The role of single-cell mechanical behaviour and polarity in driving collective cell migration

Abstract

The directed migration of cell collectives is essential in various physiological processes, such as epiboly, intestinal epithelial turnover, and convergent extension during morphogenesis, as well as during pathological events such as wound healing and cancer metastasis. Collective cell migration leads to the emergence of coordinated movements over multiple cells. Our current understanding emphasizes that these movements are mainly driven by large-scale transmission of signals through adherens junctions. In this study, we show that collective movements of epithelial cells can be triggered by polarity signals at the single-cell level through the establishment of coordinated lamellipodial protrusions. We designed a minimalistic model system to generate one-dimensional epithelial trains confined in ring-shaped patterns that recapitulate rotational movements observed in vitro in cellular monolayers and in vivo in genital or follicular cell rotation. Using our system, we demonstrated that cells follow coordinated rotational movements after establishing directed Rac1-dependent polarity over the entire monolayer. Our experimental and numerical approaches show that the maintenance of coordinated migration requires the acquisition of a front–rear polarity within each single cell but does not require the maintenance of cell–cell junctions. Taken together, these unexpected findings demonstrate that collective cell dynamics in closed environments as observed in multiple in vitro and in vivo situations can arise from single-cell behaviour through a sustained memory of cell polarity.

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Fig. 1: Experimental set-up and directed collective migration of MDCK trains.
Fig. 2: Symmetry breaking in the epithelial train leads to the emergence of single-cell polarity in migrating epithelia.
Fig. 3: Establishment of directed collective cell migration depends on cell–cell junction strength but maintenance does not.
Fig. 4: Mechanics-based simulations of epithelial cell migration on a ring.

Data availability

Source data for Figs. 1–4 and Extended Data Figs. 1–10 are available for this paper. All other data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.

Code availability

The codes for modelling and simulation are available at https://github.com/Viccach/Jain_et_al_2020.git. The custom codes for data visualization and plotting are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank D. Delacour, J. Prost, M. Sokabe, Y. Toyama, R. Voituriez, M. P. Sheetz and group members from MBI and IJM for helpful discussions and critical reading of the manuscript. We thank MBI science communication team members S. Wolf and A. Wong for proofreading the manuscript. We also thank the MBI Microfabrication Core (G. Grenci and M. Asraf) and MBI Microscopy Core (F. Margadant) for continuous support and thank H. T. Ong for her valuable suggestions in image processing and image analysis. We also acknowledge the ImagoSeine core facility of the Institut Jacques Monod, and members of IBiSA and France-BioImaging (ANR-10-INBS-04) infrastructures. We thank W. J. Nelson and F. Martin-Belmonte for their gift of MDCK cell lines. Financial support from the Mechanobiology Institute (C.T.L., B.L.), the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement 617233 (B.L.), USPC-NUS collaborative programme (R.-M.M., B.L.), Agence Nationale de la Recherche (ANR) ‘POLCAM’ (ANR-17- CE13-0013), LABEX ‘Who am I?’ (ANR-11-LABX-0071), Université de Paris IdEx #ANR-18-IDEX-0001, Ligue Contre le Cancer (Equipe labellisée) (B.L., R.-M.M.), BBSRC through grants BB/K018175/1 and BB/P003184/1 (A.J.K.) and the MERLION PhD programme (French Embassy in Singapore) is gratefully acknowledged.

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Contributions

S.J. and B.L. conceived the study and designed the experiments. B.L., R.-M.M., A.J.K. and C.T.L supervised the project. S.J., G.H.N.S.N., V.C., S.D.B., M.L.H. and T.C. performed experiments; S.J., T.C., J.A., G.H.N.S.N., S.D.B., M.L.H., R.-M.M., P.M. and B.L. carried out experimental data analysis; V.M.L.C. and A.J.K. designed and implemented the numerical model, ran the simulations and analysed the resulting data. R.-M.M. and P.M. contributed new reagents, modelling and computational tools. B.L., S.J. and V.M.L.C. wrote the manuscript. All authors critically proofread the manuscript.

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Correspondence to Benoit Ladoux.

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Extended data

Extended Data Fig. 1 Characterization of collective rotation in different conditions and different cell lines.

a, Distribution of rotation direction in rings (n=292, m=10). b, c, Coordination parameter and rotation speed evolution over time in the rings for cells under normal conditions (no-mitomycin, n=46,m=3) or proliferation blocked (with mitomycin, n=62,m=3) condition. The plateau corresponds to the emergence of a persistent rotation. d, Average speed of rotation for mitomycin treated (n=62, m=3) and untreated cells (n=46, m=3). e, Coordination parameters for different cell lines, Eph4 (n=10) and Caco2 (n=30). f, Coordination parameters D for MDCK α-catenin KD cells (n=30) and MDCK Snail-1 over expressing cells (n=30). All error bars indicated are standard deviations. Source data

Extended Data Fig. 2 Repolarization time scale of single cell and single cell polarities.

a,b, Spatio-temporal montage of MDCK-PBD-YFP cells showing repolarization of a single cell upon collision. White arrows (dashed) show the direction of cell migration. Red arrowheads indicate the time of the collision (at 2 min.) and the appearance of Rac1 repolarization signal (at 6 min.), respectively. c, Confocal image of basal plane in a 50 µm wide ring shows a PBD gradients in all the cells indicating cell scale polarized Rac1 activities. d, Line intensity profile (as visualized by PBD biosensor intensity) shows average Rac1 gradient of single cells (n=42 from 2 rings, m=2) from multiple rings (as represented in a single cell marked by line ab in panel c). e, MDCK cells expressing YFP–PBD and mcherry-AHPH. f, Saptio-temporal displacement kymograph shows the single cell (marked with white dashed line) retain the front–rear characteristics during rotations. g, Distribution of AHPH (red) and PBD (green) at single cell level from the orthogonal view at one time point. Typical example of a single cell with boundaries marked in dashed white line (XZ scale bar: 2 µm). h, Line graph showing the intensity distribution of PBD and AHPH in single cells (n=5 cells) of a rotating ring. i, Schematics showing the gradient of PBD and AHPH in single cells of a train. All error bars indicated are standard deviations. All scale bars unless mentioned specifically: 50 µm. Source data

Extended Data Fig. 3 Immunostainings of cell–substrate adhesions (paxillin) and cell–cell adhesion (E-cadherin).

a, Immunofluorescence for E-cadherin-GFP (Z-projection) in a rotating ring. Anti-clockwise direction is indicated by the white dashed arrows. The diffused E-cadherin distribution indicates the cryptic lamellipodia (indicated by white arrows). b, Basal immunofluorescent paxillin staining labelled focal adhesions. c, Nuclei labelled in blue. d, Merge E-cadherin (green), paxillin (red) and nuclei (blue). e-f, Images showing enlarged views of (e’-f’). g, Schematic showing single cells in rotating ring with cryptic lamellipodia revealed by polarized distributions of E-cadherin and paxillin. Scale bars: 50 µm (a-d), 20 µm (e, f).

Extended Data Fig. 4 Cell confinement induces large protrusive activity and high speed of cellular protrusions at the cell front.

a, Schematics explaining the experimental set-up to confine front cell extensions. b, Representative images of actin-GFP cells on fibronectin coated glass substrates. Before confinement (BC) shows cells with a free leading edge and protruding activity. After confinement (AC) shows cells whose leading edges are confined underneath the confining PDMS block (scale bar: 10 µm). c, Displacement kymographs of the cell front before (top) and after confinement (after) (scale bar: 5 µm). d, Cell edge position over time before and after confinement. Negative time correspond to the front edge position before confinement and positive time after confinement (n=10). The slope of curve (dashed magenta line) indicates the velocity of cell protrusions before (0.27 µm/min) and after confinement (1.58 µm/min), respectively. All error bars indicated are standard deviations. Source data

Extended Data Fig. 5 Measurement of cell polarity using orientation of nucleus–centrosome (NC) axis.

a, Schematic of nuclear-centrosome axis in a migrating polarized cell. The NC axis orientation is determined in cells double stained for nuclei (green) and centrosomes (red). This state of polarity is referred here as ‘CN’ when centrosome is placed ahead of nucleus in the direction of migration. When centrosome is placed behind the nucleus, then this state is referred as ‘NC’. Cells with multiple centrosomes and with centrosome either pointing radially inward or outward of the rings were categorized under “No Orientation”. b, NC axis orientation in uncoordinated subconfluent rings (n=141, 13 rings) compared to confluent coordinated rotating rings (n=131, 11 rings). c-d, NC axis orientations and representative immunofluorescence images of nucleus–centrosome positions in conditions of low calcium before the establishment of a coordinated rotation (n=216,12 rings), normal calcium conditions (n=191, 15 rings) and low calcium treatment after the initiation of coordinated rotation (n=385, 23 rings). *Conditions where rings were not rotating persistently in any direction (subconfluent and low calcium case before rotation),a fixed reference direction is chosen (clockwise in this case) in order to calculate the alignment of nucleus–centrosome axis. All error bars indicated are standard deviations. Scale bar: 50 µm. Source data

Extended Data Fig. 6 Effects of Rac1, Arp2/3 and myosin II inhibitions on rotating cell trains.

a, PBD signal distribution before (top) and after (bottom) addition of the Rac1 inhibitor Z62954982 (100 µM) on rotating rings. b, Intensity profile of PBD signal in entire rings before (top) and after (bottom) drug treatment. Expanded views (marked in red box) showing the disappearance of a single cell PBD signal gradient after drug addition. c, Displacement kymograph of the PBD images traced along the circumferential midline of the ring track; the white dashed line indicates drug addition. d, e, Coordination parameters and speeds measured before and after Rac1 inhibition. Black dashed lines indicate drug addition (n=5, m=2). f, Coordination parameters evolution before and following Arp2/3 inhibition (CK666, 100 µM, n=37) and myosin II inhibition (Blebbistatin, 80 µM, n=30). Dashed vertical lines (blue and red color) indicate the point of drug addition respectively. All error bars indicated are standard deviations. All scale bars: 50 µm. Source data

Extended Data Fig. 7 Rac1 polarity gradient perturbation using optogenetics and compromising cell–cell adhesion strength by EGTA treatment.

a, b, Time evolution of PBD signal in cells after homogeneous photoactivation of TIAM. Post activation, TIAM begins to localize at cell membrane. Within 5 minutes of the photoactivation, the PBD gradient flattens in cell indicating the loss of Rac1 polarity in the cell (n=11, from 7 cells). Intensity is measured along the dashed yellow line as indicated in representative panel a. Scale bar: 20 µm. c, Phase contrast imaging shows the loosening of cell–cell junctions after EGTA treatment. Exposed cryptic lamellipodia of migrating cells are marked by white arrowheads after cell–cell junction loosening. The dashed white arrows indicate the direction of rotation. d, Representative displacement kymograph of phase contrast imaged cells traced along the circumferential midline of the ring track before and after EGTA treatment (2 mM) (white dashed line indicates the time of EGTA addition ~ 20 h). e, Coordination parameter of rotating cell rings, before and after EGTA treatment (black dashed line indicates the time of EGTA addition, 20 h) (n=40, m=1). All error bars indicated are standard deviations. All scale bars unless mentioned specifically: 50 µm. Source data

Extended Data Fig. 8 Cell-cell junctions and focal adhesions in the presence of low and normal calcium media.

Nucleus (blue),E-cadherin (green),paxillin (red) immunofluorescence of cell monolayers. E-cadherin accumulation at cell–cell contacts detected in normal medium, disappear in low calcium. By contrast, paxillin staining reveals the presence of focal adhesions in both conditions. Scale bar: 20 µm.

Extended Data Fig. 9 Single cell migration persistency in the absence of cell–cell junctions.

a, Top panel: Phase contrast images of cell trains at low density that show spontaneous detachment of single cells at the free edges. Sequential images showing cell detachment followed by a polarized persistent migration. Expanded view of the dashed white box (last column) reveals the active lamellipodial activity on one side of the cell (marked with white dashed line). Bottom panel: Control experiment showing the behavior of a single MDCK cell with no previous contact with other cells. No preferential migration is observed. Both edges of the cell show lamellipodial activity as marked by dashed white line in the expanded view (last column) from white dashed square box. (Scale bars: 20 µm). The yellow lines indicate the distance travelled by the cells at given times. b, Persistence of cell movements for single isolated cells (n=12) and for single cells detaching from subconfluent trains (n=11). All error bars indicated are standard deviations. All scale bars unless mentioned specifically: 50 µm. Source data

Extended Data Fig. 10 Spatio-temporal distribution of velocities and forces under various condition.

a, Spatio-temporal kymograph of cell train speed for the experiment referred in Fig. 3d. b, c, Spatio-temporal tangential velocity and radial velocity kymograph for the experiment referred in Fig. 3d. d, Tangential traction force spatio-temporal profile of MDCK-PBD cells in rotating ring. e, Displacement kymograph of rotating cells with PBD intensity profile corresponding to the tangential traction force distribution shown in panel d. f, MDCK-PBD cells on ring pattern (Left). Expanded view of corresponding regions of interest for PBD gradients and traction force dipoles (marked by white rectangular box in panel d, e) (right). Scale bar, Ө: 10 µm, t: 30 minutes. g, Cross correlation of tangential traction force profile and PBD signal show a high spatial correlation (n=10,m=2). h, Tangential traction forces post EGTA treatment indicate single cell level force dipoles in a rotating ring before and after EGTA treatment (beginning of rotation is marked by the white dashed line ~ at 3 h), EGTA addition is marked by the white dashed line at ~7 h. i, Tangential traction force distribution in α-catenin KD cells. j, Tangential traction force pattern before and after Apr2/3 inhibition. The white dashed line marks CK666 drug addition (100 µM). k, Spatial velocity correlation C(x’,t) as a function of distance x’ in experiments and simulations (both with 15 cells) showed similar migration dynamics. \(C\left( {x^\prime ,t} \right) = \frac{{\left\langle {u\left( {x + x^\prime ,t} \right) \times u\left( {x,t} \right)} \right\rangle _x}}{{\sqrt {\left\langle {u\left( {x + x^\prime ,t} \right)^2} \right\rangle _x \times \left\langle {u\left( {x,t} \right)^2} \right\rangle _x} }}\), where x, x’ are curvilinear abscissas, u is the angular velocity (positive in counter-clockwise direction) and t is time. l, Ratio of contact and motile forces in 1 simulation (taken from Fig. 4b), recapitulating the force-distribution transition observed in Fig. 3g–h: before polarization starts (t<t1), and once collective rotation is established (t>t3), cells migrate as single dipole and their viscous interaction with the substrate is mainly balanced by their motility. During the coordination process (between t1 and t3), cells form larger dipoles and the contact forces contribute to viscous force balance (t1: cells start polarizing, until they are all polarized at t2, and rotate collectively from t3) (blue thick line: smoothing over time). All error bars indicated are standard deviations. All scale bars unless mentioned specifically: 50 µm. Source data

Supplementary information

Supplementary Information

Supplementary text for simulation and modelling and Fig. 1.

Reporting Summary

Supplementary Video 1

MDCK cells break symmetry and rotate collectively on 1-D ring confinement. Ring outer diameter 200 µm, track width of 20 µm.

Supplementary Video 2

MDCK cell rotation under confinements with different geometries (g1-triangle, g2-ellipse, g3-U shape), diameters(diameter d1:outer-400 µm, middle-200 µm, inner-100 µm, d2-1mm) and widths (w1-50 µm, w2-100 µm, w3-200 µm).

Supplementary Video 3

Other cell types display collective behavior: a, Eph4 and b, Caco2

Supplementary Video 4

Time scale of Rac1 based repolarization in single cells upon collision with the larger train

Supplementary Video 5

Rac1 based front–rear polarity is established in each cell of the rotating ring after symmetry breaking.

Supplementary Video 6

Optogenetics: photoactivation of TIAM at 120 minutes in a quarter region of rotating ring (marked by blue dashed line) is indicated by its junctional localization, which follows a gradual arrest of coordinated rotations in ring.

Supplementary Video 7

MDCK cells fail to initiate rotation and break symmetry a, MDCK-α-Catenin-KD cells and, b, MDCK Snail-1 over expressing cells.

Supplementary Video 8

MDCK cells fail to initiate rotation in presence of low calcium condition but breaks symmetry and begins to rotate upon addition of normal calcium. However, once symmetry is broken, MDCK cells continue to rotate even when low calcium condition is reintroduced.

Supplementary Video 9

Epithelia continuity is not required for the maintenance of rotations: Laser ablation of cells in the rotating ring maintains the directed migration of cells.

Supplementary Video 10

a, Numerical simulations reproduce the symmetry breaking process and polarity establishment in cell rings. Geometrical shapes represent cell centers. Circles: non-polarized cells. Triangles: polarized cells, pointing in their polarity direction. Cell boundaries are not represented, though blue lines indicate the intensity of contact forces on a cell. b, Cell swapping upon migrating train collisions in the case of low cell–cell junction strength-based interactions (top) when compared to cell repolarization during collision in normal cell–cell junction strength (bottom).

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Jain, S., Cachoux, V.M.L., Narayana, G.H.N.S. et al. The role of single-cell mechanical behaviour and polarity in driving collective cell migration. Nat. Phys. 16, 802–809 (2020). https://doi.org/10.1038/s41567-020-0875-z

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