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Adherens junction treadmilling during collective migration Florent Peglion1,2, Flora Llense1 and Sandrine Etienne-Manneville1,3 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. Collective migration of cohorts, sheets, groups or chains of cells is involved in morphogenesis, tissue renewal and wound healing, and has also been observed during tumour spreading (reviewed in refs 1,2). We use primary astrocytes to study directed collective cell migration. Astrocytes are major glial cells of the central nervous system that have been shown to migrate collectively over the retinal surface during eye development3–5. In the adult, reactive astrocytes undergo collective directed migration in response to inflammatory situations. Following wounding both in vivo and in vitro, astrocytes adopt a polarized morphology and migrate in the direction of the wound6,7. In addition to cell–matrix interactions, cell–cell interactions are crucial for collective movements8. These interactions are mediated by several plasma membrane protein complexes, and in particular by AJs, which are observed in a wide variety of cell types9. AJs found in epithelial cells generally form linear continuous contacts, but AJs with a punctate morphology and various molecular compositions have also been observed during the remodelling of intercellular contacts in epithelial and endothelial cells10,11. N-cadherin-mediated junctions frequently form punctate structures, but linear AJs can also be found (see herein and ref. 12). Collective movements observed during tracheal development13, mesendoderm movement14, mammary duct formation15 or neural crest migration16 require cadherin-mediated AJs, which are also involved in the collective invasion of cancer cells1. AJs mediate adhesive forces between adjacent cells17–19 and initiate crucial intracellular signals that regulate major cellular functions, such

as proliferation, differentiation or migration20,21. Classical cadherins, including E-cadherin, VE-cadherin and N-cadherin22,23, are the main transmembrane proteins of AJs. The cadherin extracellular domain is involved in homophilic cis- and trans-interactions to form dense protein clusters that constitute the core of AJs (refs 20,24). In addition to their extracellular and transmembrane domains, classical cadherins possess a cytoplasmic tail that interacts with cytosolic partners, such as β-catenin and p120-catenin (p120-ctn). Catenins connect cadherins to the cytoskeleton and to several signalling pathways. AJ association with the actin cytoskeleton is involved in mechanosensing and mechanotransduction, which contribute to the coordination required for collective migration19,25,26. Moreover, the distribution of cadherinmediated AJs at the periphery of leading cells controls front–rear polarity and influences the directionality and speed of migration16,27,28. The role of AJs in the regulation of migration is also highlighted by the fact that changes in cadherin and catenin expression are associated with epithelial–mesenchymal transition, a major process involved in carcinoma invasion and metastasis29–31. How AJs can provide a strong adhesion to maintain tissue cohesion while allowing cellular rearrangements during migration remains unclear. We have visualized AJs between adjacent astrocytes migrating in a wound healing assay27,32. Using fluorescently labelled N-cadherin, we observed that N-cadherin engaged in AJs undergoes a continuous treadmilling along the lateral sides of adjacent leading cells. This highly dynamic process, controlled by the polarized regulation of p120-ctn,

1

Institut Pasteur - CNRS URA 2582, Cell Polarity, Migration and Cancer Unit, 25 rue du Dr Roux 75724, Paris Cedex 15, France. 2Université Pierre et Marie Curie, Cellule Pasteur UPMC, rue du Dr Roux, Paris 75015, France. 3 Correspondence should be addressed to S.E-M. (e-mail: [email protected]) Received 5 May 2013; accepted 7 May 2014; published online 15 June 2014; DOI: 10.1038/ncb2985

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A RT I C L E S allows both stabilization and rapid changes in cellular interactions during collective migration. RESULTS Cadherin-mediated AJs undergo an actin-coupled retrograde flow in polarized cells To investigate the dynamics of AJs, we used primary rat astrocytes expressing a carboxy-terminal GFP-tagged N-cadherin, which colocalized with endogenous N-cadherin, β-catenin and p120-ctn (Supplementary Fig. 1a–c). In a confluent cell monolayer, N-cadherinmediated junctions showed only small movements along with the fluctuations of the cell shape (Fig. 1a and Supplementary Video 1a). In contrast, during migration, lateral AJs between wound-edge cells underwent a continuous flow from the front towards the cell rear (Fig. 1b and Supplementary Video 1b). Analysis of the corresponding kymographs indicated a continuous retrograde movement with a speed of 0.35 ± 0.01 µm min−1 (Fig. 2d). In fibroblasts expressing N-cadherin (Supplementary Video 2) and endothelial cells expressing VE-cadherin (Supplementary Video 2 and Fig. 1c), a similar retrograde flow of AJs was observed between migrating cells. AJ retrograde flow was also observed when astrocyte clusters migrated in a three-dimensional (3D) Matrigel (Fig. 1d and Supplementary Video 3), suggesting that the retrograde flow of AJs is a general feature in 2D or 3D collective migration. In follower cells, which do not present any contact-free edges, as in cells within an intact monolayer, AJs remained mainly stationary (Fig. 1e and Supplementary Video 4). Owing to the slow migration of astrocytes, the second row of cells barely moved or polarized for the first 12 h of our experiments (Supplementary Fig. 2). We thus tested whether the absence of AJ retrograde flow in these cells was a consequence of their reduced polarization and migration or of their position in the cell sheet. Astrocytes were plated on fibronectin-coated circular micropatterns surrounded by polyethylene glycol to prevent cell migration. Cells at the edge of the pattern are immobile and polarized28. In these cells, AJs flowed from the contact-free side of the cell towards the centre of the pattern (Fig. 1f and Supplementary Video 5), indicating that migration itself was not necessary, but that an asymmetric distribution of AJs and subsequent cell polarization were sufficient to promote a polarized dynamics of AJs. Taken together, our results suggest that during collective migration, the anisotropy of cell– cell contacts in leader cells triggers a highly dynamic retrograde flow of AJs. Using N-cadherin–GFP and N-cadherin–Eos-expressing astrocytes, we performed fluorescence recovery after photobleaching (FRAP) and photoconversion experiments to determine whether the AJs retrograde flow reflected an actual movement of N-cadherin molecules. An anterior region of cell–cell lateral contact was photobleached. The photobleached region remained clearly visible during 60 min and underwent a backward movement along lateral contacts (Fig. 1g). When N-cadherin–Eos was photoconverted at the front of lateral cell–cell contacts, photoconverted cadherins remained clustered for at least 2 h (Fig. 1f and Supplementary Video 6) confirming the limited diffusion of the molecules. Photoconverted N-cadherin clusters followed a retrograde movement along cell– cell contacts at a speed similar to that of AJs (∼0.30 µm min−1 ), further demonstrating that N-cadherin engaged in AJs undergoes

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a retrograde movement along the lateral sides of adjacent migrating cells. AJs are intimately coupled to the actin cytoskeleton8,33,34. In astrocytes, as well as in fibroblasts and endothelial cells, actin cables form transverse arcs that end in AJs on each lateral side of the woundedge cells (Fig. 2a). Using LifeAct–mCherry-expressing astrocytes we observed that, in non-polarized confluent cells, actin cables, like AJs, did not significantly move (Fig. 2b,e), whereas in polarized migrating astrocytes, actin cables underwent a retrograde movement (Fig. 2c and Supplementary Video 7). Flows of actin cables and AJs had identical speed and direction both in migrating (Fig. 2e) and in polarized non-migrating astrocytes plated on micropatterns (Fig. 2d,e and Supplementary Video 5). Disruption of acto-myosin cables by treatment with ROCK (Y27632) or myosin light chain (ML7) inhibitors completely stopped the retrograde movement of AJs in a large proportion of cells (Fig. 2f,g). Leading-edge accumulation of N-cadherin complexes provides material for the formation of new AJs at the front of adjacent migrating cells The continuous retrograde flow of N-cadherin led us to investigate how AJs were formed at the front of lateral contacts. Immunostaining showed an accumulation of N-cadherin at the leading edge of migrating astrocytes (Fig. 3a), as previously reported in other cell types35–37. GFP expression (Fig. 3a) and staining with a nonspecific membrane dye (Supplementary Fig. 1d) were used as volume and membrane markers and showed that the increased fluorescence intensity was not due to membrane ruffles in the lamellipodia. The AJ components β-catenin, α-catenin and p120-ctn were also visible at the leading edge of migrating cells (Fig. 3b). Time-lapse videos of N-cadherin–GFP-expressing astrocytes suggested that new AJs formed by accumulation of N-cadherin clusters emerging from the leading edge (Fig. 3c and Supplementary Video 8). Photoconversion of N-cadherin–Eos at the leading edge showed a progressive accumulation of photoconverted cadherin at the front of cell–cell contacts (Fig. 3d), confirming the incorporation of leading-edge cadherin into newly formed AJs. p120-ctn has been shown to be required for cadherin stability at the plasma membrane38–40. We used short interfering RNAs (siRNAs) directed against p120-ctn transcripts to determine p120-ctn function in N-cadherin localization at the leading edge and AJ formation. The first siRNA (p120 siRNA1) decreased p120-ctn expression by 50% and a second independent siRNA (p120 siRNA2) by 80% (Supplementary Fig. 3a,b). p120-ctn knockdown only slightly decreased the N-cadherin expression and did not notably perturb AJs between confluent cells28 (Supplementary Fig. 3b). However, p120-ctn depletion prevented the accumulation of N-cadherin at the leading edge of migrating cells (Fig. 3e), and led to a strong increase in the distance between the leading edge and the front of lateral AJs, indicating that the formation of new AJs was altered (Fig. 3f and Supplementary Video 9). Expression of a siRNA-resistant p120-ctn construct in p120-ctn-depleted cells rescued the N-cadherin accumulation at the leading edge and the formation of AJs (Fig. 3e,f), confirming the role of p120-ctn. We conclude that N-cadherin complexes accumulate in a p120-ctndependent manner at the front edge of leading cells to contribute to

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A RT I C L E S a

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Figure 1 Continuous retrograde flow of N-cadherin-mediated adherens junctions (AJs) between adjacent migrating cells. (a,b,d–h) Primary rat astrocytes expressing N-cadherin (Ncad)–GFP (a,b,d–g) or N-cadherin– Eos (h) were used to investigate N-cadherin dynamics. (a) Fluorescence image of a confluent non-migrating astrocyte (from Supplementary Video 1a). (b) Fluorescence image of a wound-edge astrocyte migrating in a 2D wound healing assay, 4 h after wounding (from Supplementary Video 1b). (c) Fluorescence image of VE-cadherin–GFP-expressing EA.hy926 endothelial cells located at the wound edge 2 h after wounding (from Supplementary Video 2). (d) Phase-contrast and fluorescence images (from Supplementary Video 3) of astrocytes migrating out of a spheroid in a 3D Matrigel. (e) Fluorescence image of astrocytes located in the first (A) and second row (B) next to the wound (from Supplementary Video 4). (f) Inverted fluorescence image of an astrocyte plated on a fibronectin-coated circular micropattern (inset), corresponding to the dashed white rectangle of the inset phase-contrast image (from Supplementary Video 5). (g) Fluorescence image of a wound-edge astrocyte 4 h after wounding and before

ch Re an d ne l

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photobleaching. The middle panels show higher-magnification images of the outlined region (white rectangle) at the indicated times before and after photobleaching (blue rectangle). (h) Fluorescence image of a wound-edge astrocyte migrating in a 2D wound healing assay. The middle panels show higher-magnification images of the outlined region (white rectangle) at the indicated times after photoconversion of N-cadherin– Eos at the front AJs (blue rectangle). Photoconverted N-cadherin–Eos molecules emit red fluorescence. (a–h) The kymographs shown on the right of the images highlight the different dynamics of cadherin-mediated AJs (red line along the lateral cell–cell contacts) in migrating, immobile, polarized and non-polarized cells. Note the absence of directed retrograde flow in the second row of migrating cells (e, B). The coloured dashed lines indicate the position of the front edge of the cells, except in e, where it indicates the limit between the first and second rows. White scale bars, 10 µm, except in d, 40 µm. Yellow scale bars, 4 µm, except in h, 2 µm. The white arrows show the direction of migration. N indicates the nucleus.

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A RT I C L E S Astrocytes

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Figure 2 Coupling between the retrograde flow of AJs and the retrograde flow of transverse actin cables. (a) Immunofluorescence images revealing cadherin (green) and actin filaments (phalloidin, red) in astrocytes fixed after 8 h of migration (left panels), in EA.hy926 endothelial cells (middle panels) and in NIH3T3 fibroblasts (right panels), both fixed after 4 h of migration. Higher magnification of the outlined area (white rectangle) is shown on the right. White arrowheads point to transverse actin fibres contacting N-cadherin-mediated AJs (yellow arrows). (b–d) Fluorescence images of astrocytes expressing the indicated constructs. (b) The inset represents the merged phase-contrast and fluorescence images of the transfected astrocyte in the monolayer. (c) Merge of a wound-edge migrating astrocyte corresponding to the dashed yellow rectangle of the merged phasecontrast and fluorescence inset image at the bottom left corner. (d) Merged fluorescence image of an astrocyte plated on a fibronectin-coated circular micropattern (white circle in the inset). The fluorescence image corresponds to the dashed white rectangle of the inset phase-contrast image where 4 cells delimited by yellow lines are visible on the pattern (Fig. 1e). (b–d) The red lines along AJs show the position where the kymographs on the

right of the images were recorded. (e) Graph showing the velocity of lateral N-cadherin clusters and transverse actin cable retrograde flow in migrating and non-migrating astrocytes. Data are mean ± standard deviation (s.d.) of n = 35 and n = 24 migrating cells, respectively for N-cadherin and actin, from 3 independent experiments, and of n = 7 (N-cadherin), n = 3 (actin) confluent cells. Almost no directed movement of either cadherin-positive clusters or F-actin transverse cables is seen for confluent cells, hence the small number of analysed cells. (f) Migrating astrocytes expressing N-cadherin–GFP and LifeAct– Cherry, treated with the indicated drugs or solvent. The yellow arrowheads point towards transverse actin cables, absent in ML-7- and Y27632treated cells. (g) Graph showing, for each condition, the percentage of cells with apparent N-cadherin retrograde flow. n = 3 (dimethylsulphoxide, DMSO) and n = 2 (Y27632 and ML-7) independent experiments. In total, 41 out of 44 cells scored positive for DMSO, 6 out of 35 for Y27632, and 18 out of 30 for ML-7. ∗∗∗ P < 0.001, unpaired Student’s t-test. NS: P = 0.62. Yellow scale bars, 2 µm. White scale bars, 10 µm. The white arrows indicate the direction of migration.

the continuous formation of new AJs at the front of lateral contacts between adjacent cells.

affect N-cadherin localization at the leading edge, even after 8 h of migration (Supplementary Fig. 4a). We thus reasoned that leading-edge accumulation resulted from N-cadherin recycling and looked for potential preferred sites of cadherin internalization. We found that cadherin co-localization with clathrin, a major player in cadherin endocytosis41,42, was significantly higher at the rear than at the front of lateral contacts (Fig. 4a). Fast time-lapse video microscopy

Anterograde vesicular trafficking of recycling N-cadherin compensates for the continuous retrograde flow of AJs Treatment of astrocytes with cycloheximide (20 µg ml−1 , 1 h before wounding) to inhibit protein synthesis did not significantly

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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. (a) Immunostaining of N-cadherin (red) in a GFP-expressing migrating astrocyte (green), 8 h after wounding. Higher-magnification images of the leading edge (outlined area) are shown on the right. The graph shows the fluorescence intensity profiles in both fluorescence channels along the three coloured lines. (b) Immunostaining of β-catenin, α-catenin and p120-ctn in migrating astrocytes 8 h after wounding. The arrowheads highlight the leading-edge accumulation. (c) An N-cadherin–GFP-expressing astrocyte 4 h after wounding. Higher-magnification images of the outlined area taken at the indicated time are shown on the right (from Supplementary Video 8). The arrowhead points to leading-edge clusters of N-cadherin, incorporated into new AJs. The yellow line marks the front of cell–cell contacts. (d) Fluorescence images of an N-cadherin–Eos-expressing astrocyte 4 h after wounding. Green and red fluorescence images of the outlined area were taken at the indicated time after photoconversion of N-cadherin–Eos in the leading-edge region (middle panel). The dotted line represents the leading

edge. The red arrows point to sites of red N-cadherin accumulation into newly formed AJs. The graph shows the changes in red fluorescence intensity in the red rectangles drawn in the red channel images. (e) Accumulation of N-cadherin at the leading edge of astrocytes nucleofected with the indicated siRNA and constructs. The histogram shows the percentage of cells with N-cadherin accumulation at the leading edge. 158, 121 and 97 cells, respectively for control siRNA, p120 siRNA + GFP and p120 siRNA + p120WT –GFP were assessed in n = 3 independent experiments. (f) AJ formation at the front of cell–cell contacts in astrocytes nucleofected with the indicated siRNA and constructs. The histogram represents the distance between the leading edge and the first AJ at the front of lateral contacts (yellow bars in the images). 55, 76 and 114 cells, respectively for control siRNA, p120 siRNA + GFP and p120 siRNA + p120WT –GFP were analysed from n = 3 independent experiments. The white arrows indicate the direction of migration. In e and f data are given as mean ± s.d. ∗∗∗ P < 0.001, unpaired Student’s t-test. NS: not significant (in e, P = 0.28, in f, P = 0.41). Scale bars, 10 µm.

showed N-cadherin–GFP-positive vesicles emerging from the AJs located at the cell rear, moving towards a perinuclear region (Fig. 4b), and then along the cell protrusion towards the cell front (Fig. 4c,d and Supplementary Video 10). Endocytosis and recycling were then inhibited by treating cells with the dynamin inhibitor dynasore43, or the microtubule-depolymerizing drug nocodazole, or by expression of a dominant-negative form of

Rab5. In each case, cadherin recruitment to the leading edge was strongly reduced (Fig. 5a,b and Supplementary Fig. 4b). Whereas dynasore had no visible effect on AJs in confluent non-migrating cells (Supplementary Fig. 4c), treatment of migrating astrocytes rapidly prevented N-cadherin accumulation at the leading edge and the formation of new AJs at the front of lateral contacts (Fig. 5c and Supplementary Video 11). We then used N-cadherin–Eos-expressing

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Percentage of clathrin-co-localized N-cadherin

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Figure 4 Polarized trafficking of N-cadherin from the cell rear to the leading edge. (a) Immunofluorescence images showing clathrin (red) and N-cadherin (green) in migrating astrocytes 8 h after wounding. Two focal planes were acquired to better image AJs at the cell front and at the cell rear. Co-localization is shown in white. The histogram shows the level of cadherin/clathrin co-localization in the rear half and in the front half of n = 8 cells from 4 independent experiments. The error bars represent s.d. The grey dashed lines between the two groups of points indicate data taken from the same cell. ∗∗ P = 0.0078, Wilcoxon matched pairs test. (b) Fluorescence images of an N-cadherin–GFP-expressing astrocyte 4 h after wounding. Higher-magnification images of the outlined area taken at the indicated time after the beginning of the experiment are shown on the right. The arrowheads point to an N-cadherin-positive vesicle emerging from the region of cell–cell contact at the cell rear, and moving towards the perinuclear region. Its trajectory (red dotted line) is depicted in the last panel. The blue line and N indicate the position of the

nucleus. (c) Stack projection (850 s) of Supplementary Video 10 showing the directional movement of N-cadherin vesicles. Images were obtained in a focal plane situated approximately in the middle of the cell height and thus do not clearly show the rear (which are located higher) and the front (which are located lower) of lateral AJs. The trajectories (red dotted lines) of N-cadherin-positive vesicles are depicted in the right panel. (d) Stack projection (850 s) of a time-lapse video of a N-cadherin–GFP-expressing migrating astrocyte. Images on the right show a time-lapse sequence of N-cadherin vesicles trafficking near the leading edge (white rectangle in the left image). Images were obtained in a focal plane situated near the bottom of the cell, near the coverslip to better visualize cadherin trafficking near the leading edge. The leading edge is delineated by a brown dashed line. The arrowheads point to an N-cadherin-positive vesicle recruited to the leading edge. Its trajectory (red dotted line) is depicted in the last panel. Scale bars, 10 µm. The white arrows indicate the direction of migration.

astrocytes and photoconverted the rear half of the lateral contacts (Fig. 5d–f). The photoconverted area shifted towards the rear of the cell, following AJ retrograde flow. The red fluorescent cadherin progressively decreased at the site of photoconversion, but became detectable in the forming AJs at the cell front (Fig. 5d–f). During the experiment, red fluorescence was never detected in the middle region

of lateral contacts, suggesting that the accumulation of photoconverted cadherin at the front results from N-cadherin intracellular trafficking (Fig. 5e). Together, these results led us to propose that during collective migration of astrocytes, AJs undergo a continuous treadmilling sustained by a polarized recycling of cadherins (Fig. 8a).

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Figure 5 Involvement of N-cadherin polarized recycling in AJ treadmilling. (a) Immunofluorescence images showing endogenous N-cadherin in migrating astrocytes treated with DMSO or dynasore (80 µM) for 2 h. Highermagnification images of the leading edge are shown on the right. The graph shows the fluorescence intensity profiles in both fluorescence channels along the three red lines. (b) Histogram showing the percentage of cells with N-cadherin accumulation at the leading edge. Quantification was performed as shown in a, with cells either treated with the indicated drugs (DMSO, 20 µM nocodazole or 80 µM dynasore) or nucleofected with the indicated constructs (GFP or Rab5DN). Data are given as mean ± s.d. of 101, 104 and 114 DMSO-, nocodazole- and dynasore-treated cells, respectively, taken from n = 3 independent experiments; and of 91 and 119 GFP- and Rab5S34N–myc (Rab5-DN)-expressing cells, respectively, from n = 4 independent experiments. ∗ P < 0.05, ∗∗ P < 0.01, unpaired Student’s t-test. (c) Fluorescence images of an N-cadherin–GFP-expressing migrating astrocyte before and after dynasore treatment (from Supplementary

Video 11). Following treatment, the distance between the leading edge and the first AJs at the front increased (blue lines compared with the red lines). (d) Fluorescence images of an N-cadherin–Eos-expressing migrating astrocyte 5 h after wounding. Green and red fluorescence images of the outlined area were taken at the indicated time after photoconversion (middle panel). The rear half of AJs (purple rectangle) was illuminated with a 405 nm laser to photoconvert green fluorescent N-cadherin–EOS to red fluorescence. (e) The graph shows the changes in red fluorescence intensity in the three rectangles drawn in the red channel image. Arrowheads in d and e highlight the enrichment of photoconverted N-cadherin at the front of lateral cell–cell junctions. (f) Quantification of N-cadherin recycling over time. Data are given as mean ± s.d. of the percentage of the red fluorescence (photoactivated cadherin) visible in the AJs at the front of the lateral cell–cell contacts. The analysis was carried on in n = 3 different experiments. Scale bars, 10 µm. The white arrows indicate the direction of migration.

p120-ctn interaction with N-cadherin is differentially regulated from the front to the rear of migrating cells In addition to its leading-edge localization (Fig. 3b), p120-ctn colocalized with N-cadherin in AJs (Fig. 6a). p120-ctn staining was constant along AJs in confluent non-migrating cells (Fig. 6c), but

strongly decreased from the front to the rear of the lateral AJs of migrating cells (Fig. 6a,b). Western blot analysis showed a strong increase in p120-ctn phosphorylation on Thr 310 in migrating cells as soon as 1 h after wounding (Fig. 6d), whereas Tyr 96 and Tyr 228 phosphorylation did not change (Supplementary Fig. 5a).

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Figure 6 Polarized regulation of p120-ctn association with N-cadherin. (a,c,h) N-cadherin (red) and p120-ctn (green) immunostaining in woundedge (a,h) and confluent astrocytes (c). Graphs in a and c show the p120-ctn/ N-cadherin ratio along cell–cell contacts. (b) Normalized p120ctn/N-cadherin ratio at the rear (last third) and front (first third) of lateral contacts of n = 25 migrating cells from 3 independent experiments. Error bars represent s.e.m. (d) Astrocyte lysates, obtained at the indicated time after wounding, analysed by western blotting using the indicated antibodies. (e) N-cadherin (red), phosphoT310-p120-ctn (Pp120-ctn(T310), green) and nucleus (blue) staining in migrating astrocytes. High magnification of the outlined area is shown on the right. The graph shows the phosphoT310-p120ctn/N-cadherin ratio along cell–cell contacts. (f) Average phosphoT310p120-ctn/N-cadherin ratios in the rear and front halves of lateral cell–cell contacts of n = 35 cells taken from 3 independent experiments. Error bars represent s.e.m. (g) Western blot of protein inputs and N-cadherin– GFP immunoprecipitates from COS-7 cells expressing N-cadherin–GFP and p120-ctnWT –HA or p120-ctnT310A –HA. The histogram shows the amount of p120-ctn bound to N-cadherin–GFP. Data are presented as ratios of

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p120-ctn–N-cadherin interaction level in p120-ctnT130A over p120-ctnWT from n = 3 independent experiments. Error bars represent s.d. ∗ P = 0.017. (h–j) p120-ctn-depleted astrocytes were nucleofected with siRNA-resistant p120ctnWT or p120-ctnT310A constructs. (h) The p120-ctn/N-cadherin fluorescence ratio at the front of lateral cell–cell contacts divided by the same ratio at the rear is shown on the right. Data are mean ± s.e.m. of n = 32 p120ctnWT cells and n = 24 p120-ctnT310A cells, from 3 independent experiments. ∗ P = 0.0314. (i) Percentage of clathrin-co-localized cadherin in the entire cell. Data are mean ± s.e.m. of n = 36 p120-ctnWT cells and n = 28 p120ctnT310A cells, from 3 independent experiments. (j) Percentage of clathrin-colocalized cadherin at the front and rear of migrating cells. The difference in co-localization between front and rear was assessed in n = 42 control siRNA, n = 26 p120 siRNA + p120-ctnWT and n = 26 p120 siRNA + p120-ctnT130A cells, from 3 different experiments. Data are mean ± s.e.m. NS: P = 0.705, ∗ P = 0.0262, ∗∗∗ P < 0.001. Except for b and f where a Wilcoxon matched pairs test was used, the statistical analysis was by an unpaired Student’s t-test. Scale bars, 10 µm. The white arrows indicate the direction of migration. See Supplementary Fig. 8 for uncropped blots.

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A RT I C L E S Thr-310-phosphorylated p120-ctn mainly localized in the rear half of AJs between migrating cells (Fig. 6e,f). The absence of Pp120(T310) staining in p120-ctn-depleted cells or in p120cnt-depleted cells transfected with a non-phosphorylatable mutant of p120-ctn (p120ctnT310A ) confirmed the specificity of the staining at cell–cell contacts but showed that the nuclear staining was irrelevant44 (Supplementary Fig. 5b). We then transfected COS cells with plasmids encoding Ncadherin–GFP and HA-tagged wild-type p120-ctn (p120-ctnWT ) or a non-phosphorylatable p120-ctnT310A mutant. Immunoprecipitation of N-cadherin showed that mutation of Thr 310 strongly increased p120-ctn binding to N-cadherin (Fig. 6g). p120-ctn binding has been shown to prevent cadherin endocytosis38–40. Expression of p120ctnT310A in p120-ctn-depleted astrocytes showed that p120-ctn phosphorylation is required for p120-ctn polarized recruitment to AJs (Fig. 6h), and for N-cadherin co-localization with clathrin, especially at the cell rear (Fig. 6i,j). Taken together these results suggest that increased phosphorylation of Thr 310 induces the local dissociation of p120-ctn from AJs and N-cadherin endocytosis at the cell rear. GSK3-dependent phosphorylation of p120-ctn is essential for the regulation of AJ dynamics during collective cell migration The serine/threonine kinase GSK3 can phosphorylate p120-ctnT310 in vitro44 and is downregulated specifically at the front of collectively migrating astrocytes45. Treatment of cells with GSK3 inhibitors (10 mM LiCl, 50 µ MSB 415286) or nucleofection with siRNA directed against GSK3 (Supplementary Fig. 6) prevented p120ctn phosphorylation on Thr 310 (Fig. 7a,b). Inhibition of GSK3 showed that the destabilization of the p120-ctn–N-cadherin complex (indicated by the decrease of the front/rear ratio of p120-ctn/Ncadherin level) at the cell rear required GSK3 activity (Fig. 7c), as well as p120-ctn phosphorylation on Thr 310 (Fig. 6h). GSK3 inhibition also perturbed N-cadherin recycling to the leading edge (Fig. 7d,e), and the formation of new AJs, increasing the distance between the front edge and the first lateral AJs (Fig. 7d,f). To confirm the role of p120-ctn phosphorylation in these events, p120ctn-depleted astrocytes were transfected with p120-ctnWT or p120ctnT310A siRNA-resistant constructs. In contrast to p120-ctnWT, p120-ctnT310A did not rescue N-cadherin recruitment at the leading edge and the formation of new AJs (Fig. 7d–f and Supplementary Fig. 7). We conclude that GSK3-dependent polarized regulation of p120-ctn promotes the polarized recycling of N-cadherin and AJ dynamics (Fig. 8a). GSK3- and p120-ctn-dependent regulation of AJs is crucial for the maintenance of cellular interactions and for cell coordination during directed collective migration Downregulation of p120-ctn does not significantly affect the formation of AJs between non-migrating astrocytes28, and cells reached confluence without leaving any free space between them (Supplementary Fig. 3c). However, p120-ctn depletion resulted in a progressive loss of intercellular interactions in leading migrating cells. Wound-edge cells lost front lateral contacts where the formation of AJs was altered (Supplementary Video 9). Moreover, 8 h after wounding, as wound-edge cells had started to move forward, free spaces appeared between the cells of the front row, which, in some cases, detached from the rest of the monolayer (Fig. 8b–d and

Supplementary Fig. 7). The mean velocity of the front row of cells was increased and cell polarity, as assessed by centrosome reorientation, was altered (Fig. 8e–g and Supplementary Fig. 3d). The cell speed was also increased in the 3D-migration assay confirming the role of p120-ctn in the regulation of astrocyte velocity (Fig. 8h and Supplementary Video 12). In contrast, GSK3 inhibition prevented the formation of free spaces between cells to a level below that of control cells (Fig. 8d) and inhibited cell migration46. Comparison of p120-ctnWT and nonphosphorylatable p120-ctnT310A expression in p120-ctn-depleted cells confirmed the role of p120-ctn phosphorylation in these events. Expression of p120-ctnWT reduced the formation of free spaces between migrating cells, rescued centrosome reorientation and slowed down cell migration to a level similar to that of control nondepleted cells, as expected (Fig. 8c–g and Supplementary Video 13). In contrast, at similar level of expression, p120-ctnT310A reduced the formation of free spaces and cell velocity to levels significantly lower than those of control and rescued astrocytes (Fig. 8c–f and Supplementary Video 14). Expression of p120-ctnT310A, which inhibits the endocytosis and recycling of N-cadherin and AJ formation at the front, also induced a wider front edge (Fig. 7e,f) and did not rescue centrosome reorientation (Fig. 8g), suggesting that the polarized recycling of N-cadherin and the maintenance of AJs along lateral contacts are required for cell orientation. These results strongly suggest that the polarized, GSK3-dependent regulation of p120-ctn is essential for AJ dynamics and maintenance between collectively migrating cells and contributes to the integrity of the cell monolayer and the coordination of cell velocity and polarity during collective migration. DISCUSSION Collective migration relies on the maintenance of cellular interactions between co-migrating cells. AJs play a pivotal role by maintaining physical interactions and counteracting the forces exerted between cells and by serving as signalling platforms controlling cell polarity and directed migration8,21. We show here that AJs that form lateral contacts between adjacent leading cells undergo a continuous retrograde movement. AJ retrograde flow is particularly evident in N-cadherinexpressing astrocytes because of the low velocity of these cells32,47. However, it is also visible in fibroblasts and in VE-cadherin-expressing endothelial cells, supporting the idea that the retrograde movement of lateral junctions may be relevant to collective migration in a wide range of cell types. The speed of the retrograde movement of AJs is independent of the speed of migration and occurs also in non-migrating cells, as long as these cells exhibit an asymmetric distribution of AJs. ROCK and myosin kinase inhibitors prevent AJs movement, suggesting that acto-myosin contractility may be involved in the generation of forces required for the retrograde movement of AJs. Stress fibres running along the lateral sides of the cells, perpendicularly to the prominent transverse actin cables, can be observed and might contribute to these forces. The polarized recycling of cadherin from the cell rear to the front edge supports AJ treadmilling. However, connexin 43, which forms gap junctions and undergoes a retrograde flow (Supplementary Video 15), tends to accumulate in the rear half of the cell, suggesting

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Figure 7 GSK3-dependent regulation of p120-ctn phosphorylation and AJ dynamics during collective cell migration. (a) Astrocytes were treated with GSK3 inhibitors or nucleofected with the indicated siRNA before wounding. Cell lysates, obtained 0 or 8 h after wounding, were analysed by western blotting using the indicated antibodies. (b) N-cadherin and phosphoT310p120-ctn immunostaining in DMSO- or LiCl-treated migrating astrocytes. (c) p120-ctn (green) and N-cadherin (red) immunostaining along lateral contacts in migrating astrocytes treated with the indicated reagents. Higher magnification of the outlined region is shown on the right. The histogram shows the ratio between p120-ctn and N-cadherin fluorescence intensities at the front of lateral cell–cell contacts divided by the same ratio at the rear. Data are mean ± s.e.m. of n = 33, 37, 26, 30 and 36 cells, respectively for DMSO, LiCl, SB415286, control siRNA and GSK3 siRNA, analysed in 2 independent experiments. Using unpaired Student’s t-test, LiCl and SB415286 groups were compared with DMSO (P = 0.020 and 0.038 respectively), and GSK3 siRNA cells were compared with control siRNA (P = 0.048). (d) N-cadherin staining in astrocytes treated with the indicated reagents

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or in p120-depleted astrocytes transfected with the indicated constructs. Yellow arrowheads point to N-cadherin accumulation at the leading edge and yellow bars show the distance between the front of lateral AJs and the cell leading edge. The asterisk marks a GFP-expressing cell. (e) Histogram showing the percentage of cells with N-cadherin accumulation at the leading edge for each of the above conditions. 97, 101, 685, 512 and 488 cells, respectively for p120WT , p120T130A , DMSO, SB41586 and LiCl were scored. Apart from DMSO- and SB41586treated cells that were collected from n = 4 independent experiments, n = 3 experiments were analysed. Error bars indicate s.d. (f) Histogram showing the distance between the leading edge and the first AJs on the lateral sides of migrating cells. Data are mean ± s.e.m. of n = 60, 36, 79, 133 and 84 cells, respectively for p120WT , p120T310A , DMSO, LiCl and SB415286 from 3 independent experiments. e,f: ∗∗ P < 0.01, ∗∗∗ P < 0.005, unpaired Student’s t-test. The white arrows indicate the direction of migration. Scale bar, 10 µm. See Supplementary Fig. 8 for uncropped blots.

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Figure 8 Role of p120-dependent AJ dynamics during collective migration. (a) Diagram depicting the AJ dynamics during collective migration. AJs follow an actin-driven retrograde flow along lateral contacts. GSK3dependent phosphorylation of p120-ctn at the cell rear promotes N-cadherin internalization and recycling to the leading edge where GSK3 is inhibited and p120-ctn is not phosphorylated. (b–g) Astrocytes were nucleofected with the indicated siRNA. (b) Phase-contrast images of migrating astrocytes taken at the indicated time after wounding. (c) Representative images of N-cadherin immunostaining of astrocytes nucleofected with the indicated siRNA and constructs. Asterisks mark the transfected cells shown in the inset. (d) Histogram showing the formation of free spaces between migrating cells, as an indicator of the destabilization of AJs at the rear. Data represented are mean ± s.e.m. of 44 control siRNA, 67 p120 siRNA + GFP, 50 p120 siRNA + p120WT , 34 p120 siRNA + p120T310A , 124 DMSO and 78 LiCl cells collected from n = 3 independent experiments. (e) Diagrams showing the migration trajectories covered in 12 h by 15 representative astrocytes

nucleofected with p120-ctn siRNA and the indicated constructs. The x and y distances are given in pixels, with 1 pixel equivalent to 320 nm. (f) Mean velocity of astrocytes nucleofected with the indicated siRNA and constructs. Data represent mean ± s.e.m. of n = 103 control siRNA, n = 235 p120 siRNA + GFP, n = 64 p120 siRNA + p120WT and n = 45 p120 siRNA + p120T310A cells analysed in 3 independent experiments. (g) Percentage of migrating cells (8 h after wounding) in which the centrosome is located in the quadrant facing the wound in front of the nucleus, as an indication of cell polarization. Data represent mean ± s.e.m. of 307 control siRNA, 299 p120 siRNA + GFP, 238 p120 siRNA + p120WT , and 93 siRNA p120 + p120T310A cells from n = 3 independent experiments. (h) Histogram showing the mean velocity ± s.e.m. of n = 50 control and n = 75 p120-ctn-depleted astrocytes in a 3D Matrigel. Data were collected from 3 independent experiments. (d,f,g,h) NS: not significant (f, P = 0.142), ∗ P = 0.041, ∗∗ P < 0.01, ∗∗∗ P < 0.005, unpaired Student’s t-test. Scale bar, 10 µm. The white arrows indicate the direction of migration.

that connexins and cadherins undergo different recycling dynamics. Cadherin endocytosis preferentially occurs in the rear half of the cells, contrasting with the recycling of focal adhesions, which seems limited to the front half of the cell48,49. Several endocytic mechanisms have been described50. Here, cadherin localizes in clathrin-coated pits and is internalized in a dynamin- and Rab5-dependent manner51. Photoconversion of cadherin near the cell rear showed that cadherin transport across the total astrocyte length, typically 100 µm, is covered in 30–40 min, even if cadherin-containing vesicles moved much faster

(0.5–1 µm s−1 ) within the protrusion. N-cadherin-containing vesicles move towards the perinuclear area and may follow a retrograde transport to the Golgi complex as reported for E-cadherin52. Inhibition of vesicular traffic prevented cadherin accumulation at the leading edge, indicating that the lateral diffusion of cadherin in the plasma membrane is not sufficient to promote cadherin recruitment at the leading edge or the formation of new lateral AJs. p120-ctn is essential for the regulation of cadherin dynamics during migration, reflecting the well-documented function of p120-

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A RT I C L E S ctn in cadherin endocytosis and degradation in fibroblasts, and epithelial and endothelial cells38–40,53–55. p120-ctn interacts with the juxtamembrane domain of cadherin on the same domain as the clathrin adaptor AP2 and thus prevents cadherin internalization53,55,56. Even though p120-ctn is dispensable for the maintenance of AJs in confluent non-migrating astrocytes28, its role is nevertheless revealed during collective migration (Fig. 8). The association of p120-ctn to N-cadherin in AJs gradually decreases from the front to the rear of the cell. This polarized distribution of p120-ctn is controlled by its GSK3-dependent phosphorylation on Thr 310. GSK3 activity is downregulated by aPKC-dependent phosphorylation of Ser 9 at the front of migrating cells46, leading to the local accumulation of p120ctn–cadherin complexes. Depletion of p120-ctn or inhibition of its phosphorylation by GSK3 prevents N-cadherin accumulation at the leading edge and AJ formation at the front of lateral contacts. This results in the progressive interruption of the retrograde flow and in the formation of a broader leading edge, associated with a perturbation of cell orientation. These observations suggest that p120-ctn-dependent polarized recycling of N-cadherin supports AJs treadmilling and thereby the maintenance of AJs during collective migration. In p120ctn-depleted cells, N-cadherin endocytosis at the rear is increased, leading to the formation of holes between the leading cells and their followers. The progressive detachment of the leading cells leads to an increased cell speed, similar to what was previously observed when astrocytes or glioma cells expressed a lower level of N-cadherin27, suggesting that AJs serve as a break for the migration of the leading cells while preventing their separation from the rest of the cell group. These results support the idea that reduction of AJs as observed during epithelial morphogenesis or during the development of astrocyteor epithelial-derived tumours promotes single-cell migration27,57. We cannot exclude the possibility that the increase in cell velocity observed following p120-ctn depletion involves p120-ctn targets other than Ncadherin, such as RhoGTPases. In contrast to p120-ctn depletion, expression of the nonphosphorylatable mutant p120-ctnT310A prevented N-cadherin endocytosis at the rear. This also led to the inhibition of AJ formation at the front of lateral contacts, the formation of a broad leading edge, the perturbation of cell orientation and the inhibition of the retrograde flow of AJs. In this case, however, the AJs at the cell rear did not disassemble, cellular interactions at the rear remained strong and cell velocity was reduced. Together our observations strongly support a model in which p120-ctn regulates directed cell migration by controlling N-cadherin recycling and AJ dynamics and by maintaining the integrity of the migrating cell group. However, we cannot totally exclude that p120-ctn phosphorylation also contributes to the destabilization of N-cadherin-mediated AJs, by a mechanism involving RhoGTPases and the regulation of the cortical actin cytoskeleton. We propose that the regulation of AJ dynamics by the actin cytoskeleton and p120-regulated recycling of N-cadherin contributes to the maintenance of AJs while providing a highly adaptable adhesive system. Together with a previous study in epithelial cells, our findings highlight the particularly active dynamics of AJs between motile cells58. The free pool of cadherin complexes at the leading edge contributes to the formation of AJs between protrusions of adjacent migrating cells and probably helps maintain a linear front of migration. The leading-edge pool of cadherin–catenin complexes is

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likely to be crucial for the formation of new AJs when migrating cells collide and for contact-induced inhibition of cell locomotion. More generally, because the distribution of AJs in migrating cells controls cell speed and polarity16,27,28, the maintenance and adaptability of AJmediated cell interactions is vital for collective directed migration. The cadherin retrograde flow requires a polarized actin flow that itself depends on the geometry of cadherin-mediated contacts. Such a feedback mechanism could maintain the polarized organization of the actin cytoskeleton during migration. Furthermore, the retrograde flow of transverse actin cables pushes the nucleus towards the rear of the cell59, and possibly contributes to cell polarization60. The increased endocytosis of AJs at the rear of wound-edge cells progressively weakens the interactions between leading cells and their followers. Weakened intercellular adhesions at the front of the second row of cells might trigger their polarization and migration together with a retrograde flow of AJs. Such a mechanism would also contribute to the coordinated migration of the cell group. The crucial role of p120-ctn in AJ dynamics together with its function in the regulation of small G proteins of the Rho family and in the organization and dynamics of the cytoskeleton61–64 strongly suggests that downregulation of this tumour suppressor65 in cancer cells may contribute to increase tumour invasion.  METHODS Methods and any associated references are available in the online version of the paper. Note: Supplementary Information is available in the online version of the paper 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. AUTHOR 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. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Published online at www.nature.com/doifinder/10.1038/ncb2985 Reprints and permissions information is available online at www.nature.com/reprints 1. Friedl, P. & Gilmour, D. Collective cell migration in morphogenesis, regeneration and cancer. Nat. Rev. Mol. Cell Biol. 10, 445–457 (2009). 2. Friedl, P., Locker, J., Sahai, E. & Segall, J. E. Classifying collective cancer cell invasion. Nat. Cell Biol. 14, 777–783 (2012). 3. Gnanaguru, G. et al. Laminins containing the β2 and γ3 chains regulate astrocyte migration and angiogenesis in the retina. Development 140, 2050–2060 (2013). 4. Chu, Y., Hughes, S. & Chan-Ling, T. Differentiation and migration of astrocyte precursor cells and astrocytes in human fetal retina: Relevance to optic nerve coloboma. FASEB J. 15, 2013–2015 (2001). 5. Fruttiger, M. Development of the mouse retinal vasculature: Angiogenesis versus vasculogenesis. Invest. Ophthalmol. Vis. Sci. 43, 522–527 (2002).

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37. Theisen, C. S., Wahl, J. K. 3rd, Johnson, K. R. & Wheelock, M. J. NHERF links the N-cadherin/catenin complex to the platelet-derived growth factor receptor to modulate the actin cytoskeleton and regulate cell motility. Mol. Biol. Cell 18, 1220–1232 (2007). 38. Davis, M. A., Ireton, R. C. & Reynolds, A. B. A core function for p120-catenin in cadherin turnover. J. Cell Biol. 163, 525–534 (2003). 39. Reynolds, A. B. & Carnahan, R. H. Regulation of cadherin stability and turnover by p120ctn: Implications in disease and cancer. Semin. Cell Dev. Biol. 15, 657–663 (2004). 40. Xiao, K. et al. p120-Catenin regulates clathrin-dependent endocytosis of VEcadherin. Mol. Biol. Cell 16, 5141–5151 (2005). 41. Troyanovsky, R. B., Sokolov, E. P. & Troyanovsky, S. M. Endocytosis of cadherin from intracellular junctions is the driving force for cadherin adhesive dimer disassembly. Mol. Biol. Cell 17, 3484–3493 (2006). 42. Levayer, R., Pelissier-Monier, A. & Lecuit, T. Spatial regulation of Dia and MyosinII by RhoGEF2 controls initiation of E-cadherin endocytosis during epithelial morphogenesis. Nat. Cell Biol. 13, 529–540 (2011). 43. Macia, E. et al. Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10, 839–850 (2006). 44. Fukumoto, Y., Shintani, Y., Reynolds, A. B., Johnson, K. R. & Wheelock, M. J. The regulatory or phosphorylation domain of p120 catenin controls E-cadherin dynamics at the plasma membrane. Exp. Cell Res. 314, 52–67 (2008). 45. Etienne-Manneville, S. & Hall, A. Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity. Nature 421, 753–756 (2003). 46. Etienne-Manneville, S. & Hall, A. Cdc42 regulates GSK3 and adenomatous polyposis coli (APC) to control cell polarity. Nature 421, 753–756 (2003). 47. Etienne-Manneville, S. & Hall, A. Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta. Cell 106, 489–498 (2001). 48. Caswell, P. & Norman, J. Endocytic transport of integrins during cell migration and invasion. Trends Cell Biol. 18, 257–263 (2008). 49. Caswell, P. T., Vadrevu, S. & Norman, J. C. Integrins: Masters and slaves of endocytic transport. Nat. Rev. Mol. Cell Biol. 10, 843–853 (2009). 50. Kowalczyk, A. P. & Nanes, B. A. Adherens junction turnover: Regulating adhesion through cadherin endocytosis, degradation, and recycling. Subcell Biochem. 60, 197–222 (2012). 51. Ulrich, F. & Heisenberg, C. P. Probing E-cadherin endocytosis by morpholinomediated Rab5 knockdown in zebrafish. Methods Mol. Biol. 440, 371–387 (2008). 52. Lohia, M., Qin, Y. & Macara, I. G. The Scribble polarity protein stabilizes Ecadherin/p120-catenin binding and blocks retrieval of E-cadherin to the Golgi. PLoS ONE 7, e51130 (2012). 53. Chiasson, C. M., Wittich, K. B., Vincent, P. A., Faundez, V. & Kowalczyk, A. P. p120catenin inhibits VE-cadherin internalization through a Rho-independent mechanism. Mol. Biol. Cell 20, 1970–1980 (2009). 54. Hoshino, T. et al. Regulation of E-cadherin endocytosis by nectin through afadin, Rap1, and p120ctn. J. Biol. Chem. 280, 24095–24103 (2005). 55. Ishiyama, N. et al. Dynamic and static interactions between p120 catenin and E-cadherin regulate the stability of cell–cell adhesion. Cell 141, 117–128 (2010). 56. Nanes, B. A. et al. p120-catenin binding masks an endocytic signal conserved in classical cadherins. J. Cell Biol. 199, 365–380 (2012). 57. Ewald, A. J. et al. Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium. J. Cell Sci. 125, 2638–2654 (2012). 58. Kametani, Y. & Takeichi, M. Basal-to-apical cadherin flow at cell junctions. Nat. Cell Biol. 9, 92–98 (2007). 59. Dupin, I., Sakamoto, Y. & Etienne-Manneville, S. Cytoplasmic intermediate filaments mediate actin-driven positioning of the nucleus. J. Cell Sci. 124, 865–872 (2011). 60. Gomes, E. R., Jani, S. & Gundersen, G. G. Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells. Cell 121, 451–463 (2005). 61. Noren, N. K., Liu, B. P., Burridge, K. & Kreft, B. p120 catenin regulates the actin cytoskeleton via Rho family GTPases. J. Cell Biol. 150, 567–580 (2000). 62. Anastasiadis, P. Z. et al. Inhibition of RhoA by p120 catenin. Nat. Cell Biol. 2, 637–644 (2000). 63. Anastasiadis, P. Z. & Reynolds, A. B. The p120 catenin family: Complex roles in adhesion, signaling and cancer. J. Cell Sci. 113, 1319–1334 (2000). 64. Charrasse, S., Meriane, M., Comunale, F., Blangy, A. & Gauthier-Rouviere, C. N-cadherin-dependent cell–cell contact regulates Rho GTPases and beta-catenin localization in mouse C2C12 myoblasts. J. Cell Biol. 158, 953–965 (2002). 65. Stairs, D. B. et al. Deletion of p120-catenin results in a tumor microenvironment with inflammation and cancer that establishes it as a tumor suppressor gene. Cancer Cell 19, 470–483 (2011).

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METHODS

DOI: 10.1038/ncb2985

METHODS DNA constructs and siRNAs. pEGFP–N-cadherin (Ncad–GFP) was kindly provided by C. Gauthier-Rouvière66, pRcCMV–p120-1A by A. Reynolds67, pEGFP–p120 by A. Ridley61, pLifeAct–mCherry by M. Piel68, and pClathrin– mCherry by B. Goud. pConnexin43–GFP (Cx43–GFP) was provided by M. Cohen-Salmon. pVE-cadherin–GFP was a gift from D. Riveline. We also used pRK5–myc–Rab5a/S34N (dominant negative) and pNcad–mEOS2. Briefly, to generate pNcad–mEOS2, we took advantage of the pVimentin–mEOS2 kindly provided L. Looger69 and exchanged EGFP and mEOS2 to obtain pNcad–mEOS2 out of the pEGFP–N-cadherin plasmid. The following siRNA (Life Technologies) sequences were used: p120-ctn siRNA1: 50 -CUCUUCCUAGGAACUUCCAC-30 ; p120-ctn siRNA2: 50 -UGAAGACUGUAAGCCACGG-30 ; human p120 siRNA: 50 -GAGGAUGACCAGCGUAGUA-30 ; GSK3b siRNA1: 50 -CUUCAGGACAAGC GAUUUA-30 ; GSK3b siRNA2: 50 -CCACCGAUUACACGUCUAG-30 . siRNA duplexes and DNA constructs were introduced into cells using the nucleofection technology70 (Amaxa Biosystems). Cells were submitted to the siRNA duplexes for 3–5 days before triggering their migration (see below). The NIH3T3 and EA.hy926 cells were transfected using Fugene6 (Promega).

Cell culture and migration assays. Primary rat astrocytes were prepared as previously described32, according to the guidelines approved by the French Ministry of Agriculture, following European standards. For scratch-induced migration assays, cells were seeded on poly-L-ornithineprecoated coverslips for immunofluorescence on 35-mm-diameter glass-bottom culture dishes (MatTek) for videomicroscopy. Cells were grown to confluence. On the day of the experiment, cells were scratched with a blunt-ended microinjection needle—creating a 300–500-nm-wide wound—to trigger their migration. For 3D Matrigel migration (spheroid assays), we used the hanging-drop cell culture method. Briefly, astrocytes cultured in 35 mm Petri dishes were washed with PBS, trypsinized for 15 min, centrifuged at 8,928g and re-suspended in 2 ml of serum-free low-glucose DMEM medium supplemented with 20 ng ml−1 of epidermal growth factor (Sigma-Aldrich) and 20 ng ml−1 of basic fibroblast growth factor (Sigma-Aldrich). Drops (20 µl) of this suspension were deposited on the underside of a 6 cm Petri dish lid. To generate hanging drops, the lid was then inverted over a dish containing 5 ml of PBS. Drops were incubated at 37 ◦ C in 5% CO2 overnight allowing cells to aggregate into spheroids ranging in size from 50 to 200 µm in diameter. For 2D experiments, spheroids were then transferred directly in a 35 mm Petri dish filled with 2 ml of the same serum-free medium to be analysed using timelapse videomicroscopy. For 3D experiments, spheroids in serum-free medium were embedded in 1:4 diluted Matrigel (BD Biosciences) and monitored for dispersion after 1 h. NIH3T3 fibroblasts and EA.hy926 endothelial cells were maintained in DMEM (Invitrogen) supplemented with 10% FBS at 37 ◦ C under a 5% CO2 atmosphere. For scratch-induced migration assays, cells were seeded on fibronectin-precoated coverslips for immunofluorescence and on 35-mm-diameter glass-bottom culture dishes (MatTek) for videomicroscopy. Cells were grown to confluence. On the day of the experiment, cells were scratched with a blunt-ended microinjection needle— creating a 300–500-nm-wide wound—to trigger cell migration and the medium was changed after wounding. Cells were imaged 2 h after wounding. COS cells were maintained in high-glucose DMEM (Invitrogen) supplemented with 10% FBS at 37 ◦ C under a 5% CO2 atmosphere. Micropattern. Primary rat astrocytes co-transfected with N-cadherin–GFP and LifeAct–Cherry were plated onto fibronectin-coated micropatterns on glass-bottom tissue culture dishes, following a previously described protocol28. N-cadherin and actin fibre dynamics in immobile polarized cells were then monitored using fluorescence time-lapse live-cell imaging.

Drug treatments. Drugs were added 1 h before wounding the monolayer except for the drugs that destabilize the cytoskeleton, which were added 3–5 h after wounding to allow protrusion formation and the initiation of cell migration. Dynasore was used at 80 µM, nocodazole at 20 µM, ML-7 at 10 µM, Y-27632 at 10 µM, cycloheximide at 20 µg ml−1 , LiCl at 20 mM and SB415286 at either 20 µM or 50 µM. Except ML-7, which was from Calbiochem (Merck), all other drugs used were from Sigma-Aldrich. In Supplementary Video 11, dynasore was added during filming. To favour its rapid dispersion in the medium, an equal volume of a 160 µM solution was added to medium covering the cells. This addition induced a change of contrast that is visible in the video.

Western blotting. For western blots, cell lysates were obtained using Laemmli buffer containing 60 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS and 50 mM dithiothreitol. Proteins were separated by 10% SDS–PAGE and analysed using mouse monoclonal anti-N-cadherin (clone 32, diluted 1:1,000; catalogue number 610921, BD Biosciences Transduction Laboratories), mouse monoclonal

anti-p120-ctn (clone 98/pp120, 1:1,000; 610134, BD Biosciences Transduction Laboratories), mouse monoclonal anti-Pp120-ctn(Y96) (clone 25a, 1:1,000; 612534, BD Biosciences Transduction Laboratories), mouse monoclonal anti-Pp120ctn(Y228) (clone 21a, 1:1,000; 612536, BD Biosciences Transduction Laboratories), mouse monoclonal anti-Pp120(T310) (clone 22/p120, 1:10 000; 558203, BD Biosciences Transduction Laboratories) and rat monoclonal anti-α-tubulin (clone YL1/2, 1:3,000; MCA77G, Serotec) antibodies. Proteins were detected with the appropriate fluorophore-conjugated secondary antibodies (IRDye 680LT or IRDye 800CW, 1:10,000; 926-68029 or 926-32212, LI-COR) and imaged with an Odyssey Fc system (LI-COR Biosciences) allowing precise quantification, or using classical HRP-conjugated secondary antibodies detected by ECL chemoluminescent substrate (Pierce, Thermo Scientific).

Immunoprecipitation. Two days after transfection with Ncad–GFP and p120ctn–HA constructs, COS cells were washed with ice-cold PBS containing 1 mM orthovanadate and were lysed at 4 ◦ C in Nonidet P-40 buffer (10 mM Tris/HCl, pH 7.5, 140 mM NaCl, 1 mM orthovanadate and 1% Nonidet P-40, 2 mM phenylmethylsulphonyl fluoride, 5 mM EDTA, and protease inhibitor mix from Roche Applied Science). Nuclei were discarded after centrifugation at 15,000g for 10 min at 4 ◦ C. The supernatant was incubated at 4 ◦ C with mouse monoclonal antiGFP antibodies (clones 7.1 and 13.1, 1:1,000; 11814460001, Roche Applied Science) and protein G–Sepharose beads for 2 h. Immunoprecipitates were collected by centrifugation and extensively washed in Nonidet P-40 buffer. Immunoprecipitated proteins were eluted with SDS-sample buffer and analysed by 9% SDS–PAGE using anti-GFP and rat monoclonal anti-HA (clone 3F10, 1:2,000;11867431001, Roche Applied Science) antibodies. The amount of p120-ctn bound to the N-cadherin–GFP (Co-IP(HA)) was normalized by the expression level of the corresponding protein (input (HA)) and the level of immunoprecipitated Ncad–GFP (IP(GFP)) and was measured in 3 independent experiments.

Immunostaining. For immunofluorescence, we used the rat monoclonal anti-α-tubulin (1:100, Serotec), rhodamine-coupled phalloidin (1:400; Molecular Probes, Invitrogen), rabbit polyclonal anti-N-cadherin (1:200; ab12221, Abcam), mouse monoclonal anti-pan-cadherin (clone CH-19 diluted 1:100, C1821, Sigma-Aldrich), mouse monoclonal anti-p120-ctn (clone 98/pp120, 1:100; catalogue number 610134, BD Biosciences Transduction Laboratories) and anti-Pp120(T310) (clone 22/p120,1:100; catalogue number 558203, BD Biosciences Transduction Laboratories), mouse monoclonal anti-β-catenin (clone 14/β-catenin, 1:100; catalogue number 610154, BD Biosciences Transduction Laboratories), mouse monoclonal anti-α-catenin (clone 5,1:50; catalogue number 5610194, BD Biosciences Transduction Laboratories) and mouse monoclonal anti-Myc (clone 9E10, 1:50, M4439, Sigma-Aldrich). DyLight-488- and DyLight-649conjugated donkey anti-mouse, anti-rat and anti-rabbit secondary antibodies were from Jackson ImmunoResearch Laboratories. Donkey anti-rabbit and donkey anti-mouse coupled to TRITC were from Chemicon (Merck Millipore). Hoechst 33342 (Life Technologies) was used to visualize nuclei. Immunostaining was realized as described previously27. Pp120(T310) staining was optimized by fixing astrocytes during 15 min with cold TCA (Sigma) on ice. Coverslips were mounted in ProLong Gold antifade reagent. Epifluorescence images were obtained on a microscope (model DM6000, Leica, Solms) equipped with ×40, NA 1.25 and ×63, NA 1.4 objective lenses and were recorded on a CCD (chargecoupled device) camera (CoolSNAP HQ, Roper Scientific) using Leica software. Image processing was performed using the freely available Mac-Biophotonics ImageJ (NIH).

Protein localization at the leading edge. AJ protein enrichment at the leading edge was quantified as follows. To prevent false positives due to volume artefacts caused by membrane ruffling we used soluble GFP or mCherry (when cells were cotransfected with GFP fusion proteins) as volume markers and diI (Molecular Probes, Life Technologies) as a membrane marker. Eight hours after wounding, cells were fixed and stained with the appropriate antibody. The fluorescence intensities of the AJ proteins and the volume marker were measured along 3 different 10 µm lines at the front of the cell. A cell was counted as positive when there was a significant difference between the two staining intensities in each of the chosen lines.

Co-localization experiments. Astrocytes were electroporated by nucleofection with N-cadherin–GFP and clathrin–mCherry and the appropriate siRNA. Eight hours after wounding, cells were fixed and the GFP and mCherry intensity signals were analysed to assess co-localization. Briefly, images were thresholded and the co-localization was measured with the ImageJ software (US NIH) ‘colocalization analysis’ plugin. Co-localization of clathrin with N-cadherin was quantified at the front and at the rear of the cell. To define the cell front and the cell rear, the cell length (from the very rear of the cell to the leading edge) was visualized using the N-cadherin staining and divided into two regions. For both the cell front and the

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METHODS

DOI: 10.1038/ncb2985 cell rear regions, the ratio between the co-localized clathrin and N-cadherin staining was calculated.

of astrocytes stained for N-cadherin and p120-ctn or after 8 h of migration were thresholded for background subtraction. Then N-cadherin, p120-ctn and Pp120ctnT310 fluorescence intensities were measured along cell–cell contacts. Front and rear cell–cell contacts were defined as follows: lateral cell–cell contact length was measured and divided in 2 for the Pp120-ctn/Ncad ratio and in 3 for p120/Ncad. The first (starting from the leading edge) half or the first third of the cell was designated as the front and the second half or the last third designated as the rear.

non-rescued p120-depleted cells not contacting the rescued cells, within the same video frames, or with GFP-rescued p120-depleted cells. Migration tracks (Fig. 8) were obtained from 12 h videos. For each condition, 15 cells with equivalent levels of protein expression (as shown by GFP fluorescence), were selected at random. The nucleus of first-row p120-depleted astrocytes, not rescued (p120 siRNA), rescued with p120WT (rescue WT) or rescued with the non-phosphorylatable p120T130A (rescue T130A) was tracked using the manual tracking plugin in Image J during wound-healing-induced migration. The position of the cells was recorded every 20 min for 12 h and plotted in the graph. All of the trajectories were calculated so that the cells are seen starting from the origin (0,0) of the graph and moving towards the top of the figure.

Live-cell imaging. Nucleofected astrocytes, NIH3T3 fibroblasts or EA.hy926

Statistical analyses. Except for experiments comparing ratios at the front and at the

endothelial cells were seeded on 35-mm glass-bottom dishes (MatTek) and grown to confluence. On the day before wounding, the medium was changed to a red-phenolfree DMEM medium supplemented with 10% serum. The monolayer was wounded and cells were monitored 4 h later, allowing them to grow a polarized protrusion. In Supplementary Videos 2, 3, 5, 6, 7 and 9, astrocytes transfected with fluorescent tag proteins (N-cadherin–GFP, LifeAct–Cherry) were imaged using the Nikon BioStation IM-Q (cell S1 version), which includes a motorized inverted microscope equipped with a high-sensitivity cooled CCD camera and a dry ×40 0.8 NA Plan Fluor objective allowing magnifications of ×20, ×40 and ×80 depending on the experiments, within an incubation chamber (5% CO2 , 37 ◦ C). A Nikon Intensilight precentred fibre mercury lamp and standard FITC (EX 465-495/DM 505/BA 515-555) and TexasRed (EX 540-80/DM595/BA 600-660) filter blocks (Chroma) were used to excite respectively GFP and mCherry fusion proteins. Images from Supplementary Videos 1, 8, 10 and 15 were obtained with the Volocity 3D image analysis software on a spinning-disc confocal microscope (Axiovert 200, Zeiss, equipped with a confocal spinning-disc head Yokogawa CSU22; Perkin Elmer Life and Analytical Sciences). Images were acquired with a ×40 1.4 NA oil-immersion objective and recorded on an electron-multiplying CCD camera (C-9100; Hamamatsu Photonics). In Supplementary Video 4, photoconversion of N-cadherin–mEOS2 was performed on the same microscope set-up, iterating 10 to 20 times a pulse of a 405 nm laser at 20% power. Non-converted N-cadherin–mEOS2 was excited at 488 nm and photoconverted N-cadherin–mEOS2 was excited at 543 nm. The objective used was a ×63 oil immersion, 1.4 NA. FRAP experiments were also performed on the same microscope. Briefly, the GFP signal of N-cadherin–GFP was monitored under a 20% 488 nm laser and areas of 10 to 15 µm2 were bleached at 100% power for a few seconds. The FRAP data were then processed using the ImageJ FRAP profiler plugin. To analyse p120-ctn-siRNA-treated and -rescued cells (Supplementary Videos 12, 13 and 14), wounded monolayers were imaged with a Zeiss Axiovert 200M microscope, equipped with a humid chamber maintained at 37 ◦ C with 5% CO2 using a ×10 0.45-NA objective and a Hamamatsu ORCA II ER CCD camera (Supplementary Video 12) or with the Nikon Biostation IM-Q for rescue experiments (Supplementary Videos 13 and 14). Videos were analysed using the ImageJ software (W.S. Rasband, ImageJ, US NIH, http://rsb.info.nih.gov/ij/). The Manual Tracking plugin was used to monitor cell migration by tracking the nucleus of non-dividing cells located at the wound edge. One hundred randomly chosen wound-edge cells were analysed per experiment. The experiment was repeated 3–5 times. For rescue experiments, rescued cells are tracked and compared with

rear of the same cell where Wilcoxon matched pairs tests were applied, an unpaired Student’s t-test was applied for statistical analyses of the data, using GraphPad Prism software.

p120-ctn (or Pp120T310)/N-cadherin ratios along cell–cell contacts. Images

Repeatability

of experiments. Still images from time-lapse movies, immunofluorescence and western-blot scans shown in this manuscript are representative of the following number of analysed cells in independent experiments. In details, the image in Fig. 1a represents n = 7 cells, from 3 independent experiments, 1b: n > 50 cells, from 5 independent experiments,1c: n = 3 0 cells, 5 independent experiments, 1d: n = 7 cells, 4 independent experiments, 1e: n = 10 cells, 2 independent experiments, 1f: n = 3 cells, 3 independent experiments, 1g: n = 6 cells, 3 independent experiments, 2a: n > 50 cells, 3 independent experiments, 2b: n = 3 cells, 2 independent experiments, 2c: n > 30 cells, 3 independent experiments, 2f: see legend, 3a: n > 100 cells, 5 independent experiments, 3b: n > 300 cells, 5 independent experiments, 3c: n > 20 cells, >5 independent experiments, 3d: n = 2, 2 independent experiments, 3e: see legend, 3f: see legend, 4a: n = 8 cells, 3 independent experiments, 4b,c et d: n > 10 cells, 3 independent experiments, 5a: n > 91 cells, 3 independent experiments, 5c: n = 3, 3 independent experiments, 5d: n = 3, 3 independent experiments, 6a,c: n = 25, 3 independent experiments, 6d: 3 independent experiments, 6e :n = 35, 3 independent experiments, 6h: see legend, 7a: 3 independent experiments, 7b: n > 100 cells, 3 independent experiments, 7c: see legend, 7d: n > 60 cells, 3 independent experiments, 8b: 3 to 6 independent experiments, 8c: 3 independent experiments 8d,8g: see legend.  66. Mary, S. et al. Biogenesis of N-cadherin-dependent cell–cell contacts in living fibroblasts is a microtubule-dependent kinesin-driven mechanism. Mol. Biol. Cell 13, 285–301 (2002). 67. Reynolds, A. B., Herbert, L., Cleveland, J. L., Berg, S. T. & Gaut, J. R. p120, a novel substrate of protein tyrosine kinase receptors and of p60v-src, is related to cadherin-binding factors beta-catenin, plakoglobin and armadillo. Oncogene 7, 2439–2445 (1992). 68. Riedl, J. et al. Lifeact: A versatile marker to visualize F-actin. Nat. Methods 5, 605–607 (2008). 69. McKinney, S. A., Murphy, C. S., Hazelwood, K. L., Davidson, M. W. & Looger, L. L. A bright and photostable photoconvertible fluorescent protein. Nat. Methods 6, 131–133 (2009). 70. Etienne-Manneville, S., Manneville, J., Nicholls, S., Ferenczi, M. A. & Hall, A. Cdc42 and Par6/PKCζ regulate the spatially localized association of Dlg1 and APC to control cell polarization. J. Cell Biol. 170, 895–901 (2005).

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S U P P L E M E N TA R Y I N F O R M AT I O N DOI: 10.1038/ncb2985

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-p120ctn (b), antibeta-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 hours 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, 10µm.

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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 hours after wounding. Results are shown as mean ± SEM of 3 independent experiments in each conditions (first row n=440 cells, second row n=307).

2

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Supplementary Figure 3 p120 depletion does not alter N-cadherinmediated cell-cell contacts in confluent non migrating astrocytes. a, Westernblot (WB) showing p120ctn and tubulin expression levels in astrocytes nucleofected with control siRNA (si ctl) or siRNA specific for p120ctn (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 +/- SEM of normalized protein expression analysed in n=4 independent experiments using the LI-COR® technology. ***: p

Adherens junction treadmilling during collective migration.

Collective cell migration is essential for both physiological and pathological processes. Adherens junctions (AJs) maintain the integrity of the migra...
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