Active wetting of epithelial tissues

Abstract

Development, regeneration and cancer involve drastic transitions in tissue morphology. In analogy with the behaviour of inert fluids, some of these transitions have been interpreted as wetting transitions. The validity and scope of this analogy are unclear, however, because the active cellular forces that drive tissue wetting have been neither measured nor theoretically accounted for. Here we show that the transition between two-dimensional epithelial monolayers and three-dimensional spheroidal aggregates can be understood as an active wetting transition whose physics differs fundamentally from that of passive wetting phenomena. By combining an active polar fluid model with measurements of physical forces as a function of tissue size, contractility, cell–cell and cell–substrate adhesion, and substrate stiffness, we show that the wetting transition results from the competition between traction forces and contractile intercellular stresses. This competition defines a new intrinsic length scale that gives rise to a critical size for the wetting transition in tissues, a striking feature that has no counterpart in classical wetting. Finally, we show that active shape fluctuations are dynamically amplified during tissue dewetting. Overall, we conclude that tissue spreading constitutes a prominent example of active wetting—a novel physical scenario that may explain morphological transitions during tissue morphogenesis and tumour progression.

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Fig. 1: E-cadherin expression causes an increase in traction forces and monolayer tension, and induces dewetting.
Fig. 2: Formation of E-cadherin junctions induces myosin phosphorylation, and hence the increase in tension that is responsible for monolayer dewetting.
Fig. 3: Active polar fluid model of tissue wetting.
Fig. 4: The wetting transition depends on substrate ligand density and monolayer radius.
Fig. 5: Evolution of monolayer morphology during dewetting.

Data availability

All the data used for this study is available upon request.

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Acknowledgements

We thank D. Sarrió and G. Moreno-Bueno for providing the E-cadherin inducible cells; N. Castro for technical assistance; A. Elosegui, V. González, E. Latorre, L. Valon and R. Vincent for stimulating discussions. R.A. thanks G. Torrents for assistance with mathematical details. C.P-G. and R.A. were funded by Fundació ‘La Caixa’. R.A. thanks J. Prost and acknowledges EMBO (Short Term Fellowship ASTF 365-2015), The Company of Biologists (Development Travelling Fellowship DEVTF-151206), and Fundació Universitària Agustí Pedro i Pons for supporting visits to Institut Curie. This work was supported by the Spanish Ministry of Economy and Competitiveness/FEDER (BFU2015-65074-P to X.T., FIS2016-78507-C2-2-P to J.C.), the Generalitat de Catalunya (2014-SGR-927 and CERCA Program to X.T., 2014-SGR-878 to J.C.), the European Research Council (CoG-616480 to X.T.), European Commission (H2020-FETPROACT-01-2016-731957 to X.T.) and Obra Social ‘La Caixa’. IBEC is recipient of a Severo Ochoa Award of Excellence from the MINECO.

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Contributions

C.P.-G., R.A., J.C. and X.T. conceived the study and designed experiments. C.P.-G. performed the experiments with the help of T.K. and E.B. C.P.-G. and M.G.-G. developed computational analysis tools. C.P.-G. processed and analysed the experimental data. R.A. developed the active wetting theory with the help of C.B.-M. and fitted the model predictions to the experimental data. J.C. and X.T. supervised the study. C.P.-G., R.A., J.C. and X.T. wrote the manuscript. All authors contributed to the interpretation of the results and commented on the manuscript.

Corresponding authors

Correspondence to Jaume Casademunt or Xavier Trepat.

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Supplementary information

Supplementary Information

Supplementary Note, Supplementary Figures 1–16, Supplementary References 1–45

Reporting Summary

Supplementary Video 1

Unconfined monolayer exhibiting a transition from wetting to dewetting.Representative example of a spreading monolayer (shown in Fig. 1f) undergoing a wetting transition.The release of confinement at t = 0 h allows the monolayer to freely spread. At ~25 h, the monolayerspontaneously starts retracting until it collapses into a spheroidal aggregate.

Supplementary Video 2

Another example of a wetting transition in a spreading monolayer. Another spreading monolayer (shown in Supplementary Fig. 2) undergoing a wetting transition.

Supplementary Video 3

Evolution of traction and tension fields during wetting and dewetting. Videos of phase contrast images (left), maps of traction (centre) and monolayer tension (right) in a monolayer with increasing concentration of E-cadherin. A wetting transition is observed at time t = 22 h.

Supplementary Video 4

Orthogonal views of monolayer dewetting. Timelapse of MDA-MB-231 cells stably expressing a cell membrane marker (CAAX-iRFP). The tissue-substrate contact area decreases pronouncedly during dewetting, while the tissue evolves from a monolayer to a spheroidal cell aggregate, resembling a droplet.

Supplementary Video 5

Calcium chelation hinders the increase of tissue forces and prevents dewetting. Phase contrast, and maps of traction forces and monolayer tension of control (left) and EGTA-treated (right)cell islands. Cells treated with EGTA move individually rather than forming a cohesive monolayer, suggesting that cell–cell junctions are efficiently abrogated. In the presence of EGTA, both tractions and monolayer tension increase much more slowly than in control islands, and the wetting transition does not occur.

Supplementary Video 6

Dewetting is inhibited and reversed when tissue contractility is externally decreased. Dewetting (left), dewetting inhibition (centre) and reversibility (right) assays. Partial inhibition of contractility with blebbistatin clearly delays the wetting transition. A sudden inhibition of contractility with Y27632 (t = 46 h) is enough to revert dewetting, inducing a rewetting of the substrate. The name of the drug indicates its presence in the cell medium.

Supplementary Video 7

Cell rearrangements in the monolayer. Phase contrast (left) and cell nuclei (right) in a 200 µm radius island during the wetting phase of the experiment. Cells incessantly exchange neighbours, a fact that provides support to the fluid behaviour of the monolayer. Moreover, cells progressively accumulate at the edge of the monolayer, which develops a gentle cell density gradient.

Supplementary Video 8

Evolution of traction and monolayer tension fields in islands of different radii. For all sizes, the magnitude of tractions and monolayer tension increase in time as E-cadherin is progressively expressed. Tractions accumulate at the edges of the monolayers, while monolayer tension has a maximum at the centre. Red frames indicate monolayer dewetting.

Supplementary Video 9

Evolution of traction and monolayer tension fields in islands on substrates of different stiffnesses. For monolayer on substrates of Young’s modulus 3 and 12 kPa, tissue forces increase in time, eventually triggering monolayer dewetting. This transition occurs earlier for the softest substrate. For the stiffest substrate (30 kPa), tissue forces keep increasing until the end of the experiment, suggesting that the critical contractility to induce dewetting is not reached. Red frames indicate monolayer dewetting.

Supplementary Video 10

The wetting transition time depends on tissue radius and substrate ligand density.Cell islands of different radii seeded on substrates with different substrate ligand densities exhibit the wetting transition at different times. Red frames indicate monolayer dewetting.

Supplementary Video 11

Symmetry breaking of monolayer shape during dewetting. A 200 µm radius cell island divided in 24 sectors. Blue = wetting, red = dewetting. Dewetting starts in diametrically opposed regions of the monolayer edge. Hence, the monolayer loses its initial circular shape and acquires an elliptic-like shape during the early stages of dewetting.

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Pérez-González, C., Alert, R., Blanch-Mercader, C. et al. Active wetting of epithelial tissues. Nature Phys 15, 79–88 (2019). https://doi.org/10.1038/s41567-018-0279-5

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