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Substrate area confinement is a key determinant of cell velocity in collective migration

Abstract

Collective cell migration is fundamental throughout development, during wound healing and in many diseases. Although much effort has focused on cell–cell junctions, a role for physical confinement in collective cell migration remains unclear. Here, we used adhesive microstripes of varying widths to mimic the spatial confinement experienced by follower cells within epithelial tissues. Our results reveal that the substrate area confinement is sufficient to modulate the three-dimensional cellular morphology without the need for intercellular adhesive cues. Our findings show a direct correlation between the migration velocity of confined cells and their cell–substrate adhesive area. Closer examination revealed that adhesive area confinement reduces lamellipodial protrusive forces, decreases the number of focal complexes at the leading edge and prevents the maturation of focal adhesions at the trailing edge, together leading to less effective forward propelling forces. The release of follower confinement required for the emergence of leader cells is associated with a threefold increase in contractile stress and a tenfold increase in protrusive forces, together providing a sufficient stress to generate highly motile mesenchymal cells. These findings demonstrate that epithelial confinement alone can induce follower-like behaviours and identify substrate adhesive area confinement as a key determinant of cell velocity in collective migration.

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Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding author upon request.

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Acknowledgements

The authors gratefully acknowledge C.-P. Heisenberg for kindly providing the GFP–UTR zebrafish line, A. Kennard for technical advice for culture of zebrafish embryos and M. Surin for technical support with AFM measurements. The authors thank M. Balland, B. L. Pruitt, M. Sixt, D. Fletcher, J. Theriot and J. Ding for insightful discussions. This work was conducted with the financial support from the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Crédit de Recherches - J009916F) and FEDER Prostem. D.M., J.L., L.A., C.B., E.V. and M.L. are financially supported by FRIA (F.R.S.-FNRS). G.C. is supported by an ERC CoG grant (MolCellTissMech, agreement 647186). K.G. is a Research Associate of the F.R.S.-FNRS.

Author information

D.M. and S.G. conceived the project. G.C. and S.G. supervised the project. D.M. and E.V. performed the experiments. C.B. helped with traction force experiments and data analysis. G.D. and O.T. performed PRIMO experiments to create new adhesive micropatterns. K.G. contributed to the silicon wafer microfabrication. M.V., J.L., L.A., C.B. and M.L. contributed resources to the project. D.M., G.C. and S.G. analysed data, wrote the main manuscript text and prepared the figures. All authors contributed to the interpretation of the results, and improved the manuscript and figure presentations.

Correspondence to Sylvain Gabriele.

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Competing interests

The authors declare no competing interests.

Additional information

Journal peer review information: Nature Physics thanks Kinneret Keren and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–9.

Reporting Summary

Supplementary Video 1

Time-lapse sequence in DIC mode of the growth of an epithelial tissue from a fish scale deposited on a glass coverslip coated with FN. Total duration: 304 min. The scale bar is 100 μm.

Supplementary Video 2

Time-lapse sequence in DIC mode of the migration of a single keratocyte migrating on a 15-μm-wide microstripe. The FN micropattern is shown in fluorescence mode at t = 5 min. Total duration: 9 min 11 s. The scale bar is 10 μm.

Supplementary Video 3

Time-lapse sequence in DIC mode of the migration of a single keratocyte migrating on a FN micropattern composed of 5 interconnected stripes of different widths: 5, 9, 13, 17 and 21 µm. The total length of the micropattern with variable widths is 320 µm. The total time is 109 min and the scale bar is 10 µm.

Supplementary Video 4

Time-lapse sequence in DIC mode of the cantilever assay to measure protrusive forces. A single keratocyte migrating on a 15-μm-wide microstripe pushed the cantilever. One can observe the cantilever deflection in response to the pushing force exerted by the leading edge of the oncoming cell.

Supplementary Video 5

Time-lapse sequence in DIC mode showing a leader cell that escaped from an epithelial tissue. The red arrow at t = 0 min shows the initial position of the leader cell. The total duration time is 25 min. The scale bar is 25 μm.

Supplementary Video 6

Time-lapse sequence in DIC mode showing a single keratocyte that escaped from a narrow microstripe. The escape process was reproduced by using micropatterns composed of adhesive lines 12 μm wide connected to a circular disc of 50 μm in diameter. The total duration time is 5 min 50 s. The scale bar is 10 μm.

Supplementary Video 7

Time-lapse sequence in SiR-actin mode showing a single keratocyte that escaped from a narrow microstripe. The escape process was reproduced by using micropatterns composed of adhesive lines 12 μm wide connected to a circular disc of 50 μm in diameter. The total duration time is 4 min 25 s. The fluorescence intensity is colour-coded on 256 levels. The scale bar is 15 μm.

Supplementary Video 8

Time-lapse sequence in epifluorescent mode of a GFP–UTR zebrafish keratocyte (in green) migrating on a FN-coated microstripe 15 µm wide connected to a circular disc of 50 µm in diameter. The FN micropattern is labelled in red.

Supplementary Video 9

Time-lapse sequence in DIC mode of the migration of a train of cells (n = 2 cells) on a FN-coated microstripe 15 µm wide.

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Further reading

Fig. 1: Epithelial cells are confined within epithelial tissues.
Fig. 2: Cell morphologies and migration velocities are regulated by the lateral confinement.
Fig. 3: 2D confinement modulates the 3D cellular morphology.
Fig. 4: Thicker lamellipodia exert less protrusive forces.
Fig. 5: Escaping a tissue requires the reorganization of the actin cytoskeleton.
Fig. 6: Migration velocity is correlated to cell–substrate adhesive area.