Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers

Abstract

Actomyosin machinery endows cells with contractility at a single-cell level. However, within a monolayer, cells can be contractile or extensile based on the direction of pushing or pulling forces exerted by their neighbours or on the substrate. It has been shown that a monolayer of fibroblasts behaves as a contractile system while epithelial or neural progentior monolayers behave as an extensile system. Through a combination of cell culture experiments and in silico modelling, we reveal the mechanism behind this switch in extensile to contractile as the weakening of intercellular contacts. This switch promotes the build-up of tension at the cell–substrate interface through an increase in actin stress fibres and traction forces. This is accompanied by mechanotransductive changes in vinculin and YAP activation. We further show that contractile and extensile differences in cell activity sort cells in mixtures, uncovering a generic mechanism for pattern formation during cell competition, and morphogenesis.

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Fig. 1: Changes from extensile to contractile behaviours in the absence of E-cadherin.
Fig. 2: Inter- and intracellular stresses dictate extensile and contractile behaviours.
Fig. 3: Knocking out E-cadherin increases cell–substrate interactions.
Fig. 4: E-cadherin removal triggers mechanotransductive changes within the monolayer.
Fig. 5: Cell sorting triggered by change in nematic behaviour of monolayers.
Fig. 6: Cell sorting is governed by activity of the system.

Data availability

Data for cell orientation detection, and Bayesian inference of tissue stress are available upon request from the corresponding author. Source data are provided with this paper.

Code availability

Codes for cell orientation detection, and Bayesian inference of tissue stress are available upon request from the corresponding author. Numerical analyses were performed using a custom-made code: CELADRO (Cells as Active Droplets), which is open source and has been deposited on GitHub (https://github.com/rhomu/celadro).

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Acknowledgements

This work was supported by the European Research Council (grant no. CoG-617233), LABEX Who Am I? (ANR-11-LABX-0071), the Ligue Contre le Cancer (Equipe labellisée), and the Agence Nationale de la Recherche (‘POLCAM’ (ANR-17-CE13-0013 and ‘MechanoAdipo’ ANR-17-CE13-0012). We acknowledge the ImagoSeine core facility of the IJM, member of IBiSA and France-BioImaging (ANR-10-INBS-04) infrastructures. A.D. acknowledges support from the Novo Nordisk Foundation (grant no. NNF18SA0035142), Villum Fonden (grant no. 29476), and funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement no. 847523 (INTERACTIONS). L.B. has received funding from the European Union’s Horizon 2020 research and innovation programme (Marie Sklodowska-Curie grant agreement no. 665850-INSPIRE). T.B.S. acknowledges support from a Lee Kuan Yew (LKY) postdoctoral fellowship and the Singapore Ministry of Education Academic Research Fund Tier 1 (R-397-000-320-114). S.G. and A.Y. were supported by project grants and fellowships from the National Health and Medical Research Council of Australia (1123816 and 1139592) and Australian Research Council (DP190102871). We would like to thank P. Marcq for help with implementation of BISM code, M. A. Glukhova for providing the vinculin antibody and S. Robine for the ZO1 antibody. A.D. and J.M.Y. acknowledge G. Zhang for helpful discussions regarding the model. We also thank the members of cell adhesion and mechanics team at Institut Jacques Monod, M. Piel and F. Gallet, for insightful discussions.

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L.B., T.B.S., A.D., J.Y., R.M.M. and B.L. designed the research. G.H.N.S.N. and T.D. developed the E-cadherin KO cell line. L.B. performed experiments and analysed the results. S.S. helped in the PA gel experiments. M.T. performed and quantified laser ablation experiments. S.G. performed the MCF10A experiments. A.D. and R.M. performed the in silico simulations. T.B.S. contributed to the analysis tools. A.S.Y., Y.T., R.M.M., A.D., J.Y. and B.L supervised the project.

Corresponding authors

Correspondence to Amin Doostmohammadi or René-Marc Mège or Julia M. Yeomans or Benoît Ladoux.

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Extended data

Extended Data Fig. 1 MDCK WT behaves as an extensile system and MDCK E-cadherin KO behave as a contractile system.

a,b, Orientation field (left) and velocity vectors (right) around a single comet-shaped (+1/2) defect (a) and trefoil (-1/2) defect obtained from WT (top) and E-cadherin KO (bottom) monolayers. c,d, Trajectory of several comet (+1/2) (left) and trefoil (-1/2) (right) shaped defects obtained from MDCK WT (c) and MDCK E-cadherin KO (d) monolayers. Scale bars: 40μm. Source data

Extended Data Fig. 2 Fibroblasts behave as a 2D contractile active nematic.

a, Average yy- and xy-components of strain rate map around +1/2 defect obtained from experiments (left and middle respectively) and corresponding average flow field (right) (n = 1489 defects from 2 independent experiments) for NIH3T3 cells. Colour code is positive for stretching and negative for shrinkage. b, Average yy (left)-, xy (middle)- and isotropic (right) components of stress around a +1/2 defect obtained from experiments for NIH3T3 (n = 1,428 defects from 2 independent experiments).

Extended Data Fig. 3 MDCK WT behave as an extensile system.

a, Kymograph of a short junction (<10μm) (top) and long junction (>15μm) (bottom) before and after laser ablation. b, Recoil velocity after laser ablation for short (<10μm) (n=9) (N=4), medium (10-15μm) (n=13)(N=4) and long junctions (> 15μm) (n=12) (N=6). n, is the number of junctions ablated and N is the number of independent experiments from which these results were obtained. Error bars represent the standard deviation. ANOVA test was performed leading to *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Scale bars, 20μm.

Extended Data Fig. 4 Characterization of MDCK E-cadherin KO cells and extensile/contractile behaviour of MCF7A cells.

a, Immunofluorescence staining (top) of E-cadherin (left), β-catenin (middle) and ZO1 (right), along with a b) representative western blot and quantification for E-cadherin (left) (n=3) and β-catenin (right) (n=3). Scale bars, 20μm. c, Western blot analysis of total MLC and quantification from 3 independent experiments normalized to α-tubulin. d, Western blot analysis of vinculin and quantification from 3 independent experiments normalized to GAPDH. Error bars represent the standard deviation. e, Western blot showing the reduced level of E-cadherin in siRNA generated E-cadherin KD cell line for MCF7A cells. f,g Average flow field for MCF7A control cells (n = 2047 defects from 3 independent experiments) (f) and siRNA E-cadherin KD MCF7A cells (n = 1256 defects from 3 independent experiments) (f). h, Uncropped blots of all the western blots shown so far. Source data

Extended Data Fig. 5 E-cadherin rescue changes the behaviour to a 2D extensile active nematic liquid crystal and flow field around trefoil shaped defects.

a, Average yy- and xy-components of strain rate map around comet (+1/2) defect obtained from experiments (left and middle respectively) and corresponding average flow field (right) (n = 1767 defects from 2 independent experiments) for MDCK E-cadherin KO cells rescued with E-cadherin GFP. b, Total number of defects obtained per 0.55mm2 as a function of time on MDCK WT and MDCK E-cadherin KO monolayers. (n=10) from 2 independent experiments. c, Average vorticity and velocity field around trefoil (-1/2) defects in WT (left) (n=1934) and E-cadherin KO (right) (n=2028) monolayers. d, Average vorticity and velocity field around trefoil (-1/2) defects in control (left) (n=3200) and condition without active intercellular forces (right)(n=3200) monolayers obtained from simulations. e, Mean square displacement (MSD) plotted against time lag for comet (+1/2) and trefoil (-1/2) defects obtained from MDCK WT and MDCK E-cadherin KO monolayers (n=11). Error bars represent the standard deviation. Source data

Extended Data Fig. 6 E-cadherin removal does not affect the contractile behaviour of single cells.

a, Mean traction force for both MDCK WT (n=31) and MDCK E-cadherin KO cells (n=27). b, Average yy- and xy-components of strain rate map around comet (+1/2) defect obtained from experiments (left and middle respectively) and corresponding average velocity flow field (right) (n = 1428 defects from 2 independent experiments) for MDCK E-cadherin KO cells plated on PDMS substrates of stiffness 15kPa from which stress maps were obtained in Fig. 3b’. Source data

Extended Data Fig. 7 Drug treatment changes 2D active nematic behaviour of MDCK E-cadherin KO cells.

a,b,c, Average yy- and xy-components of strain rate map around +1/2 defect obtained from experiments (left and middle respectively) and corresponding average velocity flow field (right) for MDCK E-cadherin KO cells treated with 5μM blebbistatin (a) (n = 2174 defects from 2 independent experiments), 20μM blebbistatin (b) (n = 1223 defects from 2 independent experiments), and 25μM Y27632 (c) (n = 1965 defects from 2 independent experiments). d,e, Average yy- and xy components of strain rate map around +1/2 defect obtained from experiments (left and middle respectively) and corresponding average velocity flow field (right) for MDCK WT cells treated with 20μM blebbistatin (d) (n = 1287 defects from 2 independent experiments), and 25μM Y27632 (e) (n = 2472 defects from 2 independent experiments).

Extended Data Fig. 8 Colocalization of vinculin and paxillin.

a, Immunostaining of basal plane of vinculin (left), paxillin (middle) and merge (right) in MDCK WT (top) and MDCK E-cadherin KO monolayers. b, Intensity of vinculin plotted against paxillin for n=15 focal adhesions in MDCK WT and n=16 focal adhesions in MDCK E-cadherin KO monolayers. Scale bars: 20μm.

Extended Data Fig. 9 Substrate rigidity alters E-cadherin KO behaviour.

a,b, Average yy- and xy-components of strain rate map around +1/2 defect obtained from experiments (left and middle respectively) and corresponding average flow field (right) for MDCK WT cells (a) (n = 1426 defects from 2 independent experiments) and E-cadherin KO cells (b) (n = 1041 defects from 2 independent experiments). Source data

Extended Data Fig. 10 Phase diagram on activity change and activity based cell sorting in a mixed culture of MDCK WT and MDCK E-cadherin KO.

a, Phase diagram showing the transition of extensile and contractile behaviour with varying values of intercellular and intracellular stresses obtained from simulations. b, Phase separation (demixing) observed from simulations where the contractile particles (orange) are surrounded by extensile particles (green). c, Cell sorting (demixing) observed for a mixture of MDCK WT and MDCK E-cadherin KO cells, where WT cells are surrounded by E-cadherin KO cells (E-cadherin, green, cadherin 6, red, actin, black). XZ and YZ projection show the height difference between the two cells when mixed. Scale bars, 20μm. d,e, Early stages of cell sorting when MDCK WT (magenta) and MDCK E-cadherin KO (green) monolayers are mixed at 30-70 (d) and 70-30 (e) ratio. Scale bars: 100μm.

Supplementary information

Supplementary Information

Supplementary Table 1 and video legends

Reporting Summary

Supplementary Video 1

Phase contrast images overlaid with orientation vectors obtained from MDCK WT monolayers. Blue arrow shows the location of a +1/2 (comet)-shaped defect and the direction in which they move. Scale bar: 50 μm.

Supplementary Video 2

Phase contrast images overlaid with orientation vectors obtained from MDCK E-cadherin KO monolayers. Blue arrow shows the location of a +1/2 (comet)-shaped defect and the direction in which they move. Scale bar: 50 μm.

Supplementary Video 3

Time-lapse demixing of extensile (magenta) and contractile (green) particles obtained from simulations.

Supplementary Video 4

Time-lapse demixing of MDCK WT (extensile-magenta) and MDCK E-cadherin KO (contractile-green) cells observed from experiments. Scale bar: 100 μm.

Supplementary Video 5

Time-lapse demixing of MDCK WT (extensile-magenta) and MDCK E-cadherin KO (contractile-green) cells before and after the addition of 20 μM blebbistatin. Scale bar: 100 μm.

Source data

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Source Data Extended Data Fig. 1

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Source Data Extended Data Fig. 4

Data used to plot graphs and uncropped western blots.

Source Data Extended Data Fig. 5

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Source Data Extended Data Fig. 9

Data used to plot graphs.

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Balasubramaniam, L., Doostmohammadi, A., Saw, T.B. et al. Investigating the nature of active forces in tissues reveals how contractile cells can form extensile monolayers. Nat. Mater. (2021). https://doi.org/10.1038/s41563-021-00919-2

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