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Stress fibres are embedded in a contractile cortical network

Nature Materials volume 20, pages 410–420 (2021)Cite this article

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An Author Correction to this article was published on 18 November 2020

An Author Correction to this article was published on 30 October 2020

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Abstract

Contractile actomyosin networks are responsible for the production of intracellular forces. There is increasing evidence that bundles of actin filaments form interconnected and interconvertible structures with the rest of the network. In this study, we explored the mechanical impact of these interconnections on the production and distribution of traction forces throughout the cell. By using a combination of hydrogel micropatterning, traction force microscopy and laser photoablation, we measured the relaxation of traction forces in response to local photoablations. Our experimental results and modelling of the mechanical response of the network revealed that bundles were fully embedded along their entire length in a continuous and contractile network of cortical filaments. Moreover, the propagation of the contraction of these bundles throughout the entire cell was dependent on this embedding. In addition, these bundles appeared to originate from the alignment and coalescence of thin and unattached cortical actin filaments from the surrounding mesh.

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Fig. 1: The stress fibre sets the magnitude of the traction force exerted by the cell but remains under tension after photoablation.
Fig. 2: Stress fibres are connected to the surrounding actin cytoskeletal network.
Fig. 3: Model with active contractile stress fibres embedded in an elastic central mesh.
Fig. 4: The cortical meshwork is contractile.
Fig. 5: The stress fibre is fully embedded in the adjacent actin cortex.
Fig. 6: Emergence and translocation of cytoplasmic bundles in the cortical meshwork.

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Raw data are available from the corresponding authors upon request. Source data are provided with this paper.

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Code is available from the corresponding authors upon request.

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Acknowledgements

We thank the live microscopy facility MuLife of IRIG/DBSCI, funded by CEA Nanobio and labex Gral, for equipment access and use. This work was supported by grants from the European Research Council (741773, AAA awarded to L.B. and 771599, ICEBERG awarded to M.T.), from Agence Nationale de la recherche ANR (ANR-14-CE11-0003-01, MaxForce awarded to L.B. and M.T.) and from the US Army Research Office (grant W911NF-17-1-0417 to A.M.). J.M. acknowledges the European Molecular Biology Laboratory (EMBL) for funding. M.T.-N. was supported by a fellowship from the EMBL Interdisciplinary (EI3POD) programme under the Marie Skłodowska-Curie Actions COFUND (664726). We finally thank A. Kawska (IlluScientia.com) for artwork in Fig.6f.

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Authors and Affiliations

  1. CytoMorpho Lab, Interdisciplinary Research Institute of Grenoble, Laboratoire de Physiologie Cellulaire et Végétale, Grenoble-Alpes University/CEA/CNRS/INRA, Grenoble, France

    Timothée Vignaud, Qingzong Tseng, Laurent Blanchoin, Manuel Théry & Laetitia Kurzawa

  2. CytoMorpho Lab, Hôpital Saint Louis, Institut Universitaire d’Hématologie, Université Paris Diderot/CEA/INSERM, Paris, France

    Timothée Vignaud, Qingzong Tseng, Laurent Blanchoin, Manuel Théry & Laetitia Kurzawa

  3. Clinique de Chirurgie Digestive et Endocrinienne, Hôtel Dieu, Nantes, France

    Timothée Vignaud

  4. Courant Institute and Department of Biology, New York University, New York, NY, USA

    Calina Copos & Alex Mogilner

  5. NeuroCyto, Institute of NeuroPhysiopathology (INP), CNRS, Aix Marseille Université, Marseille, France

    Christophe Leterrier

  6. Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

    Mauricio Toro-Nahuelpan & Julia Mahamid

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  1. Timothée Vignaud
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Contributions

T.V. and L.K. performed most of the experiments. Q.T. performed preliminary work related to fibre ablation and traction force microscopy. C.C. and A.M. conceived and ran the model. C.L. performed STORM imaging. M.T.-N. and J.M. performed the Cryo-ET experiments. L.B., A.M., M.T. and L.K. designed the experiments, supervised the project and analysed data.

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Correspondence to Alex Mogilner, Manuel Théry or Laetitia Kurzawa.

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Extended data

Extended Data Fig. 1 Variations of contractility with cell shape and architecture.

a. Actin filaments staining (SirActin 1 µM) of RPE1 live cells on homogeneous (non-micropatterned) fibronectin-coated polyacrylamide gels. N = 1 experiment. Scale bar=50 µm. b. Variation of the strain energy associated to cell traction forces with respect to the length of non-micropaterned cells. n = 63 cells, N = 1 experiment. c. Distribution of the length of RPE1 cells on homogeneous (non-micropatterned) poly-acrylamide gels. d. Traction-force maps of cells spread on various micropattern shapes. Upper images display the fibronectin coating on the micropatterns, and lower images show averaged traction-force maps of cells (scale colour bar in Pa). n-plain crossbow=46 cells, n-regular crossbow=27 cells, n-empty crossbow=32 cells, N = 1 experiment. The graph shows the scatter plot of the mechanical energies on each micropattern and associated p-value (mean and standard deviation are depicted, two-tailed Mann-Whitney t-tests, P = 0.0006 between plain and regular crossbow, P < 0.0001 between regular and empty crossbow). 2 outliers were automatically removed from the analysis (remove outliers function of Prism). e. Averaged localization of molecular components involved in cell contractility for cells displaying either two main peripheral stress fibers (when plated on dumbbell-shaped micropattern, left) or a continuous actin mesh (when plated on pill-shaped micropattern, right). For each shape, averaged Z projections of cells are displayed. From top to bottom: micropatterns labeling (fibrinogen–Cy5); paxillin (alexa-488); actin (phalloidin-ATO-488); phospho-MLC (CY3); alpha-actinin (CY3). Image scale bar = 10 µm. N = 3 experiments.

Source data

Extended Data Fig. 2 Asymmetric force relaxation after an off-centered stress fiber cut.

Images of RPE1-LifeAct-GFP cells before and after off-centered photoablation of the stress fiber (red arrow) and associated traction stress maps. Image scale bar = 10 µm. Force scale colour bar in Pa. N = 3 experiments.

Extended Data Fig. 3 Regulation of the contributions of the cortex and the peripheral stress fibers to the total force.

a. Comparison of the relative releases of strain energy following a single local ablation in the cortical meshwork (green arrowhead) or in the peripheral stress fiber (purple arrowhead). n = 1 cell, N = 1 experiment. b. Measurement of the total strain energy and the released energy following stress fiber ablation in control cells and cells treated with the Arp2/3 inhibitor CK869 (50 µM, 30 min). The total energy was increased in response to CK869, but the ablation of the fiber did not release more energy than in the control case, suggesting that the global increase was due to the hyper-contraction of the cortical network. In this experiment, cells from 2 different sizes (59 and 64 µm) were analyzed. For total mechanical energy: nDMSO=105 cells, nCK869 = 76 cells; N = 3 experiments; p-value from unpaired t-test is indicated on the plot (P < 0.0001). For ablation: nDMSO = 22 cells, nCK869 = 23 cells; N = 1 experiment. Statistical significance and p-value from a two-tailed paired t-test is indicated on the plot (P = 0.5 for released energy; P = 0.023 for relative released energy). Medians are depicted. 7 outliers were automatically excluded from the analysis for the initial energy plot and 1 for the ablation plots (remove outliers function of Prism). c. Releases of strain energy following a single ablation of peripheral stress fibers on asymmetric dumbbells (central bar off-centered by 2 µm). The top fiber was connected to a small area of cortex, spaning the space between the fiber and the anchorage on the bar. The bottom fiber was connected to a larger cortical network. The ablation of the bottom fiber released more contractile energy. nTop fiber=38, nBottom fiber=35; N = 3 experiments, median is depicted. 2 cells for which stress fiber ablation was not efficient were eliminated from the analysis. p-value from two-tailed unpaired t-test is indicated on the plot (P = 0.0194). 2 outliers were automatically excluded from the analysis (remove outliers function of Prism). d. Measurement of the total strain energy and the released energy following stress fiber ablation in cells treated with a control siRNA or with siRNA against apha-actin1 and actinin4. Down-regulation of alpha-actinins increased the total contractile energy, as well as the energy released by fiber ablation. The ratio between the two was not affected, suggesting that the contraction of the cortex increased as well. In this experiment, cells from 2 different sizes (59 and 64 µm) were analyzed. For total and released mechanical energy: n-siCTRL=18 cells, n-siACTN1 + 4 = 11 cells, for released mechanical energy % of total: n-siCTRL=19 cells, n-siACTN1 + 4 = 11 cells; N = 1 experiment. p-values from two-tailed unpaired t-tests are indicated on the plots (P < 0.0001 for initial and released energy, P = 0.6678 for relative released energy). 1 oulier was automatically excluded from the analysis (remove outlier function of Prism).

Source data

Extended Data Fig. 4 Elastic and contractile model predictions of the force loss distribution after stress fiber shaving and photoablation.

a. From top to bottom: Images of a representative RPE1-LA-GFP cell depicting the precut cell; a shaving; a consecutive cut together with the associated forces relaxed upon photoablation on the right panel. Image scale bar = 10 µm. Force scale colour bar in Pascal. N = 3 experiments. b. Spatial distribution of force loss along the stress fiber after stress-fiber shaving (purple dashed line) and off-center photoablation (red star). The loss of traction forces was calculated in partitioned area of the cell, where the orange zone included half the stress fiber and the off-centered photoablation site, and the blue zone included the other half of the stress fiber. Plot displaying the predictions of the elastic model, the contractile model and the experimental measurements (n = 80 cells, N = 14, mean is depicted). The p-value from a two-tailed paired t-test is indicated on the plot (P = 0.1178). c. Spatial distribution of force loss along the stress fiber after stress-fiber shaving (purple dashed line) and off-center photoablation (red star). The loss of traction forces was calculated in partitioned area of the cell, where the green zone included the stress fiber with photoablation site, and the purple zone included the stress fiber without photoablation. Plot displaying the predictions of the elastic model, the contractile model and the experimental measurements (n = 80 cells, N = 14, mean is depicted).

Source data

Extended Data Fig. 5 Actin filament organisation in 3D within and near the stress fiber.

a. Cryo-Scanning Electron Microscopy of a vitrified RPE1 cell adhered and spreading on a dumbbell-shape micropattern. Red line indicates the putative position of the edge of the wedge produced by cryo-FIB milling. The green line indicates the position of the stress fiber imaged by cryo-ET. The yellow dashed line represents the dumbbell shape pattern size and position. b. Cryo-TEM of the wedge of the RPE cell shown in (a), displaying the positions (P1-P4) for cryo-ET. Inset: 6.8 nm thick tomographic slice of the P4 region. Microtubules (MTs); ribosomes (R); intermediate filaments (IF). c–e. Cellular cryo-ET. Tomographic slices (6.8 nm thickness) of three positions - P1, P2 and P3 - in (b), respectively, along the same stress fiber. The three tomograms start at the basal membrane and extend the thickness of the tomograms towards the apical membrane. The distance between consecutive tomographed positions is ~ 2.5 µm (center-to-center of the tomograms), covering a total distance of ~7 µm. N = 2 experiments. f–h. 3D rendering of the actin filament network and microtubules (purple) from (c–e), respectively. Blue-to-red color map of actin filaments represent the angular distribution (ranging from 0° to 90°) relative to the z-plane of the tomogram. Most actin filaments are positioned parallel to the basal membrane. See related Video 5.

Extended Data Fig. 6 Dynamics and mechanics of cortical bundle fusion with lateral stress fibers depending on anchorage with the underlying extra-cellular matrix.

a. Live imaging of RPE1-LifeAct-GFP cells on dumbbell-shaped micropattern revealing the splitting and fusion of cytoplasmic bundles with peripheral stress fibers (left and middle columns) as well as the emergence of cytoplasmic bundles from the cortical meshwork (right column at the red arrow heads) and its connection with peripheral stress fibers. Images from top to bottom show sequential acquisitions with a time frame of 10 min. N = 1 experiment. b. Color-coded overlay of sequential images acquired every 10 min on dumbbell-shaped (left) and pill-shaped micropatterns (right). Color overlay showed static structures in white and moving structures in colors ranging from blue to yellow depending on the time frame in which they were acquired. Scale bar=10 µm. c. Transversal linescans allowing the ploting of kymographs showing the transversal motion of internal actin bundles on dumbbell-shaped (left) and pill-shaped micropatterns (right). The graphs show the quantification of bundle velocity measured on the kymographs. n-dumbbell=18 actin paths, n-pill=10 actin paths; N = 1 experiment; median is depicted. Statistical significance of two-tailed unpaired t-test is indicated on the plot (P < 0.0001). d. Measurement of the total strain energy and the released energy following stress fiber ablation in cells plated on dumbbell-shaped (left) and pill-shaped micropatterns (right). Images show, from top to bottom: Micropattern labeling (fibrinogen–CY5); Actin before stress-fiber photoablation (0 sec) and after stress-fiber photoablation (10 sec); Overlay of the traction-force map and RPE1-LifeAct-GFP image after photoablation. Image scale bar = 10 µm. Graphs show the initial mechanical energy of the cells before the photoablation (top) and the released mechanical energy following photoablation of the peripheral stress fiber (% of the initial mechanical energy) (bottom). n = 10 cells for dumbbell and n = 18 cells for pill, N = 1 experiment, means are depicted. The p-values from two-tailed Mann-Whitney t-tests are indicated on the plots (P = 0.0009 for initial energy, P = 0.0014 for released energy).

Source data

Supplementary information

Supplementary Information

Supplementary methods, references and legends of videos.

Reporting Summary

Supplementary Video 1

Illustrations of stress fibre ablation (shown with white arrowheads) in nine cells plated on dumbbell-shaped micropatterns on polyacrylamide gels. Scale bar is 10 µm.

Supplementary Video 2

Illustration of stress fibre ablation and the measurement of the corresponding traction force field relaxation in a cell plated on dumbbell-shaped micropatterns on polyacrylamide gel. Scale bar is 10 µm.

Supplementary Video 3

Illustration of stress fibre shaving (in between the two tilted arrowheads) and the measurement of the corresponding traction force field relaxation in a cells plated on dumbbell-shaped micropatterns on polyacrylamide gel. Scale bar is 10 µm.

Supplementary Video 4

Illustrations of stress fibre shaving (in between the two tilted arrowheads) followed by stress fibre ablation (single vertical arrowhead) and the measurement of the corresponding traction force field relaxation in a cells plated on dumbbell-shaped micropatterns on polyacrylamide gel. Scale bar is 10 µm.

Supplementary Video 5

Cryo-electron tomography of a stress fibre of a cryo-focused ion beam (FIB)-treated cell grown on a dumbbell-shape micropattern. Tomographic volume of a cryo-FIB generated wedge of an RPE1 cell grown on a dumbbell-shape pattern. Each slice is 6.8 nm thick. Video related to Fig. 5b and Extended Data Fig. 5c,f, wedge 1, position 1 (P1).

Supplementary Video 6

Live imaging of RPE1–LifeAct-GFP cells on tripod-shaped micropattern showing the global and permanent remodelling of network architecture, suggestive of a complex interplay of longitudinal and lateral forces on cytoplasmic bundles. Time is indicated in hours and minutes. Scale bar is 20 µm.

Supplementary Video 7

Live imaging of RPE1–LifeAct-GFP cells on tripod-shaped micropattern highlighting network reconfiguration by lateral translocation of cytoplasmic bundles in the absence of anchorage displacement (arrows). Time is indicated in hours and minutes. Scale bar is 20 µm.

Supplementary Video 8

Live imaging of RPE1–LifeAct-GFP cells on tripod-shaped micropattern revealing the emergence of cytoplasmic bundles from the cortical meshwork (in between red arrows) and the lateral expansion of a bundle, its splaying into a wider structure (orange arrows) and its re-coalescence into several adjacent bundles (magenta arrows). Time is indicated in hours and minutes. Scale bar is 20 µm.

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Vignaud, T., Copos, C., Leterrier, C. et al. Stress fibres are embedded in a contractile cortical network. Nat. Mater. 20, 410–420 (2021). https://doi.org/10.1038/s41563-020-00825-z

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