Journal of Cell Science eateto imdclEgneig nvriyo aiona ai,C 51,USA 95616, CA Davis, California, of University Engineering, Biomedical of Yamada* Department Soichiro and Shih matrix Wenting three-dimensional a in cell migration promotes adhesion cell–cell N-cadherin-mediated Article Research tegh(oe ta. 01 ekade l,21;Wi and Weis 2011; al., adhesive et adhesion provide Leckband 2011; to 2006). al., Nelson, cell–cell structure et cytoskeletal (Gomez underlying strength cadherin-mediated unique the a al., and et promotes cytoskeleton, proteins Jacinto associating 1998; purse- actin Gipson, cadherin al., and and With et 1994), (Danjo 2001). Takeichi, Sawyer healing and 2009; wound (Steinberg al., string sorting et cell (Martin al., 2009), et and constriction Rauzi (Vestweber 2008; apical al., compaction et 2010), (Cavey embryo intercalation during cell 1985), role receptors, Kemler, central adhesion a calcium-dependent play Cadherins, movement. matrix. 3D a about in known cells migrating is between little adhesion but cell–cell significantly and adhesion, can matrix dimension Kubow cell-extracellular matrix 2010). al., alter the 2001; et that (Fraley suggest al., absent studies are cases These et some in (Cukierman and molecular 2011), different Horwitz, size have matrix and 3D substrate, substrates. soft compositions stiff a a 2D the in on complexes formed from on adhesive adhesions distinct focal migration prominent is the matrix cell Unlike 3D lamellipodia-driven the a in typical, Not including migration surfaces. stiff cell (2D), surprisingly, two-dimensional matrix, in external absent unique typically extracellular provide cues matrices Yamada, (3D) and of Three-dimensional (Geiger 2011). dimensionality properties and stiffness, key organization, the a focused have is primary studies on adhesion Recent surrounding phenotype. and matrix migration substrate of cell-to-extracellular assembly determinant complex thus Many the the sites, cells. tumor onto in phenotype cancer mesenchymal grabbing prototypical step of a by metastasis with first migrate and cells critical motile tissues a of is development migration Cell Introduction migration. collective 3D efficient for required N- is the linkage words: N-cadherin– actin of Key of and expression expression N-cadherin the the that dynamic contrast, three- and bundles a By migrate, actin a that phenotype. stress-fiber-like or suggest knockdown in with junctions findings the enriched faster rescued cell–cell cells were migrated partially form clusters and epithelial domain to a cell chains, extracellular the transformed multicellular failed or addition, elongated cells cytoplasmic invasive In formed N-cadherin-knockdown cadherin isolation. cells highly forces. in transformed epithelial-to- cells contractile these these complete single provided matrix, between than a 3D persistently underwent a adhesion more In not that and cell–cell are matrix. cells cells collagen transformed MDCK analyzed (3D) these hepatocyte-growth-factor-treated dimensional but we junctions, Using cell–cell transition, proteins. specific epithelial adhesion mesenchymal lose cell–cell typically tissues of epithelial devoid from originate that cells Cancer Summary 10.1242/jcs.103861 doi: 3661–3670 125, ß Science Cell of Journal 2012 March 15 Accepted ( correspondence for *Author ctnnciearsudtekokonpeoye u niiulclswti h elcutr eels oie oehr our Together, mobile. less were clusters cell the within cells individual but phenotype, knockdown the rescued chimera -catenin 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. elcl deini eurdfrcodntdmulticellular coordinated for required is adhesion Cell–cell elmgain elcl dein -ahrn Dmti,EMT matrix, 3D N-cadherin, adhesion, Cell–cell migration, Cell [email protected] ) eeo ihyivsv hntp na3 arx These between matrix. adhesion cell–cell 3D and signaling a required pro-migratory is and in both N-cadherin for switch and phenotype collectively cadherin migrate cell invasive cells E-to-N transformed highly 3D the a or undergo HGF- develop EMT that cells al., demonstrate complete we MDCK Here, et treated induce analyzed. been (Grotegut not can has expression invasion HGF E-cadherin transcription whether a snail, regulates 2006), of upstream that an in factor epithelial acts transformed HGF of Although invasion cells. cell analyzed developmental we cells, has normal MDCK migration in progression. cancer cell in only also epithelial but not processes, transformed implications, cancers in aggressive significant understanding tumor mechanistic in N-cadherin a a observed Therefore, of 2008). as often al., is et act cancer (Wheelock switch to of E- cadherin the thought hallmark Furthermore, to-N 2004). (N)-cadherin. is Christofori, a and (Cavallaro neural E-cadherin is suppressor and E-cadherin of of development, up-regulation epithelial down-regulation the of The down-regulation and the of (E)-cadherin profile receptors: expression gene adhesion the alters cell–cell EMT 2003). and Wolf, and 2007) Friedl al., cell et has collective process Wolf invasion multicellular 2011; to a cell crest as occur Similar cancer to shown 2009). embryogenesis, been neural in al., cancer observed initiate et metastatic migration also to and Thiery of progression to 2002; the epithelial thought and (Thiery, gastrulation invasion is cell an cancer but the drives embryogenesis, epithelial-to-mesenchymal from in delamination (EMT) transition This profile phenotypic transition expression morphology. a gene mesenchymal altered in the results and factors, transcription sn eaoyegot atr(G)a nETidcrof inducer EMT an as (HGF) factor growth hepatocyte Using specific of activation the by initiated is movement Cellular nvivo in Fid n imu,2009; Gilmour, and (Friedl nvitro in Iiae al., et (Ilina 3661 Journal of Cell Science olwr(F, follower el n el nacutr() vrg pes()adedon ipaeet I fsnl el (S, cells single of (I) displacements endpoint and (H) speeds Average (G). cluster a in cells and ( cells (C). cluster the in cell oiieHFtetdMC el wiearwed)mgaeaognihoignnGPepesn el na3 olgngl e losupplementar also See gel. collagen 3D a in cells non-GFP-expressing neighboring ( along 3. migrate Movie arrowheads) (white cells MDCK HGF-treated positive oag ro)adcutrd(ht roha)HFtetdMC el irtn na3 olgnmti.Seas upeetr aeilMve1 ( 1. 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Coordinating Supplementary 12 Research after release Cancer for PMC California in of of Deposited fund Institutes Committee. University the National and the GM094798]; the number from Award; [grant program Faculty Award; EUREKA New Investigator Health Young Family Beckman Hellman a by a supported was work This Funding help for Ronco analysis. Tony K- aggregation plasmid, cell the N-cadherin-GFP with sharing the for (Northwestern for Green University) University) Kathleen antibodies, (Stanford desmoplakin and Nelson cadherin James thank We Acknowledgements trypsin with dissociated cell preserve were to 25 calcium cells of mM volume 1.8 cultured Total with receptors. supplemented analysis, surface CA) Carlsbad, clustering (Invitrogen, cell For analysis drop Hanging f1. of ae,M,Rui . en,P .adLci,T. Lecuit, and F. P. Lenne, M., Rauzi, M., Cavey, ihDn’ othcts.Tedfeec a sue ob statistically be to assumed was difference then The times, test. 5 statistic when test post-hoc triturated significant Kruskal-Wallis using the was Dunn’s thresholding using object analyzed solution with by were results determined cell drop was Hanging cell the size ImageJ. to few aggregate hour, and average allowed cells The Every single imaged. and 4C). contained dish, (Fig. initially (e.g. and adhesion clusters culture conditions cell–cell these the strong under N-cadherin with dissociate of lines or cell lid N-cadherin Some wild-type the time. specified from for aggregate upside-down hanged were aj,Y n isn .K. I. Gipson, M. and K. Y. Yamada, Danjo, and R. D. Stevens, R., Pankov, E., Cukierman, eRoj . eses . aue,G,Shat,M .adWaterman-Storer, and A. M. Schwartz, G., Danuser, A., Kerstens, J., Rooij, de elmvmn a nlzdi h ietosprle n epniua othe to perpendicular and parallel directions the in analyzed was movement Cell otakidvda el ihnacutr G-rae el eestably were cells HGF-treated cluster, a within cells individual track To n gCM ncancer. in Ig-CAMs and ahrnmdae deesjntosa h edn deo h pteilwound, epithelial the of edge movement. leading cell the coordinated at providing junctions adherens cadherin-mediated dimension. third the to adhesions matrix E-cadherin. of immobilization and stabilization scattering. M. C. P , 0.05. 20) nerndpnetatmoi otato euae pteilcell epithelial regulates contraction actomyosin Integrin-dependent (2005). p m .Cl Biol. Cell J. -ahrnmdae elmgain3669 migration cell N-cadherin-mediated imtr eeaddt h arxt iulz h matrix the visualize to matrix the to added were diameter) m area/perimeter P , 0.01. 171 a.Rv Cancer Rev. Nat. 19) ci presrn’flmnsaeacoe yE- by anchored are filaments string’ ‘purse Actin (1998). 2 153-164. , ,i hc nojc ihpretcrl a nindex an has circle perfect with object an which in ), 20) elahso n inligb cadherins by signalling and adhesion Cell (2004). D m .Cl Sci. Cell J. c746 otiigapoiaey2000cells 250,000 approximately containing l Science 4 118-132. , a 0 ececls eedfiutto difficult were cells) rescue 509 Nature 20) w-irdmcaimfor mechanism two-tiered A (2008). 294 111 1708-1712. , 3323-3332. , 453 751-756. , P 20) aigcell- Taking (2001). , .5 Statistical 0.05. Journal of Cell Science uo,K .adHriz .R. A. Horwitz, and E. K. Kubow, ait,A,Mrie-ra,A n atn P. Martin, and A. Martinez-Arias, A., Jacinto, ln,O,Bke,G . aauo . omn,R .adFid,P. Friedl, and M. R. Hofmann, A., Vasaturo, J., G. Bakker, O., Ilina, Bro M., Tusch, Y., Hegerfeldt, iz . atagl,D,Iei,C . hpnir .adGbin,G. Gabbiani, and C. Chaponnier, E., C. Iselin, D., Mastrangelo, B., Hinz, F. Lehembre, and G. Christofori, D., Schweinitz, von S., Grotegut, oe,G . caha,R .adYp .S. A. Yap, and W. R. McLachlan, A., G. Gomez, az . abr,M,Se,A,Slezn . uun . Me M. K. A., Yamada, Buguin, and B. P., Geiger, Silberzan, A., Saez, M., Lambert, A., Ganz, agoi . opr . iag-acd,C,Gos,R,Mrhl,J F., J. Marshall, R., Grosse, C., Hidalgo-Carcedo, S., Hooper, C., Gaggioli, rel .adWl,K. Wolf, and P. D. Friedl, Gilmour, Longmore, A., and Celedon, P. H., D. Friedl, Kim, R., Krishnamurthy, Y., Feng, I., S. Fraley, I. W. Weis, and J. W. Nelson, S., Yamada, S., Pokutta, F., Drees, aauh,A,Ihhr,S n skt,S. Tsukita, and S. Ishihara, A., F. Nagafuchi, E. Wieschaus, and M. Kaschube, C., A. Martin, E. K. Mostov, and P. Leroy, eo-hie,M,Mis .E,Kpa,D .adBaknuy R. Brackenbury, and D. D. Kaplan, E., T. Meigs, M., Fedor-Chaiken, D. M. Henry, and G. J. Moreland, B., T. Bair, G., Strohbehn, M., J. Drake, 3670 imn .T,Puof .S,Jhsn .R n heok .J. M. Wheelock, and R. K. Johnson, S., R. Prudoff, T., M. Nieman, D. W. Figg, and K. D. Price, H., Li, J. Rooij, de and N. Wang, Q., Duc, le E., D. Leckband, uinadrepair. and fusion htnlsrgnrtdmcorcsi Dclae atcs rnilso MMP- of principles lattices: invasion. cell collagen cancer 3D collective -independent in and dependent microtracks laser-generated photon ehncltnincnrl rnlto isecnrcieatvt n myofibroblast and activity contractile tissue differentiation. granulation controls tension beta1- Mechanical interaction, cell-cell of strategies. migration plasticity and explants: function, melanoma integrin primary in MAPK/Egr-1-mediated movement through scattering cell Snail. induces of upregulation factor growth Hepatocyte esn n oesai fcl-eljunctions. cell-cell of homeostasis and sensing adhesions. 98 B. Ladoux, acnm el ihdfeigrlsfrRoTae nlaigadfloigcells. following and leading in RhoGTPases Biol. E. for Cell roles Nat. differing Sahai, with cells and carcinoma K. Harrington, saemechanisms. escape cancer. and regeneration motility. cell D. dimensional Wirtz, and D. G. ein fcdei yolsi oan r novdi upesn oiiyo a of motility suppressing in involved line. are cell domains carcinoma cytoplasmic mammary cadherin of regions actin- regulates and E-cadherin-beta-catenin binds that assembly. switch filament molecular a is catenin of phenotype epithelial the cells. represses cancer and prostate migration transendothelial enhances ZEB1 ahrnpooe oiiyi ua ratcne el eadeso hi E-cadherin their of regardless cells cancer breast expression. human in motility catenin promotes cadherin E-cadherin-alpha of analysis functional molecules. fusion adhesion: cell cadherin-mediated constriction. apical cancer. drive network prostate actin-myosin androgen-independent and angiogenesis of Drugs Anticancer models preclinical in tubulogenesis. 1943-1952. HGF-induced of transition epithelial-mesenchymal adhesions. cadherin-mediated at matrices. 3D in adhesions 721-730. , ora fCl cec 2 (15) 125 Science Cell of Journal odSrn ab eset Biol. Perspect. Harb. Spring Cold .Cl Biol. Cell J. 20) rcinfre xre hog -ahrncontacts. N-cadherin through exerted forces Traction (2006). 9 m .Pathol. J. Am. 1392-1400. , .Cl Biol. Cell J. a.Cl Biol. Cell Nat. 18 Cell a.Rv Cancer Rev. Nat. 563-568. , o.Bo.Cell Biol. Mol. 21) itntv oefrfclahso rtisi three- in proteins adhesion focal for role distinctive A (2010). MOJ. EMBO 20) uorcl nainadmgain iest and diversity migration: and invasion Tumour-cell (2003). 123 147 a.Rv o.Cl Biol. Cell Mol. Rev. Nat. a.Cl Biol. Cell Nat. a.Cl Biol. Cell Nat. 903-915. , 631-644. , 20) lgi eurdfrcl uvvldrn partial during survival cell for required is Slug (2007). 20) olciecl irto nmorphogenesis, in migration cell Collective (2009). ce,E .adFid,P. Friedl, and B. E. ¨cker, 159 21) oeua rhtcueadfnto fmatrix of function and architecture Molecular (2011). 127 .Bo.Chem. Biol. J. 25 ur pn elBiol. Cell Opin. Curr. 21) euigbcgon loecnereveals fluorescence background Reducing (2011). 3 1009-1020. , 235-245. , 20) D1 nNcdei niio,evaluated inhibitor, N-cadherin an ADH1, (2007). E117-E123. , 3534-3545. , 20) irbatldcletv nainof invasion collective Fibroblast-led (2007). 20 3 2207-2217. , 362-374. , 12 13 acrRes. Cancer 598-604. , 3-5. , 3 19) h oe fctnn nthe in catenins of roles The (1994). 278 rnsCl Biol. Cell Trends a005033. , Nature 20) ehnsso epithelial of Mechanisms (2001). 52371-52378. , 21) rdcietnin force- tension: Productive (2011). 10 20) usdcnrcin fan of contractions Pulsed (2009). 445-457. , 21) Mechanotransduction (2011). 457 23 62 523-530. , 2125-2130. , 495-499. , hs Biol. Phys. 20) olciecell Collective (2002). 21 o.Bo.Cell Biol. Mol. g,R .and M. R. `ge, 499-505. , 20) Alpha- (2005). 21) Two- (2011). 20) Two (2003). 8 015010. , 19) N- (1999). il Cell Biol. (2006). (2001). (2009). 18 , aaa . out,S,Des . es .I n esn .J. W. Nelson, and I. S. Tsukita, W. and A. Weis, Nagafuchi, M., F., Itoh, S., Drees, Yonemura, S., Pokutta, S., Yamada, aaa .adNlo,W J. W. Nelson, and M. S. I. Yamada, Herman, and D. T. Pollard, J., A. Wong, R. K. Johnson, and Y. Fukumoto, M., Maeda, Y., Shintani, J., M. Wheelock, Weis,W.I.andNelson,W.J. A. M. Nieto, and Y. R. Huang, H., Acloque, P., J. Thiery, P. J. Thiery, hvna,E,Mrhn,L,Krym,S,Gl,M,Mep,B,Pros .and M. Parsons, B., Moepps, M., Gull, S., Kuriyama, L., Marchant, E., Theveneau, of . u .I,Lu . egr . a,E,Oeal . tc,M .adFriedl, and S. M. Stack, C., Overall, E., Tam, J., Geiger, Y., Liu, I., Y. Wu, K., Wolf, R. Kemler, and D. Vestweber, Vasioukhin,V.,Bauer,C.,Yin,M.andFuchs,E. aaa . oo . rn .P,Myzk,H,Ymsio . hmmr,T., Shimomura, J., Yamashiro, H., Miyazaki, P., C. Tran, E., Kono, H., Tanaka, aaii .J,Zag . ivra-arl,R,Bnek .P n Langille, and P. M. Bendeck, R., Silverman-Gavrila, M., Zhang, J., P. Sabatini, T. Lecuit, and F. H. P. Lenne, C. M., Rauzi, Siu, and J. Wang, N., Chen, J., Qi, P. Rørth, and A. Pacquelet, hkni . aaia .adKwuh,T. Kawauchi, and K. Nakajima, M. M., Peifer, Shikanai, and U. S. Gaul, Yamada, and C., W. Shih, K. Slep, J., N. Harris, K., J. Sawyer, P. Doherty, and G. Williams, V., F. Howell, E., Sanchez-Heras, tibr,M .adTkih,M. Takeichi, and S. M. Steinberg, 142. ucinfrainadatnflmn raiain iiaiisaddifferences and similarities organization: cells. epithelial filament polarized and actin fibroblasts non-polarized and between formation junction eosrcigtecdei-aei-ci complex. cadherin-catenin-actin the Deconstructing adhesion. cell-cell epithelial of expansion and initiation vivo. in cells endothelial vascular conundrum. Cancer Rev. olciecne elinvasion. cell cancer collective P. switching. Cadherin uvomorulin. of disease. and development in transitions mesenchymal Cell Dev. R. Mayor, agtn fNcdei niispott acrgot,mtsai n castration and metastasis growth, cancer al. prostate et L. inhibits resistance. R. N-cadherin Vessella, J., of Huang, targeting R., Wada, L., Fazli, oyeiaini h rvn oc o pteilcl-eladhesion. cell-cell epithelial for force driving the is polymerization oaiyadrglt irto fvsua mohmsl cells. muscle smooth vascular of migration regulate and polarity L. B. rspiaaai ooou ao euae ikg fteatnctseeo to cytoskeleton actin the constriction. of apical linkage during regulates junctions Canoe adherens homologue afadin Drosophila 35216. 412. remodelling. junction epithelial control beta- flows the of activation and pathway. adhesion signaling N-cadherin-mediated catenin involves cells adhesion. of melanoma types other and migration cell in function isesraig n pcfcsailptenn yqatttv ifrne ncadherin in differences quantitative by patterning expression. spatial specific and spreading, tissue neurons. locomoting 4 cortical of migration fiber-dependent glial migration. molecule. adhesion cell of all neural and N-cadherin of with interactions isoforms for major essential is the box acid receptor factor growth 326-330. , 20) ut-tpprclua rtoyi otostetasto rmidvda to individual from transition the controls proteolysis pericellular Multi-step (2007). 20) ooyi n nohla elahsosvaNcdei determine N-cadherin via adhesions cell endothelial and Homotypic (2008). 19 ipy.J. Biophys. a.Med. Nat. 20) pteilmsnhmltastosi uorprogression. tumour in transitions Epithelial-mesenchymal (2002). rc al cd c.USA Sci. Acad. Natl. Proc. 21) olcieceoai eurscnatdpnetcl polarity. cell contact-dependent requires chemotaxis Collective (2010). .Bo.Chem. Biol. J. 39-53. , 2 442-454. , MOJ. EMBO .Cl Sci. Cell J. 16 98 21) ysnIAdpnetrtord lwdie Dcell 3D drives flow retrograde dependent IIA Myosin (2010). 1414-1420. , L29-L31. , 4 20) euaoymcaim eurdfrDE-cadherin for required mechanisms Regulatory (2005). 3393-3398. , o.Bo.Cell Biol. Mol. 281 18) dniiaino uaiecl deindomain adhesion cell putative a of Identification (1985). 20) oaie oe fRoadRcatvte drive activities Rac and Rho of zones Localized (2007). 35593-35597. , a.Cl Biol. Cell Nat. 121 19) xeietlseiiaino elsorting, cell of specification Experimental (1994). Science 20) esligtecadherin-catenin-actin the Re-solving (2006). 727-735. , 21) lnrplrzdatmoi contractile actomyosin polarized Planar (2010). 91 206-209. , 219 16 20) rnedteilmgainof migration Transendothelial (2005). 4386-4397. , Nature .Cl Biol. Cell J. 867-869. , 18) ci iaetsrs iesin fibers stress filament Actin (1983). 9 893-904. , 21) -ahrnrgltsradial regulates N-cadherin (2011). Cell 468 Cell 21) oolnlantibody Monoclonal (2010). .Cl Biol. Cell J. 19) elt-eladherens Cell-to-cell (1995). .Bo.Chem. Biol. J. .Cl Biol. Cell J. 123 1110-1114. , 186 139 20) ietdactin Directed (2000). 889-901. , omn ner Biol. Integr. Commun. 57-73. , 20) h fibroblast The (2006). 871-890. , .Cl Sci. Cell J. ic Res. Circ. 20) Epithelial- (2009). Cell 170 178 100 281 20) The (2009). 803-812. , 517-527. , 209-219. , 103 108 35208- , (2005). (2008). 405- , 127- , Nat.