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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References
Friedl, P. & Gilmour, D. Collective cell migration in morphogenesis, regeneration and cancer. Nat. Rev. Mol. Cell Biol. 10, 445–457 (2009).
Friedl, P., Locker, J., Sahai, E. & Segall, J. E. Classifying collective cancer cell invasion. Nat. Cell Biol. 14, 777–783 (2012).
Gnanaguru, G. et al. Laminins containing the β2 and γ3 chains regulate astrocyte migration and angiogenesis in the retina. Development 140, 2050–2060 (2013).
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).
Fruttiger, M. Development of the mouse retinal vasculature: Angiogenesis versus vasculogenesis. Invest. Ophthalmol. Vis. Sci. 43, 522–527 (2002).
Faber-Elman, A., Solomon, A., Abraham, J. A., Marikovsky, M. & Schwartz, M. Involvement of wound-associated factors in rat brain astrocyte migratory response to axonal injury: In vitro simulation. J. Clin. Invest. 97, 162–171 (1996).
Sofroniew, M. V. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci. 32, 638–647 (2009).
Etienne-Manneville, S. Control of polarized cell morphology and motility by adherens junctions. Semin. Cell Dev. Biol. 22, 850–857 (2011).
Oda, H. & Takeichi, M. Evolution: Structural and functional diversity of cadherin at the adherens junction. J. Cell Biol. 193, 1137–1146 (2011).
Taguchi, K., Ishiuchi, T. & Takeichi, M. Mechanosensitive EPLIN-dependent remodeling of adherens junctions regulates epithelial reshaping. J. Cell Biol. 194, 643–656 (2011).
Twiss, F. et al. Vinculin-dependent Cadherin mechanosensing regulates efficient epithelial barrier formation. Biol. Open 1, 1128–1140 (2012).
Gloushankova, N. A. et al. Dynamics of contacts between lamellae of fibroblasts: Essential role of the actin cytoskeleton. Proc. Natl Acad. Sci. USA 95, 4362–4367 (1998).
Shaye, D. D., Casanova, J. & Llimargas, M. Modulation of intracellular trafficking regulates cell intercalation in the Drosophila trachea. Nat. Cell Biol. 10, 964–970 (2008).
Arboleda-Estudillo, Y. et al. Movement directionality in collective migration of germ layer progenitors. Curr. Biol. 20, 161–169 (2010).
Ewald, A. J., Brenot, A., Duong, M., Chan, B. S. & Werb, Z. Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis. Dev. Cell 14, 570–581 (2008).
Theveneau, E. et al. Collective chemotaxis requires contact-dependent cell polarity. Dev. Cell 19, 39–53 (2010).
Ganz, A. et al. Traction forces exerted through N-cadherin contacts. Biol. Cell 98, 721–730 (2006).
Smutny, M. & Yap, A. S. Neighborly relations: Cadherins and mechanotransduction. J. Cell Biol. 189, 1075–1077 (2010).
Ladoux, B. et al. Strength dependence of cadherin-mediated adhesions. Biophys. J. 98, 534–542 (2010).
Harris, T. J. & Tepass, U. Adherens junctions: From molecules to morphogenesis. Nat. Rev. Mol. Cell Biol. 11, 502–514 (2010).
Etienne-Manneville, S. Adherens junctions during cell migration. Subcell. Biochem. 60, 225–249 (2012).
Takeichi, M. Cadherins: A molecular family important in selective cell–cell adhesion. Annu. Rev. Biochem. 59, 237–252 (1990).
Angst, B. D., Marcozzi, C. & Magee, A. I. The cadherin superfamily: Diversity in form and function. J. Cell Sci. 114, 629–641 (2001).
Meng, W. & Takeichi, M. Adherens junction: Molecular architecture and regulation. Cold Spring Harb. Perspect. Biol. 1, a002899 (2009).
le Duc, Q. et al. Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II-dependent manner. J. Cell Biol. 189, 1107–1115 (2010).
Liu, Z. et al. Mechanical tugging force regulates the size of cell–cell junctions. Proc. Natl Acad. Sci. USA 107, 9944–9949 (2010).
Camand, E., Peglion, F., Osmani, N., Sanson, M. & Etienne-Manneville, S. N-cadherin expression level modulates integrin-mediated polarity and strongly impacts on the speed and directionality of glial cell migration. J. Cell Sci. 125, 844–857 (2012).
Dupin, I., Camand, E. & Etienne-Manneville, S. Classical cadherins control nucleus and centrosome position and cell polarity. J. Cell Biol. 185, 779–786 (2009).
Micalizzi, D. S., Farabaugh, S. M. & Ford, H. L. Epithelial-mesenchymal transition in cancer: Parallels between normal development and tumor progression. J. Mammary. Gland Biol. Neoplasia 15, 117–134 (2010).
Wheelock, M. J., Shintani, Y., Maeda, M., Fukumoto, Y. & Johnson, K. R. Cadherin switching. J. Cell Sci. 121, 727–735 (2008).
Baum, B., Settleman, J. & Quinlan, M. P. Transitions between epithelial and mesenchymal states in development and disease. Semin. Cell Dev. Biol. 19, 294–308 (2008).
Etienne-Manneville, S. In vitro assay of primary astrocyte migration as a tool to study Rho GTPase function in cell polarization. Methods Enzymol. 406, 565–578 (2006).
Maruthamuthu, V., Aratyn-Schaus, Y. & Gardel, M. L. Conserved F-actin dynamics and force transmission at cell adhesions. Curr. Opin. Cell Biol. 22, 583–588 (2010).
Bard, L. et al. A molecular clutch between the actin flow and N-cadherin adhesions drives growth cone migration. J. Neurosci. 28, 5879–5890 (2008).
Sabatini, P. J., Zhang, M., Silverman-Gavrila, R. V. & Bendeck, M. P. Cadherins at cell-autonomous membrane contacts control macropinocytosis. J. Cell Sci. 124, 2013–2020 (2011).
Sharma, M. & Henderson, B. R. IQ-domain GTPase-activating protein 1 regulates beta-catenin at membrane ruffles and its role in macropinocytosis of N-cadherin and adenomatous polyposis coli. J. Biol. Chem. 282, 8545–8556 (2007).
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).
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).
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).
Xiao, K. et al. p120-Catenin regulates clathrin-dependent endocytosis of VE-cadherin. Mol. Biol. Cell 16, 5141–5151 (2005).
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).
Levayer, R., Pelissier-Monier, A. & Lecuit, T. Spatial regulation of Dia and Myosin-II by RhoGEF2 controls initiation of E-cadherin endocytosis during epithelial morphogenesis. Nat. Cell Biol. 13, 529–540 (2011).
Macia, E. et al. Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10, 839–850 (2006).
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).
Etienne-Manneville, S. & Hall, A. Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity. Nature 421, 753–756 (2003).
Etienne-Manneville, S. & Hall, A. Cdc42 regulates GSK3 and adenomatous polyposis coli (APC) to control cell polarity. Nature 421, 753–756 (2003).
Etienne-Manneville, S. & Hall, A. Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta. Cell 106, 489–498 (2001).
Caswell, P. & Norman, J. Endocytic transport of integrins during cell migration and invasion. Trends Cell Biol. 18, 257–263 (2008).
Caswell, P. T., Vadrevu, S. & Norman, J. C. Integrins: Masters and slaves of endocytic transport. Nat. Rev. Mol. Cell Biol. 10, 843–853 (2009).
Kowalczyk, A. P. & Nanes, B. A. Adherens junction turnover: Regulating adhesion through cadherin endocytosis, degradation, and recycling. Subcell Biochem. 60, 197–222 (2012).
Ulrich, F. & Heisenberg, C. P. Probing E-cadherin endocytosis by morpholino-mediated Rab5 knockdown in zebrafish. Methods Mol. Biol. 440, 371–387 (2008).
Lohia, M., Qin, Y. & Macara, I. G. The Scribble polarity protein stabilizes E-cadherin/p120-catenin binding and blocks retrieval of E-cadherin to the Golgi. PLoS ONE 7, e51130 (2012).
Chiasson, C. M., Wittich, K. B., Vincent, P. A., Faundez, V. & Kowalczyk, A. P. p120-catenin inhibits VE-cadherin internalization through a Rho-independent mechanism. Mol. Biol. Cell 20, 1970–1980 (2009).
Hoshino, T. et al. Regulation of E-cadherin endocytosis by nectin through afadin, Rap1, and p120ctn. J. Biol. Chem. 280, 24095–24103 (2005).
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).
Nanes, B. A. et al. p120-catenin binding masks an endocytic signal conserved in classical cadherins. J. Cell Biol. 199, 365–380 (2012).
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).
Kametani, Y. & Takeichi, M. Basal-to-apical cadherin flow at cell junctions. Nat. Cell Biol. 9, 92–98 (2007).
Dupin, I., Sakamoto, Y. & Etienne-Manneville, S. Cytoplasmic intermediate filaments mediate actin-driven positioning of the nucleus. J. Cell Sci. 124, 865–872 (2011).
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).
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).
Anastasiadis, P. Z. et al. Inhibition of RhoA by p120 catenin. Nat. Cell Biol. 2, 637–644 (2000).
Anastasiadis, P. Z. & Reynolds, A. B. The p120 catenin family: Complex roles in adhesion, signaling and cancer. J. Cell Sci. 113, 1319–1334 (2000).
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).
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).
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).
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).
Riedl, J. et al. Lifeact: A versatile marker to visualize F-actin. Nat. Methods 5, 605–607 (2008).
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).
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).
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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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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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.
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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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DOI: https://doi.org/10.1038/ncb2985
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