1. Cell Biology
  2. Physics of Living Systems
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Myosin II isoforms play distinct roles in adherens junction biogenesis

  1. Mélina L Heuzé  Is a corresponding author
  2. Gautham Hari Narayana Sankara Narayana
  3. Joseph D'Alessandro
  4. Victor Cellerin
  5. Tien Dang
  6. David S Williams
  7. Jan CM Van Hest
  8. Philippe Marcq
  9. René-Marc Mège  Is a corresponding author
  10. Benoit Ladoux  Is a corresponding author
  1. Université de Paris and CNRS UMR 7592, France
  2. Swansea University, United Kingdom
  3. Eindhoven University of Technology, Netherlands
  4. Sorbonne Université and CNRS UMR 7636, France
Research Article
Cite this article as: eLife 2019;8:e46599 doi: 10.7554/eLife.46599
8 figures, 3 videos, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Development of an in vitro system for the study of junction biogenesis.

(a) Sequential steps for controlled initiation and visualization of junction biogenesis. The two cells are initially confined on a pair of fibronectin-coated 5 µm-away patterns (T0). When desired, the cell confinement is released by addition of BCN-RGD peptide, inducing cell spreading and kissing within a few hours. Scale Bar: 10 µm. (b) Spinning disk image sequence showing contact extension between two MDCK cells expressing GFP-E-cadherin and stained with Hoechst. Scale bar: 10 µm. (c) Kymographs of the junction forming in panel b, generated from the yellow line, shown in green and in pseudocolor to highlight GFP-E-cadherin accumulation at junction tips. The junction axis was realigned horizontally for some time points in order to generate the kymograph on a long time scale. Scale bar: 5 µm. (d) Representative confocal images of β-catenin-stained junctions from MDCK cell doublets. The arrow points at small holes frequently observed within Y27-treated junctions. The cells were fixed 20 hr after addition of BCN-RGD alone or BCN-RGD + Y27 (50 µM). Scale bar: 10 µm. (e) Graphs showing the evolution of junction length in function of time after contact initiation in Ctrl and Y27-treated MDCK cell doublets. Y27 (50 µM) was added with BCN-RGD. Data are represented as mean + /- SEM. n = 13 and 12 cell doublets from two and three independent experiments, respectively. (f) Bar graph of the percentage of cell doublets that stay in contact for more than 3 hr in Ctrl and Y27-treated MDCK cells, respectively. Data are represented as mean + /- SEM. n = 13 and 12 cell doublets from two and three independent experiments, respectively. Bonferroni statistical tests were applied for p value. (g) Spinning disk image sequence of GFP-E-cadherin-expressing MDCK cells pre-stained with Hoechst in the presence of Y27 (50 µM). The sequence starts 3 hr after addition of BCN-RGD + Y27. The arrows highlight transient contacts forming under these conditions. Scale bar: 10 μm.

https://doi.org/10.7554/eLife.46599.002
Figure 1—source data 1

Development of anin vitrosystem for the study of junction biogenesis.

https://doi.org/10.7554/eLife.46599.005
Figure 1—figure supplement 1
Reversal of nucleus-centrosome polarity axis after cell-cell contact.

(a) Scheme depicting the switchable micro-patterning strategy used for confinement release (click chemistry). (b) Spinning disk image sequence of GFP-E-cadherin and RFP-Pericentrin of doubled transfected MDCK cells pre-stained with Hoechst. Scale bar: 10 µm. (c) Plots of nucleus-centrosome axis polarization angle relative to junction axis after cell-cell contact. Data are represented as mean + /- SEM. n = 19 doublets from three independent experiments. (d) Distribution of nucleus-centrosome axis polarization angles relative to junction axis after cell-cell contact in Ctrl or Y27-treated MDCK cells at the time when junctions reach their maximal length. n = 15 and 21 cells from three and two independent experiments respectively.

https://doi.org/10.7554/eLife.46599.003
Figure 1—figure supplement 1—source data 1

Reversal of nucleus-centrosome polarity axis after cell-cell contact.

https://doi.org/10.7554/eLife.46599.004
Figure 2 with 1 supplement
NMIIA and NMIIB are both required for proper junction biogenesis.

(a, b) Left panels: Representative immunoblots showing the isoform specific knockdown of NMIIA (a) and NMIIB (b) in NMIIA KD and NMIIB KD MDCK cells. GAPDH expression levels were used as loading controls. Right panels: Bar graphs showing the relative expression level of NMIIA and NMIIB proteins in Ctrl, NMIIA KD and NMIIB KD cells normalized to GAPDH expression levels. Data are represented as mean + /- SEM from three independent experiments. Kruskall-Wallis statistical tests were applied for p value. (c) Representative epifluorescence image sequences of GFP-E-cadherin over a time course of 5 hr showing the dynamics of junction formation at low magnification in Ctrl, NMIIA KD and NMIIB KD MDCK cells. The arrows indicate the position and the orientation of the junctions. Scale bar: 10 µm. (d) Bar graph of the percentage of cell doublets that stay in contact for more than 3 hr. Data are represented as mean + /- SEM. Tukey’s multiple comparison statistical tests were applied for p value. n = 36, 37 and 31 cell doublets for Ctrl, NMIIA KD and NMIIB KD cells respectively, from three independent experiments. (e) Plots showing the evolution of junction length in function of time for Ctrl, NMIIA KD and NMIIB KD cell doublets. Data are represented as mean + /- SEM. n = 40, 43 and 35 cell doublets for Ctrl, NMIIA KD and NMIIB KD cells respectively, from four independent experiments. (f) Box and whiskers graphs representing the junction length after 3 hr after contact, for Ctrl, NMIIA KD and NMIIB KD cell doublets. n = 34, 21 and 28 cell doublets for Ctrl, NMIIA KD and NMIIB KD cells respectively, from four independent experiments. (g) Box and whiskers graphs showing the junction straightness (calculated as the euclidean/accumulated length ratio) in Ctrl, NMIIA KD and NMIIB KD cell doublets 2 hr after contact. n = 12, 15 and 17 cell doublets for Ctrl, NMIIA KD and NMIIB KD cells respectively, from three independent experiments. (h) Box and whiskers graph showing the angular deviation of junctions during the three first hours of contact in Ctrl, NMIIA KD and NMIIB KD cell doublets. n = 35, 30 and 32 cell doublets for Ctrl, NMIIA KD and NMIIB KD cells respectively, from four independent experiments. (f–h) Mann-Whitney statistical tests were applied for p value. (i) Representative spinning disk GFP-E-cadherin image sequences over a time course of 4 hr showing the dynamics of junction formation at high magnification in Ctrl, NMIIA KD and NMIIB KD MDCK cells. The red arrows point at junctional extensions typically observed in NMIIB KD doublets. Scale bar: 10 µm.

https://doi.org/10.7554/eLife.46599.008
Figure 2—source data 1

NMIIA and NMIIB are both required for proper junction biogenesis.

https://doi.org/10.7554/eLife.46599.010
Figure 2—figure supplement 1
Isoform-specific NMII Knock-down in MDCK cells.

(a, b) Original uncropped Immunoblots presented in Figure 2a (a) and 2b (b).

https://doi.org/10.7554/eLife.46599.009
Figure 3 with 2 supplements
NMIIB, but not NMIIA, localizes to early AJs.

(a, b) Representative confocal images and zoom boxes of GFP-E-cadherin-expressing MDCK cell doublets fixed 20 hr after BCN-RGD addition and immuno-stained for NMIIA (a) or NMIIB (b) Scale bar: 10 µm. (c, d) Relative intensity profiles (raw and smoothed data) of GFP-E-cadherin and NMIIA (c) or NMIIB (d) signals along the lines represented in (a) and (b) respectively. (e) Box and whiskers graphs showing the Pearson’s coefficient values that reflects the co-localization of NMIIA, NMIIB with E-cadherin quantitatively. n = 9 to 24 junctions. Mann-Whitney statistical tests were applied for p value. (f, g) Representative confocal images of WT MDCK cells plated on fibronectin-coated glass for 1 or 3 days and stained for F-actin, NMIIA (f) and NMIIB (g). Scale bar: 10 µm.

https://doi.org/10.7554/eLife.46599.012
Figure 3—source data 1

NMIIB, but not NMIIA, localizes to early AJs.

https://doi.org/10.7554/eLife.46599.017
Figure 3—figure supplement 1
NMIIA and NMIIB exhibit differential localizations in early AJs.

(a, b) Representative confocal images of MDCK cell doublets fixed 20 hr after BCN-RGD addition and stained for F-actin, NMIIA and NMIIC (a) or F-actin, NMIIB and Vimentin (b) as indicated. Scale bar: 10 µm. (c) Representative confocal images of NMIIA KD and NMIIB KD MDCK cells plated at low density on fibronectin, fixed after 12 hr and immuno-stained for NMIIA, NMIIB and E-cadherin. Scale bar: 20 µm. (d, e) Representative confocal images with zoom boxes of WT MDCK cells transfected with GFP-NMIIA (d) or mCherry-NMIIB, plated on fibronectin-coated glass for 1 day and immuno-stained for β-catenin, relative intensity profiles of β-catenin and NMIIA or NMIIB signals along the lines represented in (d) and (e) respectively. Scale bar: 20 µm in original and 5 µm in zoomed images.

https://doi.org/10.7554/eLife.46599.013
Figure 3—figure supplement 1—source data 1

NMIIA and NMIIB exhibit differential localizations in early AJs.

https://doi.org/10.7554/eLife.46599.014
Figure 3—figure supplement 2
NMIIB, but not NMIIA, localizes to early epithelial AJs.

(a) Representative confocal images of WT MDCK cells plated on fibronectin-coated glass for 1 or 3 days and stained for F-actin, NMIIA and NMIIB. Scale bar: 10 µm. (b) Representative confocal images of CaCo2 cells plated on fibronectin-coated glass and stained for F-actin, NMIIA and NMIIB. Scale bar: 10 µm. (c) Representative confocal images and zoom boxes of WT (upper panel) or α-catenin KD (lower panel) MDCK cells fixed 20 hr after BCN-RGD addition and immuno-stained for NMIIA and NMIIB. White arrow heads indicate the cell-cell contact which is depicted as a dotted line in α-catenin KD MDCK cells. Scale bar: 10 µm. (d) Relative intensity profiles of NMIIA and NMIIB signals within WT or α-catenin KD cell-cell contacts along the lines represented in (c). (e) Representative epifluorescence images and zoom boxes of WT MDCK cells fixed 20 hr after BCN-RGD addition and immuno-stained for Arp3, NMIIB and F-actin. Scale bar: 10 µm. (f) Relative intensity profiles of Arp3, NMIIB and F-actin signals along the line represented in (e).

https://doi.org/10.7554/eLife.46599.015
Figure 3—figure supplement 2—source data 1

NMIIB, but not NMIIA, localizes to early epithelial AJs.

https://doi.org/10.7554/eLife.46599.016
NMIIB localizes to a junctional actin network distinct from NMIIA-associated actin.

(a–b) SIM (Structured Illumination Microscopy) images of WT MDCK cells fixed 20 hr after addition of BCN-RGD and stained as indicated. Scale bar: 3 µm. (c) Relative intensity profiles (raw and smoothed data) of NMIIB, NMIIA and F-actin signals along the line represented in (a). (d) Box and whiskers graphs showing the Pearson’s coefficient values that reflects the co-localization of F-actin and NMIIA or NMIIB in junctional and peri-junctional areas. n = 18 to 33 junctions. For p values, pairwise t tests were applied to compare junctional vs perijunctional data for the same isoform and Mann-Whitney statistical tests to compare the two isoforms. (e, f). SIM images of nascent contacts formed between WT MDCK cells. Scale bar: 3 µm.

https://doi.org/10.7554/eLife.46599.018
Figure 4—source data 1

NMIIB localizes to a junctional actin network distinct from NMIIA-associated actin.

https://doi.org/10.7554/eLife.46599.019
Figure 5 with 1 supplement
NMIIB supports juxtamembrane actin organization and regulates α-catenin unfolding.

(a) SIM (Structured Illumination Microscopy) images of junctional areas from Ctrl, NMIIA KD and NMIIB KD cells fixed 20 hr after addition of BCN-RGD and stained for F-actin and β-catenin. Scale bar: 5 µm. (b) Representative confocal images with zoom boxes of Ctrl MDCK cells. (c) Relative intensity profiles of cortactin and NMIIA or NMIIB signals along the lines represented in (b, d). Box and whiskers graphs showing the Pearson’s coefficient values for co-localization of cortactin with NMIIA or NMIIB at cell-cell junctions n = 31 and 36 junctions respectively, Mann-Whitney statistical tests were applied for p value. (e) Ctrl, NMIIA KD and NMIIB KD cells stained for NMIIA, NMIIB and cortactin as indicated. Scale bars: 10 µm in original and 5 µm in zoomed images. (f) Relative intensity distribution profiles of cortactin signal along lines drawn perpendicular to junction in Ctrl, NMIIA KD and NMIIB KD cells, n = 15 cell-cell junctions respectively. (g) Representative confocal images of junctional area from Ctrl, NMIIA KD and NMIIB KD cells stained for α-catenin and α-cat18. Scale bar: 10 µm. (h, i) Scatter plots with mean + /- SEM showing the ratio of junctional α-cat18/α-catenin signals (h) and the mean intensity levels of α-catenin signal at the junction (i) n = 27, 20, 25 cell doublets for Ctrl, NMIIAKD and NMIIBKD, respectively from two independent experiments. Kruskal-Wallis statistical tests were applied for p value.

https://doi.org/10.7554/eLife.46599.020
Figure 5—source data 1

NMIIB supports juxtamembrane actin organization and regulates α-catenin unfolding.

https://doi.org/10.7554/eLife.46599.022
Figure 5—figure supplement 1
NMIIB supports junctional actin organization.

Related to Figure 5a: other examples of junctional actin organization in Ctrl, NMIIA KD and NMIIB KD cells. (a, b) SIM (Structured Illumination Microscopy) images of junctional area from Ctrl, NMIIA KD and NMIIB KD cells fixed 20 hr after addition of BCN-RGD and stained for F-actin and β-catenin (a) or α-catenin (b) Scale bar: 5 µm.

https://doi.org/10.7554/eLife.46599.021
Figure 6 with 3 supplements
NMIIA and NMIIB are both required for establishment of proper inter-cellular stress.

(a) Heat map with vectorial field of traction forces (left panels) and ellipse representation of intra-cellular stress (right panel, the two axes represent the direction and magnitude of the principal components of the stress tensor, positive values in red, negative values in blue) of inter-cellular stress (right panels) in Ctrl, NMIIA KD and NMIIB KD cell pairs before, during and after contact on fibronectin-coated PDMS deformable substrate (30 KPa). Cell contours are drawn in black. The red arrows indicate a hotspot of traction forces observed frequently in NMIIB KD cell doublets. Scale bar: 10 µm. (b) Linear graphs representing the resultant forces of cell doublets and individual cells before, during and after contact in Ctrl, NMIIA KD and NMIIB KD. Data are represented as mean + /- SEM. (c) The same data as in (b) were represented as bar graph with mean + /- SEM for statistical comparisons between Ctrl, NMIIA KD and NMIIB KD cells 30 min before, during and 30 min after contact. Bonferroni statistical tests were applied for p value. (d) Scatter plots with mean + /- SEM representing inter-cellular stress in the junctional area in Ctrl, NMIIA KD and NMIIB KD cells within the first 3 hr of contact. For each junction, six values corresponding to 30 min time points are plotted. The stress orientation was divided in the parallel and perpendicular components relative to the main axis of the junction. Pairwise statistical t tests (for intra-group comparisons) and Mann-Whitney statistical t tests were applied for p value. (b–d) n = 25, 26 and 28 cell doublets for Ctrl, NMIIA KD and NMIIB KD, respectively, from three independent experiments.

https://doi.org/10.7554/eLife.46599.023
Figure 6—source data 1

NMIIA and NMIIB are both required for establishment of proper inter-cellular stress.

https://doi.org/10.7554/eLife.46599.030
Figure 6—figure supplement 1
NMIIA regulates cell adhesion and traction forces on fibronectin.

(a) Representative confocal images of paxillin and F-actin staining of Ctrl, NMIIA KD and NMIIB KD single cells plated on fibronectin-coated glass coverslip for 16 hr. Scale bar: 10 µm. (b, c) Scatter plots with mean + /- SEM showing the spreading area (b) and number of focal adhesions (c) of Ctrl, NMIIA KD and NMIIB KD single cells plated on fibronectin for 16 hr. n = 23, 21 and 30 cells for (b) and 26, 39 and 34 cells for (c) from two independent experiments. Kruskal-Wallis statistical tests were applied for p value. (d) Heat map (upper panel) and vectorial field (lower panel) representing respectively the magnitude and the orientation of traction forces exerted by the single Ctrl, NMIIA KD and NMIIB KD cells, on fibronectin-coated PDMS deformable substrate (30 KPa). Cell masks used for quantification are drawn in white. Scale bar: 10 µm. (e) Scatter plots with mean + /- SEM showing the mean traction forces exerted by single Ctrl, NMIIA KD and NMIIB KD cells. n = 55, 65 and 74 cells, respectively, from three independent experiments. Kruskall-Wallis statistical tests were applied for p value.

https://doi.org/10.7554/eLife.46599.024
Figure 6—figure supplement 1—source data 1

NMIIA regulates cell adhesion and traction forces on fibronectin.

https://doi.org/10.7554/eLife.46599.025
Figure 6—figure supplement 2
NMIIB favors E-cadherin clustering on E-cadherin-coated substrate.

(a) Confocal images with zoom boxes of Ctrl, NMIIA KD and NMIIB KD cells plated on E-cadherin-coated glass for 6 hr and immuno-stained for β-catenin and F-actin. Scale bar: 10 µm. (b) Scheme depicting the experimental set-up. (c) Scatter plots with mean + /- SEM showing the cell spreading area of Ctrl, NMIIA KD and NMIIB KD cells plated on E-cadherin coated glass after 6 hr. n = 87, 58 and 58 cells respectively from two independent experiments. Kruskal-Wallis statistical tests were applied for p value. (d) Bar graph showing the number of β-catenin clusters per cell in Ctrl, NMIIA KD and NMIIB KD cells plated on E-cadherin coated glass. The clusters were classified in two categories: large clusters with area larger than 1 µm², and small clusters with area ranging from 0.2 µm² to 1 µm². Data are represented as mean + /- SEM, n = 26, 27 and 26 cells respectively from two independent experiments. Kruskal-Wallis statistical test were applied for p value (e) Heat map (top panel) and vectorial field (bottom panel) representing respectively the magnitude and the orientation of traction forces exerted by the single Ctrl, NMIIA KD and NMIIB KD cells on E-cadherin coated PDMS deformable gels (15 KPa). Cell masks used for quantification are drawn in white. Scale bar: 20 µm. (f) Scatter plots with mean + /- SEM showing the mean traction forces exerted by Ctrl, NMIIA KD and NMIIB KD cells on E-cadherin coated PDMS deformable gels (15 KPa). n = 46, 34 and 34 cells respectively from two independent experiments. Kruskal-Wallis statistical tests were applied for p value.

https://doi.org/10.7554/eLife.46599.026
Figure 6—figure supplement 2—source data 1

NMIIB favors E-cadherin clustering on E-cadherin-coated substrate.

https://doi.org/10.7554/eLife.46599.027
Figure 6—figure supplement 3
NMIIA and NMIIB are both required for establishment of proper inter-cellular stress.

(a) Scheme depicting the junction subdomains and the orientation of traction forces relative to the junction axis quantified in (b) and (c). (b, c) Scatter plots with mean + /- SEM representing the orientation of mean traction forces (b) and the ratio of parallel/perpendicular traction forces (c) in subdomains of the junction in Ctrl and NMIIB KD cells within the first 3 hr of contact. For each junction, six values corresponding to 30 min time points are plotted. Pairwise statistical t tests (for intra-group comparisons) and Mann-Whitney statistical t tests were applied for p value. n = 25 and 28 cell doublets respectively from three independent experiments. (d) Scheme depicting the orientation of inter-cellular stress relative to the junction axis quantified in (e). (e) Linear graph of parallel (left panel) and perpendicular (right panel) inter-cellular stress at the junction within the first 3 hr after contact in Ctrl, NMIIA KD and NMIIB KD cells. Data are represented as mean + /- SEM. n = 25, 26 and 28 cell doublets for Ctrl, NMIIA KD and NMIIB KD respectively from three independent experiments.

https://doi.org/10.7554/eLife.46599.028
Figure 6—figure supplement 3—source data 1

NMIIA and NMIIB are both required for establishment of proper inter-cellular stress.

https://doi.org/10.7554/eLife.46599.029
Proposed model for the role of NMIIA and NMIIB during junction biogenesis.

Upper panels: organization of early cell-cell contacts of Ctrl, NMIIA KD and NMIIB KD cells. Lower panels: proposed molecular organization of early junctions. Middle panels: distribution of intercellular stress. Ctrl cells establish stable and straight junctions maintained under an anisotropic intercellular stress preeminent parallel to the junction. NMIIB associates to- and organizes the junctional branched actin meshwork. NMIIA, which provides mechanical tugging force, sits on distant perijunctional actin bundles parallel to the junction. NMIIA KD cells fail to maintain stable cell-cell contacts exhibit shorter junctions, weak traction forces and weak intercellular stress. Perijunctional actin bundles are smaller and disorganized. NMIIB KD cells establish persistent but wavy junctions from which lamellipodial extensions and traction force hotspots arise. The junctional branched actin meshwork is disorganized which probably prevents α-catenin opening and induces the formation of lamellipodial extensions. The anchoring of perijunctional actin bundles to the junction is perturbed, despite the presence of NMIIA. There is, in these cells, no preferential orientation of intercellular stress.

https://doi.org/10.7554/eLife.46599.031
Author response image 1
NMIIB localizes to early AJs.

(a) Representative confocal images and zoom boxes of GFP-E-cadherin-expressing MDCK cell doublets fixed 20h after BCN-RGD addition and immuno-stained for NMIIB. Scale bar: 10 μm. (b) Relative intensity profiles (raw and smoothed data) of GFP-E-cadherin and NMIIB signals along the line represented in (a). Blue arrows point at NMIIB that sits on parallel perijunctional F-actin cables.

https://doi.org/10.7554/eLife.46599.034

Videos

Video 1
Dynamic of junction formation on reversible micropatterns.

Spinning disk movie showing contact formation between two MDCK cells expressing GFP-E-cadherin and stained with Hoechst. Scale bar: 10 µm.

https://doi.org/10.7554/eLife.46599.006
Video 2
Dynamic of junction formation in Y27-treated cells.

Spinning disk movie of MDCK cells expressing GFP-E-cadherin, stained with Hoechst and treated with 50 µM Y27. Scale bar: 10 µm.

https://doi.org/10.7554/eLife.46599.007
Video 3
Dynamic of junction formation in Ctrl, NMIIA KD and NMIIB KD cells.

Epi-fluorescence movies of Ctrl, NMIIA KD and NMIIB KD MDCK cells expressing GFP-E-cadherin. Scale bar: 10 µm.

https://doi.org/10.7554/eLife.46599.011

Tables

Key resources table
Reagent type
(species)
or resource
DesignationSource or
reference
IdentifiersAdditional information
Cell line (Canis familiaris, dog)MDCKATCCATCC CCL-34
Cell line (H. sapiens)Caco-2ATCCATCC HTB-37Kindly provided by S.Robine
(Institut Cuire/CNRS, Paris)
Antibodyanti-NMIIA rabbit polyclonalBiolegend9098011/100 for IF and 1/1000 for WB
Antibodyanti-NMIIA mouse monoclonalAbcamab554561/100 for IF and 1/1000 for WB
Antibodyrabbit anti-NMIIB polyclonalBiolegend9099011/100 for IF and 1/1000 for WB
Antibodyanti-β-catenin rabbit polyclonalSigma-AldrichC22061/100 for IF
Antibodyanti-β-catenin mouse monoclonalBD Biosciences6101561/100 for IF
Antibodyrecombinant anti-paxillin rabbit monoclonal antibodyAbcamAb320841/100 for IF
Antibodymouse anti-GAPDHProteinTech60004–1-Ig1/100 for IF
Antibodymouse anti-Arp3Sigma-AldrichA59791/100 for IF
Antibodymouse anti-E-cadherinBD Biosciences6101811/100 for IF
Antibodyrabbit anti-α-catenin polyclonalSigma-AldrichC-20811/100 for IF
Antibodyrat anti-α18-catenin monoclonalgenerously provided by A. Nagafuchi, (Kumamoto University, Japan)1/100 for IF
AntibodyAlexa488-Life TechnologiesA11039, A11055, A110131/250 for IF
AntibodyAlexa568-Life TechnologiesA11004, A11011, A110771/250 for IF
AntibodyAlexa647-Life TechnologiesA31571, A315731/250 for IF
Chemical compound, drugAlexa (488) -coupled phalloidinsInvitrogenA123791/250 for IF
Chemical compound, drugAlexa (555 or 647) -coupled phalloidinsLife TechnologiesA34055, A222871/250 for IF
OtherHoechst 34580ThermoFisherH35701/10000 for IF
AntibodyHorseradish peroxidase-coupled anti-mouse IgGsSigma-AldrichA90441/10000 for WB
AntibodyHorseradish peroxidase-coupled anti-rabbit IgGsPierce1/10000 for WB
Chemical compound, drugMitomycin CSigma-AldrichM248710 μg/ml for 1 hr
Chemical compound, drugY-27632 dihydrochlorideSigma-AldrichY050350 μM
OtherAPP (Azido-Poly-lysine Poly (ethylene glycol))Inspired protocol from M. van Dongen, Matthieu Pielhttps://doi.org/10.1002/adma.201204474Inspired protocol from M. van Dongen, Matthieu Piel
Peptide, recombinant proteinBCN-RGD peptide (BCN: bicyclo[6.1.0]- nonyne, coupled to RGD: peptide sequence Arg-Gly-Asp)Inspired protocol from M. van Dongen, Matthieu Pielhttps://doi.org/10.1002/adma.201204474Inspired protocol from M. van Dongen, Matthieu Piel
Commercial assay or kitDMEM (containing Glutamax, High Glucose and Pyruvate)Life Technologies31966–021
Commercial assay or kitFluorobrite DMEMThermo FisherA18967-01
Commercial assay or kitPenicillin/StreptomycinLife Technologies15140–122
Commercial assay or kitFoetal Bovine SerumLife TechnologiesS1810-50010% FBS in DMEM
Commercial assay or kitgeneticinLife Technologies10131–019
Chemical compound, drugTrypsinLife Technologies25300–054
Genetic reagent (Plasmid)pLKO.1-puroSigma-AldrichSHC002
Genetic reagent (Plasmid)MYH9Sigma-Aldrichtranscript ID: ENSCAFT00000002643.3TTGGAGCCATACAACAAATAC for NMIIA
Genetic reagent (Plasmid)MYH10Sigma-Aldrichtranscript ID: ENSCAFT00000027478TCGGGCAGCTCTACAAAGAAT for NMIIB
Genetic reagent (Plasmid)RFP-Pericentrinkindly provided M. Coppey, Institut Jacques Monod, Pariskindly provided M. Coppey, Institut Jacques Monod, Paris
Genetic reagent (Plasmid)m-Cherry cortactinkindly provided by Alexis Gautreau, Biochemisty laboratory, Ecole polytechnique, Francehttps://portail.polytechnique.edu/bioc/en/gautreaupcDNA5-FRT-GFP-mCherry-3pGW back bone (1740-pcDNAM FRTPC-mCherry Cortactine)
Genetic reagent (Plasmid)mCherry Myosin IIBAddgene55107
Genetic reagent (Plasmid)CMV-GFP-NMHC II-AAddgene11347
Chemical compound, drugprotease inhibitor cocktailRoche27368400
Chemical compound, drugphosphataseinhibitor (Phosphostop)Roche4906837001
Commercial assay or kitBradford assayBioRad500–0006
Commercial assay or kit4–12% Bis-Tris gelNovexNP0335
Commercial assay or kitSupersignal west femto maximum sensitivity substrateThermoFisher34095
Commercial assay or kitLookOut Mycoplasma PCR detection KitSigma-AldrichMP0035
Chemical compound, drugparaformaldehydeThermo Scientific22980
Chemical compound, drugFluoromount-G mounting mediaSouthern Biotech
Peptide, recombinant proteinfibronectinMerck MilliporeFC010
Chemical compound, drugAPTESSigma-AldrichA3648
Chemical compound, drugEDC-HClThermo Scientific229802 mM freshly prepared in 0.1M MES pH4.7
Chemical compound, drugNHSSigma-Aldrich1306725 mM
Peptide, recombinant proteinrecombinant human E-cadherinR and D systems8505-EC1 μg
Chemical compound, drugCy 52–276 A and Cy 52–276 B silicone elastomerDow corning
Chemical compound, drugcarboxylated red fluorescent beadsInvitrogenF8801
Software, algorithmFIJI-Image Jhttps://imagej.net/Fiji/DownloadsImage analysis were done using Fiji-Image J and plugins
Software, algorithmMATLAbMATLABTraction force, PIV analysis were done using alogorithms developed in lab to analyse traction force
Software, algorithmPhotoshop and IllustratorAdobeImages were mounted using these softwares
Software, algorithmGraphPad prismGraphPad PrismGraphs and statistical tests were done using GraphPad Prism

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for the main figures and figure supplements.

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