Japan. inln,RKNCne o Development for Center RIKEN Signaling, Ato o orsodne([email protected]) correspondence for *Author Ok Studies, Advanced rdaeSho fMdcn,Kb 5-07 Japan. 650-0017, Kobe Medicine, of School Graduate uaoOda Yukako Cdc42 tricellular at through cells contacts epithelial of tension junctional regulates Tricellulin ARTICLE RESEARCH ß eevd2 aur 04 cetd1 uy2014 July 11 Accepted 2014; January 24 Received The 5 2007). Nelson, 3 and Yamada 2005; 2010; Hall, 1 and al., (Jaffe process et this II signaling their in myosin Smutny and and involved The Rho polymerization are Cdc42, F-actin activation, 2011). including regulate proteins, which Yonemura, G effectors, small 2005; of tensile pathways generate (Gumbiner, to II myosin The circumferential force with called cooperate 1963). filaments, which Palade, actin bundles, and thick apical actin by (Farquhar are the delineated membrane circumscribe is which lateral AJC and junctions, the other of tight each part and with by associated junctions crucial closely AJC play adherens processes the to vertebrates, of In thought consists biological behavior. is cell actomyosin actomyosin-related (AJC) in of complex cells, roles of epithelial junctional In variety apical contraction behavior. the and organization a by cellular regulating generated controls force filaments tensile The INTRODUCTION cell Epithelial Tuba, F-actin, MARVELD2, Cdc42, Tricellulin, junction, in Cell–cell complex WORDS: roles KEY junctional crucial system. tricellulin–Tuba–Cdc42 apical play the through actomyosin-mediated contacts tension tricellular the that regulating These A cells. indicate tricellulin-knockdown Cdc42. of findings the activates phenotype rescue border cannot Tuba and cell bind to curved ARHGEF36), ability the lacks and that mutant tricellulin (also DNMBP Tuba as junction (GEF) factor known to cell–cell binds guanine-nucleotide-exchange tricellulin during Cdc42 of F- the domain contacts cytoplasmic of N-terminal tricellular organization The impaired formation. around and polygonal fibers borders at cell irregular actin curved localized exhibit with is cells shapes Cdc42. which through epithelial organization MARVELD2), Tricellulin-knockdown F-actin regulates as contacts, known tricellular that show we (also Here, studied. its epithelial tricellulin well been in whereas not roles have junctions, a whose morphogenesis with contacts, cell cell–cell tricellular compared to to is correspond correspond cell vertices epithelial sides each its of polygon, view surface the When ABSTRACT 4-77 Japan. 444-8787, iiino eerlSrcue ainlIsiuefrPyilgclSine,Okazaki Sciences, Physiological for Institute National Structure, Cerebral of Division Sciences of Faculty Biology, of Department iiino elBooy eateto Phys of Department Biology, Cell of Division 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,40–22doi:10.1242/jcs.150607 4201–4212 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 4 RSO aa cec n ehooyAec,Siaa3201,Japan. 332-0012, Saitama Agency, Technology and Science Japan PRESTO, 6 eateto hsooia cecs h rdaeSho for School Graduate The Sciences, Physiological of Department 1 eshs Otani Tetsuhisa , zk 4-55 Japan. 444-8585, azaki lBooy hok,Kb 5-07 Japan. 650-0047, Kobe Chuo-ku, Biology, al ysuUiest,Kuh,812-8581, Kyushu, University, Kyushu , ooyadCl ilg,Kb University Kobe Biology, Cell and iology 2 uih Ikenouchi Junichi , 2 aoaoyfrMorphogenetic for Laboratory 3,4 n ii Furuse Mikio and optrsmltosbsdo hsnto erdc h hp of shape the reproduce notion this cell–cell Consistently, on along 2011). based actomyosin Yonemura, simulations of 2009; computer Lecuit, force and tensile the (Cavey be the junctions to support considered be that can three points contacts of Tricellular corners meet. the cells where epithelial contacts, correspon tricellular sides to correspond its vertices polygon, a with Zallen, and Vichas 2011; al., et development Lecuit 2011). during 2007; cells Lenne, epithelial and of (Lecuit cellular remodeling epithelial of the of shape and morphogenesis the sheets the the controls border, of cell AJC tension epithelial the the the at and bundles adhesion actomyosin underlying cell–cell strong of combination oswt eeeaino arclsatrbrhaddisorganized and birth after cells hearing hair shows of of DFNB49 one recessive degeneration in mimics with mutations that a loss gene al., mouse tricellulin cause knock-in et human gene the (Riazuddin gene tricellulin a (DFNB49) tricellulin and 2006) deafness 2008). human al., familial the et nonsyndromic junction (Ikenouchi in tight fibroblasts claudin-based Mutations the in of increases reconstituted morphology and structures the membrane of plasma of four the components complexity within intramembrane junctions, major Tricellulin have tight Marveld3, region. claudins, and cytoplasmic with occludin all N-terminal to associates long contrast al., a In and et has 2009). Raleigh tricellulin al., 2005; Marveld3, et al., Steed of et 2010; and (Ikenouchi consists domains which tight- transmembrane tricellulin the family, of member al., protein occludin, a et is Marvel of Tricellulin Ikenouchi function 2011). 2013; junction-associated al., barrier al., et full are et Masuda the (Higashi angulins 2005; as sheets and well cellular as tricellulin epithelial formation that tTJ cultured RNA in revealed in involved and have studies tTJs cells to knockdown epithelial tricellulin al., (RNAi)-mediated recruit tight tight et interference tricellular specialized Angulins Masuda named at (tTJs). contacts, 2005; localized contacts junctions tricellular are al., at tricellular proteins structures et of junction Ikenouchi of been types 2013; Both have constituents 2011). ILDR2), al., as et molecular known (Higashi proteins, as (also family angulin-3 known identified and angulin (also angulin-2 ILDR1) LSR), and as as known MARVELD2) (also angulin-1 as including known level. (also molecular the at demonstrated been not have junctions implications generat the the in However, contacts are tricellular 2009). for Lecuit, contacts and (Cavey tricellular durin rearrangement 1982). sheet that al., cells cellular idea epithelial et the of rearrangement Honda within allows contacts 1980; shor tricellular Eguchi, between and sheets and elongation cellular epithelial Honda Coordinated true 1983; in observed (Honda, cells epithelial polygonal hnteaioltrlbre fec pteilcl scompared is cell epithelial each of border apico-lateral the When eety w ye fitga ebaepoen,tricellulin proteins, membrane integral of types two Recently, 1,5,6, * opoeei,laigt the to leading morphogenesis, g eigo h ce the of tening o ftniefreaogcell–cell along force tensile of ion ocl–eljntosadits and junctions cell–cell to d euaoyuisfrepithelial for units regulatory lcl junctions ll–cell 4201

Journal of Cell Science h hp n eairo pteilclstruhactomyosin through cells epithelial regulating of in thereby contacts behavior tricellular and Cdc42, for shape role contacts. the a activates tricellular suggest at findings fibers and Our F-actin of ARHGEF36), organization accelerating as and detailed known (also the DNMBP Tuba (GEF) However, factor guanine-nucleotide-exchange 2013). elusive. remains al., function tricellulin et of mechanism (Nayak molecular structures tTJ ARTICLE RESEARCH 4202 low-Ca a from switched were epithelial of gland cells organization mammary after the EpH4 (tricellulin- in cells mouse in cells features observed same these were The F-actin markedly 2A). in re- (Fig. were tricellulin cells) after rescue HA-tagged restored fibers and of PEC–PEC cells, expression tricellulin-knockdown These in S2A). decreased (supplementary PEC Fig. another These to 2A). material PEC (Fig. one plating connected cell of in fibers h F-actin prospective (PECs), 24 of corners after cells region cell MTD-1A F-actin subconfluent the epithelial at premature identified transiently designated further up vertices, came we that investigations, fibers intensive of region During the at vertices organization cell F-actin in involved is Tricellulin is tricellulin during cells that epithelial formation. suggest of junction configuration cell–cell junctional findings the Similar These in involved 1F–I). 1A). after (Fig. h (Fig. 48 at plating tricellulin-knockdown line, remarkable adenocarcinoma colorectal in human a observed cells, less Caco-2 were became changes morphological cells tricellulin-knockdown of MTD-1A changes h morphological 72 the at confluence plating, reached mostly after had 1E). cells (Fig. the When 1A–E). cells effects (Fig. these rescued tricellulin- parental cells tricellulin-knockdown the in the into transgene circle, tricellulin than (RNAi)-resistant interference a RNA lower an the resembles of Overexpression which was cells to cells extent the knockdown the of indicating shape cells, polygonal the the structures, of rosette-like (supplementary index the apoptosis cell circularity with Consistent after by assays S1B). sheet caused Fig. cellular material TUNEL not the were S1A). structures from rosette-like extrusion Fig. the in that material apprarent revealed supplementary more were 1D; meet, (Fig. cells cells MTD-1A parental the many to compared of tricellulin-knockdown cells vertices the structures, ‘rosette-like’ tricellulin-knockdown Second, where 1C). of (Fig. curved AJC indeed the was lengths cells that junctional relative revealed distorted were the vertices AJC of between the analyses of level Quantitative the 1A). after at (Fig. cells h 1A–E). the (Fig. of 48 outlines aspects the at two First, gland in density morphology saturation altered mammary exhibited reaching plating, MTD-1A before cells, mouse epithelial found was we tricellulin-knockdown AJC, tricellulin the that of of (shRNAs). staining expression immunofluorescence RNAs analyzing the hairpin By small which tricellulin-specific by in several suppressed generated lines we AJC, cell the epithelial along force tensile the of in maintenance contacts tricellular of involvement possible the investigate To cells cell–cell epithelial apical in of junctions configuration the regulates Tricellulin RESULTS tension. eeaino pcaie -ci iesrnigbtenPECs between the running formation. in junction fibers involved cell–cell F-actin is during specialized tricellulin that of indicate generation findings These S2A). Ca ntepeetsuy eso httiellnbnst h Cdc42 the to binds tricellulin that show we study, present the In 2+ cnann eim(i.2,;splmnaymtra Fig. material supplementary 2B,C; (Fig. medium -containing 2+ eimit a into medium efrhrfudta vrxrsino rclui nmouse in tricellulin of overexpression that found further IIB myosin We and F-actin recruits Tricellulin ihdmi ewe mn aci amino between domain rich Tuba. C- of the the with domain that interacts SH3 indicate tricellulin terminal findings of these region tricellulin cytoplasmic together, whereas tricellulin N-terminal Taken cells, 4B,C). (Fig. HEK293 and not in Tuba tricellulin endogenous expressed An exogenously C-terminus. full-length that the which revealed at Cdc42, domain assay SH3 immunoprecipitation for the sixth in SH3 activity the obtained DH contained fragments two GEF SH3 The screening Tuba Both 4A). the and four formation. (Fig. for F-actin domain N-terminus contains regulates responsible the BAR Tuba is from one 2003). order domain (Otani that domain, al., 4A) in (Fig. DH et domains, Tuba Salazar one GEF 2006; domains, Cdc42 of al., the 1–124 C- et of the acids encoding fragments amino clones independent terminal containing two- two bait identified yeast we a By tricellulin, tricellulin. with of screening of region hybrid F- interactions cytoplasmic molecular tricellulin-mediated N-terminal the the the investigated behind we organization, mechanism actin the Tuba examine GEF Cdc42 To the with interacts suggest Tricellulin recruitment. findings F-actin These induce tricellulin 3C). of (Fig. 1–124 not acids did amino that 26 those whereas connexin F-actin, native recruited of contacts cell–cell amino connexin of bicellular which N-terminus at the in to 26 fused construct were chimeric tricellulin (tricellulin of a 1–124 acids tricellulin of N-terminal clusters the the of in Conversely, 1–124 region acids amino cytoplasmic the lacking in observed mutant not also tricellulin was aggregation F-actin 3C). (Fig. clusters F-actin cells, tricellulin C-terminal into EpH4 concentrated in or was overexpressed tricellulin were N-terminal 3B), (Fig. named its respectively domain, lacking tricellulin cytoplasmic deletion of When 3A). constructs that (Fig. suggesting recruitment produced concentrated, F-actin were contacts, promoted sometimes IIB tricellulin cell–cell myosin bicellular and cells on F-actin where aggregates EpH4 tricellulin of epithelial clusters gland mammary rclui sivle ntegnrto fatmoi tension junction actomyosin cell–cell during of stage generation subconfluent the that formation. the at suggest in PECs results between involved These is in 2F). labeling tricellulin (Fig. F-actin the from cells distinguished the of be tricellulin-knockdown not of could fibers and Immunolabeling decreased 2F). much PEC–PEC (Fig. cells the pulling-force-dependent MTD-1A had cells in density, the when disappeared plating, saturation after h tricellulin- 72 in reached At not 2E). but around (Fig. cells, cells contacts tricellulin-rescue knockdown cell–cell and labeled MTD-1A in strongly PECs 2010) al., et (Yonemura tricellulin-knockdown epitope an in recognizes of that not antibody clearly monoclonal but a Furthermore, was cells, cells. MTD-1A tricellulin-rescue chain parental and the in light cells PECs of staining between immunolabeling fibers regulatory F-actin 2D, along Fig. immunofluorescence II detected in myosin shown by As diphosphorylated 2007). al., contraction, et the indicator (Watanabe examined an actomyosin we chain, tension, light under regulatory of were II plating myosin after of h di-phosphorylation 24 at cells 1A h -emnlctpamcrgo ftiellncnan proline- a contains tricellulin of region cytoplasmic N-terminal The oivsiaewehrteFatnfbr omda Esi MTD- in PECs at formed fibers F-actin the whether investigate To a ctnnta sdpneto -ci-eitdpligforce pulling F-actin-mediated on dependent is that -catenin ora fCl cec 21)17 2141 doi:10.1242/jcs.150607 4201–4212 127, (2014) Science Cell of Journal a ctnneioei T-Aclswas cells MTD-1A in epitope -catenin s4–7(LPPPQP nmice in (PLPPPPLPLQPP) 46–57 ds D lses u o tricellulin not but clusters, C D D n tricellulin and N neatdwith interacted C D 2)(i.3C). (Fig. 124) D did N D D C, N

Journal of Cell Science G muoltigo eiso ao2clssoni sn nioisaanttiellnadGPH H uniiaino ucinlnaiyof linearity junction of Quantification (H) GAPDH. and tricellulin against antibodies na using its F by detected GFP-tag * in was F. expressing shown GFP in antibody. cells stably anti-ZO-1 shown Caco-2 clone an cells of Caco-2 and Caco-2 series phalloidin siRNA-treated labeled a tricellulin fluorescently of using a Immunoblotting plating and after (G) KD) h (Tricellulin 48 at cells (KD/Tricellulin-GFP) Caco-2 tricellulin tricellulin-siRNA-treated cells, Caco-2 of * A. in shown EERHARTICLE RESEARCH fedgnu uawt rclui-4-3 a o eetdin detected not was residues tricellulin-P48-53A with alanine interaction Tuba with endogenous cells, of HEK293 replaced in were residues expressed proline was 48–53 (tricellulin-P48-53A) the acids of an amino all which When within in Tuba. al., mutant and tricellulin tricellulin et the HA-tagged of of of (Feller cluster region domains proline PxxP cytoplasmic the between N-terminal proline-rich of binding the motif with examined we minimum 1994), interact the to containing SH3 proteins known many that are Given shown). conserved domains not is sequence (data acid species amino vertebrate the among of feature this and 4D), (Fig. anti-ZO with junction of h Quantification 72 * (C) at A. GAPDH. in and cells tricellulin shown these against plating of antibodies after with staining flu h A with immunofluorescence 48 in plating shows shown at after cells cells panel h MTD-1A K MTD-1A right the 48 Tricellulin of The of at series and series (KD1/Tricellulin-HA) (left). a KD1 tags tricellulin of (Tricellulin HA Immunoblotting and cells three (B) formation. ZO-1 antibody. with MTD-1A junction against tricellulin tricellulin-knockdown cell–cell shRNA-resistant antibodies of during expressing and clones cells cells phalloidin stable KD1 epithelial Tricellulin two in of cells, organization clone MTD-1A stable actomyosin parental and of configuration staining junctional immunofluorescence regulates Tricellulin 1. Fig. P , .1 E uniiaino iclrt nasre fMD1 el t4 fe ltn hw nA * A. in shown plating after h 48 at cells MTD-1A of series a in circularity of Quantification (E) 0.01. P , .01 I uniiaino iclrt nasre fCc- el hw nF * F. in shown cells Caco-2 of series a in circularity of Quantification (I) 0.0001. P , .01 D uniiaino oet-iesrcue nasre fMD1 el t4 fe plating after h 48 at cells MTD-1A of series a in structures rosette-like of Quantification (D) 0.0001. ugs httepoierc oani h N-terminal the in C-terminal domain the results Tuba. to of proline-rich binds These domain directly SH3 4F). tricellulin (Fig. the of region residues cytoplasmic 48–53 that acids alanine amino with suggest within residues replaced its proline to the were not but which GST, with in tricellulin mutant of 1–150 fusion acids a amino to of bound and protein protein fifth maltose-binding the with containing domains Tuba SH3 mouse sixth of 1291–1580 acids amino of 4D,E). vitro (Fig. antibody anti-HA with assays immunoprecipitation uldw sasas hwdta atra uinprotein fusion bacterial a that showed also assays pull-down ora fCl cec 21)17 2141 doi:10.1242/jcs.150607 4201–4212 127, (2014) Science Cell of Journal P , .01 cl as 10 bars: Scale 0.0001. P , .01 F muoloecnestaining Immunofluorescence (F) 0.0001. m m. rsetylabeled orescently iefluorescence. tive e mouse ged A Triple (A) 2 n a and D2) iert na in linearity -1 eisof series a 4203 In

Journal of Cell Science EERHARTICLE RESEARCH 4204 panel ‘Merge’ the (Tricellul in tricellulin shown MT shRNA-resistant is in HA-tagged area present boxed expressing are the cells cells of KD of view vertices Tricellulin enlarged the and An low-Ca bridge cells. KD) subconf in that KD1 (Tricellulin cultured a fibers Tricellulin cells F-actin in in EpH4 phalloidin. clear cells labeled not tricellulin-knockdown (KD1/Tricellulin-HA) are fluorescently cells, cells fibers and tricellulin-rescue F-actin antibody These and anti-ZO-1 cells. an cells cells. KD1/Tricellulin-HA with epithelial KD1) plating in (Tricellulin assembly after cells junction h cell–cell MTD-1A 24 during tricellulin-knockdown formation fiber of actin clone in stable tricellulin of Role 2. Fig. oet-ieptena 8hatrpaigwsntrescued reduced much not were of force generation pulling was F-actin the the plating plating, by mediated of after epitope catenin the after of immunolabeling h and and F-actin 24 AJC of h fibers PEC–PEC at 48 curved Moreover, the 5A,B). at of (Fig. phenotype of pattern the strains cells, rosette-like two these generated In we P48-53A. tricellulin- cells, expressing stably epithelial cells of MTD-1A tricellulin-knockdown behavior the in a of relevant. showed interaction physiologically the is cells Tuba that with suggesting Caco-2 tricellulin 1F–I), been junctional (Fig. Tricellulin-knockdown of phenotype already 2006). similar regulation has (Otani al., the cells It Caco-2 in et in organization involved cells. between F-actin is through epithelial interaction configuration Tuba an in Caco-2 that is in Tuba reported there and and that cells indicating tricellulin MTD-1A 4G), in (Fig. Tuba cells and tricellulin between n nioisaantd-hshrltdmoi 2-ysn n O1 EF rpeimnfursec tiigo T-Aclsa 4h()ada 72 at and (E) h 24 at phalloi cells labeled MTD-1A fluorescently of with staining of h immunofluorescence epitope ant 24 Triple and an for (E,F) the cell anti-tricellulin recognizes culture ZO-1. with KD/tricellulin-HA with after and plating Tricellulin cells cells (2P-myosin) and after KD/tricellulin-HA KD1/tricellulin-HA myosin Tricellulin cells (F) and di-phosphorylated and EpH4 KD1 against KD in Tricellulin antibodies Tricellulin observed MTD-1A, EpH4, and are of of vertices staining analyses cell Immunofluorescence blotting between (D) Western fibers (C) F-actin cells. of KD patterns Tricellulin Geometric F-actin. visualize to ofrhrdmntaeterl ftetiellnTb interaction tricellulin–Tuba the of role the demonstrate further To interaction an showed experiments Immunoprecipitation 2+ eimadtesm el t3 n 2hatrasic noaCa a into switch a after h 12 and 8 3, at cells same the and medium a a 1 anti- -18 ctnnta sdpneto h -ci-eitdpligfre cl as 10 bars: Scale force. pulling F-actin-mediated the on dependent is that -catenin a ctnnrtmncoa nioy nanti- an antibody, monoclonal rat -catenin a - a ctnnrbi oylnlatbd n loecnl aee phalloidin. labeled fluorescently and antibody polyclonal rabbit -catenin rclui n uaato h elcl ucinconfiguration pathway. junction same cell–cell the that the using suggest organization on F-actin results act and Tuba These cells and S2B). was single-knockdown tricellulin Fig. effect Tuba material or Caco-2 additive double-knockdown tricellulin (supplementary Tuba with no disorganization and compared the cells, tricellulin cells F-actin the When these and in between effects. observed AJC compared the knockdown junctional additive were of individual the configuration an and the and curved Tuba exhibit of F-actin If would phenotype on cells. double-knockdown Caco-2 independently configuration, in act down tricellulin and/or knocked Tuba were pathway, tricellulin Tuba-independent a through organization cells. epithelial in formation and junction F-actin cell–cell tricellulin and during AJC between organization the of configuration binding normal the for the required is Tuba that These suggest 5C). (Fig. strongly cells results MTD-1A tricellulin-rescue with compared oeaietepsiiiyta rclui euae F-actin regulates tricellulin that possibility the examine To 2+ cnann eimwr aee ihfursetylbldphalloidin labeled fluorescently with labeled were medium -containing ora fCl cec 21)17 2141 doi:10.1242/jcs.150607 4201–4212 127, (2014) Science Cell of Journal A muoloecnesann fMD1 el,a cells, MTD-1A of staining Immunofluorescence (A) m m. nKD/tricellulin-HA) in -AD antibodies. i-GAPDH un odto at condition luent ,btntin not but s, -Aclsand cells D-1A i (F-actin) din .()EpH4 (B) s. a -18 h

Journal of Cell Science EERHARTICLE RESEARCH osrcswt n ihu -ci lsesaeidctdb h rosand arrows the by 10 indicated bars: are Scale respectively. clusters tricellulin F-actin arrowheads, the anti-HA without an of and with Aggregates with B phalloidin. constructs in labeled shown fluorescently tricellulin and of antibody constructs expressing chimeric cells and transmembrane EpH4 deletion of represent the staining boxes immunofluorescence tagged black Double were The (C) constructs C-termini. domains. All their tricellulin 26. at of connexin HA 1–124 of with acids N-terminus amino the of Tri- with construct respectively. connected chimeric domain, a cytoplasmic is and N-terminal 26 1–175 the N124-connexin acids in amino 1–123 and acids domain, amino cytoplasmic C-terminal the in full-length 396–555 HA-tagged a is Tricellulin Tricellulin-full tricellulin. deletion tricellulin. various of of constructs drawings chimeric Schematic and by (B) (arrows). accompanied F-actin sometimes junctions of tricellulin cell–cell accumulation Exogenous bicellular HA. within for aggregates antibody forms fluorescently an with and tricellulin phalloidin HA-tagged the labeled in full-length tricellulin expressing cells of domain EpH4 clusters. cytoplasmic actomyosin N-terminal of the formation of Role 3. Fig. D ,tricellulin C, D n tricellulin and N A muoloecnesann of staining Immunofluorescence (A) m m. D 2 akaioacids amino lack 124 Esi rclui-ncdw T-Acls(i.6B), (Fig. cells tricellulin. by MTD-1A influenced at not is localized tricellulin-knockdown localization still Tuba was concentration that Tuba suggesting Tuba in that of found region we PECs contrast, the By around 6B). with up (Fig. anti-ZO-1 PECs cells at came and formed 2A,B), were MTD-1A which (Fig. anti-Tuba fibers, F-actin of and that showed labeling antibodies phalloidin Triple labeled These tricellulin. fluorescently cells. against and a antibodies tricellulin-knockdown our Tuba of and in specificity the detected S3D) demonstrate Fig. observations not material Fig. (supplementary were blotting material S3E) western (supplementary in tricellulin band PECs Furthermore, tricellulin at S3A,B). added staining Fig. was immunogen material different excessive observed (supplementary when a also disappeared recognizing was and Tuba antibody epitope of anti-mouse-Tuba pattern staining another This with h). 24 formation junction 6A, at cell–cell antibody (Fig. colocalized during stage transiently subconfluent anti-tricellulin were the at tricellulin PECs and the the Tuba of that and revealed one Double we with antibodies tricellulin. cells Tuba, mouse anti-mouse-Tuba MTD-1A against of of Tuba antibody staining polyclonal mouse immunofluorescence localization against rat antibodies subcellular a polyclonal and the rabbit two examine generated to Next, cell–cell during regions formation vertex junction cell at localizes Tuba h d4 ciainmdae ytiellni dose-dependent a reduced Tuba, in domains, tricellulin of SH3 by sixth 1288–1581 mediated and activation acids fifth Cdc42 amino the the and only endogenous Furthermore, contained Tuba-N Tuba-N. in which of of reduction addition addition the sole the by than caused Tuba-N extent activity of co- greater Cdc42 addition by to by inhibited 7E,F) was (Fig. Cdc42 blocked cells Tuba-C-induced HEK293 was Notably, with in binding. activation competes interaction Tuba-C Tuba-N for that this Tuba-N tricellulin suggesting 7D, and tricellulin, Fig. of when in Tuba-C expression Tuba-C shown to al., and As et cells. (Tuba-N) bound between HEK293 Yu Tuba binding in 2004; of the al., overexpressed examined al., fragment et we et N-terminal Chen idea, Sondermann the this 2001; 2007; evaluate al., To activity al., et 2010). et half GEF (Bi Mitin proteins autoinhibition its C-terminal GEF 2011; by various suppresses its of modulated activities and the are with Indeed, 2006). domain al., interacts et DH (Otani Tuba the of containing the that half in an activation, is Cdc42 N-terminal there in full-length that Tuba implies the of observation E- mechanism of This autoinhibitory of 2006). expression al., concentration et than (Otani greater contacts Tuba in cell–cell results at domains cells SH3 cadherin of two Caco-2 half C-terminal in C-terminal and BAR (Tuba-C) the DH, of the These expression containing It Tuba that 7B,C). Tuba. shown (Fig. through previously Cdc42 Cdc42 Tuba activates was tricellulin increased of that indicate decreased knockdown findings also significantly was shRNA-mediated activation tricellulin by Cdc42 7B,C). this the of (Fig. and cells with activation HEK293 expression compared in Cdc42 overexpressed Exogenous active of was We level was Tuba performed. background Cdc42 its endogenous were when of Tuba, form tricellulin increased active overexpressing GTP-bound or the cells that 7A) found (Fig. HEK293 mutants CRIB in the deletion WASP with To assays of Tuba. of at pulldown of formation domain activity independent conventional GEF F-actin idea, Cdc42 is this in the examine of PECs tricellulin activation of be at might role PECs localization possible Tuba a tricellulin, the that Tuba Given through Cdc42 activates Tricellulin ora fCl cec 21)17 2141 doi:10.1242/jcs.150607 4201–4212 127, (2014) Science Cell of Journal 4205

Journal of Cell Science EERHARTICLE RESEARCH 4206 sh is (N (lysates) tricellulin lysates of 1–150 whole-cell acids the amino of of proteins Immunoblotting resi fusion immunoprecip GST-tagged HA. proline a were (MBP–Tuba-SH3-5/6). and five P48-53A Tuba immunoprecipitation Tuba of tricellulin which in domains and against in SH3 P48-53A tricellulin antibodies mutant (F) HA-tagged tricellulin with prol proline for panels. with the immunoblotting vectors its bottom not expression of by of the but or sequences followed and (mock) tricellulin acid (HA-IP), vector (wild-type) with Amino antibody control tricellulin Tuba a (D) anti-HA mouse of with panels. of transfected Co-precipitation bottom cells domain (E) the HEK293 cytoplasmic (P48-53A). of in N-terminal residues shown the alanine is tri in with (lysates) tricellulin. tricellulin, wit 46–57 replaced lysates HA-tagged mouse tricellulin acids for whole-cell of of amino vectors the 1–124 Interactions at expression of acids (C) region or Immunoblotting amino C-termini. (mock) bands. of their vector non-specific bait bars), at control a indicates (black HA a using 1427–1581 with with screening and transfected tagged two-hybrid cells 1375–1581 were tricellulin HEK293 yeast acids constructs of by amino All Lysates obtained encoding constructs. assays. Tuba were tricellulin immunoprecipitation mouse domain, of of SH3 representation fragments sixth Schematic the C-terminal (B) independent contained Two which domains. of SH3 each Tuba. six GEF has Cdc42 Tuba the to domain. binds Tricellulin 4. Fig. omlrtsrm(tlIG n nat-rclui a oylnlatbd TiellnA)frCc- el eeimnbotdwt nat-uaa anti-Tuba an with immunoblotted were cells immunopre Caco-2 (l or fusi for lysate cells GST Ab) whole-cell MTD-1A (Tricellulin or A for antibody MBP assays. polyclonal Ab) input immunoprecipitation rat (Tricellulin antibody. the anti-tricellulin in antibody monoclonal of an cells polyclonal rat staining and Caco-2 rabbit Blue anti-tricellulin IgG) and anti-tricellulin Brilliant (ctrl cells an Coomassie serum MTD-1A and rat indicates in serum normal panel Tuba rabbit bottom and preimmune The tricellulin with antibody. endogenous immunoprecipitates anti-MBP of or anti-GST Interaction an (G) with proteins. resin amylose to bound 3 elcmn (N replacement 53A D eeimnpeiiae iha niH nioy(AI) olwdb muoltigwt nioisaantTb n A h asterisk The HA. and Tuba against antibodies with immunoblotting by followed (HA-IP), antibody anti-HA an with immunoprecipitated were C P48-53A nvitro In n mn cd 9–5 ftielln()wr nuae ihMPTb-H-/,floe yimnbotn fteproteins the of immunoblotting by followed MBP–Tuba-SH3-5/6, with incubated were (C) tricellulin of 396–555 acids amino and ) ulonasy fGTtiellnfso rtisadafso rti fmloebnigpoen(B)wt h it n sixth and fifth the with (MBP) protein maltose-binding of protein fusion a and proteins fusion GST–tricellulin of assays pulldown A ceai ersnaino ua H,Schmlg oan H b oooydmi;BR BAR BAR, domain; homology Dbl DH, domain; 3 homology Src SH3, Tuba. of representation Schematic (A) ora fCl cec 21)17 2141 doi:10.1242/jcs.150607 4201–4212 127, (2014) Science Cell of Journal WT ,aioais110o rclui ihteP48– the with tricellulin of 1–150 acids amino ), uain Tuba h sy.Lysates ssays. tbd n an and ntibody cellulin ttdwt an with itated iiae with cipitates st)and ysate) usare dues w in own ine-rich D and N on

Journal of Cell Science EERHARTICLE RESEARCH nteFatnmdae uln oc.Saebr:10 bars: Scale force. pulling F-actin-mediated the on phalloidin. the labeled with plating anti- after an h antibody, P48- 24 Tricellulin at and cells cells, P48-53A1 53A2 immunofluorescence Tricellulin cells, Triple A Tricellulin-HA in (C) of shown antibodies. staining lysates anti-GAPDH cell and the of anti-HA tricellulin- blotting with in Western phenotype (B) junction cells. MTD-1A cell–cell knockdown curved Tricellulin the antibody. rescue anti-HA cannot an anti-ZO-1 P48-53A and an phalloidin using labeled P48-53A2) fluorescently Tricellulin HA antibody, of an and clones with P48-53A1 stable P48-53A (Tricellulin tricellulin two tag RNAi-resistant and expressing (Tricellulin-HA) cells tag KD1 HA tricellulin full- an RNAi-resistant with expressing tricellulin KD1) length (tricellulin cells MTD-1A knockdown 5. Fig. A muoloecnesann fasal ln ftricellulin- of clone stable a of staining Immunofluorescence (A) a ctnnrbi oylnlatbd n fluorescently and antibody polyclonal rabbit -catenin a 1 eonzsa ptp of epitope an recognizes -18 a 1 anti- -18 a a ctnnta sdependent is that -catenin ctnnrtmonoclonal rat -catenin m m. eurdfrisascainwt nui aiypoen,which proteins, family is angulin tricellulin with of association domain previously its cytoplasmic have for contact C-terminal We required tricellular regulation junctions. the the cell–cell that the in of shown role whether configuration its for investigate the required of to is tricellulin interest of localization of is It junctions contacts cell–cell tricellular of at configuration the regulates Tricellulin 7G). (Fig. GEF autoinhibition with its Tuba-C this C- of suppresses cancel the interaction to and the with Tuba-N blocks domain tricellulin interacts DH that Tuba and a activity, of containing findings half half These N-terminal terminal S4A–E). the that Fig. suggest material (supplementary manner 10 bars: Scale cells. KD1 tricellulin in KD1/Tricellulin-HA and not cells but MTD-1A fibers cells, in F-actin observed F-actin. strongly are visualizes PECs which antibody bridging phalloidin, monoclonal labeled anti-ZO-1 antibody, fluorescently condition polyclonal and subconfluent rabbit a anti-Tuba in an and cells with cells (KD1/Tricellulin-HA) stable KD1) cells a (Tricellulin MTD-1A cells, cells rescue PECs MTD-1A MTD-1A at of tricellulin-knockdown colocalized staining of transiently immunofluorescence clone and are Triple 24 tricellulin (B) 12, and h). at Tuba (24 fixed plating. were after Cells h antibody. 48 monoclonal antibody mouse polyclonal anti-ZO-1 rat and anti-tricellulin antibody, junction polyclonal cell–cell rabbit during anti-Tuba cells MTD-1A in Tuba formation. of Localization 6. Fig. ora fCl cec 21)17 2141 doi:10.1242/jcs.150607 4201–4212 127, (2014) Science Cell of Journal A rpeimnfursec tiigo T-Aclswt an with cells MTD-1A of staining immunofluorescence Triple (A) m m. 4207

Journal of Cell Science hscnesteatihbto fTb eitdb h neato ewe t -emnladCtria avs hrb nbigCc2t cest Tuba access of to change Cdc42 conformational enabling thereby a halves, induces C-terminal Tuba and of N-terminal domain its SH3 between C-terminal interaction the the to by Tuba. (PPPPLP) mediated of Tuba tricellulin domain of of autoinhibition domain the cytoplasmic cancels N-terminal This the in region rich d4 a eetda hw nB F uniaieaayi fteatv d4 hw nE h ai nmc-rnfce el a dutdt .Data 1. to adjusted was cells mock-transfected in ratio The of E. form in active shown The Cdc42 Cdc42. active and the vec FLAG of expression HA, analysis the against of Quantitative combinations antibodies (F) HA, with with B. against transfected mean immunoblotted in antibodies were the were cells shown with represent HEK293 lysates as immunoblotted Tuba. cell detected were of Whole was half immunoprecipitates were Tuba-C. a N-terminal Cdc42 the the cells FLAG-tagged with and by HEK293 and immunoprecipitation lysates activation Tuba-N by tricellulin. Cdc42 Whole-cell followed HA-tagged by of FLAG). Inhibition tricellulin, inhibition (IP: (E) GFP-tagged its GFP. antibody and and and anti-FLAG Tuba-C FLAG Tuba and FLAG-tagged of HA) Tuba-N, halves (IP: HA-tagged C-terminal for antibody and vectors N-terminal expression the with between transfected Interaction (D) experiments. independent EERHARTICLE RESEARCH 4208 introduced we Thus, contacts. tricellulin tricellular 2013; that to al., tricellulin localize suggesting et to 2011), (Higashi unable al., contacts et tricellular Masuda to tricellulin recruit as defined was lysates cell total mean the represent in Data those 1. to to samples adjusted pulldown was GST–WASP-CRIB which antibody. Quanti the cells, anti-Cdc42 (C) in mock-transfected (lysate). an signals in proteins with Cdc42 con these ratio blotting for of a the western antibodies ratio with to with by The transfected relative analyses detected B. were K blotting Cdc42, was (Tuba in western cells Cdc42/total protein, in siRNA shown HEK293 active fusion Tuba shown Cdc42 tricellulin. are and GST–WASP-CRIB active by lysates tricellulin a the whole-cell Cdc42 HA-tagged to the of of of bound in combination Activation Cdc42 which the (B) and Cdc42), and Tuba C-termini. (active tricellulin Tricellulin, their HA-tagged Cdc42 at or of Tuba HA form FLAG-tagged or active for FLAG vectors with Tuba. expression of tagged (mock), autoinhibition were vector the constructs cancelling by All Tuba Tuba. through of Cdc42 activates Tricellulin 7. Fig. yolsi oan hc neat ihTb Fg 8A,B). contacts. (Fig. tricellular at Tuba configuration AJC the the regulates with of tricellulin that interacts tricellulin indicate findings which These that domain, fact cytoplasmic tricellulin, the full-length RNAi-resistant despite membrane expressing tricellulin-knockdown with these cells plasma of compared MTD-1A junctions curved the cell–cell remained the still throughout of cells configuration located The 8A,B). was (Fig. and tricellulin contacts the expected, D notiellnkokonMD1 el.As cells. MTD-1A tricellulin-knockdown into C 6 ..* s.d. P , D .5 ** 0.05; i o ocnrt ttricellular at concentrate not did C P D , otisteN-terminal the contains C 0.01; n 5 needn xeiet.()Mdlfrtiellnmdae ciaino ua h idn fteproline- the of binding The Tuba. of activation tricellulin-mediated for Model (G) experiments. independent 4 D ol be would C ehns nwihatasebaepoenascae with peculiar associated a protein here transmembrane report a we which Furthermore, in in cells level. mechanism involved epithelial molecular of are the determination contacts shape at tricellular have and that tension we time junctional a Notably, the first formation. the tricellular tricellulin, junction and, for cell–cell Cdc42 along shown that actomyosin during activate at of to AJC tension found Tuba and the localizing organization GEF the we Cdc42 controls the hence, protein study, to binds molecular present contacts, spanning the is at the little function tetramembrane cells, In and epithelial organization polygonal level. intriguing their of as about behavior regarded known the been for have regions contacts tricellular Although DISCUSSION ora fCl cec 21)17 2141 doi:10.1242/jcs.150607 4201–4212 127, (2014) Science Cell of Journal A ceai igaso h ullnt n eeinconstructs deletion and full-length the of diagrams Schematic (A) 6 ..* s.d. P , .5 ** 0.05; P , 0.01; aieanalysis tative n 5 anti-HA n h DH the o 4 ) The D). osfor tors trol .

Journal of Cell Science EERHARTICLE RESEARCH o ece h elcniuainpeoyei tricellulin- in phenotype configuration cell C- the the lacking rescued mutant contacts tricellular tricellulin nor to mouse located neither a domain cytoplasmic study, terminal mutants, present these the 2013). al., of In et of (Higashi contacts none tricellular truncation at localize that could a terminus, with found one We the including cells. all C-terminal epithelial cultured of the which system mouse model encoding a tricellulin using region for tricellulin, of gene of domain DFNB49, cytoplasmic the behaviors within with occur the associated mutations examined proteins previously mutant We database. designated are isoforms corresponding short and long tricellulin- acids, These amino reported. independent been 27 shorter with has replaced a and truncated isoform, are tricellulin form C-terminal Schlu longest the 2006; the which in to al., isoform, addition et in Riazuddin humans, 2013; al., et (Nayak several splicing alternative are by generated there transcripts tricellulin but other mutants, its and (NM_001038602) tricellulin o region this proline-rich whether manner. unclear intermolecular a the although to autoinhibition, remains mediate contains bind to it Tuba might of also which domains SH3 609–619), Tuba C-terminal The acids Tuba. amino of of half (PPPPRPRTPTP, C-terminal half the to the binding N-terminal for that Tuba t of and of half terminal activation, domain Cdc42 cytoplasmic for terminal domain cancels Tuba, DH interaction of tricellulin–Tuba half a auto C-terminal the contains is and Tuba which of Tuba half the N-terminal of the that between suggests activity study Our GEF 1994). and Cdc42 al., domains SH3 et between (Feller interaction domains the of proline-rich case typical a the be when to w impaired seems replaced was were for binding residues this proline that the SH3 and target tricellulin), sixth tricellulin, mouse of binding of the 46–57 domain prol contains cytoplasmic a the contains N-terminal which which the Tuba, is to amino mouse bound that domain, Tuba of found We 1291–1580 of tricellulin. acids of domain C-terminus cytoplasmic N-terminal the F- at control to GEF protein G small organization. a actin to binds junctions cell–cell u es w-yrdsreigsoe httesxhS3domain SH3 sixth the that showed screening two-hybrid yeast Our nti td,w nlzdtelnetioomo mouse of isoform longest the analyzed we study, this In a n - and b b sfr nmc sntpeeti h genome the in present not is mice in isoform epciey(Schlu respectively , n-ihrgo,PPPLLP aioacids (amino PLPPPPLPLQPP region, ine-rich , iellncmee ihteN- the with competes ricellulin hsatihbto.Ide,teN- the Indeed, autoinhibition. this t lnn eius hsfinding This residues. alanine ith ccursinanintramolecularor 3 mn cd ftelongest the of acids amino 130 , tre l,20) odt,the date, To 2007). al., et ¨ter niie yteinteraction the by inhibited 0aioaisa h C- the at acids amino 60 tre l,20) In 2007). al., et ¨ter otcsad ec,b novdi h euaino F-actin of regulation the in involved be hence, observations and, these contacts Taking tricellulin- domain. human together, cytoplasmic by N-terminal mediated function tricellulin its above the of for localization required is contact tricellulin tricellular that indicating cells, knockdown 99 ahsye l,19) sapasbeefco for effector plausible a al., is et 1999), (Rohatgi al., polymerization et actin Machesky nucleate to 1999; lateral activation complex Arp2/ activates Cdc42 the is which 3 N-WASP, the F-actin Tuba, of and along whether tricellulin Downstream clarify by mediated direction tTJs. to along interest tTJs basal organized of also that be the would Given although it toward elusive. membranes, PECs, remain contacts deeply tricellular mechanism between with extend molecular F-actin bridges of its connection the and in the as that of well roles manner as F-actin contacts the crucial tricellular of play to fibers formation might F-actin of system to linkage cell–cell connected Tuba–Cdc42 the be polygonal the should of control through organization they F-actin side tricellulin-triggered to force, Thus, contacts. tensile the tricellular likely by along is stretch contacts. bundles junctions tricellulin tricellular F-actin at contrast, work Tuba–Cdc42 When to By through seem tension AJC. they actomyosin cell– membranes, 2012). the at plasma located al., are lateral along et Lulu or and (Fanning junctions Willin Shroom3, shape cell least cell at that epithelial Given affect and are tension which actomyosin perijunctional regulate respectively), negatively to reported and TJP2, been have and ZO-1 TJP1 configuration. similarity, structural with as proteins junctional tight-junction-associated scaffold known at the F-actin (also of influence ZO-2 regulation and (Epb41l5)– the in AJC Lulu involved the are pathways Terry and (FRMD6)–aPKC– 2011) 2011; al., 2011), Tanoue, et Willin and Takeichi, (Nakajima Shroom3–ROCK 2008), and p114RhoGEF–Rho–ROCK the (Ishiuchi Takeichi, addition, ROCK In and 2006). the al., (Nishimura (N- et mediating regulate cell–cell protein (Otani syndrome along by to WASP) Wiskott–Aldrich assembly neural F-actin junctions reported through by junctions cell–cell followed previously activation signaling epithelial been Cdc42 the of of has epithelial terms configuration of and Tuba In shape polygonal 2010). (Cunningham the cells, determine al., to junctions et required pathways Ivanov cell–cell 2012; epithelial the Turner, affects of and actomyosin behavior perijunctional of contraction induces configuration. junction cell–cell and organization xesv tde aervae htmoi Iphosphorylation II myosin that revealed have studies Extensive ora fCl cec 21)17 2141 doi:10.1242/jcs.150607 4201–4212 127, (2014) Science Cell of Journal rmteclssoni ihat-Aadanti- and anti-HA with A antibodies. in GAPDH shown cells the 10 from bar: anti- Scale an antibody. and HA phalloidin labeled fluorescently antibody, anti-ZO-1 (Tricellulin an HA domain tricellulin with cytoplasmic of tagged C-terminal clones deleted the stable with two tricellulin and expressing (Tricellulin-HA) KD1 tag HA tricellulin an cells full-length with MTD-1A RNAi-resistant tricellulin-knockdown expressing of KD1) clone (tricellulin stable a of staining in phenotype cells. junction MTD-1A cell–cell tricellulin-knockdown deleted curved cytoplasmic the C-terminal rescue the cannot with Tricellulin 8. Fig. b sulkl ob oae otricellular to located be to unlikely is m .()Wsenbotn flysates of blotting Western (B) m. D 1adTricellulin and C1 A Immunofluorescence (A) D 2 using C2) 4209

Journal of Cell Science pteilcls(tn ta. 06.Hwvr twudbe would it of However, to contacts. that contribution tricellular and 2006). junctions the cell–cell bicellular between al., to discriminate Tuba–Cdc42–N-WASP et to difficult (Otani technically of configuration cells the assembly and epithelial junctions F-actin cell–cell the cadherin-based along controls pathway that report previous Tuba–Cdc42–N-WASP the to similar organization, F-actin modulating ARTICLE RESEARCH 4210 cochlear Na the of affect gradient might steep a but which the the mice, in affect barrier, of that not growth does indicating and contacts tricellulin contacts, tricellular development for from tricellular case tricellulin the to of was absence to locate similar protein in cannot is cells mutant which mutation This epithelial cochlear mice. tricellulin of knock-in tricellulin contacts The the tricellular the 2006). human corresponding from to al., absent similar the et deafness, (Riazuddin containing from of suffered patients but mutations a normally, protein the mutant had grew of tricellulin that of a truncation one mice generate a to with on mimics predicted Recently, reported is that and 2013) function. DFNB49 tricellulin al., barrier et for (Nayak epithelial knock-in in al. the role et crucial of Nayak a play F-actin maintenance also tricellulin-mediated might the contacts is account, tricellular 2011). into at which al., regulation et studies throughout Masuda function, these 2013; tricellulin al., barrier Taking et of (Higashi distribution membrane epithelial plasma diffuse the the a by EpH4 reduces in accompanied is sheets angulin-1/LSR also of cellular tricellulin Knockdown epithelial cells 2005). of that al., function et barrier indicating (Ikenouchi full flux, the mouse for paracellular required also in resistance increased electrical are tricellulin transepithelial and function reduces of cells barrier epithelial depletion at EpH4 epithelial organization RNAi-mediated the F-actin intriguing. in tricellulin-mediated contacts to of tricellular studies The dynamic roles future processes. such developmental for potential during to interest cells contribute epithelial great of contacts behaviors of tricellular and be (Cavey how will rearranged investigate dynamically It withstand be 2009). to to only Lecuit, also not but appear forces contacts tensile tricellular 2009). aspects, al., et al., such Fernandez-Gonzalez et In 2006; (Bertet actomyosin al., AJC the of et of Blankenship contraction polygons 2004; within the sides given of cell– on because filaments of change lengths junctions the action cell processes, developmental its in packed. but highly morphogenesis sheet, become cortical cells cellular cells the generating the the when within decrease when by to packed the borders appears tightly cells cell to not along epithelial are actomyosin contributes through of system tension shape as tricellulin–Tuba–Cdc42 polygonal contacts, pulling-force-dependent the tricellular the that at of tension of immunolabeling epitope the the at by also fibers F-actin evaluated but the only boundary not our density, cell mature cell In in became junction increase junctions the cells. cell–cell cell–cell with the during between epithelial after observed However, bridging transiently formation. of were fibers shape corners context, F-actin cell the the well-developed on determine depending pushing observations, factors, 2007). to cells two Lenne, packed and these appears Lecuit of of 1984; combination al., tension et been A (Honda surface have other a each borders cell– against and along cell actomyosin junctions, by straight generated cell with tension cortical cells a proposed: epithelial of shape uigteitraaino pteilclsdrn dynamic during cells epithelial of intercalation the During polygonal the of generation the underlying mechanisms Two a ctnn a o sntcal.Tu,w speculate we Thus, noticeable. as not was -catenin, , 0aioaisa h -emns h mice The C-terminus. the at acids amino 60 + n K and + ocnrtosis concentrations D C, h ioetmn lsRaet(nirgn codn othe to according (Invitrogen) Reagent Plus using instructions. performed limiting manufacturer’s was Lipofectamine by supplemented transfection once DNA F12 cloned cells. Ham’s the were uniform and cells obtain MTD-1A DME to serum. of dilution calf cells were mixture fetal Caco-2 1:1 10% serum. cells a with calf EpH4 fetal in 10% cultured respectively. Reichmann with were Ernst supplemented Japan), DME from Kobe, in cultured gifts Takuji CDB, and kind (RIKEN Switzerland) were Zurich, Tanoue Zurich, cells Hospital Caco-2 Children’s (University and cells EpH4 transfection and culture Cell METHODS AND MATERIALS Aesa isine) abtpbat-Pmoi Cl Signaling (Cell conjugate anti-2P-myosin HRP tricellulin anti-GST pAb rabbit Ab rabbit human Biosciences), Biolabs), Biosciences), (BD against England (Amersham Cdc42 (New pAb against anti-MBP mAb pAb rabbit pAb mouse Stratagene), rabbit and (Trevigen), (MBL), (Sigma, (Invitrogen) DDDDK FLAG G3PDH against against (pAb) against M2 antibody against polyclonal mAb 16B12 mAb were rabbit mouse mouse 2005) (Roche), (Covance), HA al., GFP against HA et against and 3F10 7.1/13.1 monoclonal mAb (Ikenouchi 2006) mAb rat mouse al., tricellulin (Roche), and mouse previously; et mouse described (Otani used: as against generated Tuba N54 were human mAb against rat antibodies 5G6A3 (mAb) primary antibody following The reagents and 2005). Antibodies al., et (Ikenouchi previously described as used oligonucleotide was the pSUPER knockdown, 5 tricellulin was mouse and For effective sequence study. most were this controls, the Tuba in negative was used human as HSS135886 well Invitrogen. and as from 118490), purchased 135886–135888) HSS select HSS stealth (DNMBP select into subcloned stealth tricellulin were human (MARVELD2 against which oligonucleotides 1– 396–555), siRNA acids The acids vectors. (amino pGEX6P-1 (amino N-terminus C-terminus tricellulin fusion or mouse by GST 150) the generated for of were vectors amplification pGEX-Tri-C, expression PCR and The pGEX-Tri-N pGEX. fragments, an they and tricellulin with pCA Briefly, tag tag, HA 2006). HA or al., FLAG N-terminal C-terminal et a with (Otani pCA into by previously subcloned residues were described alanine as deletion to its were changed and residues were mutants Tuba for 53) proline expression vectors and expression (P48-53A), the The 51 mutagenesis. tricellulin 50, site-directed create 49, of mutagenesis 48, To mutant acids acids. proline site-directed (amino the amino by for encoded targeted C- disrupted vector the expression, a were changing tricellulin with without tricellulin 1991) siRNA-resistant of al., For and et sequences PCR tag. by (Niwa described HA generated mouse neodelEcoRI as terminal were of pCAGGS 26 1–124 constructed into connexin acids subcloned mouse (amino were with Tric-N124 2011) fused and tricellulin) tricellulin) al., mouse N- of et tricellulin 555 for its vectors (Masuda tag expression and HAs The tag three previously. 2005) C-terminal GFP a al., with tag, or mutants et GFP deletion C-terminal or (Ikenouchi or HA terminal fragments C-terminal a tricellulin with tricellulin GST-fused for vectors expression The siRNAs and construction Plasmid ebae ed ob lrfe ttemlclrlvli future in level molecular the at clarified studies. plasma be the to to connection of needs its membrane, leak including F- contacts, the of tricellular organization at prevents detailed actin that the contacts, barrier tricellular through paracellular solutes actomyosin the of role in the understand contraction better actomyosin To in of contacts. contacts regulation tricellular by tricellular at controlled is of be It closing would 2008). phenomena and such al., opening et the Shum the that 2009; sense reasonable al., to et lumen tricellular (Kubo the environment into that external penetrate beneath reported sheets cells the cellular of been epithelial protrusions across wherein the has windows as it perilymph used are addition, and contacts In endolymph sheet. the cellular between generated ora fCl cec 21)17 2141 doi:10.1242/jcs.150607 4201–4212 127, (2014) Science Cell of Journal 9 -GAACAAACTCTCTCACATA-3 9 D 2 aioais125– acids (amino 124 K1 lndinto cloned (KD1)

Journal of Cell Science S,1m T)fr2ha 4 at h 2 for DTT) mM 1 BSA, niZ- A 874(the l,19)adrtanti- rat and Mouse 1991) 561–710. al., tricellulin. et and (Itoh mouse 1376–1518 T8-754 acids mAb of proteins anti-ZO-1 amino fusion 1–150 human GST encoding also against acids fragments raised of was were amino with Tuba 1–148 tricellulin to mouse against acids mouse fused pAbs Rabbit amino against GST against pAb and raised Rat (amino tricellulin domain respectively). mouse N-terminal tricellulin, of the with 1–150 proteins acids were fusion tricellulin GST the human against and from mouse raised purchased against pAbs were Rabbit (sigma) sources. indicated anti-catenin pAb rabbit Technology), ARTICLE RESEARCH rti xrsinwsidcdb diino . MIT obacterial to IPTG mM 0.1 of in addition by expressed induced was were expression protein GST–Tricellulin-C and DH5 GST–Tricellulin-N GST, purification protein Recombinant complexes The 2 buffer. lysis in ice, with boiling on times h by three 2 eluted washed for were proteins were fusion beads GST the to and coupled Healthcare) (GE glycerol). beads 10% and 4B EDTA glutathione–Sepharose with mM incubated were 1 lysates NP-40, the 0.5% centrifugation, Tris- NaCl, After mM mM (50 150 buffer 7.5, lysis ice-cold pH 2009). HCl in al., lysed were et cells (Hogan assays, Hokkaido pulldown previously For Fujita, described Yasuyuki as the from Japan) sample with gift Sapporo, Laemmli incubated University, (a were in domain lysates boiling CRIB cell WASP by assays, GST-fused eluted activation and Cdc42 Healthcare) For buffer. (GE Flow were lysates Fast Cell glycerol). 4 protein-G–Sepharose 10% antibody-bound anti-FLAG and EDTA and anti-HA mM with 1 treated NaCl, Hepes-KOH mM mM 150 25 7.5, deoxycholate, sodium pH buffer 0.2% RIPA SDS, with 0.05% NP-40, solubilized (1% were cells assays, immunoprecipitation For assays pulldown and Immunoprecipitation Briefly, (Clontech). library cDNA embryo mouse 11-day 7.16 an Two-Hybrid Matchmaker and a with System performed was screening two-hybrid Yeast screening two-hybrid Yeast 40 Apo UPlanS using an photomicroscope, with h. fluorescence observed 72 and or IX71 (Calbiochem) 48 Olympus FluorSave 24, on in for cells mounted culture The were after coverslips. Samples immunostaining containing 10- for dish a used 35-mm from were a cells coverslips on confluent was plated the were of cells dish thirtieth cm a epithelial experiments cells, For epithelial 2011). cultured cultured al., et using and (Masuda previously sections described as performed frozen of Immunostaining staining Immunofluorescence eeaddit h ecinmxueadicbtdfr2ha 4 at h 2 for incubated and mixture reaction the into added were eepce padpamd eeotie rmpstv lnsuiga using clones sequenced. positive were plasmids from the of colonies obtained inserts 120 The were Research). of (Zymo plasmids Kit total Zymoprep and A up adenine. picked and were tryptophan leucine, histidine, lacking ahn ihbnigbfe,tepeiiat eeeue ihbinding SDS-PAGE. with to eluted subjected and were maltose, precipitants mM 20 the containing buffer, buffer binding with washing isine) esrmnso h iert ne n iclrt were circularity software. and J index Image linearity camera using (BD the performed CCD software of cooled processing Measurements image ORCA-ER IPLab Biosciences). an and K.K.) of Photonics combination (Hamamatsu a using 60 performed FLN Uplan or 0.9 NA visualized was F-actin Healthcare). (Invitrogen). anti- phalloidin (GE Alexa-Fluor-488-conjugated using anti-rabbit-IgG anti-rat-, and horseradish-peroxidase-conjugated mouse- and Immunoresearch Cy5-conjugated (Jackson or anti-rabbit-IgG Laboratories); anti-rat-, and Cy3- anti-mouse- donkey anti-rat-, (Invitrogen); goat 488-conjugated anti-rabbit-IgG Alexa-Fluor- and anti-mouse- used: secondary following were The respectively. antibodies Japan), (Nara Nagafuchi Nara, Akira University, and Itoh Medical Japan) Masahiko Tochigi, by University, provided Medical kindly were (Dokkyo 1994) Tsukita, and (Nagafuchi 18 eeicbtdwt S-ue rtisi 200 in proteins GST-fused with incubated were 10% rsHlp .,5m MgCl mM 5 7.5, pH Tris-HCl 6 a b 10 mratehnl For -mercaptoethanol. shrci coli Escherichia 6 es rnfrat eeplated were transformants yeast el n uiidb tnadpoeue.Briefly, procedures. standard by purified and cells 6 N .5olojcie.Iaeaqiiinwas acquisition Image objectives. oil 1.25 /NA 6 nvitro in 2 aml apebfe upeetdwith supplemented buffer sample Laemmli ˚ 0 MNC,1%Gyeo,05mg/ml 0.5 Glycerol, 10% NaCl, mM 100 , .AyoeRsn(e nln Biolabs) England (New Resin Amylose C. ulonasy,MPfsdproteins MBP-fused assays, pulldown nsnhtccmlt medium complete synthetic on m idn ufr(0mM (50 buffer binding l a ctnnmAb -catenin ˚ .After C. 6 a - / utrs n rtiswr xrse t37 at expressed were proteins and cultures, h ucinlnt,wihwsidctdb h egho O1staining ZO-1 of length the by indicated was which length, junction The shape cell of Quantification Student’s indicated The respective legends. The figure calculated. were s.d. and Means analysis Statistical (Fujifilm). and system imaging SDS-PAGE mini Healthcare) Signals (GE LAS-3000 by system (Millipore). an chemiluminescence and ECL separated membranes an using PVDF were detected were Immobilon-P proteins onto blotting, transferred western For blotting Western i . ercn,B,Zu . aai . v,A n hn,Y. Zheng, and A. Eva, B., Salani, K., Zhu, B., Debreceni, T. F., Lecuit, Bi, and L. Sulak, C., Bertet, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.150607/-/DC1 online available material Supplementary Y.O.]. to material 23570229 Supplementary number [grant JSPS from Grant- a (C) and for Research M.F.]; to Grant-in-Aid Scientific 26291043 a for number M.F.]; in-Aid [grant to JSPS the from LS084 (B) for number Research Generation Society [grant Scientific Next Japan (JSPS) for the Science Program from of Funding Program) Promotion the (NEXT by Researchers part Leading in World supported was work This Funding the performed T.O. and materials. Y.O. the manuscript. provided the J.I. wrote experiments. and designed M.F. and Y.O. contributions Author interests. competing no declare authors The interests Competing discussions. helpful their for the laboratory all Furuse and Masatoshi the Honda and of Hisao assistance members Yonemura, technical Shigenobu for Nagafuchi, Nagafuchi Kato Akira Tomoko Akira Takeichi, reagents; and Reichmann and Ernst cells Fujita, for Yasuyuki Tanoue, Takuji thank We Acknowledgements vertices cell five than 5- 32400 more of circle in a where within diameter together crowded regions were staining ZO-1 of by delineated number cell–cell the in structures rosette-like junctions, of number junction the quantify the To which were measured. of and of vertices each ratio in experiments the independent these performed, Three were by vertices. the between defined between distance the distance was to length index the linearity and The measured. vertices, two between eepromd h iclrt ne a eie ytertoof Student’s by ratio followed which measured, of experiments the independent each Three in by performed, shape. were cell defined each of was measure compactness index circularity 4 The performed. were rsplmne ih1%gyeo n trdat MWCO; stored immediately and (10K mM glycerol used 20 cassette 10% either with were Dialysis 8.0, supplemented proteins pH or Slide-A-Lyzer purified Tris- The a Tris-HCl mM Scientific). 20 using mM Thermo proteins against (50 7.5) dialyzed bound (pH were The buffer proteins HCl buffer PBS. eluted elution wash the with and with twice with glutathione), twice and eluted PBS, NaCl) with M were times 1 three containing were washed (PBS and supernatants were h, 1 beads the for Healthcare) the (GE Subsequently, beads glutathione–Sepharose with centrifugation. incubated by clarification oiae yae,floe yicbto o 0mna 4 at min 30 for incubation the to by added was followed 1%) concentration, lysates, (final X-100 sonicated phosphate-buffered Triton in (PBS). sonication by saline lysed and centrifugation by collected p uoniiinmcaimo proto-Dbl. of mechanism Autoinhibition elongation. axis and intercalation cell 667-671. planar controls remodelling 6 pclsraeae otesur fteprmtr hc elcsa reflects which perimeter, the of square the to area surface apical ora fCl cec 21)17 2141 doi:10.1242/jcs.150607 4201–4212 127, (2014) Science Cell of Journal t -test. m m 2 a one.Treidpnetmeasurements independent Three counted. was P auswr bandwt one-tailed a with obtained were values t ts analysis. -test . . 0jntoswr admyselected randomly were junctions 30 0clswr admyslce and selected randomly were cells 30 o.Cl.Biol. Cell. Mol. 20) ysndpnetjunction Myosin-dependent (2004). ˚ o .Teclswere cells The h. 3 for C n ausaesoni the in shown are values 2 21 80 1463-1474. , ˚ ni use. until C Nature ˚ and C (2001). 4211 429 m m ,

Journal of Cell Science ii,N,Bts . oe .E,Dr .J,Sne,J n osa,K L. K. Rossman, and J. Sondek, J., C. Der, E., M. Yohe, L., Betts, N., Mitin, E. Nishi, M., Akashi, T., Higashi, J., Ikenouchi, H., Sasaki, Y., Oda, S., Masuda, May, L., Blanchoin, A., D. Kaiser, N., H. Higgs, D., R. Mullins, M., L. Machesky, unnhm .E n unr .R. J. Turner, and E. K. C. Cunningham, P. Sternweis, and R. S. Sprang, L., Guo, Z., Chen, A. J. Zallen, T. Lecuit, and and O. M. Cavey, Weitz, S., J. Sanny, T., S. Backovic, T., J. Blankenship, ARTICLE RESEARCH 4212 Dupre and C., Y. Hogan, Oda, H., Nakamura, S., Masuda, S., Kitajiri, S., Tokuda, T., Higashi, Gumbiner,B.M. Ro M., S. Simoes, R., Fernandez-Gonzalez, eut . en,P .adMno E. Munro, and F. P. Lenne, T., Lecuit, S. Tsukita, and M. Furuse, S., Tsukita, H., Sasaki, J., Ikenouchi, od,H n gci G. Eguchi, and H. Honda, H. Honda, D. Baltimore, and H. Hanafusa, R., Ren, M., E. S. G. Feller, Palade, and G. M. Farquhar, M. J. Anderson, and M. C. Itallie, Van S., A. Fanning, eut .adLne .F. P. Lenne, and T. Lecuit, A. Hall, A. and Nusrat, B. A. and Jaffe, A. C. Parkos, I., A. Ivanov, S. Tsukita, and S. Tsukita, A., Nagafuchi, S., Yonemura, M., Itoh, M. Takeichi, and T. Ishiuchi, knuh,J,Frs,M,Frs,K,Ssk,H,Tuia .adTuia S. Tsukita, and S. Tsukita, H., Sasaki, K., Furuse, M., Furuse, J., Ikenouchi, K. Watanabe, and M. Dan-Sohkawa, H., Honda, K. Kani, and S. Higuchi, Y., Ogita, H., Honda, uo . aa,K,Ykuh,M,Ssk,H n mgi M. Amagai, and H. Sasaki, M., Yokouchi, K., Nagao, A., Kubo, 20) ees fatihbto fAE yAClast D4 activation CDC42 to leads APC by ASEF suppression. of tumor autoinhibition and of Release (2007). cells. epithelial in formation M. Furuse, and D. complex. Arp2/3 T. the Pollard, by USA filaments and Sci. actin E. of nucleation M. activates Hall, C., R. 157-184. rclui erimn,eihla are ucinadipiaini deafness in implication and function barrier pathogenesis. epithelial recruitment, tricellulin M. Furuse, morphogenesis. cells. intercalating in tension by regulated are Cell dynamics II Myosin (2009). function. junction tight epithelial of strings the activity exchange nucleotide guanine intrinsic p115-RhoGEF. the of of tissue autoinhibition switch: to GEF polarity cell planar dynamics. links formation rosette morphogenesis. Multicellular (2006). cells. nerto uigcl n isemorphogenesis. tissue and cell during integration hrceiaino h nefc ewe omladtasomdeihla cells. Biol. epithelial transformed Cell and Nat. normal between interface Baena-Lo the of E., Characterization Piddini, E., A. Abl. and Crk of interactions the adhesives: Sci. molecular Biochem. as domains in cytoskeleton epithelia. adherens zonula the and structure cell epithelia. apical polarized regulate -2 and 1 hp,tsu atrsadmorphogenesis. and patterns tissue shape, inflammation. during function barrier epithelial cell- sites. cadherin-based at adhesion localization cell specific its protein: undercoat-constitutive kD phosphorylation. ROCK aPKC-mediated Biol. Cell through Nat. constriction apical epithelial tricellulin. of localization tricellular affects occludin 20) rclui osiue oe are ttiellrcnat fepithelial of contacts tricellular at barrier novel a constitutes starfish. Tricellulin the (2005). in wall, blastular the sheet, tensile a Differentiation into organized becoming cells corneal wounded in cats. cells of individual of endothelia observation long-term tissue: mammalian sheet? cell monolayered nie paeb agrasclswt eraiaino pdra tight epidermal of reorganization with cells Langerhans barriers. by junction uptake antigen Biol. Dev. Cell Rev. 17 .Cl Biol. Cell J. 736-743. , 18) emtia oesfrclsi tissues. in cells for Geometrical models (1983). .Cl Biol. Cell J. 96 odSrn ab eset Biol. Perspect. Harb. Spring Cold 21) nlsso h agln rtisLR LR n LR – ILDR2 and ILDR1 LSR, proteins ‘angulin’ the of Analysis (2013). 3739-3744. , 11 -rce,S,Nra,M,Kjt,M,Zmemn,C,Pelling, C., Zimmermann, M., Kajita, M., Norman, S., ´-Crochet, 13 25 19 .Cl Sci. Cell J. 460-467. , a.Rv o.Cl Biol. Cell Mol. Rev. Nat. Cell Dev. 860-866. , 16-22. , 453-458. , .Ep Med. Exp. J. 21) S eie elcresfrtiellrtgtjunction tight tricellular for corners cell defines LSR (2011). rti Sci. Protein 171 20) euaino ahrnmdae deinin adhesion cadherin-mediated of Regulation (2005). .Morphol. J. o.Bo.Cell Biol. Mol. .Cl Biol. Cell J. 21 939-945. , 17 20) oeua ae fcl-eljntossaiiyand stability junctions cell-cell of bases Molecular (2009). 247-269. , 20) h Tae:bohmsr n biology. and biochemistry GTPases: Rho (2005). a.Src.Ml Biol. Mol. Struct. Nat. 375-412. , 18) o uhde h elbudr otati a in contract boundary cell the does much How (1980). .Ter Biol. Theor. J. 20) elsraemcaisadtecnrlo cell of control the and mechanics surface Cell (2007). 126 11 .Cl Sci. Cell J. pz .A,Vnet .P,Io,Y tal. et Y. Itoh, P., J. Vincent, A., L. ´pez, 459-470. , 966-977. , 20 21) ilnadPr oprtvl regulate cooperatively Par3 and Willin (2011). 206 174 115 107-117. , 23 2937-2946. , 25-39. , 21) oc eeain rnmsin and transmission, generation, Force (2011). 1449-1462. , 577-590. , 16) ucinlcmlxsi various in complexes Junctional (1963). 21) ysnlgtcankns:pulling kinase: chain light Myosin (2012). 124 19) cr Aprltdprotein, WASp-related a Scar, (1999). 84 e,J . ao,S n aln .A. J. Zallen, and S. Eaton, C., J. per, ¨ 6 575-588. , 622-634. , a.Rv o.Cl Biol. Cell Mol. Rev. Nat. 548-555. , n.N .Aa.Sci. Acad. Y. N. Ann. 21) yokltlrglto of regulation Cytoskeletal (2010). 18) elmvmnsi living a in movements Cell (1982). 1 m .Pathol. J. Am. a002998. , 14 18) emtia nlssof analysis Geometrical (1984). nu e.Cl e.Biol. Dev. Cell Rev. Annu. 814-823. , o.Bo.Cell Biol. Mol. n.Rv Cytol. Rev. Int. 21) ouaoccludens- Zonula (2012). 21) ouaino a of Modulation (2011). 19) H n SH3 and SH2 (1994). 177 rc al Acad. Natl. Proc. 20) External (2009). 19 20) osof Loss (2008). 19) 220- A (1991). 512-524. , 4687-4693. , 1258 81 8 633-644. , 191-248. , 34-42. , Trends (2009). Annu. Dev. 27 , ihmr,T n aeci M. Takeichi, and T. Nishimura, aa,G,Le .I,Yua,R,Eemn,S . rno,C,VnIale .M., C. Itallie, Van C., Trincot, E., S. Edelmann, R., Yousaf, I., S. Lee, G., Nayak, T. Tanoue, and H. Nakajima, tn,T,Ihi . oo .adTkih,M. Takeichi, and S. Aono, T., Ichii, T., Otani, aauh,A n skt,S. Tsukita, and A. Nagafuchi, oag,R,M,L,Mk,H,Lpz . ichue,T,Tknw,T and T. Takenawa, T., Kirchhausen, M., Lopez, H., Miki, L., Ma, R., Rohatgi, Ramzan, S., Kitajiri, A., Lagziel, S., A. Fanning, M., Z. Ahmed, S., Riazuddin, Y., Wang, H., Sasaki, L., Shen, Y., Zhang, M., A. Marchiando, R., D. Raleigh, J. Miyazaki, and K. Yamamura, H., Niwa, aaae . ooa .adYnmr,S. Yonemura, and H. Hosoya, T., Watanabe, A. J. Zallen, and A. Vichas, Balda, V., I. San, Chong Leefa E., Vitiello, A., Elbediwy, C., Zihni, J., S. Terry, K. Matter, and S. M. Balda, T., N. Rodrigues, E., Steed, u . atn,I . i . mrsnh,G . mtn,J,Fernandez-Zapico, J., Umetani, K., G. Amarasinghe, P., Li, R., I. Martins, B., Yu, M. Shibata, and A. Nagafuchi, T., Watanabe, Y., Wada, S., Yonemura, S. Yonemura, aaa,M . wakwk,A . elgii . eta . ulr .H., M. Butler, G., Cestra, L., Pellegrini, V., A. Kwiatkowski, A., M. Salazar, aaa .adNlo,W J. W. Nelson, and S. Yamada, and D. Bar-Sagi, S., S. Yang, S., Boykevisch, M., S. Soisson, H., Sondermann, Ferguson, A., M. Conti, M., E. Kovacs, M., J. Leerberg, S. L., Breton, H. and Cox, D. M., Smutny, Brown, J., P. Smith, M., McKee, N., Silva, Da W., W. Shum, Schlu iae oteaia eljntosrgltseihla n ereihla planar neuroepithelial and epithelial regulates junctions remodeling. cell apical the to kinases ih,G . aeq . oe,S . eynsv,I .e al. et A. cells. I. hair Belyantseva, cochlear and M., architecture S. Invest. junction Clin. Jones, tight J. M., affects deficiency Rafeeq, Tricellulin P., p114RhoGEF. G. through Sinha, cells epithelial in system Biol. tensile actomyosin h 0 Dcdei soitdpoen ncnrlnrostsusduring tissues nervous central in protein, associated cadherin development. kD 102 the ope ik d4-eedn inl oatnassembly. actin to signals Cdc42-dependent links complex W. M. Kirschner, al. et hearing. M. for J. necessary Anderson, protein L., 79 P. tight-junction Friedman, a P., is Chattaraj, Tricellulin N., S. Khan, K., functions. overlapping but distinct Cell have Biol. occludin and tricellulin, R. marveld3, J. Turner, and M. Long, cells. epithelial simple of 146. configuration junctional the vector. eukaryotic novel a with transfectants expression pteiljntosb 14hGFdie ucinfrainand Biol. formation Dev. Cell junction Semin. drives p114RhoGEF by morphogenesis. junctions epithelial K. Matter, and S. M. occludin the of protein transmembrane junction-associated family. tight a as MarvelD3 tutrladeegtcmcaim fcoeaieatihbto n activation K. and Vav1. M. autoinhibition Rosen, of cooperative junction and of R. mechanisms D. energetic Tomchick, adherens and M., Structural Machius, D., induces D. Billadeau, E., that M. transducer tension a development. as alpha-Catenin Biol. Cell Opin. adhesion. cell-cell epithelial of expansion 517-527. and initiation drive oan,lnsdnmnt euaino h ci cytoskeleton. actin P. the Camilli, De of and regulation B. to homology F. dynamin Dbl Gertler, and links bin/amphiphysin/Rvs J., containing domains, Sondek, protein novel M., a D. Tuba, (2003). Serna, L., K. Rossman, 231. yaisb hshrlto n ehshrlto fislgtcanin chain light its of dephosphorylation and cells. phosphorylation epithelial by dynamics sevenless. of S. J. A. Kuriyan, Yap, and the S. of integrity R. support adherens. Adelstein, that zonula modules G., functional epithelial distinct R. in identify Parton, isoforms II sensors A., Myosin N. luminal Hamilton, are C., cells basal from epithelia. projections pseudostratified Transepithelial (2008). stratified other and metaplasia. epidermal cell in 655. squamous proteins including cells, junction epithelial tight containing structures 278 1040-1051. , e,H,Ml,I,Wlug .adFak,W W. W. Franke, and H. Wolburg, I., Moll, H., ter, ¨ 49031-49043. , 195 M elBiol. Cell BMC ora fCl cec 21)17 2141 doi:10.1242/jcs.150607 4201–4212 127, (2014) Science Cell of Journal Cell 245-261. , 21 20) tutrlaayi fatihbto nteRsatvtrSon activator Ras the in autoinhibition of analysis Structural (2004). 1200-1213. , Development 140 21) ahrnatnitrcin tahrn junctions. adherens at interactions Cadherin-actin (2011). Cell e.Got Differ. Growth Dev. a.Cl Biol. Cell Nat. 123 o.Bo.Cell Biol. Mol. 23 a.Cl Biol. Cell Nat. 246-256. , 515-522. , 119 4036-4049. , 19) h neato ewe -APadteArp2/3 the and N-WASP between interaction The (1999). 21) ptal etitdatvto fRo inligat signalling RhoA of activation restricted Spatially (2011). 393-405. , 10 22 95. , 858-864. , Cell 21) rnltn elplrt notsu elongation. tissue into polarity cell Translating (2011). 135 a.Cl Biol. Cell Nat. 21) ih ucinascae AVLproteins MARVEL junction-associated Tight (2010). 19) h oso h xrsino lh catenin, alpha of expression the of loss The (1994). 12 20) oaie oe fRoadRcactivities Rac and Rho of zones Localized (2007). 1493-1502. , 18 135 13 533-542. , 20) hom-eitdrcuteto Rho of recruitment Shroom3-mediated (2008). 21) uu euae h circumferential the regulates Lulu2 (2011). 605-616. , 159-166. , 36 1108-1117. , 59-71. , 12 19) fiin eeto o high- for selection Efficient (1991). 20) d4 E uaregulates Tuba GEF Cdc42 (2006). 696-702. , 20) euaino ysnII myosin of Regulation (2007). u.J elBiol. Cell J. Eur. .Cl Biol. Cell J. 20) dniiainof Identification (2009). Gene 20) h different The (2007). m .Hm Genet. Hum. J. Am. .Cl Biol. Cell J. 108 .Bo.Chem. Biol. J. Cell 193-199. , 175 97 86 (2013). (2006). (2010). (2010). (2010). .Cell J. 221- , 135- , 645- , Curr. 178 Mol. ,

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