A935 USA. 94305, CA hcg,I 01,USA. 60611, IL Chicago, USA. ol Lowndes Molly integrity complex structural and assembly the the of in of roles Different ARTICLE RESEARCH ß eevd1 oebr21;Acpe eray2014 February 4 Accepted 2013; November 15 Received 5 cadherins, 2 classical 1 cadherins the atypical four 2009). are Weis, and cadherins: and There (Shapiro 2011). of al., al., cadherins, et et desmosomal subfamilies Simpson Runswick cancers 2012; 1995; metastatic al., major in Garrod, et common Saito 2007; 2001; is Dawson, generally and and binding defects, (Chidgey tissue of in and Oda Disruption results 2009; 2011). (Franke, organization essential Metazoa Takeichi, is the and the throughout formation adhesion tissue The cell–cell for for organisms. of multicellular superfamily essential in cadherin tissues complex is of maintenance adhesion Cell–cell INTRODUCTION , Adhesion, Desmosome, WORDS: KEY . mature in adhesion force Single-molecule binding. formed Dsg2, not but Ca adhesive Dsc2a, monomeric and that showed spectroscopy strong puncta recruit desmosome to sufficient produce into and necessary was desmosome-specific Dsg2Fc, that not show we but IV; Dsc2aFc, collagen micro-patterned and Dsg2Fc used and/or Dsc2aFc we of complexes, substrates assembly desmosome adhesion desmosome cell–cell in and uncouple other To roles from (Dsc) understood. combinatorial not desmosomes. are their cadherinassembly proteins, but the as adhesion (Dsg), of cell members such two of of superfamily complexes, isoforms contain Desmosomes junctional specialized of intercellular formation the by established is cells between Adhesion ABSTRACT USA. Ato o orsodne([email protected]) correspondence for *Author USA. 94305, CA Stanford, University, ih eivle ae nrgltn wtht Ca to switch a regulating in later involved be might htDca u o s2 srqie o emsm assembly desmosome for required is was Dsg2, Ca Dsg2W2A, homophilic not through indicate results but not These Dsc2a, cells. but that MDCK in Dsc2aW2A, desmosomes from and excluded cadherins, classical el ehrh cetfqe n Universite and Scientifique, Recherche la de needn eeohlcbnswt s2.AWAmtto in mutation W2A Ca A inhibited Dsc2a. with Dsc2a bonds heterophilic independent ahenJ Green J. Kathleen eateto ahlg,Nrhetr nvriyFibr colo Medicine, of School Feinberg University Northwestern Pathology, of Department 50011, IA Ames, University, State Iowa USA. Astronomy, 94305, and CA Physics Stanford, of University, Department Stanford Program, Biology Cancer 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,23–30doi:10.1242/jcs.146316 2339–2350 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 2+ dpnethmpii od,adta s2fre Ca formed Dsg2 that and bonds, homophilic -dependent 4 3 ntttJcusMnd Unite Monod, Jacques Institut msLbrtr,Uie ttsDprmn fEeg,Ae,I 50011, IA Ames, Energy, of Department States United Laboratory, Ames 7 eateto oeua n ellrPyilg,Stanford Physiology, Cellular and Molecular of Department 1 aysciRakshit Sabyasachi , 2+ 6 2+ eateto ilg,Safr nvriy Stanford, University, Stanford Biology, of Department dpnethmpii idn,smlrto similar binding, homophilic -dependent 5 ajeiSivasankar Sanjeevi , n 2dpnetbnig n htDsg2 that and binding, W2-dependent and - ´ it eRcece79,Cnr National Centre 7592, Recherche de Mixte ´ ai-ieo,703Prs France. Paris, 75013 Paris-Diderot, 2,3 mrShafraz Omer , 2,3 2+ -independent n .JmsNelson James W. and 2+ - 2,3 ioa Borghi Nicolas , iiaiyi emsmlcdeist eiusrqie o the for required residues which inserts to sequence of W2 cadherins X-dimer. no is latter desmosomal which there but in the into 2011), similarity al., A80, et pocket (Nie and swapping hydrophobic strand W2 during the in to mutations 2010; the contributes by al., facilitates Dsc2 of et blocked that binding (Harrison homophilic is intermediate Importantly, dimer 2009). an al., strand-swap et be W2 Sivasankar the to of thought formation is and sites, aebe apdt h xrclua oano s2adDsc2 mutations and (SPPK) hereditary Dsg2 of these keratoderma domain extracellular of the palmoplantar to Several mapped 2009). been al., striate have al., et et mild Heuser ventricular (Simpson 2009; and right al., et arrhythmogenic 2006) and (Bhuiyan as (Dsc2 (ARVC) cause Dsg2 such subtype cardiomyopathy and Dsc2 each heart diseases in mutations of connect and hereditary 2009), isoform that al., et one discs (Delva Dsg2) contain intercalated cells al., the et muscle example, Green 2007; For Simpson, that and 1998). organs (Green and stress tissues - in mechanical a required resist is form that that complex adhesion (Dsc), dense desmocollins and (Dsg) desmogleins ieiain eeldascn ofgrto emdteX- Ca the EC1–EC2 the termed near configuration residues An involves second 2002). which dimer, a al., swap revealed et strand swap inhibits (Boggon dimerization, ‘strand which (W2A), cadherin a mutation replacement opposing N- forms alanine first the that the with in repeat (W2) dimer’ (EC1) 2 position extracellular at terminal tryptophan crucial a identified 2003; al., et 1999). He 2007; al., al., et plaque et North Al-Amoudi filaments dense 2011; intermediate al., to a et connects (Al-Amoudi that that and the 1988b) cadherins, trans in of structure Nelson, opposing sites contacts to and between 2003). corresponding cell–cell interactions Pasdar midline al., intercellular at 1988a; an comprise Nelson, et structures and punctate binds (Patel (Pasdar that form proteins domain a Desmosomes cytoplasmic adaptor repeats, a ‘cadherin’ desmosome-specific and extracellular ( domain five similarity transmembrane include sequence that low cadherins, play with Dsg domains, and (Kottke Dsc differentiation 2006). and that al., adhesion et disruption cell–cell suggests by in Waschke, caused roles adhesion defects important 2012; skin desmosomal and and Stanley, heart of of the and range in The (Amagai adhesion 2008). of membranes loss of causing mucosal cell-type- binding Dsg3, extracellular a as and target in Dsg1 such vulgaris pemphigus expressed diseases and autoimmune are epidermis, foliaceus and isoforms the In manner, Dsc 2011). specific and al., et Dsg Gehmlich different 2012; al., et (Gaertner emsmscmrs w ifrn ahrn,termed cadherins, different two comprise Desmosomes nlsso h tutr fcasclcdeisbudi trans in bound cadherins classical of structure the of Analysis conserved evolutionarily contain cadherins Desmosomal 1,6,7, * 4 oetM Harmon M. Robert , , 5 , 0)t classical to 30%) 2+ -binding 2339

Journal of Cell Science oe fDcaadDg ndsooeorganization. desmosome in Dsg2 and Dsc2a of roles EERHARTICLE RESEARCH nope emsm sebyfo te eladhesion cell other 2340 from assembly to 2010) assay This desmosome IV. al., collagen and/or protein et uncouples Dsc2a matrix extracellular (Borghi of the surfaces and method micro-patterned Dsg2, to a adhesion binding desmosome adapted cell in analyze we Dsc2a and assembly, Dsg2 and of roles distinguish structures To desmosomal the punctate for of cadherins formation desmosomal of requirements Different RESULTS aeidctdta s2cnas omhmpii bonds homophilic in form homophilic assays only Cell-based found also 2010). reagents form al., cross-linking can et using Schlegel not 2013; Dsg2 al., does et proteins that force (Hartlieb fusion Dsg2 atomic indicated Fc contrast, dimeric of In have with 2002). experiments domain al., (AFM) with et microscope EC1–EC2 bonds (Syed heterophilic bonds of the al., homophilic and domain but bonds et EC1–EC2 homophilic the Dsg2, (Nekrasova form that can strength showed each Dsc2 studies adhesive trafficking, by Cell-free weakened junctions 2011). (MT)-mediated from in depleted microtubule expression be resulting can Dsg2 their desmosomes. Dsc2 of functional and blocking of knockout loss Dsg2 a addition, or in In of results 1998) 2002) Knockdown al., al., et 2008). Dsc2) (Eshkind et (Waschke, and used (Roberts (Dsg2 assay isoforms Dsc2 is the expressed bonds al., on ubiquitously assigned adhesive depending et been most form have Runswick and the to properties 1998; to combine 2004; binding al., different Dsg (Chitaev al., because et and et unclear Dsc Roberts cells Getsios How 1998; 2007; 2001). al., al., adjacent et et Marcozzi Dusek between 1997; Troyanovsky, interactions trans integrity adaptor structural desmosome. the cytoplasmic the to stabilize of the thought these and is clustering in of Dsc–Dsg filament promote complexes recruitment intermediate the Dsc–Dsg The and proteins to 1994b). Troyanovsky 1994a; al., recruited al., evidence et et be also (Troyanovsky is can There of absence I/II 1994a). Kowalczyk al., DP et 1996; that Troyanovsky major al., 1997; two et proteins, al., et the (Bornslaeger protein al., I/II, (cytokeratins) desmosome-specific filaments (DP et adaptor other by desmoplakin encoded Troyanovsky desmosomal proteins and binds 1996; (PKP) core al., which the et have 1994b), Dsg (Kowalczyk for and Dsc studies plakoglobin both sites Biochemical of 2013). domain 2007; al., binding cytoplasmic al., et the Harmon et that additional 2009; indicate Brennan have cytoplasmic al., 2009; might et the Mahoney, and Getsios and whereas longer (Brennan are b), isoforms functions (a, Dsg events of domains splicing different two raiainadsaiiyo idtp n 2 uatDsg2 mutant the force W2A desmosomes examine and in mutant single-molecule to wild-type Dsc2a assay of W2A and using cell-based stability and and a Dsc2a single- organization and a wild-type a and (SMFS); or IV; with spectroscopy Dsg2 collagen Dsc2a, assay and monomeric Dsg2, Dsg2, binding plus purified molecule dual- Dsc2a using of containing assay combination surfaces assembly desmosome patterned a assays: the unclear. independent Thus, organization remains 2011). structural complex desmosomal the al., the in et of Dsc (Nie maintenance and and Dsc2 Dsg both and for Dsg2 requirement both for binding odsigihterlso s2 n s2 eue three used we Dsg2, and Dsc2a of roles the distinguish To extracellular in involved be to thought are Dsc and Dsg Both from derived are isoforms Dsc of domains cytoplasmic The DSP ,ta iktecmlxt intermediate to complex the link that ), nsitu in u eut dniyunique identify results Our . te emsmlcmlxpoen uha PII,wihlinks which I/II, DP recruit as such and and/or proteins stripes complex homophilic Dsg2Fc desmosomal and form 1988a; other Dsc2aFc could Nelson, with they and bonds MDCK 1988b) (Pasdar heterophilic that Nelson, Dsg2 ventral Given and and 1). the before Dsc2a Pasdar (Fig. both on Dsc2aFc h express I/II 2 plus cells Dsg2Fc, Dsg2Fc DP microscopy containing least stripes or cellular to dual-patterned Dsc2aFc, attached fluorescence at detect cells MDCK to the of reflection for membrane used onto adhere internal was (TIRF-M) to seeded Total allowed were Fig. fixation. were material and showing cells stripes (supplementary coverslips stripes IV proteins MDCK both the collagen S1C). with of the stripes, staining that uniform Dsc2a/Dsg2 so Dsg2Fc to or (50:50) juxtaposed Dsc2aFc Dsg2Fc, Dsc2aFc of plus amounts saturating by followed s2i o eurdo tlatoeo h poigsurfaces. opposing the of one whereas least puncta, at on desmosome-like required into not is organization necessary Dsg2 is the I/II Dsc2a (supplementary in that DP distributed indicate Dsg2Fc for results evenly or These were S1E). Dsc2aFc them Fig. of material of plus distribution both to punctate as appear Dsg2Fc a not stripes, desmosomes does to I/II over DP due fide material of be localization (supplementary bone puncta punctate stripe cells The MDCK S1D). to I/II Fig. Dsc2aFc of similar pairs plus DP adherent appeared the between Dsg2Fc the over stripes punctate and Dsc2aFc Indeed, Dsc2aFc more 1C) but 1D). (Fig. diffuse (Fig. plus 1B), was (Fig. stripe I/II stripe Dsg2Fc DP collagen Dsc2aFc Dsg2Fc of the the organization over or Dsg2Fc the over areas Significantly, from I/II containing stripe. 1C) excluded (Fig. DP IV generally stripes and cellular 1D), contrast, Dsc2aFc over (Fig. In restricted 1A). 1B), (Fig. (Fig. was Fc and distribution IV collagen of cytoskeleton. the to complex the n aee ihC5det iulz rti atrso micro- on patterns S1B). protein affinity Fig. visualize material (supplementary by to coverslips dye 2005), patterned purified Cy5 al., with medium, et labeled and (Drees the Protein-A–Sepharose Fusion into with modifications. chromatography secreted post-translational were HEK293T correct mammalian proteins ensure in constant to expressed the were cells extracellular to Dsc2aFc and fused and S1A), complete Fig. was Dsg2Fc material (supplementary Dsc2a (Fc) The IgG1 and human of Dsg2 region E-cadherin. of (EC1–5) as domain such complexes tahdt ufc,adaTVrmvbeHstg for tag, His TEV-removable a and biotin surface, binding for a tag, at Avi fused to an Dsg2 comprising tag or attached a Dsc2a 2009), to al., of E-cadherin C-terminal et domain the Zhang for 2009; extracellular al., previously the et comprising Sivasankar described and 2012; al., as Dsc2a data et (Rakshit proteins, monomeric in fusion designed artifacts single- between Dsg2 we introduce from Fc Therefore, interactions might distinguished interpretation. which because thus, be interactions cannot 2009); experiments cis molecule pairs al., a cadherin in SMFS et proximity opposing close Fc (Zhang the in the cadherins orientation use not the in did positions We dimerization construct AFM. an measured with fusion SMFS we Dsg2 using interactions, and binding heterophilic Dsc2a their whether and/or address homophilic to directly form sufficient To not puncta. Dsg2. cellular were these not occurred, form but desmosome-like they Dsc2a, if interactions, punctate surfaces of Heterophilic binding micro-patterned of homophilic to involved formation structures binding that MDCK indicate from results properties binding The distinct have cadherins Desmosomal ellrD /Iwsdsrbtdeel vratraigstripes alternating over evenly distributed was I/II DP Cellular atre 10 Patterned ora fCl cec 21)17 3925 doi:10.1242/jcs.146316 2339–2350 127, (2014) Science Cell of Journal m tie eecnatpitdwt olgnIV, collagen with printed contact were stripes m

Journal of Cell Science EERHARTICLE RESEARCH fCa of interact (Dsg2pATH–Dsc2apATH) Dsc2apAT or (Dsg2pATH–Dsg2pATH to due conditions. kept occur experimental were all frequency densities for adjusted constant unbinding the this and of unbinding), 95% at single-molecule than events more that measured predicts statistics frequencies the (Poisson binding range the yield and 5% to on the adjusted in rates), densities were coverslip Dsc2apATH (loading and and tip AFM rates Dsg2pATH S2). pulling (supplementary Fig. compiled were different material were forces measurements unbinding 11 the of 1000–2000 histograms at Approximately out 2009). carried al., to (Zhang used et binding was non-specific polymer from stretched interactions the 1992) specific of al., distinguish length et contour (Smith the surface 2D); the polymer to (Fig. the proteins the of anchored stretching that that were chain tether interactions freely-jointed so Single-molecule the surface bond. from the the identified and from to withdrawn applied tip was then AFM force was The 2012; tip cadherins 2009). opposing The al., that al., so interacted. contact et et into brought Zhang first (Rakshit were 2C), 2009; coverslip E-cadherin (Fig. al., tip for et cantilever (PEG) Sivasankar AFM previously an glycol described and coverslip polyethylene as glass a via to immobilized linkers the and likely biotinylated is slower and a *). 2B, 2B); occasionally (Fig. (Fig. detected precursor tag uncleaved was an same the protein had to migrating proteins fused purified domain The extracellular of 2A). mass Fig. molecular expected Ni- (pATH; a column using medium NTA conditioned cell HEK293T from purification h oc eurdt utr h odbtenhomophilic between bond the rupture to required force The were monomers Dsc2apATH and Dsg2pATH Purified 2+ rET,t hlt Ca chelate to EGTA, or , 0ka iia oteE-cadherin the to similar kDa, 90 2+ oswsmaue ntepresence the in measured was ions Fg E.Nnseii binding Non-specific 2E). (Fig. –s2pT)o heterophilic or H–Dsc2apATH) En3 Bl,17;EasadRthe 97 odetermine to unbinding 1997) the that Ritchie, Ca showed and of results model rates Our Evans Bell–Evans rates. the 1978; unbinding used we intrinsic (Bell, 2F), 3) (Fig. rate (Eqn loading the of log of inactivity to due not is binding Dsg2pATH. Dsg2pATH homophilic of lack thro the Dsc2apATH to bound Dsg2pATH Ca vnswssmlri h rsneo bec fCa of absence binding or those presence of the in frequency similar between the was events and events binding Dsg2pATH, heterophilic and detected Dsc2apATH We 2E). (Fig. events was homo Dsg2pATH the contrast, In mechanism. niaeta ooei s2,btntDg,frsCa forms Dsg2, not results but These Dsc2a, other. monomeric each that from indicate indistinguishable statistically n emsmlcdeiscnan rpohna oiin2 position at tryptophan a classical contains of domain cadherins extracellular desmosomal the of and sequence acid amino The Ca Dsc2a for required is W2 fubnigeet novdhmpii neatosbetween interactions homophilic , involved ( events Dsc2apATH number unbinding greatest the that of showed (percentage) frequency binding decoraThe not were that coverslips identical using measured were rates s2AHmnmr were monomers Dsg2pATH eedn oohlcitrcin,adta s2 om Ca Dsg2. forms with Dsc2a interactions that heterophilic and independent interactions, homophilic dependent Ca n erimn nodsooepuncta desmosome into recruitment and % pnadto fET,idctn Ca a indicating EGTA, of addition upon 2%, eas h nidn oc nrae ierywt h natural the with linearly increases force unbinding the Because 2+ 2+ idpnetbnigmcaim(i.2) ie that Given 2E). (Fig. mechanism binding -independent idpnethtrpii idn ewe s2pT and Dsc2apATH between binding heterophilic -independent ora fCl cec 21)17 3925 doi:10.1242/jcs.146316 2339–2350 127, (2014) Science Cell of Journal 2+ iedsgae h elotie cl a:10 bar: Scale outline. white cell The the the stripes). designates as (black line marked surfaces are printed stripes IV location. control non-collagen and Fc functionalization and stripe Dsg2Fc to Dsc2aFc, of respect recruitment with specific I/II show DP images merged condition; each eevsaie sn IFmcocp.Ms el showed cells ( Most behavior microscopy. this (Cy3), TIRF location using cell– stripe visualized the IV were at collagen I/II and DP interface, of substrate Localization I/II). and protein (DP fixed adaptor desmoplakin Dsg2Fc were desmosomal or cells the (C), h, for Dsc2aFc 4–6 immunostained or After (B) (D). Dsg2Fc Dsc2aFc (A), plus Fc either collagen and with IV functionalized substrates micro-patterned onto cadherin engagement. surface desmosomal to on I/II depends desmoplakin complexes of Localization 1. Fig. dpnethmpii idn fDcaAHand Dsc2apATH of binding homophilic -dependent . %,adta idn a infcnl eue,to reduced, significantly was binding that and 4%), , % hc a eo hto o-pcfcbinding non-specific of that below was which 1%, n DKclswr ltda igecl density cell single at plated were cells MDCK . )adarpeettv elwscoe for chosen was cell representative a and 5) e ihDgpT rDsc2apATH. or Dsg2pATH with ted 2+ , dpnethmpii binding homophilic -dependent hlcbnigfeunybetween frequency binding philic ypoesdAMcnieesand cantilevers AFM processed ly .3s 0.23 g eeohlcinteractions, heterophilic ugh 2 1 n htte were they that and 2+ dpnetbinding -dependent 2+ m niaiga indicating , m. 2341 2+ 2+ - -

Journal of Cell Science EERHARTICLE RESEARCH a hltdb GA(i.3) nietewild-type the unlike 3B), (Fig. EGTA 2342 by chelated (Dsc2aW2ApATH, was tag pATH 3A). (Fig. domain the SMFS for extracellular used were monomeric containing Dsg2W2ApATH) and (Dsg2W2A), mutants (Dsc2aW2A) Dsc2a Dsg2 of in role mutants and a substitution for alanine test made To 2011). we al., W2, et use (Nie cadherins mechanism desmosomal been binding that opposing similar not suggested a been has has cadherins al., between it desmosomal a but et of reported, forms (Boggon structure binding crystal it cadherins A because opposing 2002). trans 1995) classical between al., dimer in for et strand-swap W2 (Shapiro crucial (EC1). domains domain extracellular is extracellular first cadherins the in (W2) h ahrnbnigfeunyddntcag hnfe Ca free when change not because did SMFS, frequency binding by cadherin measured the binding homophilic Dsc2apATH h 2 uainrsle nls fCa of loss in resulted mutation W2A The 2+ -dependent 2+ s2pT seFg E.Hwvr h rqec fCa of frequency the However, 2E). Fig. (see Dsc2apATH s2 idn sCa Dsc2a– homophilic is protein that binding wild-type indicate results the Dsc2a These to 2E). similar (Fig. binding was 3B) and (Fig. Dsc2aW2ApATH Dsg2W2ApATH between binding heterophilic independent ewe s2 n s2i Ca binding is heterophilic Dsg2 contrast, and In Dsc2a W2. between involving mechanism lna esspntt)wr oprdt rninl expressed transiently to distributions compared their were and punctate) cells versus MDCK in protein (linear in puncta mutant expressed GFP-tagged transiently desmosome C-terminal was endogenous cells. into MDCK contacting incorporated be are 2012). could residues al., K14 et and (Rakshit W2 binding E- both inhibit to of to contrast required mutations in which is in This cadherin, mechanism. W2-independent a through etse hte h s22 n s2WAmutants Dsc2aW2A and Dsg2W2A the whether tested We ora fCl cec 21)17 3925 doi:10.1242/jcs.146316 2339–2350 127, (2014) Science Cell of Journal 2+ eedn,adrqie strand-swap a requires and dependent, s2AHadDcaAHwas Dsc2apATH and of Dsg2pATH combinations different of frequency binding Single-molecule for (E) used analysis. were event a binding showing single the events on only protein and and coverslip, tip AFM the on protein between single- interactions of molecule hundreds measure of to thousands times raised and lowered was tip cantilever AFM The (D) interactions. single-molecule measure to Dsc2apATH or Dsg2pATH monomeric with functionalized (5% and PEG coverage), with coated tip coverslip cantilever and AFM an of (*). Schematic sample (C) Dsc2apATH low the at in present is levels cadherins the of form masses ( molecular similar have which control), (EcadpATH, cadherinpATH E- and Dsc2apATH purified Dsg2pATH, of gel SDS-PAGE Coomassie-blue stained (B). (pATH). Tev His Avadin, and comprising tags to fused cadherins desmosomal monomeric properties. binding trans extracellular have distinct cadherins Desmosomal 2. Fig. diino h Ca the of Addition cadherins). desmosomal the lacking cantilevers functionalized binding (identically non-specific events to all compared of and percentage a as plotted s2pT eeohlcbinding. heterophilic Dsg2pATH–Dsc2apATH and EGTA) without (with or Dsg2pATH of both binding for homophilic plotted was bond force and rupture (pN/s) rate loading relationship between The (F) events. binding a sdt etCa test to used was , 5ka;a nrcse precursor unprocessed an kDa); 95 2+ needn n occurs and independent A ceai fthe of schematic A (A) 2+ 2+ hltrEGTA chelator dpnec of -dependence 2+

Journal of Cell Science EERHARTICLE RESEARCH l fwihwr noprtdit emsmlpuncta. desmosomal used into of was incorporated (FRAP) mobility photobleaching were higher after Dsg2W2A, recovery which a and Fluorescence Dsg2 in of Dsc2a, than endogenous result all membrane into the incorporation would in of Dsc2aW2A puncta lack the desmosome that speculated at We Dsc2a wild-type than contacts mobile cell–cell more is a mutant mostly as Dsc2aW2A was act it puncta. not desmosome that endogenous did and from excluded Dsc2aW2A protein that (dominant-negative) presence the suggesting disrupting in cadherins contacts Dsc2aW2A, cell–cell and at of both 4B,D). puncta proteins (Fig. in that normally cytoplasmic indicate localized contacts results desmosomal These endogenous mutant cell–cell more 4C,E). than (Fig. and Dsc2a–GFP and Dsc2aW2A–GFP punctate, same Dsc2a–DsRED also wild-type to was similar the staining in plakoglobin localized Endogenous along all wild- Dsc2a–GFP wild-type 8/18, puncta and cytokeratin Dsc2a–DsRED I/II, type DP distribution endogenous and punctate Dsc2aW2A–GFP, Dsc2a, or linear mutant a W2A had 4B–E). or (Fig. localization wild-type the proteins the whether endogenous whether with determine were colocalized protein to Line- each used staining. for and each intensity normalized for fluorescence shown measured is that cell scans cell–cell expressing representative an A with the 4C,E). 8/18) contact I/II, (Fig. cytokeratin plakoglobin DP (hereafter or 4B,D) proteins pair 18 (Fig. desmosome cytokeratin the and for 8 4D,E) cytokeratin stained (Fig. and Dsc2a–GFP only fixed or desmosome were 4B,C), Dsc2aW2A–GFP into (Fig. co-expressing Dsc2a–DsRED components and transiently cells endogenous MDCK of puncta. localization recruitment the and affected levels contacts cell–cell along Dsc2a–GFP Dsc2aW2A–GFP control to similar 4F). were (Fig. and levels efficiency transfection wild- the expression since to plasma overexpression to compared due the not C) Dsc2aW2A–GFP was of Mislocalization 4B, along 4D,E). (Fig. (Fig. However Dsc2a–GFP distribution contacts type cell-cell 4A). linear (Fig. at a membrane Dsg2–GFP proteins had wild-type cell–cell Dsc2aW2A–GFP to along desmosomal puncta similar to endogenous restricted contacts, was Dsg2W2A–GFP and/or 4). (Fig. protein wild-type etw netgtdwehrteaoaoslclzto of localization anomalous the whether investigated we Next ncnrs otelna lsammrn tiigof staining membrane plasma linear the to contrast In oprsn h irto aeo el ltdo olgnI and ( IV collagen stripes on substrate plated cells Dsg2Fc of rate migration The comparison. Fg ) s22 a lgtyhge oiefato (0.354; fraction model mobile recovery higher slightly exponential a P single had Dsg2W2A a 5). and on wild-type (Fig. of based rate proteins recovery and mutant fraction mobile the measure to idtp s2(.1 n 2 ,rsetvl)(i.5B; (Fig. respectively) s, 127 and (0.312 Dsg2 wild-type h s2WAmbl rcinwssgiiatygreater significantly was fraction 3), mobile Movie material ( versus Dsc2aW2A supplementary (174 recovery 5B; Dsg2W2A Fig. the of the that respectively; to Although s, similar 167 2). was Dsc2aW2A 1, of rate Movies material supplementary omlzdt 10 to the normalized examine addition, to time In over 6C). traced 6B). (Fig. was (Fig. dynamics cell lamellipodia representative direction a in migration of contour changes and velocity and migration the 6A) h, determine (Fig. 6–10 to tracked cell for were to quantify position recorded adhesion nucleus To that were effect. found images significant we movements, a 2010), had al., migration cadherins et cell desmosomal (Borghi of rate IV the collagen on 2010). on effect al., little et cell–cell (Borghi had ECM E-cadherin of adhesion on Although effects migration different cell the on to test proteins adhesion substrates recruitment patterned protein dual testing complexes, to addition migration cell In MDCK inhibits formation Desmosome 2 uatwslkl ihrnticroae rrtie in retained or the incorporated that not and either structures. puncta likely these desmosome was mutant into required W2A incorporation are lipid-anchored dimers Dsc2a a strand-swap to and for similar W2 that fraction confirming mobile protein, a plasma In the had along puncta. and distributed reduced membrane desmosome was are mutant into a dimers Dsc2aW2A incorporation the strand-swap had contrast, Dsg2 and for mutant W2 required Dsg2W2A that not intermediate indicating The a and fraction into 4). proteins mobile Fig. incorporation reduced adaptor their (see a with to filaments in complex due results be protein puncta might dense that desmosome fraction into mobile Dsg2 and lipid-anchored Dsc2a a 5B). Lyn–GFP, Fig. respectively; of 0.75, that and of (0.714 to fraction protein mobile similar the was Indeed Dsc2aW2A 4). Movie material supplementary P , , irto eoiiso DKclso l ufcswere surfaces all on cells MDCK of velocities Migration hs RPdt niaeta h erimn fwild-type of recruitment the that indicate data FRAP These .)adsoe eoeyrt ( rate recovery slower and 0.1) .01 hnta fDca(.1 ess031 i.5B; Fig. 0.351; versus (0.714 Dsc2a of that than 0.0001) ora fCl cec 21)17 3925 doi:10.1242/jcs.146316 2339–2350 127, (2014) Science Cell of Journal m / n lte nantrllg(n cl for scale (ln) log natural a on plotted and m/h o eeso Ca of showed levels SMFS low (B) line). simplification (dashed for purposes deleted been the has between samples lane A (**). products degradation (*; dimers by possible contamination low-level molecular and expected mass, the of band protein major a shows SDS-PAGE Coomassie-stained in Dsc2aW2ApATH. (W2A) and residue Dsg2W2ApATH alanine an was to (EC1) mutated the repeat in binding (W2) extracellular 2 first position at residue tryptophan disrupts Dsc2a binding. in trans mutation single-molecule W2A A 3. Fig. s2WAAH.Ca . Dsc2aW2ApATH and Dsg2W2ApATH of binding idtp Fg 2E). (Fig. in type that wild to comparable was and Dsc2aW2ApATH Dsg2W2ApATH of binding heterophilic , 9 m /)wssmlrt hto cells of that to similar was m/h) t 1/2 2+ 5 idpnethomophilic -independent , 6 s; 167 0 D)and kDa) 200 2+ -independent P , A A (A) .5 than 0.05) 2343

Journal of Cell Science VadDcacsrpso olgnI n s2cstripes Dsg2Fc and IV collagen or stripes collagen Dsc2aFc ( on cells plus and of Dsg2Fc that and between IV IV in was collagen stripes on substrate plated Dsc2aFc cells of rate migration the EERHARTICLE RESEARCH 2344 (Borghi IV ( collagen stripes stripes on cells substrate substrate than Dsc2aFc control faster significantly and Fc was and and 2010), al., IV et collagen on plated 4. Fig. , 7 m e etpg o legend. for page next See m/h; P , 0.1). , 5 m m/h; P , .1.Importantly, 0.01). rvosyfrclsmgaigo olgnI n E-cadherinFc reported and as IV 6B), biased collagen (Fig. on showed stripes migrating the stripes cells of for Dsg2Fc direction previously and the IV in movement collagen on migration el rfrnilymv ntedrcino h tie ( stripes the of direction the in move preferentially cells iie ytepredclrvlct ( stripes velocity the perpendicular of direction the the by in velocity divided cell as plotted were surfaces h ietoaiyo elmvmnso h dual-patterned the on movements cell of directionality The ora fCl cec 21)17 3925 doi:10.1242/jcs.146316 2339–2350 127, (2014) Science Cell of Journal x / y ;ln( ); x / y spstv if positive is ) x . y .Cell ).

Journal of Cell Science emsm-iepntt pt tcl–elcnat.Saebr 10 bar: Scale contacts. cell–cell at characteristic spots Wild-type in punctate methanol. localized desmosome-like with Dsg2W2A–GFP fixed mutant and and Dsg2W2A Dsg2–GFP GFP-tagged punctate or to Dsc2a Dsg2 of tagged localization disrupts mutation complexes. W2A The 4. Fig. ARTICLE RESEARCH oDcacaoeadsihl infcn oprdt Dsg2Fc to compared the significant of slightly ( and direction alone alone the Dsc2aFc towards to significant biased Dsc2aFc ( slightly plus stripes significantly Dsg2Fc and a was IV almost collagen stripes small 6B; on showed (Fig. were migration the cell only and Dsg2Fc cells MDCK stripes from contrast, movement Dsc2aFc difference In and of IV stripes. amount 2010)], collagen IV al., on et collagen stationary Borghi also the (see S3 to Fig. restriction to –GFP material as due [supplementary such is machinery, bias adhesion migration-based Directional the 2010). of al., et (Borghi stripes akr oa o ocet n issbtentetasetdGFP-tagged transfected the the as between 5 proteins biases bar: endogenous any Scale of create proteins. to staining not the as using so note, set marker, Of was plakoglobin). with plane and colocalized 8/18 focal and cytokeratin the contacts I/II, (DP cell–cell proteins at desmosomal spots the punctate Dsc2a– into Wild-type localized a (D,E) GFP had distribution which punctate of desmosome-like all characteristic Dsc2a_DsRED, plakoglobin) and wild-type 8/18, co-transfected co- cytokeratin or not I/II, (DP did proteins and desmosomal membrane with a plasma localize and in the maximum along accumulated distribution in Dsc2aW2A–GFP non-punctate difference (B,C) linear, the range). by (pixel intensity dividing values average and minimum the contact subtracting whole by the cell– normalized over representative was the for Intensity plotted contact. were cell I/II DP of and scans (PG) plakoglobin line Dsc2a– DsRED, Dsc2a–GFP, Bottom: Dsc2aW2A–GFP, for contacts. intensity cell–cell fluorescence representative normalized zoomed- the Middle: white. of in images outlined in contact wide- representative a Top: the proteins. with desmosomal image wild-type endogenous field RFP-tagged for stained without and or fixed with Dsc2a, Dsc2aW2A Dsc2a, GFP-tagged GFP-tagged with wild-type transfected or transiently were cells MDCK (B–E) u otedlto fDcachmpii neatosby interactions homophilic Dsc2aFc of smaller was Dsg2Fc dilution the cell plus that the Dsc2aFc strong suggest of to in presence We the due result stripe. in 1) Dsc2aFc decrease Fig. velocity the (see to binding adhesion 2010). Dsc2a al., Dsg2Fc et homophilic (Borghi and substrates or 7) IV control greater 6, Fc Movies collagen and 6B; a IV (Fig. on stripes collagen with Dsc2aFc 6C; and than IV (Fig. consistent collagen bias stripes, movement 5) persistent was migration cell Movie which more directional stripes, processive material and IV highly supplementary Dsg2Fc, on collagen by membrane on the Cells accompanied had on cells 6A). stripes projections (Fig. to Dsc2aFc membrane rate plus similar migration Dsg2Fc dynamics and overall IV significant 6C; the collagen (Fig. the in with projections consistent reduction 7) membrane membrane Movie exhibited persistent material the stripes supplementary few Dsc2aFc along 6). but and Movie generally IV dynamics, material collagen supplementary was on 6C; which (Fig. Cells stripes movement, IV the cell collagen in but of predominantly contour, occurred stripes cell direction projections Dsg2Fc whole and membrane the IV over persistent collagen dynamics on membrane Cells exhibited the experiment. during the migrated membrane of cells onto the course of because images attempted quantification not all was 6C); dynamics superimposing (Fig. and substrate time dual-patterned over contour cell hto noeosDcai DKcells. MDCK in Dsc2a endogenous of that 2 with efficiencies (similar cells hs eut niaeta emsm ucafre by formed puncta desmosome that indicate results These the tracing by activity lamellipodia membrane examined We P P , , 0.1). A DKclswr rninl rnfce ihwl-yeGFP- wild-type with transfected transiently were cells MDCK (A) .01 upeetr aeilMve5 compared 5) Movie material supplementary 0.0001; m .()Epeso eeso rninl transfected transiently of levels Expression (F) m. . 0 rnfce)Dca2–F were Dsc2aW2A–GFP transfected) 60% P , .) h ieto of direction The 0.1). m m. , 1/ ye A-asre l,21) ee eue reductionist used we Here, 2013). al., et sub- (Al-Jassar cadherin desmosomal in high- two types of E- the dissociation lack of of the structures to (e.g. and crystal complexity 2011), resistance resolution al., cadherins the et their (Nie other conditions and adhesion, non-denaturing desmosomes of cell–cell in roles epithelial proteins overlapping in and the cadherin) assembly establish to to desmosome difficult been owing binding has in identified extracellular cadherins desmosomal the Dsc of not in properties specificity and is have there Dsg Whether however, adhesion. for al., different assays, roles using et blistering cell-based specific Studies Kottke 2010). and 2013; epidermal al., al., binding in et and et Thomason cadherins (Al-Jassar defects, 2006; syndromes obligate structural extracellular and heart are Dsg the diseases or causes Dsc Dsc maintain either and interactions of to impairment Dsg and required tissues. desmosomes, are of and integrity cells, opposing eiiino igemlcl neato rorueof use our addition, Ca our In the in domains. induced differences or strict extracellular of of findings of our possibility interaction clustering to the (cis) excluded due single-molecule lateral which be proteins, a might monomeric experiments of SFMS al., et definition in our Schlegel 2013; and binding al., et Differences 2010) (Hartlieb 2002). Dsg2–Dsg2 results al., SMFS et homophilic other (Syed between results SMFS weak Dsc2a– our with and heterophilic 2011), agreement al., indicate binding et assays (Nie only membrane. Dsg2 biochemical Dsg2 the cross-linkers and bulk Dsc2 at chemical whereas by I/II binding using homophilic DP heterotypic studies found of promote other recruitment surfaces Interestingly, diffuse and Dsg2Fc Dsg2 and SMFS that between binding However, interactions suggesting heterotypic 2). and Dsc2a, (Fig. only interactions Dsg2 indicates homophilic data with forms SMFS interactions the Dsc2a from on heterophilic results that of our both Indeed, indicated or cell. assembly Dsg2 assay the of Dsc2a, the surface exogenous cellular ventral initiated binding the the by 1991), have puncta Dsg2 al., could desmosome and et Dsc2a Dsc2a Pasdar both 1989; substrate-bound express Nelson, cells and desmosome-specific MDCK and (Pasdar that structures a necessary cellular Given punctate of 1). was into (Fig. I/II) recruitment DP Dsg2Fc, in (e.g. the protein not cytoplasmic induce functions but to Dsc2aFc, sufficient and that properties in revealed Dsg2 binding adhesion. and and distinct and Dsc2a organization that desmosome Dsc2a have indicate results Our absence of Dsg2 adhesions. the cell–cell in roles other adhesion of and organization distinguish assembly, desmosome to approaches tutr Hrio ta. 00.Atog h tmcstructures atomic the Although dimer 2010). strand-swap al., X-dimer weak EC1 et a a (Harrison by N-terminal formed an structure of also 2002); the is al., formation structure et in adhesion (Boggon intermediate the (W2) domains extracellular for 2 opposed between crucial position is at domain tryptophan a below). (see that assays cellular between or differences bulk and revealed data have single-molecule binding protein heterophilic emsmsasml naCa a in assemble Desmosomes DISCUSSION olgnI usrt n eue deinsrnt from strength adhesion Dsg2. of reduced incorporation the the and to on due assembly substrate complexes desmosome -based IV of requires collagen adhesion bias direction anisotropic migration both Thus, thereby migration. and strength increased decreased in results which Dsg2Fc, ir-atre usrtso uiidDcaco Dsg2Fc or Dsc2aFc purified of substrates Micro-patterned nlsso h rsa tutr fcasclcdeisrevealed cadherins classical of structure crystal the of Analysis ora fCl cec 21)17 3925 doi:10.1242/jcs.146316 2339–2350 127, (2014) Science Cell of Journal 2+ 2+ eednyo oohlcand homophilic of dependency dpnetmne between manner -dependent nvitro in 2345

Journal of Cell Science uca n htbnigt yolsi dpo rtisi not is proteins adaptor cytoplasmic desmosome to into binding sufficient. Dsc2a that mechanism of strand-swap incorporation and adaptor W2 the puncta, the for desmosome that necessary of likely also is is localization it punctate 4), (Fig. the proteins and affect desmosomes not from Furthermore, excluded did assembly. mostly desmosome was Dsc2aW2A for because required is mechanism EERHARTICLE RESEARCH 2346 X-dimer. Ca an inhibited of Dsc2a in formation mutation the W2A for have the neither residues but 2011) Significantly, al., necessary et proteins (Nie both 2 solved, the position been at not tryptophan have a Dsg2 have or Dsc2a full-length of noprto nodsooe custruhaCa Dsg2 a that through indicate results occurs These desmosomes 5). into (Fig. nor 4) incorporation fraction (Fig. cells mobile MDCK in its puncta desmosome colocalization 3), endogenous (Fig. with Dsc2a to binding heterophilic independent s2F ln a eesr n ufcett induce binding micro-patterned to homophilic on Dsc2a Ca sufficient that cells a suggest and in we through 1), puncta necessary (Fig. I/II substrates increased was DP significantly that and desmosome-like Given alone 5). 4), (Fig. (Fig. Dsc2a Dsc2aFc wild-type cells to compared same fraction linear mobile the its desmosome the in endogenous from to inferred puncta compared endogenous we Furthermore, staining from which membrane excluded plasma cells, 3). MDCK be (Fig. in to desmosomes appeared binding mutant Dsc2aW2A homophilic dependent ncnrs,teWAmtto nDg fetdnihrCa neither affected Dsg2 in mutation W2A the contrast, In 2+ n 2 srn-wpdmr dependent dimer) (strand-swap W2- and - 2+ and - 2+ 2+ - - ifrne nCa in Differences el nmcoptendsbtae Fg ) htrl could role what 1), (Fig. MDCK substrates in Ca puncta micro-patterned Dsg2-dependent I/II on DP desmosomal-like not cells did of with alone Dsg2Fc assembly that interactions Given induce desmosome. the cytoplasmic in on proteins and/or other relies that interactions mechanism heterophilic independent dimer) (strand-swap W2- 08.Orrslsrietepsiiiyta uhCa such that possibility the raise results Our 2008). eeldltl rn feto -ahrno h irto rate IV migration the collagen on E-cadherin and of E-cadherinFc effect Dsg2Fc no 6A; with or (Fig. contrast, controls little coated studies revealed to previous Dsc2aFc substrates compared in Our 6). migration Movie with of material cell 7); supplementary 6A; inhibit (Fig. not amounts Movie did the substrates saturating material substrates infer by dual-patterned supplementary with on adhesion can cell inhibited MDCK cell We Significantly, coated severely migration. on 2). was cell of migration assembly 1, rate (Figs the desmosome measuring Dsg2Fc of and contribution dual- IV Dsc2aFc on collagen of migration stripes and cell alternating mechanisms comprising examined substrates adhesion we patterned other cadherins), of classical contribution (e.g. the from migration binding. Dsc2a–Dsg2 heterophilic by mediated be might adhesion ugse htmtr emsm opee r nahyper- a Ca in is are been that complexes has state it desmosome and adhesive mature studies, cell-based that and suggested tissue- in identified been odsrmnt h fet fdsooeahso ncell on adhesion desmosome of effects the discriminate To ora fCl cec 21)17 3925 doi:10.1242/jcs.146316 2339–2350 127, (2014) Science Cell of Journal 2+ 2+ dpnec fdsooa deinhave adhesion desmosomal of -dependency idpnethtrpii deinplay? adhesion heterophilic -independent 2+ B oiefcin n eoeyrts( rates recovery and factions Mobile (B) mean as plasma Plotted the membrane. to proteins lipid-anchored for are reference that a used is differences. Lyn–GFP slight only mutant showed whereas Dsg2W2A Dsg2, wild- and and Dsc2 Dsc2aW2A type of rate in recovery difference the significant a cell–cell showed at contacts cadherins Dsg2 mutant and W2A Dsc2a and wild-type GFP-tagged mutant. of W2A the Dsc2a in of increases mobility Membrane 5. Fig. sn oprsno is xr sum-of- extra fits, squares of comparison a using different significantly ( are pairs both of rates r essgiiat( significant less are Dsg2W2A–GFP and Dsg2–GFP ( whereas different significantly are Mobile Dsc2aW2A– GFP A. and in Dsc2a–GFP shown of data fractions the to exponential fitted single curve a using compared are P , needn Gro n Kimura, and (Garrod independent .5.Sgiiac a calculated was Significance 0.05). F test. P , .) h recovery The 0.1). 2+ 6 -independent s.d. P A FRAP (A) , 0.0001), t 1/2 )

Journal of Cell Science n iua 08.WeesDcamgthv pcfcrl in role specific a have might Dsc2a Whereas 2008). Kimura, and ogtr tblt fdsooe,adas ehp the perhaps also and Ca desmosomes, a of of the formation for stability required be might Dsg2 long-term surfaces. cell Dsc2a opposing between on proteins trans-dimerization homophilic dependent dimer) EERHARTICLE RESEARCH xrclua otcsfre yDcai h structural of the assembly Ca The in on desmosomes. depends Dsc2a of puncta by function desmosome and formed organization contacts extracellular detail. in hypothesis this test However, to sheets. complex needed epithelial desmosome are studies in the further migration of cell dynamics modulating thereby and adhesion strength the cell–cell regulate might the desmosomes of in in differences Dsg2 and that Dsc2a suggest of We ratio 6A,C). in (Figs reduction rate on the migration rescued effect partially cell substrate little Dsc2aFc of the had addition to Interestingly, they Dsg2Fc 6C). a that (Fig. dynamics found lamellipodia than overall we migration, rather cell inhibited substrates dual substrate, Dsc2aFc-containing whereas that Indeed, and 2003). machineries (Pollard Borisy, migration Dsc2aFc cell actomyosin and dynamics membrane and in the involved Arp2/3 are the E-cadherin to of strong downregulation unlike forming bonds by movement structures, adhesive cell resists desmosome-like mechanically adhesion, of the that propose we Therefore, formation 2010). al., et (Borghi cells MDCK of [Ln( scale log natural a ( on rate plot migration box-and-whisker Cell tukey (A) a time. as over plotted position and nuclear surfaces, in IV substrates. changes collagen dual-patterned monitoring only on by on migration tracked movement cell was MDCK condition regulates substrate engagement dual-patterned cadherin Desmosomal 6. Fig. oeetwspotda ue o-n wikrpo nantrllgsaea ucino oeetprle osrps( stripes of to Directionality parallel (B) movement values. of minimum function and a maximum as the scale show log Dsg2Fc+Dsc2aFc, Whiskers natural median. a the on marks plot box -whisker the box-and inside tukey band a the as and plotted data was the movement of percentile 25th–75th the mgsadpoetn h ulnsot h tiepten C.Saitclaayi sdteMn–hte,two-tailed Mann–Whitney, the used analysis Statistical (C). patterns stripe the onto outlines the projecting and images ( movement nsmay u eut aeucvrdaseii oefor role specific a uncovered have results our summary, In y .* ). P , n .;** 0.1; 5 27). 2+ idpnethprdeiesae(Garrod state hyperadhesive -independent P , .1 **** 0.01; P , .01 C ebaeatvt uigmgainasy a sesdoe ieb rcn h elcnorfo DIC from contour cell the tracing by time over assessed was assays migration during activity Membrane (C) 0.0001. 2+ n 2 (strand-swap W2- and - ehnsso noprto n ucino s n Dsg and differentiation. Dsc and functions migration desmosome of adhesion, of and cell diversification function in the a (Brennan in and allow differences might roles incorporation cadherins Thus, 2007). of additional al., mechanisms et have Brennan 2009; might Mahoney, Dsg2 adhesion, eotdpeiul Brh ta. 00.Frt h olgnI was IV collagen the First, 2010). al., et as process (Borghi two-step previously a using reported prepared were surfaces cadherins Dual-micropatterned desmosomal of immobilization Surface USA). CO, Aurora, functionalization LLC, surface Avidity for kit, enzyme (BirA500 BirA and using beads, agarose biotinylated Ni-NTA was over protein tag medium His conditioned and proteins from the and sequence purified cells, of were Tev HEK293T in tag, C-terminal expressed Avi were the constructs the Fusion where (pATH). to fused as 2009), was also al., domain was extracellular et design for (Zhang construct USA) previously Dsc2a CA, described and Carlsbad, Dsg2 Technologies, Monomeric labeled Life visualization. then (Invitrogen were dye Proteins Cy5.5 Sweden). with Uppsala, (GE Sciences, Sepharose A Life Protein Healthcare over of medium and conditioned region cells from HEK293T constant purified in the proteins the expressed were to of constructs Fusion fused described C-terminal (Fc). was IgG1 The was human domains 2005). extracellular proteins Dsc2a al., fusion or et Dsg2 Fc (Drees E-cadherinFc desmosomal for for previously design cloning The purification Protein METHODS AND MATERIALS ora fCl cec 21)17 3925 doi:10.1242/jcs.146316 2339–2350 127, (2014) Science Cell of Journal h oeeto igeMC el neach on cells MDCK single of movement The v /10 m t /).Tebxmrsternefrom range the marks box The m/h)]. ts (Dsg2Fc, -test v a omlzdt igecell single to normalized was ) x iie yperpendicular by divided ) n 5 3 Dsc2aFc, 13; n 2347 5 31;

Journal of Cell Science where izeeti rnltradwsmvda ifrn eoiis( velocities different at moved thermal was on the and mounted measure was translator with to cantilever piezoelectric the order measured rates, a In loading different 1993). were at Bechhoefer, forces cantilevers unbinding and (Hutter AFM method the fluctuation of constants H-CL-itn(0m nwtr aefehec ie,then in mg/ml time), (0.3 A each Protein fresh biotinylated then made PBS), in water; sulfo- mg/l link in (5 EZ neutravidin with mM incubated (50 first 2005). NHS-LC-LC-biotin were coverslips al., coated (200 Stamped et were IV coverslips. groves collagen (Drees striped Cy3.5-tagged previously with with described stamps were (PDMS) as or surfaces Polydimethylsiloxane Dsc2aFc Dsc2aFc Dsg2aFc, non-ECM–coated IgG1 plus human the 2003; the Dsg2aFc then with al., fusion and by et with 2002), (Cuvelier functionalized al., protocols et standard Nishizawa using printed micro-contact ARTICLE RESEARCH 2348 CaCl were mM NaCl, 2.5 mM AFM. either 100 experiments 5500 7.5, in Agilent pH KCl Tris-HCl mM an spectroscopy mM 10 using 10 in velocities performed force were pulling Experiments different dynamic at performed molecule experiments spectroscopy Single force dynamic molecule Single room at H 25% h a 1 with cleaned density. first for low were at coverslips (control) glass coverslips the protein between to added Fc were room times Cy5.5- cells or at with MDCK three incubated Dsc2aFc performed temperature. were were PBS Dsg2aFc, coverslips Finally, washes tagged dark. with and the in incubations washed temperature all for DMSO) were and in mM incubations Coverslips (15 protein, solution Dsg2aFc D-biotin min. or a 30 Dsc2aFc with with blocked incubation were Before h. coverslips 1 for each PBS), where 07 h equation: the 2007) ehd(ct,17) h itgaswr itdt Gaussian a to fitted ( were forces probable statistical histograms most a the The to obtain according to 1979). distribution calculated (Scott, width each for bin histogram method a with as plotted velocity were eliminating forces multiple retracting After the 2012). hidden events, al., multiple or et hidden bonds all (Fuhrmann previously chemical used described in protocol as heterogeneity bonds a the followed identifying we for events, unbinding single indistinguishable oe sn h olwn qain(mt ta. 1992): al., (FJC) chain et jointed (Smith freely equation the following with the fitted using and model selected were curves force ucinlzdwt E pcr PG50,Lya i,Aa,AL, Arab, Bio, then Laysan were 5000, amine-reactive coverslips an (PEG and containing spacers USA) cantilevers PEG v/v The with 2% acetone. functionalized with in functionalizing dissolved MO, Louis, by USA) St. (Sigma-Aldrich, reactive 3-aminopropyltriethoxysilane of amine and solution cantilevers made AFM cleaned were The coverslips acetone. hydroxide and potassium water M 1 deionized water, solution, deionized with washed subsequently and force odn ae( rate loading h F i n oesi ufcs olwn s2 rDsg2a or Dsc2a min. 30 Following 10 streptavidin. surfaces. for on in surface sites incubated coverslip biotin-binding streptavidin were free on non-specific and surfaces streptavidin mg/ml the minimize to tip 0.1 immobilization, bound to were AFM with monomers h, the Dsg2a 12 or then Dsc2a for PEG Biotinylated the and BSA of single-molecule incubated 7% interactions, measure mg/ml were and 0.1 to surfaces 5% biotinylated order with monomers, The in respectively. at mutant low biotin, biotin and presented contained kept wild-type PEG was of for and fraction events; known end A other surface. the the to bind to end v 1 F o igemlcl oc pcrsoy(MS,AMcnieesand cantilevers AFM (SMFS), spectroscopy force single-molecule For nodrt lmnt idnmlil nidn vnsfo visibly from events unbinding multiple hidden eliminate to order In ~ F k k lF 1 c , ðÞ c v L z stesrn osato h cantilever. the of constant spring the is c stecnorlnt fPGand PEG of length contour the is , steedt-n itneo h E ehrsbetdt a to subjected tether PEG the of distance end-to-end the is 1 v v lF dl F dF a siae sn Mrhl ta. 06 a tal., et Ray 2006; al., et (Marshall using estimated was ) ðÞ ~ lF ðÞ k 1 c v ~ "# L 1 c z coth k k c B L T c 2 a N k r2m GA igeunbinding Single EGTA. mM 2 or Fa ! m B hdoyucnmd se tone at ester -hydroxysuccinimide /l n rne nosilanized onto printed and g/ml) T k Fa B { T a k Fa steKh egh Spring length. Kuhn the is B 2 2 T { F O mp 2 csch , 7%H :75% o ahretracting each for ) m itnt block to biotin M 2 k Fa 2 B SO T 4 solution v , ;the ); ð ð 1 2 Þ Þ e,splmne ih2 MHpsa 37 at Phenol Hepes without DMEM mM in 25 Technology, imaged with 2005; were (Andor al., supplemented cells et system Live Red, (Spiliotis 2005). laser previously al., described et FRAP as Yamada USA), MicroPoint CT, a Widsor, South and Cy5, and an drive USA), interline harmonic AZ, HQ with Coolsnap Tuscon, a (Photometrics, light changer, Xenon camera filter Watt CCD galvanometric 175 dual a a including with with source Innovations equipped inverted USA] TIRF 200M Imaging CO, Axiovert Denver, [Intelligent and (3i), Zeiss microscope custom-built (FRAP) epifluorescence a Microscopy imaging widefield on AxioVision acquired live-cell were with Fluorescence images acquired (Zeiss). and Software Japan) Tokyo, (Olympus, ifrne ewe oee aaeeswr acltduiga using calculated were sum-of-squares exponential parameters extra single fits, of modeled a Significant comparison 2010). with and al., between et fitted (Borghi J and differences previously described Image normalized as two-tailed model was with Mann–Whitney, recovery data a performed FRAP using was performed test. was analysis outlines analysis contour cell Image cell statistical whereas monitor and to migration, fluctuations. used recruitment cell were scales of and protein time measurement long assess for at substrates to images scales DIC micro-patterned time used long was and at The dye) position (GFP) software. Cy5.5 cells (3i) or Slidebook (Cy3.5 from using signal acquired fluorescence were images movement; ai–ab aiekde MC)tp IGclswr rw in grown were cells G 200 II and glucose type low with (MDCK) DMEM kidney canine Madin–Darby constructs and culture Cell muoloecnewspromdo DKclsfxdwt %PFA 25 4% at with min fixed 20 cells for MDCK on performed was Immunofluorescence microscopy Fluorescence ih01 rtnX10fr5mnbfr lcigi B containing PBS in blocking before NH min mM 5 50 for BSA, X-100 0.2% Triton 0.1% with irsoeeupe ihaMruylm n 100 a and lamp Mercury a inverted 200 with Axiovert Germany) equipped (Jena, Labs, Zeiss a microscope (Vector on Immunofluorescence Vectashield acquired imaging). TIRF were using for images PBS mounted (or USA) and CA, Burlingame, USA) Laboratories, PA, ImmunoResearch Westgrove, Jackson (1:200; with Red-X/Cy5 incubated Rhodamine were Cells anti- anti- ab17151]. (Fab England), – USA), secondary Cambridge, (Cy90) MA, [Abcam Abcam, anti-plakoglobin 8/18 Waltham, – cytokeratin 1988a), anti- Scientific, (ab72792 Fisher interest: Nelson, (Dsc2a) Thermo of desmocollin2 and – proteins (Pasdar 15F11 the (clone I/II) against (DP desmoplakin with incubated were hnfte Bl,17;EasadRthe 97 nodrt esr the measure to order in 1997) ( Ritchie, off-rate and force-independent Evans 1978; (Bell, fitted then eoiy otpoal oc ( force probable Most velocity. etduigaStudent’s a using tested betv FA n IF n 40 a and TIRF) and (FRAP objective fterato oeta otepa fteptnilbriruigthe using potential-barrier the of peak the to potential equation: reaction the of otiigtepitmtto o ooei uinproteins fusion monomeric for GFP-tagged Dsc2aW2A–GFP). full-length and mutation (Dsg2aW2A–GFP and versions point fragments Dsc2aW2A_pATH) PCR the (Dsg2aW2A_pATH, with the USA) from (Aligent Medicine, CA, mutagenesis containing Clara, (gift of Santa site-directed School Stratagene, construct using Feinberg Technologies, IL) Troyanovsky, Dsc2a–GFP University, M. 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Journal of Cell Science ev,E,Tce,D .adKwlzk .P. A. Kowalczyk, and K. D. Tucker, E., Delva, skn,L,Ta,Q,Shit . rne .W,Wnofr .adLeube, and R. Windoffer, W., W. Franke, A., Schmidt, Q., Tian, J. L., K. Eshkind, Green, and M. L. Godsel, L., R. Dusek, J. W. Nelson, and A. Reilein, F., Drees, ndsgigteeprmnsadwiigtemanuscript. the writing involved and were W.J.N. experiments and the S.S. was K.J.G., designing experiments; N.B. in of SMSF proteins; design the fusion initial Dsc2-Fc performed the in and S.S. involved Dsg-Fc and the O.S. provided S.R., R.M.H. the manuscript; experiments; assembled the experiments, wrote the and interpreted figures, and performed designed, M.L. contributions Author interests. competing no declare authors The interests Competing ARTICLE RESEARCH ueir . ose,O,Bseeu .adNso,P. Nassoy, and P. Bassereau, O., Rossier, M. D., Cuvelier, S. Troyanovsky, and A. N. C. Chitaev, Dawson, and M. Chidgey, O’Brien, D., Whitaker-Menezes, W., Y. Choi, S., Joubeh, Y., Hu, D., Brennan, G. M. Mahoney, and D. Brennan, Dı A., Al-Amoudi, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.146316/-/DC1 online available material Supplementary material months. Supplementary 12 after release predoctoral for grant Association PMC training Heart in T32 American Deposited NIH an fellowship. a and by GM08061]; Mason supported number and was [grant Grant R.M.H. Training Fellowship. Biology Graduate Cancer Case by Fellowship, supported Human Graduate was and M.L. Genentech GM35527] RGP0040/2012]. a number number [grant [grant Program W.J.N. NIH Science the the Frontier Health in by of Work supported Institutes AR41836]. was National R01 laboratory the R37AR043380, from numbers grants [grant the by American (NIH) in supported the Work from was 12SDG9320022). grant laboratory a Grant K.J.G. by Development supported (Scientist partially Association was Heart laboratory S.S. the in Work Funding onlee,E . ocrn .M,Sapnek .S n re,K J. K. Green, and S. T. Stappenbeck, M., C. Corcoran, A., E. Bornslaeger, Castan A., Al-Amoudi, ogi . one,M,Mrtauh,V,Gre,M .adNlo,W J. W. Nelson, and L. M. Gardel, V., Maruthamuthu, M., Lowndes, M. N., B. Borghi, Gumbiner, E., Wong, S., Chappuis-Flament, J., Murray, M., J., P. T. Boggon, Lombardi, J., Smagt, der van H., D. J. Jongbloed, A., Z. Bhuiyan, I. G. Bell, R. M. J. Stanley, Chidgey, and M. and Amagai, M. Overduin, H., Bikker, C., Al-Jassar, pigHr.Prpc.Biol. Perspect. Harb. Spring 342-354. .E. 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