and inelastic mechanical responses of actin and talin-C to single stretch-plateau patterns.

a, Fluorescence image of paxillin-GFP in a MEF on the 10 µm PDMS stretching device before stretching (right direction) (top left). Projection of all actin-mEos2 super‐resolution intensity images of a single stretch-plateau pattern time-lapse (stretching 2.9 %, 2 Hz, 40 s) (bottom left). Scale bar, 5 µm. Right, actin-mEos2 kymographs generated from the single stretch-plateau pattern time-lapse (as shown in the left, dashed lines). Horizontal axis, space (500 nm); vertical axis, time (5 s). The magenta kymograph corresponds to the reference bead, and the green kymographs correspond to actin-mEos2. b, Displacement versus time plot for elastic responses of actin-mEos2 (green lines) after normalization to the initial position of the magenta reference bead (magenta line). c, Same as b for inelastic responses of actin-mEos2. d, same as b for plastic deformation responses of actin-mEos2. e, Fluorescence image of paxillin-GFP in a MEF on the 10 µm PDMS stretching device before stretching (right direction) (top left). Projection of all talin-C-tdEos super‐resolution intensity images of a single stretch-plateau pattern time-lapse (stretching 3.8 %, 2 Hz, 40 s) (bottom left). Right, talin-C-tdEos kymographs generated from the single stretch-plateau pattern time-lapse (as shown in the left, dashed lines). Horizontal axis, space (500 nm); vertical axis, time (5 s). The magenta kymograph corresponds to the reference bead, and the green kymographs correspond to talin-C-tdEos. f, Displacement versus time plot for elastic responses of talin-C-tdEos (green lines) after normalization to the initial position of the reference bead (magenta line). g, Same as b for inelastic responses of talin-C-tdEos. b-g, Data pooled from independent experiments: beads (n = 52 kymographs pooled from 3 independent experiments); talin-C-tdEos (n = 117 kymographs pooled from 3 independent experiments); actin-mEos2 (n = 65 kymographs pooled from 3 independent experiments).

Extended Data Fig. 6 Elastic and inelastic mechanical response of actin to trapeze-like patterns after fixation and blebbistatin treatment.

a, Fluorescence image of paxillin-GFP in a fixed and permeabilized MEF on the 10 µm PDMS stretching device before stretching (right direction) (top left). Projection of all actin-mEos2 super‐resolution intensity images of a trapeze-like pattern time-lapse (stretching 2.8 %, 2 Hz, 40 s) (bottom left). Right, actin-mEos2 kymographs generated from the trapeze-like pattern time-lapse (as shown in the left, dashed lines). Horizontal axis, space (500 nm); vertical axis, time (5 s). The magenta kymograph corresponds to the reference bead, and the green kymographs correspond to actin-mEos2. b, Displacement versus time plot for elastic responses of actin-mEos2 in fixed/permeabilized cells (green lines) after normalization to the initial position of the reference bead (magenta line) (top). Corresponding mean displacement ± s.e.m. (bottom). c, Same as b for inelastic responses of actin-mEos2 in fixed/permeabilized cells. d, same as a for a MEF treated with blebbistatin. e, same as b for a MEF treated with blebbistatin. f, same as c for a MEF treated with blebbistatin. b-f, Data pooled from 2 or 3 independent experiments: beads (n = 52 kymographs pooled from 3 independent experiments; actin-mEos2 (n = 65 kymographs from 3 independent experiments); actin-mEos2 blebbistatin (n = 83 kymographs from 2 independent experiments). Source data can be found at ED Source Data Fig. 6.

Source data

Extended Data Fig. 7 Vinculin is not recruited to mature integrin-based adhesions by large scale (10 %) stretching.

a, Fluorescence image of paxillin-GFP in a MEF on the 10 µm PDMS stretching device before stretching (right direction). b, Projection of vinculin-mEos2 super-resolution intensity images for the three phases of a trapeze-like pattern (stretching 12.6 %, 2 Hz, 80 s per phase): before (left), plateau (middle) and relax (right). c, Variation of trajectory density (trc/µm2) for vinculin-mEos2 (n = 5 cells pooled from 3 independent experiments) throughout trapeze-like patterns (mean ± s.d.). The density of trajectories was normalized to the value obtained before stretching. Individual cells (green), mean trajectory ratio ± s.d. (bold green), increase > 1.1 (plain), < 1.1 (dotted). a,b, Data are representative of 3 independent experiments. Statistical significance was calculated using two-tailed Wilcoxon matched-pairs signed rank tests. Statistical source data can be found at ED Source Data Fig. 7.

Source data

Extended Data Fig. 8 Elastic and inelastic mechanical response of talin-C to trapeze-like patterns in nascent integrin-based adhesions.

a, Fluorescence image of paxillin-GFP in a spreading MEF on the 10 µm PDMS stretching device before stretching (right direction) (top left). Projection of all talin-C-tdEos super-resolution intensity images of a trapeze-like pattern time-lapse (stretching 3.7 %, 2 Hz, 40 s) (bottom left). Scale bar = 5 µm. Right, talin-C-tdEos kymographs generated from the trapeze-like pattern time-lapse (as shown in the left, dashed lines). Horizontal axis, space (500 nm); vertical axis, time (5 s). The magenta kymograph corresponds to the reference bead, and the green kymographs correspond to talin-C-tdEos. Scale bar = 5 µm. b, Displacement versus time plot for elastic responses of talin-C-tdEos (green lines) after normalization to the initial position of the reference bead (magenta line) (top). Corresponding mean displacement ± s.e.m. (bottom). c, Same as b for inelastic responses of talin-C-tdEos. d, Fractions of elastic and inelastic responses inside and outside FAs for talin-C-tdEos. Values represent the average of fractions obtained from three independent experiments (mean ± s.e.m.). e, Box plots displaying the median (line), mean (square) ± percentile (25%–75%) and s.d. (whiskers) of local displacements for talin-C-tdEos for elastic (E) and inelastic (IE) responses after subtraction of the extrapolated PDMS displacements.b-e, Data pooled from independent experiments: beads (for e, n = 52 kymographs from 3 independent experiments); talin-C-tdEos NAs (for d, n = 3 stretches and for e, n = 111 kymographs, both pooled from 3 independent experiments).; talin-C-tdEos FAs (for d, n = 4 stretches and for e, n = 117 kymographs, both pooled from 3 independent experiments). f, Schematic representation of the acute mechanical response of talin-N and talin-C compared to β3-integrin Statistical significances were obtained using two-tailed unpaired Student’s t-tests. Statistical source data can be found at ED Source Data Fig. 8.

Source data

Extended Data Fig. 9 Vinculin does not exhibit region-selective recruitment in nascent adhesions after small-scale (4 %) stretching.

a, Separated color-coded projection of vinculin-mEos2 intensity images during the plateau phase of stretching (inset, paxillin-GFP) (red 0–15 s, green 15-30 s, blue 30-45 s, yellow 45-60 s). This cell corresponds to the cell displayed in Fig. 7a–f. b–d, same as a for other cells displaying increased detections of vinculin-mEos2 during the plateau phase. Scale bar = 3 µm. a–d, Data are representative of 3 independent experiments.

Extended Data Fig. 10 Trapeze-like patterns (∼ 4 %) are not triggering stress fiber ruptures and repair.

a, Low resolution fluorescence image of SiR-Actin in a MEF on the PDMS stretching device before stretching (top), after a ∼ 4% small stretch (middle), merge image (bottom) (0 out of 8 cells exhibited ruptures). b, Low resolution epifluorescence image of SiR-Actin in a MEF on the PDMS before stretching (top), after a ∼ 30% large stretch (middle), merge image (bottom). Arrows display stress fibers (SFs) ruptures (3 out of 3 cells exhibited ruptures). c, same as a (∼ 4% stretch) for a MEF expressing α-actinin-GFP (0 out of 6 cells exhibited recruitment). d, same as b (∼ 30 % stretch) for a MEF expressing α-actinin-GFP. Arrows display major recruitment of α-actinin-GFP to SFs (2 out of 2 cells exhibited recruitment). e, same as a (∼ 4% stretch) for a MEF expressing zyxin-mEmerald. f, same as a (∼ 4% stretch) for a MEF expressing zyxin-mEmerald and displaying a local recruitment of zyxin-mEmerald on SFs (arrow) (3 out of 12 cells exhibited recruitment). g, same as b (∼ 30 % stretch) for a MEF expressing zyxin-mEmerald. Arrows display major recruitment of zyxin-mEmerald to SFs (2 out of 2 cells exhibited recruitment). Scale bar = 5 µm. a-d, g, Data are representative of 2 independent experiments. e, f, Data are representative of 4 independent experiments.

Supplementary information

Supplementary Information

Supplementary Notes 1 and 2

Reporting Summary

Supplementary Video 1

Assembly of the micromechanical device. This video displays the schematic assembly of the 3D-printed micromechanical stretching device composed of the glass–glycerol–PDMS assembly in between the holding arm (being part of the chip base) and a clamp. The stretching arm attached to the surface of the thick elastomer frame (40 µm) is connected to the piezo motor.

Supplementary Video 2

Fluorescent micro-pattern stretching. Stretching of a fluorescent micro-pattern (100 µm × 100 µm) imprinted on a 10-µm glass–glycerol–PDMS assembly. We applied trapeze-like patterns, composed of stretch–plateau–relax phases (90% stretch in this example). Scale bar: 50 µm.

Supplementary Video 3

Unprocessed sptPALM stretching acquisition of β3-integrin–mEos2 during a trapeze-like pattern. Unprocessed sptPALM stretching acquisition (50 Hz; 20 ms; duration: 40 s) of β3-integrin–mEos2 during a trapeze-like pattern (2% stretching). Adsorbed 0.1-μm fluorescent beads (stronger fluorescent signal) were used to register and test the elasticity of the stretching device and measure the percentage of stretch. Blinking fluorescent single molecules (weaker fluorescent signal) correspond to individual β3-integrin–mEos2. One frame over four frames is displayed. Data are representative of three independent experiments. Scale bar: 5 µm.

Supplementary Video 4

Super-resolution time lapse of β3-integrin–mEos2 during a trapeze-like pattern (entire imaged field). sptPALM stretching acquisition (Supplementary Video 3; 50 Hz; duration: 40 s) processed into a super-resolution time lapse (2 Hz) of β3-integrin–mEos2 during a trapeze-like pattern (2% stretching). The white dot is above the position of the origin bead used for registration (upper left bead in Supplementary Video 3). The magenta dot is above the position of the reference bead used to measure the percentage of stretch (most central bead in Supplementary Video 3). The white rectangle corresponds to Supplementary Video 5. Data are representative of three independent experiments. Scale bars: 5 µm.

Supplementary Video 5

Super-resolution time lapse of β3-integrin–mEos2 during a trapeze-like pattern (higher magnification). Same as Supplementary Video 4, but with a higher magnification. The left magenta dot is above the position of the reference bead. The other magenta dots are above positions of β3-integrin–mEos2 used to generate kymograph 1 (central dot) and kymograph 2 (right dot) in Fig. 2d. Data are representative of three independent experiments. Scale bars: 1 µm.

Supplementary Video 6

Super-resolution time lapse of talin-C–tdEos during a trapeze-like pattern (close elastic and inelastic responses). Super-resolution time lapse (2 Hz) of talin-C–tdEos during a trapeze-like pattern (3.7% stretching) obtained from a sptPALM sequence (50 Hz; duration: 40 s). The left magenta dot is above the position of a talin-C–tdEos displaying an elastic response to the trapeze-like pattern (kymograph 3 in Fig. 3a). The near right cyan dot is above the position of a talin-C–tdEos displaying an inelastic response to the trapeze-like pattern (kymograph 3 in Fig. 3a). Data are representative of three independent experiments. Scale bars: 1 µm.

Supplementary Video 7

Super-resolution time lapse of talin-C–tdEos during a trapeze-like pattern (remote elastic and inelastic responses). Same as Supplementary Video 6, but for kymograph 1 (left magenta dot; elastic response) and kymograph 2 (right cyan dot; inelastic response) in Fig. 3a. Data are representative of three independent experiments. Scale bars: 1 µm.

Supplementary Video 8

Super-resolution time lapse of actin–mEos2 during a trapeze-like pattern. Super-resolution time lapse (2 Hz) of actin–mEos2 during a trapeze-like pattern (2.9% stretching) obtained from a sptPALM sequence (50 Hz; duration: 40 s). The left magenta dot is above the position of an actin–mEos2 displaying an elastic response to the trapeze-like pattern (kymograph 1 in Fig. 4a). The right cyan dot is above the position of an actin–mEos2 displaying an inelastic response to the trapeze-like pattern (kymograph 2 in Fig. 4a). Data are representative of three independent experiments. Scale bars: 1 µm.

Supplementary Video 9

Super-resolution time lapse of zyxin–mEos3.2 in mature FAs during a trapeze-like pattern. Super-resolution time lapse (2 Hz) of zyxin–mEos3.2 during a trapeze-like pattern (4.3% stretching) obtained from a sptPALM sequence (50 Hz; duration: 80 s). A walking average of six was applied to the entire stack. Data are representative of three independent experiments. Data are representative of four independent experiments.

Supplementary Video 10

Super-resolution time lapse of vinculin–mEos2 in mature FAs during a trapeze-like pattern. Super-resolution time lapse (2 Hz) of zyxin–mEos3.2 during a trapeze-like pattern (4.3% stretching) obtained from a sptPALM sequence (50 Hz; duration: 80 s). A walking average of six was applied to the entire stack. Data are representative of three independent experiments.

Supplementary Video 11

Super-resolution time lapse of vinculin–mEos2 in nascent adhesions during a stretch–plateau-like pattern. Super-resolution time lapse (2 Hz) of vinculin–mEos2 during a trapeze-like pattern (4.2% stretching) obtained from a sptPALM sequence (50 Hz; duration: 80 s). A walking average of six was applied to the entire stack. Data are representative of four independent experiments.

Source data

Source Data Fig. 2

D coefficient distributions and diffusion/immobilization fractions for β3-integrin–mEos2 before and after 30% stretch.

Source Data Fig. 3

Elastic (squared) and parabolic displacement amplitudes of β3-integrin–mEos2 for stretching.

Source Data Fig. 4

Displacement amplitudes and inelastic versus elastic fractions of β3-integrin–mEos2, tdEos–talin-N, talin-C–tdEos and mEos2–talin–THATCH for stretching. Rise and recoil times for talin and actin are given.

Source Data Fig. 5

Displacement amplitudes and inelastic versus elastic fractions of actin–mEos2 in controls, fixed/permeabilized cells and cells treated with blebbistatin for stretching. Rise and recoil times for talin and actin are given.

Source Data Fig. 6

Variation of the trajectory number and density (trc µm−2; absolute and normalized to before stretching) of zyxin–mEos3.2 and vinculin–mEos2 for 4% stretching in mature FAs.

Source Data Fig. 7

Variation of trajectory number and density (trc µm−2; absolute and normalized to before stretching) of vinculin–mEos2 and talin-C–tdEos for 4% stretching in nascent adhesions. Vinculin–mEos2 detections per frame before and during the stretching plateau are shown.

Source Data Extended Data Fig. 3

PSFs and mEos2 detections for spatial resolution and mEos2 density in glass and 10 µm and 50 µm PDMS. D coefficient distributions and diffusion/immobilization fractions for β3-integrin–mEos2 in 10 µm and 50 µm PDMS. Trajectory accumulation of β3-integrin–mEos2 in 10 µm and 50 µm PDMS. Densities of β3-integrin–mEos2 detections in 10 µm and 50 µm PDMS are given.

Source Data Extended Data Fig. 4

Elastic (squared) and parabolic displacement amplitudes of tdEos–talin-N for stretching.

Source Data Extended Data Fig. 6

Elastic (squared) and parabolic displacement amplitudes of actin–mEos2 in fixed/permeabilized cells and cells treated with blebbistatin for stretching.

Source Data Extended Data Fig. 7

Variation of trajectory number and density (trc µm−2; absolute and normalized to before stretching) of vinculin–mEos for 10% stretching in mature FAs.

Source Data Extended Data Fig. 8

Displacement amplitudes and inelastic versus elastic fractions of talin-C–tdEos for stretching in mature FAs and nascent adhesions.

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Massou, S., Nunes Vicente, F., Wetzel, F. et al. Cell stretching is amplified by active actin remodelling to deform and recruit proteins in mechanosensitive structures. Nat Cell Biol 22, 1011–1023 (2020). https://doi.org/10.1038/s41556-020-0548-2

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