research critique paper-1200 wordsAnalyses the article attached titled "Cadherin Switch during EMT in Neural Crest Cells Leads to Contact Inhibition of Locomotion via Repolarization of Forces" and write a critically analysed essay answering the questions in the paper critique .pdf- this powerpoint explains the assignment in detail and how each section should be answered and what the markers are looking for. Make sure to summarize findings of the article and talk about the strength and weaknesses of the article.
Cadherin Switch during EMT in Neural Crest Cells Leads to Contact Inhibition of Locomotion via Repolarization of Forces Article Cadherin Switch during EMT in Neural Crest Cells Leads to Contact Inhibition of Locomotion via Repolarization of Forces Graphical Abstract Highlights d Neural crest cells acquire contact inhibition of locomotion (CIL) during EMT d An E- to N-cadherin switch controls CIL d E-cadherin represses CIL by controlling Rac1-dependent protrusions via p120 d During CIL, forces are redistributed from intercellular junctions to cell matrix Scarpa et al., 2015, Developmental Cell 34, 421–434 August 24, 2015 ª2015 The Authors http://dx.doi.org/10.1016/j.devcel.2015.06.012 Authors Elena Scarpa, András Szabó, Anne Bibonne, Eric Theveneau, Maddy Parsons, Roberto Mayor Correspondence
[email protected] In Brief Cell-cell adhesions are transiently formed during contact inhibition of locomotion. Scarpa et al. show that an E- to N- cadherin switch controls CIL acquisition upon EMT. E-cadherin loss leads to repolarization of protrusions via p120 and Rac1, resulting in redistribution of forces from intercellular tension to cell-matrix adhesions, triggering junction disassembly. mailto:
[email protected] http://dx.doi.org/10.1016/j.devcel.2015.06.012 http://crossmark.crossref.org/dialog/?doi=10.1016/j.devcel.2015.06.012&domain=pdf Developmental Cell Article Cadherin Switch during EMT in Neural Crest Cells Leads to Contact Inhibition of Locomotion via Repolarization of Forces Elena Scarpa,1 András Szabó,1 Anne Bibonne,2 Eric Theveneau,1,2 Maddy Parsons,3 and Roberto Mayor1,* 1Cell and Developmental Biology Department, University College London, Gower Street, London WC1E 6BT, UK 2Centre de Biologie du Développement–UMR5547, Centre National de la Recherche Scientifique and Université Paul Sabatier, Toulouse 31400, France 3Randall Division of Cell and Molecular Biophysics, Kings College London, London SE11UL, UK *Correspondence:
[email protected] http://dx.doi.org/10.1016/j.devcel.2015.06.012 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). SUMMARY Contact inhibition of locomotion (CIL) is the process through which cells move away from each other after cell-cell contact, and it contributes tomalignant inva- sion and developmental migration. Various cell types exhibit CIL, whereas others remain in contact after collision and may form stable junctions. To investi- gate what determines this differential behavior, we study neural crest cells, a migratory stem cell popu- lation whose invasiveness has been likened to can- cer metastasis. By comparing pre-migratory and migratory neural crest cells, we show that the switch from E- to N-cadherin during EMT is essential for acquisition of CIL behavior. Loss of E-cadherin leads to repolarization of protrusions, via p120 and Rac1, resulting in a redistribution of forces from intercel- lular tension to cell-matrix adhesions, which break down the cadherin junction. These data provide insight into the balance of physical forces that con- tributes to CIL in cells in vivo. INTRODUCTION More than 50 years ago, Abercrombie and Heaysman discov- ered that the direction of migration of chick heart fibroblasts cultured in vitro was modified by their interaction with other cells (Abercrombie and Heaysman, 1953). This process was defined as contact inhibition of locomotion (CIL). Its potential importance emerged as Abercrombie and colleagues showed that invasion of normal fibroblasts bymalignant mesenchymal cells was linked to a modified CIL response, linking CIL to invasive metastasis (Abercrombie, 1979; Abercrombie and Ambrose, 1962; Aber- crombie and Heaysman, 1954). More recently, CIL was shown to regulate the invasiveness of prostate malignant cells toward stromal fibroblast (Astin et al., 2010). Furthermore, the require- ment of CIL in guiding complex migratory processes during em- bryonic development has been demonstrated in vivo for neural crest (NC) cells and macrophages (Carmona-Fontaine et al., Develop 2008; Stramer et al., 2010). The molecular pathways underlying CIL remained poorly understood for decades. However, in both prostate cancer cells and Xenopus NC cells, the CIL response seems to rely on cell-cell contact-dependent signaling. In particular, Eph-Ephrin signaling has been found to be respon- sible for CIL in cancer cells (Astin et al., 2010), while in NC cells, activation of Wnt-PCP pathway leads to recruitment of Frizzled to the cell-cell contacts and activation of RhoA-ROCK, which is required for cell separation (Carmona-Fontaine et al., 2008). In addition, it has been suggested that cadherin-dependent cell-cell adhesion is required for CIL (Becker et al., 2013; Theve- neau et al., 2010, 2013). During Xenopus neural crest-neural crest (NC-NC) and neural crest-placode (NC-PL) cell-cell inter- actions, N-cadherin is functionally required for CIL (Theveneau et al., 2010, 2013), and a classical cell adhesion complex formed by N-cadherin, p120, a-catenin, and b-catenin is transiently assembled upon these cell-cell interactions (Theveneau et al., 2010, 2013). However, both the NC-NC and the NC-PL junctions have a short half-life and eventually disassemble (Theveneau et al., 2013). Many pending questions remain. Why do certain cell types undergo CIL, whereas others cells do not? Why do some cell-cell interactions lead to the formation of a stable adhe- rens junction while during CIL these junctions are transient? Here we have used NC cells, a migratory embryonic stem cell population, to address these questions. We show that NC cells acquire CIL at the same time that they activate their epithe- lial-to-mesenchymal (EMT) program and start migrating. By comparing premigratory and migratory NC cells, we show that switching E- to N-cadherin during EMT is essential for CIL. We demonstrate that prior to EMT E-cadherin inhibits contact- dependent cell polarity via p120 and Rac1. Culturing NC on micropatterns, photoactivating different forms of Rac and measuring traction forces during CIL, we conclude that the cad- herin switch leads to cell-cell junction breakdown by generating higher forces resulting from cell repolarization. RESULTS CIL Is a Developmentally Regulated Property of NCCells Acquired during EMT Xenopus NC cells are an archetypical model for CIL, whose CIL response is well characterized, and it is essential for their mental Cell 34, 421–434, August 24, 2015 ª2015 The Authors 421 mailto:
[email protected] http://dx.doi.org/10.1016/j.devcel.2015.06.012 http://creativecommons.org/licenses/by/4.0/ http://crossmark.crossref.org/dialog/?doi=10.1016/j.devcel.2015.06.012&domain=pdf directional migration in vivo and in vitro (Carmona-Fontaine et al., 2008; Moore et al., 2013; Theveneau et al., 2010). To investigate whether CIL is an intrinsic property of NC or whether it is acquired during NC development, we cultured Xen- opus laevis premigratory NC (Premig-NC) before they undergo EMT and compared them with migratory NC (Mig-NC) after EMT has taken place. Nearly 80% of observed cell-cell collisions of Mig-NC showed typical CIL by forming a transient contact, stopping migration and moving away, while only 40% of Pre- mig-NC collisions exhibited CIL (Figures 1A and 1B; Movie S1, collision assay) with most Premig-NC forming a stable contact and their nuclei remaining within a short cell-cell distance (Fig- ure 1C). This differential behavior is not due to a difference in cell motility as the speed of migration is the same between Pre- mig-NC and Mig-NC (Figures S1A and S1B). At the cell popula- tion level, CIL is known to prevent cell mixing, as has been shown in Mig-NC explants exhibiting CIL (Carmona-Fontaine et al., 2008). While our observations in Mig-NC explants confirm this result (Figures 1D and 1E), the Premig-NC intermingled readily indicating a lack of CIL (Figures 1D and 1E; Movie S1, overlap assay). At migratory stages, NC explants are known to undergo EMT in vitro (Kuriyama et al., 2014) and disperse due to CIL (Car- mona-Fontaine et al., 2011;Woods et al., 2014). Such dispersion was observed in Mig-NC explants but not in Premig-NC (Figure 1F; Movie S1, dispersion assay), as shown by Delaunay triangulation analysis (Carmona-Fontaine et al., 2011) (Figures 1G and 1H). During CIL, cell protrusions are polarized via small GTPase activity leading to the formation of lamellipodia away from the cell contact in migrating NC clusters (Carmona-Fon- taine et al., 2008; Theveneau et al., 2010). In Mig-NC explants, most protrusions pointed away from the cell contact and toward the free space, while in Premig-NC, most lamellipodia were beneath the cell-cell contacts, as shown by cell membrane (Fig- ures 1I and 1J; Movie S1, protrusion analysis) and F-actin distri- bution (Figures 1K and 1L; Movie S1, LifeAct-GFP). Consistently, Rac1 activity in Mig-NC was high at the free edge (Figures 1M, top, white arrows, and 1N) and low at cell-cell contacts (Figures 1M, top, black arrowheads, and 1N), as detected by FRET (Itoh et al., 2002; Theveneau et al., 2010). Importantly, Rac1 activity in Premig-NC was reversed, being low at the free edge (Figures 1M, bottom, white arrows, and 1N) and high at cell-cell contacts (Figures 1M, bottom, black arrowheads, and 1N). Interestingly, the difference in protrusive activity between Premig-NC and Mig-NC, which is likely to be a consequence of the differential distribution of active Rac1, affected higher order features of CIL such as intermixing between cells (Figures S1C–S1F). High-resolution imaging of explant overlap assays shows that boundaries between differentially labeled Mig-NC clusters are significantly straighter (Monier et al., 2010) than in Premig-NC (Figures S1C and S1D). In addition, the duration of protrusions at the boundary (Figure S1E, arrowheads) was significantly higher in Premig-NC (Figures S1E and S1F), while Mig-NC tended to collapse protrusions upon contact (Figures S1E and S1F, arrows).In summary, these results show that NC cells acquire CIL at the time of their EMT. Analysis of Cell Junctions during CIL The distinct behavior of Mig-NC and Premig-NC in response to cell-cell interactions might arise from differential dynamics of 422 Developmental Cell 34, 421–434, August 24, 2015 ª2015 The Au junction formation. To test this, we expressed p120-GFP or a-Catenin-GFP in Mig-NC or Premig-NC and imaged cell colli- sions with high time resolution. Expression of p120-GFP or a-Catenin-GFP did not per se affect the CIL response of Mig- NC (Figures S2A and S2B). BothMig-NC and Premig-NC formed junctions containing p120 (Figures 2A–2C; Movie S2) and a-cat- enin (Figures 2D–2F; Movie S2) with similar dynamics. However, in Mig-NC, cell-cell contacts were rapidly disassembled while they persisted in Premig-NC (Figures 2C and 2F). Indeed, the duration of contact in Premig-NC was strongly increased when compared with Mig-NC (Figures 2G and 2H). Taken together, these findings suggest that Mig-NC is unable to stabilize their junctions. Based on this, we postulated that the composition of endogenous adherens junctions might be different in Mig-NC and Premig-NC. Indeed, a-catenin and b-catenin levels of immu- nostaining were higher in Premig-NC adhesions than in Mig-NC junctions (Figures 2I–2K). Second, we analyzed the expression of classical cadherins in Mig-NC and Premig-NC since cadherin switching has been observed during EMT in cancer cells and NC development in other organisms (Dady et al., 2012; Wheelock et al., 2008), and our data demonstrate that the acquisition of CIL correlates with EMT. We found that Mig-NC predominantly expressed N-cadherin, while Premig-NC expressed E-cadherin (Figures 2L–2N). The differential cadherin expression suggests