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Cortical F-actin stabilization generates apical–lateral patterns of junctional contractility that integrate cells into epithelia

Nature Cell Biology volume 16, pages 167–178 (2014)Cite this article

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Abstract

E-cadherin cell–cell junctions couple the contractile cortices of epithelial cells together, generating tension within junctions that influences tissue organization. Although junctional tension is commonly studied at the apical zonula adherens, we now report that E-cadherin adhesions induce the contractile actomyosin cortex throughout the apical–lateral axis of junctions. However, cells establish distinct regions of contractile activity even within individual contacts, producing high tension at the zonula adherens but substantially lower tension elsewhere. We demonstrate that N-WASP (also known as WASL) enhances apical junctional tension by stabilizing local F-actin networks, which otherwise undergo stress-induced turnover. Further, we find that cells are extruded from monolayers when this pattern of intra-junctional contractility is disturbed, either when N-WASP redistributes into lateral junctions in H-RasV12-expressing cells or on mosaic redistribution of active N-WASP itself. We propose that local control of actin filament stability regulates the landscape of intra-junctional contractility to determine whether or not cells integrate into epithelial populations.

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Figure 1: Distinct levels of contractile tension coexist within individual E-cadherin junctions.
Figure 2: E-cadherin clusters exchange between the apical and lateral junctions.
Figure 3: Actomyosin contractility generates different patterns of motion on apical and lateral E-cadherin clusters.
Figure 4: E-cadherin establishes the contractile lateral cortex through actin assembly.
Figure 5: Myosin II drives oscillatory fluctuations in the lateral F-actin network.
Figure 6: Local F-actin stabilization by N-WASP determines the apical–lateral pattern of junctional contractility.
Figure 7: Redistributing N-WASP into the lateral cortex drives apical extrusion.
Figure 8: Dysregulated apical–lateral patterns of junctional contractility drive oncogenic extrusion.

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Acknowledgements

We thank our laboratory colleagues for their unstinting support and advice; A. Langendijk and B. Hogan for exploratory experiments; and our many other colleagues who generously provided reagents and helpful suggestions. This work was supported by the National Health and Medical Research Council of Australia, through grants and research fellowships to A.S.Y. (631383, APP1010489, APP1037320, 1044041) and R.G.P. (511055, 569542, APP1037320), the Australian Research Council (DP120104667) and the Kids Cancer Project of The Oncology Children’s Foundation. S.W. was supported by a University of Queensland Research Scholarship. Confocal and optical microscopy was performed at the ACRF Cancer Biology Imaging Facility, established with the generous support of the Australian Cancer Research Foundation.

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Authors and Affiliations

  1. Division of Molecular Cell Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia

    Selwin K. Wu, Guillermo A. Gomez, Magdalene Michael, Suzie Verma, Hayley L. Cox, Robert G. Parton, Nicholas A. Hamilton & Alpha S. Yap

  2. Division of Genomics and Computational Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia

    James G. Lefevre & Nicholas A. Hamilton

  3. School of Mathematics and Physics, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia

    Zoltan Neufeld

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Contributions

S.K.W., G.A.G., Z.N. and A.S.Y. conceived the project; S.K.W. performed most of the experiments except for the nanoablation and western analyses, which were shared with G.A.G., M.M. and S.V.; R.G.P. contributed to caveolin analysis; S.K.W., G.A.G., J.G.L. and H.L.C. quantified the data; S.K.W., G.A.G., N.A.H. and Z.N. performed quantitative modelling; S.K.W., G.A.G. and A.S.Y. analysed the data and wrote the paper.

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Integrated supplementary information

Supplementary Figure 1 Regional disparity in apical and lateral junctional tension.

a, Cartoon of apicolateral imaging at slanted (en face) cell-cell contacts. b, Representative image at the cell-cell contact of cells expressing GFP-UtrCH (F-actin, green) and immunostained for Myosin IIB (red). c, d, Representative confocal images at various time points before and after nanoablation at either apical (c) or lateral (d) regions of cell-cell contacts. Scale bars: 5 μm.

Supplementary Figure 2 Dynamic lateral E-cadherins are trans-interacting clusters.

a, Surface expression of E-cadherin was measured with trypsin protection assays. Cells were lysed immediately (Total) or after trypsinisation in the presence (+Ca) or absence (–Ca) of extracellular calcium. Cell lysates were immunoblotted for E-cadherin and GAPDH. Uncropped images of blots are shown in Supplementary Fig. 9. b, Representative immunofluorescence images of the interface between Ecad-GFP and Ecad-tdTomato cells, and the corresponding merged image segmented by OBCOL to resolve individual E-cad clusters. c, Mean square displacement (MSD) of apical and lateral cadherin clusters. d,e, Radial velocity distribution of E-cad clusters in planar and tilted contacts (d). Effect of contact inclination on Mean Square Displacement (MSD) measurements of E-cadherin clusters (e). Simulation of tracking data to produce MSD values at 20°,30°,40°,50°,60°and70° tilt angles respectively. f, Pooled density fluctuations of E-cad clusters within 1 μm2 regions of analysis at the lateral interfaces of Ecad-GFP and Ecad-tdTomato expressing cells (average Pearson’s coefficient = 0.67). Data are means ± SEM calculated from n = 9 individual cell-cell contacts (28 individual cadherin clusters) from 2 independent experiments (statistical information and source data in Supplementary Data 1). Scale bars: 5 μm.

Supplementary Figure 3 Actomyosin network exerts contractile forces on lateral E-cad clusters.

a, Immunofluorescence image (a, merged, a’, in detail) of cell-cell contact immunostained for E-cad (blue), Myosin IIA (red) and Myosin IIB (green). A high magnification view (a”) of the indicated area in a is shown. b, F-actin integrity and active myosin support lateral cadherin oscillations. Fourier analysis of lateral E-cad after treatment with DMSO (Control, green), latrunculin A (Lat A, red) or Y-27632 (black). Power spectrum amplitude for the full-range of frequencies are shown. c,c’, Myosin IIA (c) or Myosin IIB (c’) with GAPDH (loading control) immunoblots of lysates from control, Myosin IIA KD and Myosin IIB KD cells. Uncropped images of blots are shown in Supplementary Fig. 9. d, Myosin II isoforms support lateral cadherin oscillations. Power spectrum amplitude for the full-range of frequencies from Fourier analysis of lateral E-cadherin radial velocity in cells expressing either pLL5.0 empty vector (Control, green), shRNA against Myosin IIA (NMIIA KD, red) or shRNA against Myosin IIB (NMIIB KD, black). All data are means ± SEM calculated from n = 3 independent experiments (statistical information and source data in Supplementary Data 1). Scale bars: (a) 5 μm, (a’,a”), 0. 5 μm.

Supplementary Figure 4 E-cadherin supports junctional contractility through Arp2/3 based actomyosin assembly.

a, Ecad-GFP and Caveolin-1-mCherry expressed at the lateral junctions do not colocalize from Supplementary Video 4. b,c, Representative images of Caveolin-1-GFP (Cav1-GFP, b) and power spectrum obtained from Fourier analysis of Cav-1-GFP puncta radial velocity (c) at the lateral junctions in control (green) and E-cadherin knockdown cells (E-cad KD, red). d, E-cad, Arp3 and GAPDH (loading control) immunoblots of lysates from control or E-cadherin KD cells. e, Fourier analysis of lateral E-cadherin in control (green) or ArpC2 knockdown (red) cells. f, ArpC2, Arp3, E-cadherin, Myosin IIA, Myosin IIB and GAPDH (loading control) immunoblots of lysates from control or ArpC2 knockdown cells. Uncropped images of blots in (d) and (f) are shown in Supplementary Fig. 9. All data are means ± SEM calculated from n = 3 independent experiments (statistical information and source data in Supplementary Table 1). Scale bars: 5 μm.

Supplementary Figure 5 Lateral F-actin turnover determines E-cad cluster oscillation.

a, Time series of actin network condensation from isotropic cortical network with superimposed tracks (white) of cadherin clusters. b, Average lateral F-actin cable intensity and the corresponding normalized distances between E-cad clusters plotted against time. c, d, Jasplakinolide inhibits lateral cadherin oscillations. Representative radial velocity traces (c) and Fourier analysis (d) of lateral E-cadherin in cells treated with either DMSO (Control, green) or jasplakinolide (Jasp, red). All data are means ± SEM calculated from n = 3 independent experiments (statistical information and source data in Supplementary Table 1). Scale bars: 0.5 μm.

Supplementary Figure 6 N-WASP regulates junctional contractility.

a, Recoil curves of apical and lateral junctions in control or N-WASP KD cells. b, b’, Representative radial velocity traces of apical (red: Control Apical, black: N-WASP KD Apical) or lateral (green: Control Lateral, blue: N-WASP KD Lateral) E-cad in cells transduced with lentivirus bearing either pLL5.0 empty vector (b) or shRNA against N-WASP (b’). c, Fourier analysis and power spectrum amplitude for the full-range of frequencies of ZA and lateral E-cad cluster radial velocity in control and N-WASP knockdown cells. d, Schematic of Ecad-GFP-N-WASPΔVCA fusion protein. e, GFP and GAPDH (loading control) immunoblots of lysates from Ecad-GFP and E-cad-GFP-N-WASPΔVCA cells. Uncropped images of blots are shown in Supplementary Fig. 9. f, Representative images of junctional Ecad-GFP, E-cad-GFP-N-WASPΔVCA and WIRE. Lower detailed panels show colocalization of lateral E-cad-GFP-N-WASPΔVCA with WIRE. However, lateral Ecad-GFP does not colocalize with WIRE. Scale bars are 5 μm except for magnified view: 0.5 μm. All data are means ± SEM calculated from n = 3 independent experiments except for (a) which were technical replicates (means ± SD, n = 19 contacts) from one out of two independent experiments (statistical information and source data in Supplementary Table 1).

Supplementary Figure 7 N-WASPΔVCA promotes junctional contractility by F-actin stabilization.

a, b, Representative radial velocity traces (a) and Fourier analysis (b) of both lateral Ecad-GFP and Ecad-GFP-N-WASPΔVCA. c, Recoil of the lateral junctions of Ecad-GFP and Ecad-GFP-N-WASPΔVCA expressing monolayer. d, Lateral recoil in DMSO (Control) or Jasplakinolide (Jasp) treated cells. e, f, Recoil at apical (e) and lateral (f) interfaces of cells either expressing Ecad-GFP/WT or Ecad-GFP-N-WASPΔVCA/WT in N-WASP + or N-WASP KD monolayer. All data are means ± SEM; calculated from n = 3 independent experiments (statistical information and source data in Supplementary Table 1).

Supplementary Figure 8 Characterization of junctional contractility at the interface of WT/WT, H-RasV12/H−RasV12 and WT/ H-RasV12 cells.

a, Design of a HA-tagged H-RasV12 construct co-expressing Ecad-GFP and E-cad shRNA in the pLL5.0 vector by introduction of an IRES sequence. b, E-cadherin trans-interactions are unaffected at the interface of wild-type and transformed cells. Pooled density fluctuations of E-cad clusters within 1 μm2 regions of analysis at the lateral interface of Ecad-GFP-IRES-HA-H-RasV12 and Ecad-tdTomato expressing cell (average Pearson’s coefficient = 0.74). c, d, Recoil of apical (c) and lateral (d) junctions at the interface of WT/WT, H-RasV12/H-RasV12 and WT/H-RasV12. e, f, Radial velocity traces (e) and Fourier analysis (f) of lateral E-cad at the interface of WT/WT, H-RasV12/H-RasV12 and WT/H-RasV12. g, Recoil of lateral junctions at interface between H-RasV12/WT cells in either control or N-WASP KD monolayers. All data are means ± SEM; calculated from n = 3 independent experiments (statistical information and source data in Supplementary Table 1).

Supplementary Figure 9 Uncropped Western Blots related to Supplementary Figs 2a,3c,c’,4d,f, and 6e.

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Distinct levels of contractile tension coexist within individual E-cadherin junctions.

Nanoablation of apical and lateral cell-cell junctions in Caco-2 cells expressing Ecad-GFP in E-cad knockdown background. Arrowhead indicates the point of laser ablation with a Ti:Sapphire laser (Chameleon Ultra, Coherent Scientific, US) tuned to 790 nm. Scale bar: 5 μm. (MP4 77 kb)

Dynamic exchange of E-cadherin between apical and lateral cell-cell contacts.

Ecad-mRFP-PA-GFP photoactivation at the lateral (upper panels) or apical (lower panels) regions of a cell-cell contact. mRFP fluorescence images are in red and activated PAGFP fluorescence is in green. Scale bar: 5 μm. (MP4 3932 kb)

E-cad clusters do not colocalize with endosomal markers.

Dual-colour time-lapse imaging of Ecad-GFP with either internalized Alexa Fluor 546 conjugated Transferrin with or mCherry-2XFYVE at cell-cell contacts. Scale bar: 5 μm (MP4 1865 kb)

E-cad clusters and caveolin-1 puncta do not colocalize but display similar oscillatory motion.

Dual-colour time-lapse imaging of Cav1-mCherry (red) and Ecad-GFP (green) at a cell-cell contact. Scale bar: 5 μm (MP4 3080 kb)

E-cad clusters display different patterns of movement at apical versus lateral regions of cell-cell contacts.

Live imaging of Caco-2 cells expressing Ecad-GFP in an epithelial monolayer (left). Magnified timelapse movie of an E-cad cell-cell contact (right). Note the pulsatile movement of the lateral E-cad clusters. Each image is a projection of a 6 μm Z-stack. The initial still frame outlines the apical (red) and lateral (green) regions of the cell-cell contact. Scale bar: 5 μm (MP4 10503 kb)

E-cadherin puncta represent adhesive clusters.

Mixed population of Ecad-GFP and Ecad-tdTomato expressing cells (left). Magnified timelapse movie of a lateral cell-cell contact at the interface between Ecad-GFP and Ecad-tdTomato expressing cells. Scale bar: 5 μm (MP4 10548 kb)

Myosin II drives oscillatory fluctuations in the lateral F-actin network to drive E-cad cluster motion.

Live imaging of Caco-2 cells co-expressing Ecad-GFP (green) and TagRFP-T-UtrCH (F-actin, red) at cell-cell contacts between control, NMIIA knockdown or NMIIB knockdown cells. Upper panels show E-cadherin-GFP fluorescence images in grayscale. Scale bar: 5 μm (MP4 9100 kb)

E-cad is required for oscillatory motion at lateral cell-cell contacts.

Time-lapse live cell imaging of Cav1-GFP at a cell-cell contact in control and E-cad RNAi cells. Scale bar: 5 μm (MP4 1672 kb)

Arp2/3 complex is required to generate oscillatory motion of lateral E-cad clusters.

Live imaging of Ecad-GFP at a cell-cell contact in control or ArpC2 knockdown cells. Scale bar: 5 μm (MP4 1483 kb)

Actin cables connecting E-cad clusters condense from a low intensity isotropic F-actin network.

Ecad-GFP (green) and TagRFP-T-UtrCH (F-actin, red) at a cell-cell contact (left) and F-actin only (center and right panels). Note that a low intensity isotropic F-actin network condenses into brighter F-actin cables connecting E-cad clusters. Scale bar: 5 μm (MP4 4183 kb)

F-actin stabilization by jasplakinolide reduces oscillation of E-cad clusters.

Live imaging of Ecad-GFP at a cell-cell contact after 10 minutes of DMSO (control) or jasplakinolide treatment. Scale bar: 5 μm (MP4 1168 kb)

Distinct apicolateral regions of F-actin stability at cell-cell contacts.

Time-lapse imaging of F-actin (GFP-UtrCH, hot cyan) at a cell-cell contact (left) and the corresponding heat map (right). The top region corresponds to the zonula adherens. Scale bar: 5 μm (MP4 2245 kb)

N-WASP restrains oscillatory motion of apical E-cad clusters.

Ecad-GFP at a cell-cell contact of control or N-WASP knockdown cells. Note the pulsatile behaviour of apical E-cad in N-WASP knockdown cells. The apical and lateral regions of the cell-cell contact are labelled. Scale bar: 5 μm (MP4 688 kb)

Fusion with N-WASPΔVCA reduces oscillatory motion of lateral cadherin clusters.

Live cell imaging at cell-cell contacts on cells expressing either Ecad-GFP or Ecad-GFP-N-WASPΔVCA. Scale bar: 5 μm. (MP4 2006 kb)

Homophilic clustering of lateral E-cad persists at the interface of a presumptive extruding H-RasV12 expressing cell, however the oscillatory motion of lateral clusters is reduced.

Live imaging of either a WT/WT or a WT/H-RasV12 interface, where an Ecad-GFP (Green, Wild-Type or Transformed) cell is surrounded by Ecad-tdTomato (Red) expressing cells. Scale bar: 5 μm. (MP4 1474 kb)

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Wu, S., Gomez, G., Michael, M. et al. Cortical F-actin stabilization generates apical–lateral patterns of junctional contractility that integrate cells into epithelia. Nat Cell Biol 16, 167–178 (2014). https://doi.org/10.1038/ncb2900

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