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.

Extended Data Fig. 1. MDCK WT behave as an extensile system

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. 2. 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.

Extended Data Fig. 3. 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. 4. Characterization of MDCK E-cadherin KO cells and extensile/contractile behaviour of MCF7A cells.

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 and 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.

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.

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 Ecadherin 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 Figure 3b’.

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 Ecadherin 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).

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.

Figure 1. Changes from extensile to contractile behaviours in the absence of E-cadherin.

a) Top, left and right: typical examples of traction force magnitude maps for a single MDCK WT and E-cadherin KO cell cultured on deformable PDMS surfaces. Bottom, left and right: vectorial maps of traction forces for a single MDCK WT and E-cadherin KO cell on a soft PDMS substrate. Scale bars, 20μm. b) Schematic showing the defect movement based on force balance for an extensile active nematic system (left) and contractile active nematic system (right) with an inset of forces exerted on neighbours by an extensile (left) and contractile (right) nematic particle. c) Schematic (left) and experimental (right) images of +1/2 defect (left, comet configuration) and -1/2 defect (right, trefoil configuration). Scale bars, 20μm. d) 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 (top) (n = 1934 defects from 2 independent experiments) and MDCK E-cadherin KO cells (bottom) (n = 1,884 defects from 2 independent experiments). Schematic on the extreme right illustrates the movement of defects. Colour code is positive for stretching and negative for shrinkage. e, f) Experimental data for MDCK WT (e) and MDCK E-cadherin KO (f) monolayers. Top panels: phase contrast images of the cells overlaid with the average local orientation of the cells (red lines). Bottom panels: average local orientation of the cells (red lines). The blue circle shows the location of a +1/2 defect and the corresponding arrow indicates the direction of motion of this defect over time. Dashed lines have been added for better reading of defect movement. Scale bars, 40μm.

Figure 2. Inter and intracellular stresses dictate extensile and contractile behaviours.

a) Schematic illustrating the model used in numerical simulations which incorporates cell-cell interaction through active intercellular forces. The direction of cell elongation is denoted by the headless vector ŝ, which is found from the eigenvector corresponding to the largest eigenvalue of the shape tensor 𝐬 for each cell. b) Numerical simulations for the case without active intercellular stresses, showing: (top), phase contrast images of the cells overlaid with the average local orientation of the cells (red lines) and (bottom), average local orientation of the cells (red lines). The blue circle shows the location of a +1/2 defect and the corresponding arrow indicates the direction of motion of this defect over time. c) Average yy- and xy-components of strain rate map around +1/2 defect obtained from simulations (left and middle respectively) and corresponding average velocity flow field (right: n = 2,083 defects) for the control condition (top) and the condition without active intercellular forces. Colour code is positive for stretching and negative for shrinkage. d) RMS velocity, and e) the velocity correlation length in the monolayer normalized to the individual cell size obtained from n=30 different simulations for the control condition and the condition without active intercellular forces.

Figure 3. Knocking out E-cadherin increases cell-substrate interactions.

a) Average isotropic stress around a +1/2 defect obtained from simulations for the control condition (left) and condition without intercellular forces (right) (n = 2,083 defects). b,c) Average yy (left)-, xy (middle)- and isotropic (right) components of stress around a + 1/2 defect obtained from experiments for (b) MDCK WT (n = 1,899 defects) and (c) E-cadherin KO (n = 1,428 defects) from 2 independent experiments. For a and b colour code represents the strength of the stress with positive for tensile state, negative for compression. d, e, f) velocity correlation length (d) (n=10), velocity (e) (n=10) and mean traction force (f) (n=12) of cells within a monolayer for both MDCK WT and MDCK E-cadherin KO cells. g, h) Cell spreading area (g) and aspect ratio (h) of cells within the monolayer obtained from n=10 different images for MDCK WT and E-cadherin KO cells as a function of time from 2 independent experiments. The error bars represent the standard deviation. Unpaired t-test was performed resulting in *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Figure 4. E-cadherin removal triggers mechanotransductive changes within the monolayer.

a) pMRLC (left), zoom of pMRLC (middle), actin (right) staining of MDCK WT (top) and E-cadherin KO (bottom) monolayers. b) Evolution of mean traction force of MDCK WT and E-cadherin KO monolayers before and after 20μM blebbistatin treatment (n=10 from 2 independent experiments). c, d, e) actin (red) and paxillin (green) (c), vinculin (d), YAP (green), and nucleus (blue) (e), staining within a monolayer for both MDCK WT and E-cadherin KO cells. c) Area of focal adhesion (left) and length of focal adhesion within the monolayer for n=106 focal adhesions. d) Mean intensity of vinculin at the cell-cell junction in the middle plane (n=54). e) Distribution of YAP in nucleus, cytoplasm, or uniform distribution calculated for n=1162 cells (MDCK WT) and n=1008 cells (MDCK E-cadherin KO). Error bars represent the standard deviation. Unpaired t-test was performed leading to *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Scale bars, 20μm and 10μm for the figure zooms.

Figure 5. Cell sorting triggered by change in nematic behaviour of monolayers.

a,b) Time lapse sorting of extensile and contractile cells observed over time represented by mixing index in simulations (a) and experiments (b) of MDCK WT (magenta) and E-cadherin KO cells tagged with LifeAct GFP (green). In (a) ζs/Rα = 0.042, ζQ/Rα = -0.062 for the extensile cells and ζs/Rα = 0.0, ζQ/Rα = -0.062 for the contractile cells. Mixing index was obtained from two independent simulations and the error bars mark the standard deviation. Mixing index in experiments (b) was obtained from n=5 different clusters from 2 independent samples. Error bars represent the standard deviation. Scale bars: 100μm.

Figure 6. Cell sorting is governed by activity of the system.

a) Demixing of MDCK WT and E-cadherin KO at different starting densities, WT (30%) and E-cadherin KO (70%) (left) and WT (70%) and E-cadherin KO (30%) (right). b) Demixing of extensile and contractile particles obtained from simulations at different starting densities. Extensile and contractile particles are mixed at 50-50 (left), 30-70 (middle) and 70-30 (right) respectively. In (b) ζs/Rα = 0.016, ζQ/Rα = -0.016 for the extensile cells and ζs/Rα = 0.0, ζQ/Rα = -0.016 for the contractile cells. c) Demixing phase observed before and after the addition of 20μM blebbistatin characterized by mixing index (left) (n=5) and circularity of several colonies (right) (n=5). Error bars represent the standard deviation. Scale bars: 100μm.