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Adherens junction treadmilling during collective migration

Nature Cell Biology volume 16, pages 639–651 (2014)Cite this article

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Abstract

Collective cell migration is essential for both physiological and pathological processes. Adherens junctions (AJs) maintain the integrity of the migrating cell group and promote cell coordination while allowing cellular rearrangements. Here, we show that AJs undergo a continuous treadmilling along the lateral sides of adjacent leading cells. The treadmilling is driven by an actin-dependent rearward movement of AJs and is supported by the polarized recycling of N-cadherin. N-cadherin is mainly internalized at the cell rear and then recycled to the leading edge where it accumulates before being incorporated into forming AJs at the front of lateral cell–cell contacts. The polarized dynamics of AJs is controlled by a front-to-rear gradient of p120-catenin phosphorylation, which regulates polarized trafficking of N-cadherin. Perturbation of the GSK3-dependent phosphorylation of p120-catenin impacts on the stability of AJs, and the polarity and speed of leading cells during collective migration.

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Figure 1: Continuous retrograde flow of N-cadherin-mediated adherens junctions (AJs) between adjacent migrating cells.
Figure 2: Coupling between the retrograde flow of AJs and the retrograde flow of transverse actin cables.
Figure 3: Leading-edge accumulation of N-cadherin–catenin complexes and formation of new AJs at the front of lateral cell–cell contacts depend on p120-ctn.
Figure 4: Polarized trafficking of N-cadherin from the cell rear to the leading edge.
Figure 5: Involvement of N-cadherin polarized recycling in AJ treadmilling.
Figure 6: Polarized regulation of p120-ctn association with N-cadherin.
Figure 7: GSK3-dependent regulation of p120-ctn phosphorylation and AJ dynamics during collective cell migration.
Figure 8: Role of p120-dependent AJ dynamics during collective migration.

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Acknowledgements

We are particularly grateful to M. Piel (Institut Curie, France), A.B. Reynolds (Vanderbilt University, USA), A. Ridley (King’s College, UK), C. Gauthier-Rouvière (CRBM, France), B. Goud (Institut Curie, France), M. Cohen-Salmon (College de France, France), D. Riveline (IGBMC, France) and L. Looger (Janelia Farm, USA) for plasmids and reagents. We thank J-Y. Tinevez and E. Perret from the Plate-Forme d’Imagerie Dynamique/IMAGOPOLE, of Institut Pasteur, and J-B. Manneville for critical reading of the manuscript. F.P. is financially supported by the University Paris VI and VII, the Association pour la Recherche contre le Cancer and the Fondation pour la Recherche Medicale; and F.L. by the Institut National du Cancer. This work was supported by the Institut National du Cancer, l’Association pour la Recherche contre le Cancer, and La Ligue contre le Cancer. We also would like to thank D. Porquet and G. Porquet for contributing to the funding of this study.

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Authors and Affiliations

  1. Institut Pasteur - CNRS URA 2582, Cell Polarity, Migration and Cancer Unit, 25 rue du Dr Roux 75724 Paris Cedex 15, France,

    Florent Peglion, Flora Llense & Sandrine Etienne-Manneville

  2. Université Pierre et Marie Curie, Cellule Pasteur UPMC, rue du Dr Roux Paris 75015, France,

    Florent Peglion

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Contributions

F.P. conceived and performed all experiments except those shown in Figs 1c, 2a, 6h and 8g and Supplementary Figs 1d, 2 and 5b, which were conceived and performed by F.L. S.E-M. contributed to the conception of the experiments, the interpretation of the data and wrote the manuscript.

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Correspondence to Sandrine Etienne-Manneville.

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Integrated supplementary information

Supplementary Figure 1 N-cadherin-GFP forms cadherin/catenins complexes with the endogenous proteins.

Fluorescence images showing the immunostaining of endogenous N-cadherin (a), anti-p120-ctn (b), anti-beta-catenin (c) in N-cadherin-GFP (Ncad-GFP) expressing astrocytes. (a) Note the colocalization of Ncad-GFP with the endogenous adherens junction proteins both at cell-cell contacts (arrows) and at the leading edge (arrowheads). (d) Immunostaining of endogenous N-cadherin (left) together with a membrane dye DiI (right) of migrating astrocytes 8 h after wounding. Higher magnification images of the leading edge (boxed area) are shown in the respective coloured rectangles on the right. The graph shows the fluorescence intensity profiles in both fluorescence channels along the two coloured lines. White arrows: direction of migration. Scale bar, 10 μm.

Supplementary Figure 2 Cell polarity in first and second row cells migrating in a 2D wound-healing assay.

Histogram showing the percentage of cells with a correctly oriented centrosome in the first and second row near the wound, 8 h after wounding. Results are shown as mean ± s.e.m. of 3 independent experiments in each conditions (first row n = 440 cells, second row n = 307).

Supplementary Figure 3 p120 depletion does not alter N-cadherin-mediated cell-cell contacts in confluent non migrating astrocytes.

(a) Westernblot (WB) showing p120-ctn and tubulin expression levels in astrocytes nucleofected with control siRNA (si ctl) or siRNA specific for p120-ctn (si p120#1, si p120#2). (b) Histogram showing an average 54% and 79% decrease in p120 expression respectively for si p120#1 and si p120#2 expressing cells, compared to control. Data are mean ± s.e.m. of normalized protein expression analysed in n = 4 independent experiments using the LI-COR technology. ∗∗∗P < 0.001, unpaired Student t-test. (c) Immunofluorescence images showing N-cadherin (green) and Hoechst (nuclei, magenta) staining of confluent astrocytes 4 days after nucleofection with si ctl and si p120-ctn (si p120#1, si p120#2). The yellow arrows highlight the similar organization of AJs in si CTL and si p120-ctn cells. (d) Primary astrocytes were nucleofected with the indicated siRNA and submitted, 3 days later, to a wound healing assay. The graph shows the percentage of migrating cells, in which the centrosome is located in the quadrant facing the wound in front of the nucleus, as an indication of cell polarization. Data are mean ± s.e.m. of more than 150 cells from n = 3 independent experiments. ∗P < 0.05,∗∗P < 0.01, unpaired Students t test. Scale bar, 10 μm.

Supplementary Figure 4 Role of protein synthesis and membrane traffic in N-cadherin dynamics.

(a) Immunofluorescence images showing N-cadherin immunostaining in DMSO- or cycloheximide-treated astrocytes 8 h after wounding. Yellow arrowheads point to sites of N-cadherin accumulation at the cell leading edge. The histogram on the right shows, for both conditions, the percentage of cells with an N-cadherin accumulation at the leading edge. Data are given as mean ± s.e.m. of more than 100 cells from n = 3 independent experiments. The difference in N-cadherin accumulation is not statistically significant: ns: P = 0.065, unpaired Student t-test. (b) Immunostaining of endogenous N-cadherin in DMSO- or Nocodazole-treated astrocytes, and GFP or Rab5S34N-myc (Rab5-DN) transfected cells, after 8 h of migration. The images and the corresponding analytical graphs showing the intensity at the leading edges in each representative cells refer to the data given by the graph in the main Fig. 5b. (c) Immunofluorescence images (from time-lapse acquisition) of dynasore (80 μM)-treated confluent astrocytes. Scale bar, 10 μm.

Supplementary Figure 5 p120-ctn tyrosine phosphorylation is not changed during collective cell migration.

(a) Cell lysates were obtained at the indicated time after wounding and were analysed by westernblotting (WB) using antibodies specifically recognizing phosphorylated Tyr96 or Tyr228 of p120-ctn and p120-ctn independently of its phosphorylation state (p120 total). (b) Immunofluorescence images showing N-cadherin and phospho T310-p120-ctn in migrating astrocytes nucleofected with the indicated siRNA or p120-ctn construct. Scale bar 10 μm.

Supplementary Figure 6 GSK3 depletion by siRNA.

Primary astrocytes were nucleofected with the indicated siRNAs. Three days later, cells were lyzed and analysed by westernblotting using anti-GSK3 and anti-tubulin. The quantitative analysis, using the LI-COR technology, of GSK3 expression level compared to tubulin is shown under the westernblot.

Supplementary Figure 7 Table summarizing the effects of p120-ctn depletion, GSK3 inhibition and inhibition of GSK3-dependent phosphorylation of p120-ctn on AJs maintenance and dynamics during collective cell migration.

The + or− signs in the table represent the level of the evaluated biological process (+++ being the highest level of activity and −−−, the lowest).

Supplementary Figure 8 Original Western blots referring to main figures 6d, g, 7a and Supplementary Figs 3.a, 5.a and 6.

Red dotted-line boxes highlight the regions of the Western Blots shown in the figures. All Western blots were named according to the main figure to which they relate to. The blot marked by a black asterisk was used for the main figure 6d and the Supplementary Fig. 5a since the kinetics of p120-ctn phosphorylation on T130, Y228, Y96 during cell migration was assessed during the same experiments, using the same cell lysates.

Supplementary information

Supplementary Information

Supplementary Information (PDF 2643 kb)

AJs dynamics in confluent and migrating astrocytes (2D).

Fluorescence time-lapse acquisition of N-cadherin-GFP expressing astrocytes in a confluent monolayer (a, left) or migrating in a wound healing assay (4 h after wounding) (b, right). Note the absence of apparent lateral AJs movement in confluent cells, as opposed to migrating cells in which a treadmilling of AJs is observed. 1 frame/3 min for 2 h (a, left) and 1 frame/5 min for 3h20 (b, right). Scale bar, 10 μm. (AVI 4738 kb)

AJs dynamics migrating fibroblasts and endothelial cells (2D).

Phase contrast (left) and fluorescence (right) time-lapse acquisition of N-cadherin-GFP expressing NIH3T3 fibroblasts (higher panels) and VE-cadherin-GFP expressing EA.hy926 endothelial cells (lower panels) migrating in a wound healing assay (2 h after wounding) 1 frame/3 min for 2 h (a, left) and 1 frame/5 min for 3h20 Scale bar, 10 μm (AVI 1146 kb)

AJs dynamics in astrocytes migrating in a 3D matrix.

Fluorescence time-lapse acquisition of migrating astrocytes transfected with N-cadherin-GFP (1 frame/5 min). N-cadherin-GFP expressing astrocytes seeded within a thick layer of matrigel (3D), migrating out of a coherent group of cells (white arrows). Note the AJs retrograde flow (coloured arrowheads) in collectively migrating astrocytes. Scale bar, 50 μm. (AVI 4055 kb)

AJs dynamics in the first and second row of cells (2D).

Fluorescence time-lapse acquisition of N-cadherin-GFP expressing astrocytes migrating in a wound healing assay (4 h after wounding). Note the absence of AJs movement in the cell in the second row, compared to wound edge cells in which a treadmilling of AJs is observed. Scale bar, 10 μm. (AVI 3779 kb)

AJs and F-actin dynamics in polarized immobile astrocytes.

Fluorescence time-lapse acquisition of astrocytes transfected with N-cadherin-GFP (green) and Lifeact-Cherry (red) (1 frame/5 min). Astrocytes were plated on circular micropatterns to allow polarization in the absence of migration. Note the similar retrograde flow of transverse actin cables and AJs. Scale bar, 10 μm. (AVI 60100 kb)

N-cadherin molecules undergo an actual retrograde movement on the lateral junctions of collectively migrating cells.

Fluorescence time-lapse acquisition of migrating N-cadherin–EOS-expressing astrocytes. Photoconverted (red) N-cadherin–EOS at the front of the lateral junction (Fig. 1e) undergo a retrograde movement, opposite to the direction of migration (white arrow). 1 frame/5 min. Scale bar, 2 μm. (AVI 2433 kb)

AJs and F-actin dynamics in 2D migrating astrocytes.

Fluorescence time-lapse acquisition of migrating astrocytes transfected with N-cadherin-GFP and Lifeact-Cherry (1 frame/5 min). Note that AJs and transverse actin filaments undergo a similar retrograde flow, opposite to the direction of migration (white arrow). Scale bar, 10 μm. (AVI 15139 kb)

Leading edge N-cadherin moves to the side of the cell and participates in the formation of new AJs.

Fluorescence time-lapse acquisition of migrating astrocytes transfected with N-cadherin-GFP (1 frame/5 min) focusing on the front region of the cell. Note the lateral movement of cadherin clusters (colored arrows) participating in the formation of new AJs at the front of lateral intercellular contacts. Scale bar, 5 μm. (AVI 1133 kb)

p120-ctn depletion affects N-cadherin dynamics and formation of new AJs at the front side of migrating cells.

Phase contrast (left) and fluorescence (right) time-lapse acquisitions of migrating p120-ctn-depleted astrocytes expressing N-cadherin-GFP (1 frame/10 min). The acquisition starts a few minutes after wounding and shows that, in contrast to the continuous treadmilling of AJs observed in control cells (movie 1), new AJs formation at the front of lateral cell-cell contacts is altered while the retrograde flow of preexisting AJs is initiated (white arrowheads). Scale bar, 10 μm. (AVI 6429 kb)

Polarized N-cadherin vesicular trafficking from the rear to the front of migrating astrocytes.

Fluorescence time-lapse acquisition of N-cadherin-GFP expressing migrating astrocytes 8 h after wounding (1 frame/10 sec, inverted contrast) reveals trafficking of N-cadherin vesicles. Images were obtained in a median focal plane (that is situated approximately in the middle of the cell height) and thus do not clearly show the rear (which is located higher) and the front (which is located lower) of lateral AJs. Note the active trafficking of N-cadherin positive vesicles preferentially moving from the rear to the front of the cell. Scale bar, 10 μm. (AVI 12358 kb)

Altered membrane trafficking affects N-cadherin polarized recruitment to the leading edge and the formation of new AJs at the front side of migrating cells.

Fluorescence time-lapse acquisition of migrating N-cadherin-GFP expressing astrocytes (1 frame/5 min). Migration was initiated in DMSO containing cell culture medium. The dynamin inhibitor Dynasore was added after 170 min of time-lapse acquisition. The addition of new medium leads to a change of contrast in the movie. Scale bar, 7 μm. (AVI 7407 kb)

p120-ctn depletion increases astrocyte migration in 3D matrix.

Phase contrast time-lapse acquisitions of control(left) and p120-ctn-depleted (right) astrocytes seeded in a 3D matrigel. 1 frame/15 min for 6 h. Scale bar, 10 μm. (AVI 1374 kb)

p120-ctn reduces collective cell migration.

Phase contrast (left) and fluorescence (right) time-lapse acquisitions of migrating p120-ctn-depleted astrocytes expressing siRNA-resistant WT-p120-ctn-GFP (1 frame/20 min). The acquisition starts 6 h after wounding. Note that wound-edge p120-ctn-depleted cells, but not the cell expressing WT-p120-ctn-GFP, tend to separate from the rest of the monolayer. (AVI 2924 kb)

Inhibition of p120-ctn phosphorylation perturbs AJ dynamics and inhibits cell migration.

Phase contrast (left) and fluorescence (right) time-lapse acquisitions of migrating p120-ctn-depleted astrocytes expressing siRNA-resistant T310A-p120-ctn-GFP (1 frame/20 min). The acquisition starts 6 h after wounding. Note that wound-edge p120-ctn-depleted cells, but not the cell expressing T310-p120-ctn-GFP, tend to separate from the rest of the monolayer. In contrast to WT-p120-ctn-expressing cells (Supplementary Video 13), T310-p120-ctn-GFP expressing cells have a broader and less polarized leading edge, a sign of altered AJ formation at the front of lateral cell-cell contacts (Fig. 7e, g and Supplementary Videos 9 and 14). (AVI 2449 kb)

Gap junction dynamics in migrating astrocytes (2D).

Fluorescence time-lapse acquisition of Connexin43-GFP expressing astrocytes migrating in a wound healing assay (4 h after wounding). Note the absence of apparent retrograde movement of connexin-mediated gap junctions along the lateral sides of migrating astrocytes. 1 frame/3 min for 2 h Scale bar, 10 μm. (AVI 850 kb)

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Peglion, F., Llense, F. & Etienne-Manneville, S. Adherens junction treadmilling during collective migration. Nat Cell Biol 16, 639–651 (2014). https://doi.org/10.1038/ncb2985

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