Abstract
Mechanical stress is increasingly being shown to be a potent modulator of cell–cell junctional morphologies in developmental and homeostatic processes. Intercellular force sensing is thus expected to be an important regulator of cell signalling and tissue integrity. In particular, the interplay between myosin contractility, actin dynamics and E-cadherin recruitment largely remains to be uncovered. We devised a suspended cell doublet assay to quantitatively assess the correlation between myosin II activity and local E-cadherin recruitment. The single junction of the doublet exhibited a stereotypical morphology, with E-cadherin accumulating into clusters of varied concentrations at the rim of the circular contact. This local recruitment into clusters derived from the sequestration of E-cadherin through a myosin-II-driven modulation of actin turnover. We exemplify how the regulation of actin dynamics provides a mechanism for the mechanosensitive response of cell contacts.
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Acknowledgements
This work was supported by MBI seed funding (NRF grant). V.V. acknowledges additional support from the joint Singapore/France research laboratory LIA CAFS. J.P.T acknowledges IMCB A-star core funding.
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W.E. performed the experiments and handled the data analysis. B.A. prepared the microwells. L.L.Y. prepared the plasmids. W.E., J.P.T. and V.V. conceived the experiments. W.E. and V.V. wrote the manuscript. V.V. supervised the work.
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Integrated supplementary information
Supplementary Figure 1 Digital procedure for rotation compensation and comparison of E-cadherin junctional distribution for different cell types.
(a) Top view (TV1-4) and side view (SV1-4) of a 3D stack used for determining the coordinates of the contact plane. TV1-SV1: deconvoluted stack as imaged in confocal microscopy. TV2-SV2: deconvoluted stack after rotation along the z-axis. TV3-SV3: deconvoluted stack in the referential of the contact. TV4-SV4: raw data in the referential of the contact (scalebar 5 μm). (b) Kymograph of a portion of the E-cad ring before and after image registration (scale bar 5 μm). (c) Typical ring of a MDCK-E-cadGFP doublet (n = 24 cell doublets, 4 days) (left) and an embryonic stem cell E-cadGFP doublet (n = 26 cells, 3 days) (right) (scalebar 5 μm). (d) Western blot of alpha catenin for control S180 cells and cells after alpha catenin knockdown. Cells used to look at the doublets had all the known phenotype of alpha catenin knockdown junctions displaying transient and dynamic cadherin clusters.
Supplementary Figure 2 Measurements of doublet deformation.
(a) Five rings and their anisotropy vector (top line). Same rings after rescaling and alignment of the anisotropy (bottom line) (scalebar 5 μm). (b) Superimposition of n = 5, 20 and 110 rings after image processing taken. (c) Side view schematic of the doublet deformation. (d) Images of the 2 slices taken 1.5 μm above and below the contact used to determine the contour of the cell (top and bottom). Image of the junction with anisotropy and deformation vectors (middle) (scalebar 5 μm). (e) Extreme cases where d1 and d2 have opposite directions (isotrope ring) and same direction (anisotrope ring) (scalebar 5 μm). (f) Anisotropy orientation as a function of the deformation orientation taken during contact formation and at steady state (n = 28 doublets with a total number of 572 time-points).
Supplementary Figure 3 Typical distribution of different junctional proteins.
Typical distributions at the junction of E-cad, phospho-MLC, -catenin, p120, actin, EPLIN, myosin VI and vinculin. The S180-Ecad-GFP cells were transfected with different junctional proteins tagged with m-cherry. The distribution of these proteins are compared to that of E-cad. A total of 116 doublets were observed (scalebar 5 μm).
Supplementary Figure 4 FRAP measurements on E-cadherins and actin.
(a) Typical E-cad FRAP signal for a doublet treated with Y27632 at 5 μM, in control conditions and treated with nocodazole at 10 μM. (b) E-cad recovery time for doublets treated with latrunculin at 1 μM (n = 15 doublets), with Y27632 at 5 μM (n = 21 doublets), in control conditions (n = 24 doublets), with nocodazole at 10 μM (n = 15 doublets) and with jasplakinolide at 100 nM (n = 12 doublets). The p-values are calculated from unpaired Students t-tests. (c) Typical actin FRAP signal for a doublet treated with Y27632 at 5 μM, in control conditions and treated with nocodazole at 10 μM. (d) E-cad recovery time in the dim region and the bright region of the same doublet (n = 22 doublets). The p-value is calculated from a paired Students t-test.
Supplementary Figure 5 Features of the E-cadherin distribution at the junction and cortical tension of single cells.
Data in graphs a–c correspond to the same drugs and concentrations: Y27632 at 2 μM (n = 49 doublets), 5 μM (n = 24 doublets), 10 μM (n = 44 doublets) and 20 μM (n = 23 doublets); blebbistatin at 1 μM (n = 22 doublets), 5 μM (n = 17 doublets) and 50 μM (n = 12 doublets); nocodazole at 10 μM (n = 40 doublets); latrunculin at 1 μM (n = 11 doublets), and 5 μM (n = 22 doublets) and jasplakinolide at 100 nM (n = 18 doublets) (*, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 10 − 4). P-values are calculated from paired Students t-test. The whole distributions of the data values are also shown. a Average full-width at half maximum (FWHM) of the E-cad puncta on the dim side and on the bright side in control conditions (grey) and upon drug treatment. The error bars represent the standard deviation. The whole distribution is also shown. b Inter-cluster distance, contact radius and mean E-cadherin signal along the ring in control conditions (grey) and upon drug treatment. The same statistical tests as a. were applied. c E-cadherin density on the ring, at the center of the contact and on the membrane (as depicted in the schematic) in control conditions (grey) and upon drug treatment. d Image of a single cell deformed by negative pressure (left). Cortical tension of single cells for different drug treatment: latrunculin 1 μM (n = 8 doublets), Y27632 5 μM (n = 8 doublets), control (n = 12 doublets), nocodazole 10 μM (n = 9 doublets) and jasplakinolide at 100 nM (n = 10 doublets) (right).
Supplementary Figure 6 Model of E-cadherin immobilization by actin turnover.
We propose that the immobilization of E-cadherin is regulated by the turnover dynamics of the underlying actin cortex. In turn the turnover dynamics is modulated by the tension of the cortex generated by the actomyosin contractility. This mechanism provides a regulatory pathway for enhanced adhesion in cortex under tension.
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Digital correction of residual rotations.
Illustration of the different stages of correction for the residual rotations of the doublets. The doublets are imaged by confocal imaging with z-stacks spaced by 0.5 μm during the expansion of the contact (E-cad labeled with GFP). The movie displays the maximum intensity projection. From left to right: raw image, correction for azimuthal rotation, additional correction for rotation in the plane of the junction. (AVI 2277 kb)
Protocol for digital correction during contact expansion.
llustration of the effect of correction (Movie 1) projected along the ZY plane. (AVI 426 kb)
Dynamics of contact formation.
Simultaneous dynamics of E-cad (green) and actin (magenta) localization at the rim of the contact during the contact expansion. Time t = 0 corresponds to the initial physical contact between both cells. (AVI 887 kb)
Cell contact shrinkage upon myosin II inhibition.
Dynamics of the shrinkage of the contact after inhibition of myosin II activity by Y27632 at 10 μM. (Ecad GFP). (AVI 195 kb)
Time correlation of cadherin anisotropy and doublet deformation.
Correlated time variations of the E-cad distribution anisotropy a (red) and the doublet deformation at the level of the contact d (green). (AVI 259 kb)
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Engl, W., Arasi, B., Yap, L. et al. Actin dynamics modulate mechanosensitive immobilization of E-cadherin at adherens junctions. Nat Cell Biol 16, 584–591 (2014). https://doi.org/10.1038/ncb2973
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DOI: https://doi.org/10.1038/ncb2973
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