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Mechanical stress driven by rigidity sensing governs epithelial stability

Nature Physics volume 19, pages 132–141 (2023)Cite this article

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Abstract

Epithelia act as barriers against environmental stresses. They are continuously exposed to various mechanical stress and abrasion, which impact epithelial integrity. The impact of the environment on epithelial integrity remains elusive. By culturing epithelial cells on two-dimensional hydrogels, we observe a loss of epithelial monolayer integrity on soft substrates through spontaneous hole formation. These monolayer ruptures are associated with local cellular stretching and cell-division events. Substrate stiffness triggers an unanticipated mechanical switch of epithelial monolayers from compressive stress on stiff substrates to highly tensile on soft, favouring hole formation. In agreement with an active nematic model, hole-opening events occur preferentially near spontaneous half-integer topological defects, which underpin large isotropic stress fluctuations triggering stochastic mechanical failure. Our results thus show that substrate stiffness provides feedback on the mechanical state of epithelial monolayers with potential application towards a mechanistic understanding of compromised epithelial integrity during normal and pathological human ontogenesis.

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Fig. 1: Rigidity-based hole formation within MDCK monolayers.
Fig. 2: Hole characteristics.
Fig. 3: Gel stiffness modulates tissue stress within MDCK monolayers.
Fig. 4: Hole formation is triggered by tensile stresses around topological defects.
Fig. 5: Characterization of hole dynamics and cells around the hole.

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Acknowledgements

This work was supported by the European Research Council (grant no. Adv-101019835 to B.L.), LABEX Who Am I? (ANR-11-LABX-0071 to B.L. and R.-M.M.), the Ligue Contre le Cancer (Equipe labellisée 2019 to B.L. and R.-M.M.), the Agence Nationale de la Recherche (‘MechanoAdipo’ ANR-17-CE13-0012 to B.L., ‘Myofuse’ ANR-19-CE13-0016 to B.L., ANR-20-CE30-0023 to J.-F.R.) and France 2030, the French Government programme managed by the French National Research Agency (ANR-16-CONV-0001), and the Excellence Initiative of Aix-Marseille University – A*MIDEX. We acknowledge support from the ImagoSeine core facility of the IJM, a member of IBiSA and France-BioImaging (ANR-10-INBS-04) infrastructures. L.B. has received funding from the European Union’s Horizon 2020 research and innovation programme (Marie Skłodowska-Curie grant agreement no. 665850-INSPIRE), La Ligue Contre le Cancer and an EMBO Postdoctoral Fellowship. We thank J. Nelson and S. Robin for gifting us MDCK and Caco2 cell lines, respectively. We also thank the members of the ‘Cell Adhesion and Mechanics’ team, M. Piel, F. Gallet, D. Delacour, M.-A. Fardin and S. Tlili, for insightful discussions.

Author information

Author notes

  1. Surabhi Sonam

    Present address: D Y Patil International University, Pune, India

  2. Lakshmi Balasubramaniam

    Present address: Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK

  3. These authors contributed equally: Surabhi Sonam, Lakshmi Balasubramaniam, Shao-Zhen Lin.

Authors and Affiliations

  1. Université Paris Cité, CNRS, Institut Jacques Monod, Paris, France

    Surabhi Sonam, Lakshmi Balasubramaniam, Irina Pi-Jaumà, Cecile Jebane, René-Marc Mège & Benoît Ladoux

  2. Aix Marseille Univ, Université de Toulon, CNRS, CPT, Turing Centre for Living Systems, Marseille, France

    Shao-Zhen Lin, Marc Karnat & Jean-François Rupprecht

  3. Mechanobiology Institute, National University of Singapore, Singapore, Singapore

    Ying Ming Yow Ivan, Yusuke Toyama & Jacques Prost

  4. Department of Biological Sciences, National University of Singapore, Singapore, Singapore

    Yusuke Toyama

  5. Physique et Mécanique des Milieux Hétérogènes, CNRS, ESPCI Paris, PSL University, Sorbonne Université, Université de Paris, Paris, France

    Philippe Marcq

  6. Physico-Chimie Curie, Institut Curie, CNRS UMR 168, Paris, France

    Jacques Prost

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Contributions

S.S., L.B., R.-M.M. and B.L. designed the research. S.S., L.B., Y.M.Y.I. and C.J. performed experiments. S.-Z.L., J.-F.R. and J.P. developed the theoretical vertex and analytical models. S.S. and L.B. analysed data with help from I.P.-J. M.K. helped with segmentation. Y.T. participated in discussions. P.M. provided the BISM code and helped with analysis. S.S., L.B., S.-Z.L., J.-F.R., R.-M.M. and B.L wrote the paper. J.-F.R., R.-M.M. and B.L. oversaw the project. All authors read the manuscript and commented on it.

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Correspondence to Jean-François Rupprecht or Benoît Ladoux.

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Extended data

Extended Data Fig. 1 Hole formation is a dynamic process influenced by cell division.

(a) Number of holes formed on unconfined and confined monolayers on 2.3 kPa and 55 kPa gels. (For confined, n2.3 kPa = 23 and n55 kPa = 15 from 3 independent experiments. For unconfined n2.3 kPa = 19 and n55 kPa = 19 from 3 different experiments, two-tailed unpaired t-test, **P = 0.0071, n.s.=not significant). (b) Hole formation in PA gels (2.3 kPa) evenly coated with fibronectin. Fibronectin coating before and after the hole formation does not change. Scale bars, 50 µm. Two independent experiments were performed with same results. (c) Representative image of hole formation due to cell stretching on soft (2.3 kPa) gels (scale: 50 µm). Four independent experiments were performed with same results. (d) Illustration of hole formation after a cell division event. Scale bar, 5 µm. Red line represents the division axis, green line, hole major axis and blue encloses the location of hole formation. Four independent experiments were performed with same results. (e) Angle of division axis in degree with respect to the hole major axis (n = 20 holes from 3 independent experiments) on soft (2.3 kPa) gels. Green line represents major axis of the hole and red line the division axis. (f) Probability distribution as a function of distance from a newly nucleated hole to the nearest cell division within 25 mins of hole nucleation on soft (2.3 kPa) gels, blue, experiments (n = 14 holes), green, simulation with cell divisions at random locations (identical number of divisions as in experiments). (g) Representative immunostaining images of EdU positive cells on 2.3 kPa and 55 kPa gels. Blue panel denotes cell nucleus and green panel denotes EdU stain. (h) Percentage of cells stained positive for EdU (marker of proliferation) on 2.3 kPa and 55 kPa gels (n2.3 kPa = 4 and n55 kPa = 4 from 2 independent experiments, *P < 0.05) with scatter points for each individual circle (coloured per experiment), encircled coloured symbols for the mean per experiment and solid bars for the mean over experiment. (i) Number of holes formed in a circular monolayer of MDCK (n = 33 different circles) and Caco2 (n = 17 different circles, *P < 0.05) on soft (2.3 kPa) gels from 2 independent experiments with scatter points for each individual circle (coloured per experiment), encircled coloured symbols for the mean per experiment and solid bars for the mean over experiment. (j, k) Change in perimeter as a function of time for (j) short-lived (n = 35 holes from 3 independent experiments) and (k) long-lived holes (n = 9 holes from 3 independent experiments) on soft (2.3 kPa) gels. Solid lines represent the mean and error bars the standard deviation. Red triangles represent the hole opening and closing process.

Source data

Extended Data Fig. 2 Rigidity alters focal adhesions but not cell-cell adhesions.

(a) Number of holes formed/mm2 on different concentrations (5 µg and 20 µg) of fibronectin-coated samples (n2.3kPa-5µg = 17, n2.3kPa-20µg = 23, n55kPa-5µg = 23, n55kPa-20µg = 15; two-tailed unpaired t-test, ****P2.3kPa-5µg-55kPa-5µg = 0.000032, ****P2.3kPa-20µg-55kPa-20µg = 0.000003, n.s.=non-significant). (b) Paxillin staining for focal adhesions formed within a MDCK monolayer on 2.3 kPa (top) and 55 kPa (bottom). Inset shows the zoomed image of the representative focal adhesions. Scale bars, 5 µm; inset scale bars, 5 µm. Two independent experiments were performed with same results. (c) Length (n = 69 from 2 independent experiments ; with scatter points for each individual circles (coloured per experiment), encircled coloured symbols for the mean per experiment and solid bars for the mean over experiment; ***P < 0.001) and (d) area (n = 50 from 2 independent experiments; ***P < 0.001) of focal adhesion on 2.3 kPa and 55 kPa gels. (e) Immunostaining of actin (green) and paxillin (magenta) on monolayers grown on glass at low (top) and high (bottom) density. Panels represent nucleus, actin and paxillin. Merge and the inset are an overlay of actin and paxillin panels. Scale bars, 20 µm. (f, g) Quantification of area (f) and length (two-tailed unpaired t-test, ***P = 0.0003) (g) of paxillin at these two different densities for n = 30 focal adhesions. (h) E-cadherin localisation at junctions on 2.3 kPa and 55 kPa gels. Scale bars, 10 µm. (i) Mean intensity plot of E-cadherin localisation at junctions (n = 30 from 2 independent experiments). (j) Averaged E-cadherin intensity of new junctions formed after a cell division event on soft (2.3 kPa) and stiff (55 kPa) gels for n = 33 division events from 2 independent samples for each condition. (k) Averaged E-cadherin intensity over time just before hole formation on soft (2.3 kPa) gels (n = 12 junctions from 2 independent experiments). (l) Immunostaining of vinculin (red) and actin (green) at different planes of monolayer (bottom and mid) of monolayers grown on soft (2.3 kPa) and stiff (50 kPa) gels. Last panel and inset are a merge of actin and vinculin panels. Scale bars, 20 µm and inset 5 µm. (m) Quantification of vinculin intensity at cell junctions obtained from n2.3 kPa = 151 and n55 kPa = 166 junctions from 2 independent experiments. Solid lines represent mean and error bars standard deviation.

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Extended Data Fig. 3 Cell-substrate adhesion and tissue stress contributions in hole lifetime.

(a) Vertex-model for the differential fibronectin experiment (as Fig. 3a), where a circular spot with lower friction (green, 10% of the friction within the rest of the tissue) mimics an area of low fibronectin concentration. (b) Probability distribution of isotropic stress per cell in the low friction region (green) as compared to the rest of the tissue region (blue). Value of the isotropic stress in the spot with respect to the rest of the tissue: mean = 87 Pa.µm (resp. 37 Pa.µm). 95th percentile value = 397 Pa.µm (resp. 240 Pa.µm) obtained from 10 independent simulations, n = 85,715 cells within the spot and n = 422,347 cells in the rest of the tissue. Simulation parameters are specified in Supplementary Table 1, SI. (c) Average traction force magnitude in MDCK unconfined monolayers (n = 102 time averaged for n2.3 kPa = 11 different circles and n = 112 time averaged for n30 kPa = 11 different circles; **P < 0.05 from 2 independent experiments). (d) Traction force quiver overlaid on phase contrast images for soft (2.3 kPa, top) and stiff (30 kPa, bottom) gels. Scale bars, 100 µm. Three independent experiments were performed with same results. (e) Average isotropic stress for no-hole regions of unconfined monolayers on 2.3 kPa and 30 kPa PA gels. Green dashed line represents 0 Pa-µm on the y-axis (For unconfined, n2.3 kPa = 12 and n30 kPa = 12 different circles obtained from 2 independent experiments). (f-g) Divergence of the velocity field and traction forces overlaid on phase contrast images on 2.3 kPa gel prior to hole formation. Three independent experiments were performed with same results. Scale bars, 50 µm. (h, h’, h”) Representative graphs of evolution in the hole area (blue) and averaged local isotropic stress (red) around the hole initiation site as a function of the time where t = 0 indicates hole initiation on soft (2.3 kPa) gels. Each panel represents an independent hole formation event. (i) E-cadherin localisation at junctions around the hole and within the monolayer. Scale bar, 10 µm. Three independent experiments were performed with same results. (j) Junctional straightness (ratio between Euclidean and actual lengths) around the holes and within the monolayer (n = 35 different circles; two-tailed unpaired t-test, ****P = 0.000000049) on soft (2.3 kPa) gels. Error bars represent standard deviation.

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Extended Data Fig. 4 Hole formation around defects from experiments.

(a) Averaged number of defects on soft and stiff gels obtained from n2.3 kPa = 6 circles for soft (2.3 kPa) gels and n55 kPa = 5 circles for stiff (55 kPa) gels. Solid lines represent average values and error bars represent the standard deviation. (b) Average velocity flow field around +1/2 defects demonstrating the extensile nature of MDCK monolayers on soft (2.3 kPa) gels (n = 2646 defects) and stiff (30 kPa) gels (n = 2765 defects) obtained from experiments. (c) MSD of +1/2 and -1/2 defects on both soft and stiff gels (n = 15 defects for -1/2 defects and +1/2 defects on 55 kPa and n = 17 defects for -1/2 defects and +1/2 defects on 2.3 kPa gels). Error bars represent standard deviation. (d) Lifetime of random tensile regions on both soft (2.3 kPa) and stiff (30 kPa) regions obtained from TFM for n = 30 regions (two-tailed unpaired t-test, ****P = 0.00007). Solid lines represent mean and error bars represent standard deviation.

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Extended Data Fig. 5 Defect mediated tensile stress and its association with hole formation.

(a, b) Averaged isotropic stress, averaged stress xy (a’, b’) and averaged stress yy (a”, b”) around +1/2 defects on soft (2.3 kPa) gels (a, a’, a”) (n = 2924 defects) and stiff (30 kPa) gels (b, b’, b”) (n = 3632 defects) from 2 independent experiments. (c, c’) Averaged isotropic stress around -1/2 defects on (c) soft (2.3 kPa) gels (n = 2810 defects) and (c’) stiff (30 kPa) gels (n = 3716 defects) from 2 independent experiments. White dots indicate the location of the defect core. (d) Number of holes per mm2 that are triggered by each defect type in the preceding frame in comparison to regions where defects do not lead to hole formation (n = 20 circles from 4 independent experiments; two-tailed unpaired t-test, *P+ 1/2_-1/2 = 0.0210, ***P-1/2_none = 0.0004, ***P+ 1/2_none = 0.0004). Solid line represents mean and error bars represent standard deviation. (e) Averaged peak isotropic stress around a region of 25.6×25.6 µm obtained from a random defect that does not lead to a hole site and defects that lead to hole formation (n-1/2 random no-hole and hole = 16, n + 1/2 random and hole = 17; two-tailed unpaired t-test, *P-1/2random_-1/2hole = 0.0399, **P+ 1/2random_+1/2hole = 0.0052) from 3 independent experiments. While a region around the defect core was averaged for -1/2 defects, for +1/2 defects, stresses were averaged in the tail region of the defect. Solid line represents the mean and error bars represent the standard deviation. As shown in (e’) a region around the defect centre was averaged for -1/2 defects, while for +1/2 defects, stresses were averaged in the tail region of the defect. Peak areal averaged stress within a time frame of 20-40 minutes prior to hole formation was obtained as shown in (e”). Error bars represent standard deviation. (f) Box plot distribution of the average tensile stress around defect that led to cell division or stretching-related hole formation (ncell_div = 19, nstretching = 13; n.s.= not significant) from 4 independent experiments. Solid lines represent the mean and the error bars represent the standard deviation. Horizontal black line denotes the zero value. (g) Percentage of + /- 1/2 linked division and stretched cell linked hole formation on 2.3 kPa gels from 4 independent experiments. Error bars represent standard deviation. (h-h’) Scatter plot in the location of the nearest cell division (with the cell separation detected in the next frame) to (h) a + 1/2 defect and (h’) a + 1/2 defect. The spatial pattern displays no bias towards a particular region of space, as in the case of a random distribution of points. Red and blue cores represent the defect location and orientation. (i) Cumulative distribution of distances of cell divisions to their nearest defect, showing non-significant differences between experiments (solid blue curve) and random (Poisson) distributions (dashed green curve). Relatively minor differences arise at large distances (100-150 μm).

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Extended Data Fig. 6 Activity and tension drive hole formation.

(a) Inhibition of arp2/3 by 100 µM CK666 treatment prevents hole formation on 2.3 kPa gels. Scale bar, 100 µm. Upper panel represents control, and lower panel represents CK666 treated monolayer at 0 h and 16 h. Three independent experiments were performed with same results. (b) Number of holes/mm2 under control, CK666 and blebbistatin (5 µM) treated samples averaged over n = 33 for control, n = 24 for CK666 and n = 23 for blebbistatin (two-tailed unpaired t-test, ****Pctrl_CK666 = 0.00000039, *Pctrl_bbn = 0.04). Solid lines represent mean and error bars represent standard deviation. (c) Area of holes formed under control (n = 49) and 5 µM blebbistatin treated (n = 14) samples (n.s. = non-significant). (d) Averaged velocity profile around +1/2 defects with CK666 treatment (left) (nCK666 = 2294) and WT (right) (nWT = 2646) monolayers on soft (2.3 kPa) gels. Solid lines represent mean and error bars represent standard deviation. (e) Simulations with a lower active stress (left; n = 4750 defects) as compared to the control case (right; n = 8553 defects, soft gel set of parameters, SupplementaryTable 1 SI). Colour bars refer to the magnitude of the average velocity field. Black arrows represent the direction of the average velocity vectors. White symbols represent the centre and orientation of the +1/2 defect.

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Extended Data Fig. 7 Maps of the average von Mises stress near half-integer topological defects.

(a, a’) Experiments: (a) −1/2 defects (n = 2810); (a’) +1/2 defects (n = 2924), from n = 2 independent experiments. (b, b’) Simulations: (b) −1/2 defects (n = 8555); (b’) +1/2 defects (n = 8553, n = 2 independent simulations, soft gel case set of parameters, see Table 1Supplementary Information). White dots for defect core locations and black dots for hole initiation sites.

Extended Data Fig. 8 Stress maps and hole opening locations near topological defects.

(a, b) Sketches of the two mechanisms of hole initiation at a tri-cellular vertex: (a) stress-dependent mechanism (model 1) where a large tension at cell-cell junctions favours hole initiation, and (b) strain rate-dependent mechanism (model 2), where a junctional elongation rate favours hole initiation. (c-f) Average isotropic stress comparisons of hole opening locations near half-integer topological defects: (c, e) model 1 and (d, f) model 2. (c, e) Average isotropic stress (c) and 95 th percentile of isotropic stress (e) overlaid with the location of holes in the frame of reference of the closest (left) -1/2 defect (n = 8555 defect and n = 55 holes) or a (right) +1/2 defect (n = 8553 defects and n = 30 holes). (d, f) Average isotropic stress (d) and 95 th percentile of elongation rate (f) overlaid with the location of holes in the frame of reference of the closest (left) -1/2 defect (n = 8555 defect and n = 69 holes) or a (right) +1/2 defect (n = 8553 defects and n = 90 holes). White symbols represent the centre and orientation of the defects. The black spots indicate the locations of opening holes. The 95th percentile is the value above which lies 5% of the field distribution. Domain size = 200 µm. The initial state of the simulations is under tension (soft gel case, see Supplementary Information Sec. I. G. for more details).

Extended Data Fig. 9 Lifetime of holes and cell organisation around hole periphery.

(a) Time-lapse of a typical tissue-scale vertex model simulation. The grey regions refer to holes. See Supplementary Information Sec. I for model details and Supplementary Table S1 for parameter values. (b,c) Time-dependent change in hole area normalised over maximum area of the hole for simulation of (b) short-lived holes (<5 hr) (n = 9 different simulations) and (c) long-lived holes (n = 9 different simulations). Red triangles on top of the graphs represent the hole opening and closing lifetime. (d) Evolution in the estimated hole radii in experiments, mean (red squares) ± standard deviation (red shaded area) and simulations, mean (black diamonds) ± standard deviation (black dashed lines); n = 14 in experiments and n = 10 in simulations. (e) Time-lapse of a typical opening and closing process on soft (2.3 kPa) gels. Orange dot in the first panel shows the location of a hole opening event. White arrows in the third panel show the cells stretched during the opening process. Scale bar, 20 μm. Red triangle at the bottom shows the hole opening and closing lifetime. (f) Cell alignment along the tangent of the hole obtained from experiments is highly co-related during the maximum hole area. The scatter plot between cell angle and tangent angle shows the distribution during the hole opening phase (blue dots), maximum hole area (red dots) and hole closing phase (green dots). A linear line (black line) fits the maximum hole area distribution with a Pearson’s coefficient, r = 0.83, and slope = 0.88. The majority of the maximum hole area data falls inside the pink ellipsoid area showing the small spread of their distribution.

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Supplementary information

Supplementary Information

SupplementaryNotes I and II.

Reporting Summary

Supplementary Video 1

MDCK monolayer on 2.3 kPa and 55 kPa PA gels recorded at 6 frames h−1. Scale bar, 50 µm.

Supplementary Video 2

MDCK monolayer on circular fibronectin patterns on 2.3 kPa and 55 kPa recorded at 6 frames h−1. Scale bar, 100 µm.

Supplementary Video 3

Video obtained from confocal imaging. MDCK cells on circular fibronectin patterns (Cy3 conjugated) on 2.3 kPa recorded at 6 frames h−1. Scale bar, 20 µm.

Supplementary Video 4

Modes of hole formation of MDCK monolayer on 2.3 kPa PA gels. Recorded at 6 frames h−1. Scale bar, 10 µm. Part 1: hole opening by division; Part 2: hole opening by cell stretching; Part 3: feature analysis: cell (blue) and hole (red) contours, cell divisions (yellow spot at the last frame preceding cytokinesis).

Supplementary Video 5

Caco2 monolayer within circular fibronectin patterns on 2.3 kPa PA gels recorded at 6 frames h−1. Scale bar, 20 µm.

Supplementary Video 6

Lifetime of hole formation. Recorded at 6 frames h−1. Scale bar, 10 µm.

Supplementary Video 7

MDCK cells migrating and forming hole on area with low fibronectin. Recorded at 6 frames h−1. Scale bar, 20 µm.

Supplementary Video 8

Laser ablation of MDCK monolayer plated on 2.3 kPa and 30 kPa PA gels. Recorded at 1 frame s−1. Scale bar, 20 µm. White contour indicates location of ablation.

Supplementary Video 9

Cell shape main axis of elongation (yellow) and half-integer topological defect detection in a MDCK monolayer plated on 2.3 kPa recorded at 6 frames h−1.

Supplementary Video 10

Brightfield movie of a MDCK tissue with the overlay of: (yellow bars) coarse-grained cell orientation main axis field; (red symbols) +1/2 defects; (blue symbols) −1/2 defects; (green dots) position of detected cytokinetic ring, marking cell division events.

Supplementary Video 11

Vertex-model simulations and topological defect detection without hole opening (blue, −1/2 defects; red, +1/2 defects) without (left) or with (right) cell divisions. Parameters are those used to model the soft case; see Supplementary Table 1.

Supplementary Video 12

Part 1: Vertex-model simulations and topological defect detection with hole opening process for the control value of the active stress level (β = 0.8) or an increased active stress value (right: β = 1.0). Other parameters are those used to model the soft case; see Supplementary Table 1. Part 2: Zoom on the hole opening process near a −1/2 defect (standard set of parameters).

Supplementary Video 13

Free boundary simulations; (left) with no polarity forces at the edges; (right) with additional normal polarity forces at the edges. See Supplementary Information for model definition.

Supplementary Data S5

Statistical source data for Supplementary Notes Fig. 5.

Supplementary Data S10

Statistical source data for Supplementary Notes Fig. 10.

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Statistical source data of Fig. 1.

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Statistical source data of Extended Data Figure 6.

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Statistical source data of Extended Data Fig. 3.

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Sonam, S., Balasubramaniam, L., Lin, SZ. et al. Mechanical stress driven by rigidity sensing governs epithelial stability. Nat. Phys. 19, 132–141 (2023). https://doi.org/10.1038/s41567-022-01826-2

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