Journal of Cell Science odnE1 1,UK 71B, EC1A London UK 2AT, E1 London Street, Newark 4 o:10.1242/jcs.084152 doi: 2853–2861 125, Science Cell of Journal 2012 February 23 Accepted iaM Cabral M. keratinocytes Rita desmosome- HaCaT for in crucial adhesion is mediated variant splice DSPII The Article Research deinjntossc scmlxsahrn ucin in junctions adherens the complexus IFs. other highlighting as for in such components literature attachment desmosomal junctions other published providing adhesion and DSP protein is of importance plakin 2009; there linker Jonkman, Additionally key and a 2012). (Bolling al., et both Brooke 2011; instances al., et some Chalabreysse in-vitro-engineered heart in or and skin compromise natural of and of which genes, by importance flexibility desmosomal highlighted in and The mutations strength is 2004). the tissues Green, of these number and maintenance a for (Yin and desmosomes proteins (DSP) other desmoplakin of plakoglobin (PKPs), through the network of plakophilins IF cadherins halves the two (PG), to desmosomal the linked couple are to and transmembrane space desmosome extracellular The the in family plakins. interact armadillo the the gene cadherins, and three resilient desmosomal of products mechanically and the the and by superfamilies: formed large epithelial is desmosome a The (IFs) generating network. of filaments cells, intermediate adjacent junctions connect of that intercellular tissues cardiovascular are Desmosomes Introduction is dosage DSPII key levels that and expression the function the in being on words: redundant DSPII skin. Key completely DSPII than the not influence are in with greater variants adhesion splice a stress DSP desmosomal having major DSPI cellular for reduced two with expression important the upon DSC3 DSP found that particularly and in suggest adhesion we 2 arrhythmogenic results reduction siRNA, (DSC) These addition, keratinocyte severe DSC3. desmocollin In DSPII-specific with of and 1, to adhesion. a plakophilin desmosome-mediated hair associated respect additionally of and woolly amount stability is and with the PPK, (IF) mutations and filament isoforms epidermolytic two intermediate haploinsufficiency recessive DSP in these component autosomal DSP mimic between with DSPIa to total isoform differences associated treatments minor in the striking is siRNA and results and DSPI Using of DSPII, cardiomyopathy. mutations absence complete dilated of the to levels leads of other varying normal The be the One (PPK). but to keratoderma remain by mechanisms. palmoplantar mechanisms striate evidenced these molecular dominant of as autosomal majority their mechanisms the but compared molecular in protein, disease-causing mutations and same two the different studied of we have domains Here, different Defective mutations determined. affecting muscle. ( Different mutations heart desmoplakin from and both. gene resulting epidermis desmosomal instances phenotypes the constitutive some in the prominent in in mutations are and inherited that to adhesion due strong function for desmosomal specialised junctions intercellular are Desmosomes Summary work this to equally ` contributed authors *These 4 3 2 1 oii .Abrams J. Dominic uhrfrcrepnec ( correspondence for Author iiino acrRsac,Uiest fDne,NnwlsHsia n eia col udeD19Y UK 9SY, DD1 Dundee School, Medical and Hospital Ninewells Dundee, Smithfield, UK West of 2ES, London, University E1 of Research, University London Mary Cancer Street, Queen of Hospital, Newark Bartholomew’s Division London, 80 St of Trust, Trust, NHS University NHS London Mary London The Queen and The Dentistry, Barts and and Department, Research Medicine Barts Cardiology of Pathology, School Cellular London of The Department and Barts Institute, Blizard The Research, Cutaneous for Centre 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. S stems bnatcmoeto h emsm and desmosome the of component abundant most the is DSP dein empai,Dsooe eaioye pieisoform Splice Keratinocyte, Desmosome, Desmoplakin, Adhesion, 1, ,Dne Tattersall Daniel *, [email protected] 3 nrwP South P. Andrew , ) 1, ,Vsa Patel Vishal *, DSP 4 n ai .Kelsell P. David and htla odfeetdsgso h w ao S pievrat,DP n DSPII, and DSPI variants, splice DSP major two the of dosages different to lead that 1 rhmD McPhail D. Graham , ale eosrto fteiprac fDPi h er by 2001). heart al., the et in (Gallicano DSP experiments of rescue the embryonic importance confirming partial the 2006) of severe to al., demonstration due et earlier onwards E11 (Garcia-Gras from abnormalities the lethality in cardiac in water DSP results of mice extensive ablation of Conditional and heart to 2001). al., blistering leads et epidermis skin (Vasioukhin the loss through to (Gallicano targeting (E6.5) lethality of conditional 6.5 germline postnatal and day the 1998), embryonic in al., at DSP et lethality Jonkman, of in Ablation and results 2012). (Bolling mice al., early loss cardiomyopathy et water Brooke to from subsequent 2009; death and through blistering sudden skin or (SPPK) severe to skin keratoderma due non-syndromic lethality palmoplantar from range cardio-cutaneous striate as phenotypes designated resulting These phenotypes date, to clinical syndromes. identified of been range mutations have a human DSP in Numerous encoding gene 2004). the al., in et (Zhou tissue vascular n SI r xrse tnal qiaetlvl nstratified have in levels we equivalent nearly DSPI which at 2010). expressed al., (DSPIa), are et DSPII Ia (Cabral and characterised desmoplakin and identified and isoform, recently (DSPII), II minor desmoplakin and a (DSPI) I desmoplakin variants, Alternative 1, ` DSP 2 lzbt Hatzimasoura Elizabeth , RAtasrpsecd w ao splice major two encode transcripts mRNA DSP asssi rhatdisorders heart or skin causes ) 1 , 2853 Journal of Cell Science Results the into insight keratinocyte-associated on further dosages desmosomes. DSPII gain We and phenotype. DSPI to clinical of Heterozygous influence mutations no 2006). show these mutation al., investigated DSPI and this et DSPII) of (Uzumcu carriers early affecting and cardiomyopathy hair (without woolly PPK, DSPI- lethal isoform epidermolytic mild the DSPI recessive 1999; a alongside causes the complete al., modelled in of results were absence which et causing p.R1267X, 1999), mutation DSPII nonsense (Armstrong al., specific and et SPPK DSPI Whittock both non-syndromic of dominant haploinsufficiency to lead eitdi i.1 n hw S-elto pattern ( blots mutations. western DSP-depletion the in specific of shown a quantification is is Densitometry these result 1D. shows 1987) Fig. the of confirming al., and analysis modelling Immunofluorescence et 1C DSPII effective Fig. (Parrish and indicating (11-5F) in DSPI antibody of depicted a with DSP downregulation cells siI/II partial C-terminal in mutation) and haploinsufficiency the cells (mimicking of downregulation siI showing homozygous in negative the blot a mimics western as (which A used DSPI was cells). siRNAs (NT the (NT) A control non-targeting with cells. siI four transfected as of designated Cells were pool (siI) 1A,B. siRNAs Fig. DSPI-specific of as in pool the well shown as are si4) of and p.R1267X) the si3 position (si2, target siRNAs three the specifically these to of sites designed targeting was siRNAs DSPI three nonsense of p.R1267X pool The this cells. only with siI/II targets transfected mutation as Cells designated all keratinocytes. were downregulate HaCaT siRNA in transiently Cabral 50% to 2007; about al., 2010) et (Wan al., previously and et described was p.Q331X that as siRNA a such mutations c.939+1G haploinsufficiency DSP ablation The DSPI-specific and haploinsufficiency effectively DSP keratinocytes models HaCaT in knockdown siRNA c.939+1G and p.Q331X biology (PTC)- as keratinocyte codon such in mutations termination generating premature variants Heterozygous splice disease. the DSPII and for role(s) and functional the DSPI into major insight further gain of elucidated. to mechanism and molecular action be their investigate can to human to of keratinocytes gene remains classes HaCaT different same variants functional two model the the splice we Here, Additionally, in DSP phenotypes. mutations of different different significance and such why understood to fully clear and/or lead not not skin are is to DSP it lead with associated that disease mechanisms 2006). heart al., molecular predominant et the the Uzumcu mutations, 1990; is al., DSPI et (Angst but heart epidermis, the in the isoform as such epithelia 2854 mut fDP n SI ay ntesIcls SIis DSPI 45% (by cells, downregulated siI/II siI partially respectively). the both 81%, the in are and and DSPII In relative and unchanged, vary. DSPI DSPII the cells with this DSPII but 90% and knockdowns, by using downregulated both DSPI in that of DSP 40–50% of amounts amounts by demonstrates total levels reduced The DSP and mutations. are the specific replicate these effectively 1E,F by to Fig. produced able are in we methodology shown is lhuhteeaei ioado nvtodt nsome on data vitro in and/or vivo in are there Although seii uloie c36 oc55) h eune and sequences The c.5659). to (c.3861 nucleotides -specific . ora fCl cec 2 (12) 125 Science Cell of Journal ,wihcuesraePK eemdle yusing by modelled were PPK, striate cause which A, DSPI DSPI RA.T oe hsmtto,a mutation, this model To mRNAs. nnes uain(c.4079C mutation -nonsense DSPI nnes mutation) -nonsense DSP DSP uain in mutations sfrsto isoforms . ,which A, n DSP . 5 T; 6) e seik hwsaitclsgiiac fdfeigDP eesbetween levels DSPI differing of * cells, significance knockdowns. NT statistical the show with compared asterisks asterisks reduction red protein Black of both respectively). significance are 81%, statistical DSPII and and show DSPI 45% cells (by siI/II the downregulated in partially and unchanged, DSPII with 90% by r eue y4–0 nbt ncdws()btteaayi fDP and DSPI of analysis the but ( (E) knockdowns levels both DSPII DSP in of 40–50% amounts by total reduced the are reveal 10 (mean+s.e.m.) blots bar: western Scale of both siI/II. quantification of with downregulation knockdown partial after show analysis isoforms (bottom) siI; antibody with anti-DSPI/II knockdown the upon with downregulation complete near reveals mutants. siI/II, specific with these ( knockdown of upon modelling partial DSPII effective and and demonstrating siI DSPI with both knockdown of upon downregulation unchanged levels DSPII with sqec hw nA agt xn1 hc scmo oalDPsplice DSP all to common is which 11 exon ( targets variants. A) in shown (sequence nlsso ain kn orsl nls fcl–elcontacts, cell–cell of loss in result to skin, patient DSP of causing ultrastructural analysis by mutations shown, previously site been have splice haploinsufficiency and nonsense Heterozygous DSC3 in decreases DSP-isoform specific reveal the mutation and nonsense haploinsufficiency DSPI-specific DSP modelling cells HaCaT the shows also (B) variants splice DSPII (c.4079C and p.R1267X DSPI of the diagram the of schematic of position The 24 siI. as to designated 22 pool target exons a specifically as used to were designed siRNAs si4) three and si3 (si2, siRNAs keratinocytes. HaCaT siRNA in ( transient isoforms by DSP mutation of nonsense haploinsufficiency downregulation DSPI-specific DSP homozygous the the and of mutation modelling Effective 1. Fig. D A muoloecneaayi ihteat-SIseii nioy(top) antibody anti-DSPI-specific the with analysis Immunofluorescence ) , B uloiesqecs()adtreigsts(,rdsas fthree of stars) red (B, sites targeting and (a) sequences Nucleotide ) C etr ltsoigna opeedwrglto fDSPI of downregulation complete near showing blot Western ) n P 5 # )()rva ntesItetdcls SIi downregulated is DSPI cells, siI-treated the in reveal (F) 6) .5 ** 0.05, P # .1 *** 0.01, . )nnes uain h i/IsiRNA siI/II The mutation. nonsense T) P # 0.001. m .(,)Densitometry (E,F) m. DSPI RA.The mRNAs. Journal of Cell Science he rtishv o enivsiae ntesi rmthe from skin these the but DSC3, in 2004), investigated al., and been et not DSC1 (Wan have layer PKP1, proteins spinous three of the in expression particularly mutations decreased these harbouring immunohistochemical patients showed addition, from biopsies In Whittock skin 2004). 1999; of al., staining et IF al., Wan et keratin 1999; (Armstrong al., the et nucleus of the towards retraction network and desmosomes under-developed infcnl ifrn ewe i n i/Icls ncontrast, than In ( cells cells siI/II cells. siI in not higher in siI/II are significantly siI/II levels are and in levels DSC2 expression siI 4:1 and DSC3 to PKP1 between way, cells different similar siI a significantly in In 1:8 DSPI:DSPII 2A). ( from (Fig. the cells cells average, significant, on statistically siI control changed, not between ratios was levels NT DSP cells total siI/II to in difference and the compared in expression Although 40% 1E). to DSP when and Fig. cells Total siI cells in cells. 50% to siI/II average, control on siI/II reduced, NT and were siI levels with both in the compared DSC3 in and cells DSC2 reductions PKP1, significant of as levels and well expression consistent as confirmed 2A), (Fig. analysis DSG3 desmosomal the and of PG DSG2, levels PKP3, expression shows PKP2, the in 2A components protein cells Fig. control NT experiment. assess and each protein. to four analysed in each out for to protein blots carried western two each representative were and for blots siRNA levels other conducted independent western and were Two replicate partners blot. experiments DSP-binding western knockdown of by mutations levels proteins these nonsense junctional expression of DSPI-specific influence the the the of on investigated first effects we the mutation, with and keratinocytes them in compare mutations haploinsufficiency DSP of effects mutation. nonsense DSPI-specific the for homozygous patient odtcal ifrne eeosre ewe i rsiI/II or siI between observed were differences detectable No downstream these reproduce could we whether investigate To b ctnnadkrtn1 dt o hw) Densitometry shown). not (data 14 keratin and -catenin P # .5.Teerslssgetta K1adDSC2 and PKP1 that suggest results These 0.05). P , 0.001, ofre ylwpwreeto irsoy(M analysis (EM) microscopy junctions was electron cell–cell space at low-power intercellular DSP in by increase of confirmed [This expression arrowheads). regions intercellular no 3C, in (Fig. of pronounced was 3A–C). more widening there (Fig. were where cells, which which periphery observed, cell IFs, siI/II were the keratin spaces some to of among nucleus network However, the normal from a extended exhibited cells time. DSP siI/II of periods different monolayers for to cell stress HaCaT mechanical oscillating subjected leading mimic to we To and pressure soles. and intense mechanical palms mutations to such as such exposed pressure, sites mechanical (PTC) in constant prominent is codon haploinsufficiency premature heterozygous harbouring termination patients of keratoderma The adhesion stress an mechanical and upon stability defect IF in deficiencies haploinsufficiency demonstrate DSP mimicking cells HaCaT oepoieti h eiula eino i/Icls(i.3F, (Fig. cells even siI/II of were region IFs the perinuclear thicker the around These in particularly arrows). prominent more IFs, open 3D,E, through keratin (Fig. compact other nucleus more each and thicker to connected attachment, appeared IF of junctions. still intercellular loosening cells and spaces where these Despite intercellular regions area). boxed a wider in 3C, (Fig. being the stronger slightly higher were appeared it appeared levels expression attachment IFs of DSP IF Keratin possibility and contraction.] the thicker IF eliminating of below), consequence 5, Fig. (see h SIioomi oe,sgetn htDP a greater a has DSPI DSC3. that on suggesting DSPII lower, than is influence of expression isoform when lower DSPI is ratios expression the the ratios, Its by isoforms. DSPI:DSPII influenced DSP these is the of expression by DSC3 contrast, influenced in not whereas, are levels expression eoeaysrthwsapid( or) Tcnrl i and siI control, NT hours), (0 applied was stretch any Before olwn 0mnt tec,N n i el a slightly had cells siI and NT stretch, 30-minute a Following SI n emsmlahso 2855 adhesion desmosomal and DSPII eesbtenkokon.N,ntsaitclysignificant; statistically not * DSPI NS, differing knockdowns. of between significance levels cells; statistical NT show the asterisks with red compared reduction statistical protein show of asterisks significance Black DSC3. on influence yteDPrto,bigsgiiatyhge nsII el than cells ( siI/II cells in siI higher significantly in influenced being are ratios, DSP levels the expression by DSC3 and contrast, siI In between cells. different siI/II significantly not are ( (mean+s.e.m.) cells. NT with compared cells siI/II S3 ossetdcessi rti eeswr observed were ( levels PKP1 and protein for PG in PKP3, decreases PKP2, Consistent components DSG3. expression desmosomal the the in of observed levels were differences detectable No DSPI. of levels with ( linked closely more desmosome being other not but DSC3 components. and DSC2 levels PKP1, protein of in decreases demonstrate mutation DSPI-specific nonsense homozygous DSP the mimicking and cells haploinsufficiency HaCaT siRNA-treated 2. Fig. A P ersnaiewsenboso ahpoeninvestigated. protein each of blots western Representative ) # .5 ** 0.05, n P 5 P # h eraei S3i S-sfr specific, DSP-isoform is DSC3 in decrease The ) S2( DSC2 7), # .5 ugsigta SIhsagreater a has DSPI that suggesting 0.05) .1 *** 0.01, n P 5 # )adDC ( DSC3 and 5) 0.001. B K1adDC levels DSC2 and PKP1 ) n 5 )i i and siI in 6) Journal of Cell Science n elahso eet ntesII el eemr eeeand severe more were instability cells IF siI/II the that the and revealed in cells, amplitude hours) defects 4 NT adhesion Hz, to the cell up in and 5 of observed duration to thickening and IF 18% up the to 0 frequency to (a up range stress mechanical severe expressed that DSP. deficiency. cells of siI/II adhesion levels in lower an prominent were and suggesting with observations defect, These spaces stability bundles exhibited IF intercellular cells IF an wider suggesting siI/II keratin bundles, stretch, filaments of mechanical thinner to thickening exposure showed cells, increased which some arrowheads). cells, 3L, among (Fig. stretch observed of hours were were 2 star). after they gaps 3L, particularly cases (Fig. cells nucleus intercellular some the IFs in towards siI Large had retracted and cells and 3I,L) for siI/II deformed although (Fig. most even contrast, IFs, observed thinner thick in appeared showed were but, that still arrows) cells results closed siI/II and some Similar open 3H,K, 2004). (Russell (Fig. elsewhere published al., as around arrow) denser open et were 3G, which cells (Fig. IFs, NT nuclei hours), keratin the 2 the a and of After (1 thickening arrowhead). stress showed 3F, mechanical of (Fig. period absent time was longer wider edges DSPI cells, where particularly free siI cells, regions and these in along NT among observed with particularly were contrast spaces In intercellular thinner, arrow). siI/II double were of 3F, membrane (Fig. IFs plasma the keratin to cells close However, arrows). open 2856 usqeteprmnso h i/IadN el sn more using cells NT and siI/II the on experiments Subsequent siI and NT with contrast in that, demonstrate results These ora fCl cec 2 (12) 125 Science Cell of Journal alisfiinymtnswr nbet epn to respond the to mimicking bundles. keratin cells of unable thickening the by observed were that as stress siI/II mechanical result in mutants observed not the was haploinsufficiency increase confirming bundle this filament but cells, keratin cells in siI increase in similar mechanical ( bundle thickness a pixels of filament suggested 2.9 data hours to keratin The pixels 2 average 2.6 upon from the stress, increased cells significantly control confirmed thickness NT This in acquired CA). using Devices, that images performed was (Molecular in view software of cells Metamorph fields high-power of random thickness ten from bundle filament of keratin amount relative the on dependent stretch. were cell differences these so Gsann ttepam ebaewsol rsn tteless the at present and only contacts, was membrane cell–cell plasma of the number siI/II at the stretch, staining in PG in Following decrease some mostly a expression. showed showed membrane, DSP cells cells plasma reduced the siI/II with at and regions staining siI PG both discontinuous contrast, showed plasma In cells the NT around membrane. applied, staining was immunofluorescent stretch in PG non- Before characteristic localisation some types. cellular and cell their three desmosomal all investigate of and to panel proteins stretched a desmosomal of using staining cells unstretched immunofluorescence PG performed of re-localisation We most partial of but localisation proteins normal junctional to leads stress Mechanical ovrf hs bevtos uoai esrmn of measurement automatic observations, these verify To xrsinlvli ihr cl a:10 DSP bar: the Scale where higher. region is a level in expression C IFs to added thicker been the has showing panel adhesion magnification an high suggesting A stretch, defect. of periods particularly longer cells, after siI/II in (arrowheads) predominantly gaps observed intercellar were Large to (star). retracted nuclei and the deformed IFs and cell-free arrow), the (double along edges cells particularly siI/II IFs but thinner more arrows) demonstrate and (open thicker IFs displayed keratin cells compact siI and NT stretch, eso ytmfr0hus(ntece)( (unstretched) ( hours minutes 0 FX-4000 for Flexcell System Hz the Tension 2 using frequency 11–14% of amplitude stress and mechanical cyclic were to haploinsufficiency subject DSP a modelling and cells mutation siI/II nonsense DSPI-specific the mimicking cells. siI not HaCaT but siI/II cells in instability cell–cell IF reduced increased and causes adhesion stress Mechanical 3. Fig. D – F ,1hu ( hour 1 ), TcnrlHCTcls i cells siI cells, HaCaT control NT P G # – .5 dt o shown). not (data 0.05) I n or ( hours 2 and ) m m. A J – – L C .After ). ,30 ), Journal of Cell Science nldn S12 S2 S3 K2 K3 -ahrnand E-cadherin PKP3, PKP2, b DSC3, DSC2, DSG1/2, in including increase mechanical S1). cells following slight Fig. siI/II predominantly material some A (supplementary cells, in stretch NT observed adhesion. with also was compared cell–cell staining PG of intracellular sites frequent h medn rcs,peual eas fteadhesion the DSPII. of of depletion because the by presumably caused during defect process, detached becoming cells embedding the consistently defect). to the were adhesion owing stretched imaged an be been of to had unable indicative that cells, cells siI/II siII Additionally, and intercellular siII in in increase spaces the (e.g. (5000 studies stressed EM immunofluorescence mechanically power the in were Low hours. that desmosomes 4 cells of for and analysis cells unstretched ultrastructural both performed have We siII cells in siI/II defects and adhesion. reveals cell analysis in ultrastructural greater DSPI Desmosome a of is that there with that IF compared suggest the DSPII and in 4B) for changes (Fig. similar role as cells well siII as in 3A–L), compared structure (Fig. cells cells in siI/II siI observed sheet the the are defects with in results monolayer the assay These of stretch-immunofluorescent the severity shown). the increased not as the (data with sheet defect consistent distinct adhesion a cells as siI/II cell–cell monolayer, remained and siII cell a the cell contrast, control In siI showed NT clump. The small the sheet. a into cell to condensing the identically by of acted dish upon condensation culture monolayer siI/II elastic tissue visualised of the the degree from empirically monolayer the in cell be the observed of could detachment those ( adhesions than intercellular cells statistically greater larger were haploinsufficiency-mimicking much defects a ( adhesion showed The of intercellular which in number decrease cells, the the significant to siI/II in contrast and in increase agitation) siII small upon a produced fragments by monolayer cell– DSPI- in (indicated the decrease adhesion small DSP simulating adhesion a cell cells only of dispase showed The mutation 4C). a effects nonsense (Fig. specific adhesion, the performed cell was quantify assay on and downregulation confirm isoform independently To HaCaT for important adhesion are cellular DSPII of levels nuclei observed The the also were to gaps retracted 4B, intracellular arrowhead). (Fig. and large 4B, IFs (Fig. and deformed thinner star), IFs including 4B, (Fig. and cells, siI/II arrow) in defect double of complete adhesion observed similar analysis a that near was Immunofluorescence to there remained showed that 3). showed isoform filaments lane cells DSPII-specific DSPI keratin 4A, larger siII (Fig. the a performed. whereas unaffected was of DSPII, DSPI, strategy of analysis depletion intercellular site of blot in trans-splice that DSPII Western a for using with role knockdown significant compared more suggest a cells adhesion knockdown is siI/II there and that siI disease-modelling DSPII- the a As with further knockdown investigated siRNA was specific either DSPII of cells role siI/II The or siI stretch. and after NT or before between localization cellular ctnn(aantson hwdn ao ifrne in differences major no showed shown) not (data -catenin te emsmladahrn ucincomponents, junction adherens and desmosomal Other 6 ofrstefnig from findings the confirms ) P # .1.Additionally, 0.01). P # .1 i.4C). Fig. 0.01, eeti h iIcl ooae sgetrta htosre nsiI/II in observed that than greater is monolayer ( cell monolayer siII the in defect eosrtn eraei neclua dein ** adhesion. intercellular which in fragments monolayers, decrease of cell number a siI/II significant) demonstrating and (statistically larger siII much the a to into disintegrated contrast number decrease in the small adhesion) in a cell–cell (suggesting increase in agitation small upon a produced only fragments monolayer showed integrity of cells monolayer siI cell 10 of stress. bar: degree mechanical the Scale upon by cells. adhesion siI/II intercellular assess in to adhesion observed assay Large an that (star). suggesting to nuclei (arrowheads), similar the observed defect to also retracted were and gaps deformed intracellular IFs IFs thinner and displayed arrow) morphology (double filament ( keratin cells. for siII immunofluorescence in DSPII of knockdown i.4 pcfckokono SI ofrsakyrl o hsisoform this for role key a adhesion. confirms DSPII desmosomal of in knockdown Specific 4. 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C Dispase ) Journal of Cell Science n fteDPsRAtetdcls ncnrs otepositive the to contrast in cells, siRNA-treated in DSP with compared the differences cells of NT No any the in S2). between differences observed Fig. were for proliferation material responsible being (supplementary eliminate number adhesion to cell an performed in 2007), was differences in al., assay reported et proliferation been cell (Wan has MTT-based haploinsufficiency proliferation DSP cell mimicking in cells increase observed an phenotypes that siRNA Given not the are of cytotoxicity cause or the proliferation cell of rates 5H, in Changes (Fig. cells siI and area control objectively unit NT statistically per P with desmosomes independently a compared of have cytoplasm number of was cells the in siI/II to decrease result and significant available siII when latter Unstretched cells quantified. stretched This in particularly structures analyse. desmosome cells, distinctive these of number in total the in decrease 2858 # .5and 0.05 ora fCl cec 2 (12) 125 Science Cell of Journal P # .1 respectively). 0.01, iseacietr n omlfnto,b tdigterl of the role into the studying insight by gain function, to isoform-specific contribute two normal to isoforms and DSPII was architecture and tissue DSPI investigation the which this by mechanism of focus The Discussion cell in decrease no as observed. observed, the was phenotypes are number siRNA cytotoxicity the in also of differences data cause This that 2010). possibility a al., the et display eliminates (Bishop to rate known proliferation are in which decrease cells, siRNA-treated Cbx7 control eei eet edn oteedsae r nw,the known, not part, are most diseases the encoding the for these genes Although are, to mechanisms in discovered. pathogenic leading mutations been molecular defects by have genetic caused proteins are desmosomal that diseases disease. cardio-cutaneous or disease skin vrteps 3yas nicesn ubro monogenic of number increasing an years, 13 past the Over ( ttsial infcn eraei ohsI ( siII both a in revealed decrease structures significant desmosome statistically distinctive of number the ucinta a enple pr.Saebr:lwpwr mm; 5 power, ( low nm. bars: 200 Scale power: apart. pulled high failed been a has suggesting that a half-desmosomes junction indicates to a appears arrow of IDP The indicative the rudimentary. structure and and observed electron-dense not siI/II less is stretched be ODP in defined prominent a particularly where is cells defined This less-well ODPs. including and which deficiencies IDPs desmosomes structural of and have number siII the to both in appear increase contrast, an In exhibit desmosomes cells. cells of unstretched siI/II ultrastructure or the stretched change in to either appear not does cells defect. (120,000 adhesion EM the the power during of High detached because becoming presumably to cells process, unable the embedding consistently to were owing stretched cell–cell imaged been increased be had of that way cells by the cells siII confirms siI/II gaps. and and siII staining in citrate defect lead adhesion (5000 and EM acetate power uranyl Low stressed equal bottom). mechanically (II, were hours that four cells for and top) both (I, in cells cells (siI/II) unstretched and mutation respectively), haploinsufficiency siII, the DSPI- and mimicking (NT), (siI cells cells control DSPII-depleted siRNA or non-targetting in performed was eet nsI n i/Icells. desmosomal siI/II reveals and EM siII in by defects analysis Ultrastructural 5. Fig. P # .1 el ncmaio ihteN oto cells. control NT the with comparison in cells 0.01) DSP uain hc as non-syndromic cause which mutations H needn betv uniiainof quantification objective Independent ) 6 hw htdpeino SIi siI in DSPI of depletion that shows ) ( A – G Maayi fdesmosomes of analysis EM ) P # .5 n siI/II and 0.05) 6 demonstrates ) Journal of Cell Science ofrigadetnigterslspbihdeswee(Wan elsewhere cells, siI/II published and results siI the both extending in and knockdown confirming DSPI upon observed was HaCaT on effects similar cells. cause would mutation nonsense specific ta. 04.Peiul ulse tde aesonta under that shown have Ca studies low published Previously 2004). al., et yeo uain eody hr salc fcreainbetween correlation the of lack on a depending is manifestations there Secondly, skin, heart mutation. severity of and of of type combination skin degrees a in varying in observed Additionally, are result disease. some heart and and others hair heart hair, and/or the skin only to affect Some restricted clear. phenotypes not cause is mutations patients in presentations in clinical different mutations Why understood. completely a ta. 07.Hr,w se hte oneuainof downregulation whether asked we Here, 2007). 2004; DSPI al., al., et et normal (Wan skin Wan SPPK in from and influence derived knockdown keratinocytes DSPI/DSPII primary upon cells might HaCaT in components ratios that and DSPI:DSPII properties function. desmosomal signalling in might and/or variants human changes splice adhesion This DSP disease. different different heart and have that and abnormalities suggests hair hair data skin, skin and mutation of cause skin combination can of a in protein combination also the mutations a of only, whereas N-terminus disease the cardiomyopathies, for C-terminal to coding and regions N- regions lead the the in as can well and mutations as domains domain instance, DSP rod central For the of encoding outcome. domain clinical functional resulting mutation-harbouring the tesb sesn Fmrhlg.I epnet cyclic to response In morphology. 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DSC2 but knockdown of partially, lack DSP PKP1, compensate less can upon a adhesion, DSPII extent cell that that has cell–cell some fact including to DSPI maintaining downregulated that in for the functions DSPII indicate cadherins although than other turn layer role in have upper important could the This might than possible signalling. is but important it 2007), less adhesion, al., Simpson, and is et Green DSC3 less 1989; (North and al., keratinocytes smaller et cadherin suprabasal has expression (Skerrow of epidermis basal that of cell-type-specific than layer desmosomes a basal electron-dense to the primarily as relate Also, is 1996). might DSC3 it DSPI because the and but DSC3 why is between clear link isoform not a functional is expression apparent has DSC3 It this DSPI DSPII. is in that than DSC3 there expression suggests increase DSC3 cells on the siI/II This effect to greater ratios. siI DSC2, from DSPI:DSPII levels and expression the by PKP1 influenced with other contrast to of high sufficiently levels levels junctions. in in present post-translational expression stabilisation not their the is enable in DSP at because see either or level occur we therefore components We changes 2004). desmosomal South, (Pasdar their the 1989; degradation) al., and et less believe Penn desmosome by 1988; the Nelson, evidenced and within (as contact decreases cell–cell stabilised upon turnover are high However proteins is degraded. proteins rapidly these desmosomal are of they turnover and the contacts, cell–cell euto nepeso eeso K1 S2adDSC3 and DSC2 PKP1, of levels expression in reduction A rvosrprshv hw oneuaino desmosomal of downregulation shown have reports Previous hrfr enx xmnddrcl h nlec fDSPI of influence the directly examined next we Therefore in that, revealed cells siI/II and siI between Comparison wtottargeting (without 2+ odtoso odtosta rvn h omto of formation the prevent that conditions or conditions DSPII nclsmmcigteDSPI- the mimicking cells in ) DSP a iers to rise give can DSP opnns scnitn ihamaual feton effect measurable a all of with subtle disruption global These consistent more tested. a is components than other staining components, rather the the effects, of change observed not any does of in but pattern changes localisation, causes intracellular depletion other PG DSP the Similarly, not but tested. desmosomal DSC3, components some and DSC2 of PKP1, including stabilisation adhesion components, and/or an expression is gaps decreased is there intercellular that DSP suggests increased of junctional which hours with no defect. cells, 2 these after where coincides prominent among regions and most thin in is of observed, cells especially retraction siI/II using and stretch, in thickness by Deformation IFs bundle confirmed software. keratin filament membrane were analysis keratin cell results image the of These to scoring thinner. close automatic are thickening nucleus, exhibit the filaments also around cells these IFs siI/II observed some keratin also although of is but effect cells this siI Crucially, imposed in stresses stretch. the mechanical for the compensate by to regions, IFs attempt perinuclear keratin an in as its normal particularly presumably of bundles, conditions, wrinkling of and condensation experimental and thickening exhibit these cells in HaCaT stretch mechanical deindfc otesII el a bevdb a of way by the similar observed to disruption A was similar a strategy. cells as network. well IF siI/II siRNA as gaps the site intercellular ablating increased to specifically trans-splice siRNA defect by DSP a confirmed adhesion disease-related was using two siI/II) the DSPII and on (siI focus models initial the from (Godsel assembly plakophilin. 2010). desmosome al., of through et contributes regulation PKP2 possibly actin-dependent actin that to the demonstrate or regulate studies to published directly ability Previously their possibly to due network, be might isoforms keratinocytes DSP cell–cell among of spaces loss DSP 2006). intercellular al., of to maintaining et large C-terminus (Norgett leads the and in attachment in adhesion IF by mutation importance idea compromises recessive this which a support its also that possible colleagues showing desmosome of and is for Norgett force driving desmosomes. because It the is al., tension et adhesion. IF disassembly (Russell attachment of colleagues loss and cell–cell that IF–desmosome Russell 2004), by decreased suggested weakened as therefore, cause of in which levels resulting DSP the similar a of withstand in In mutations disassembling. As the to started fragmented. way, junctions cells and cell unable collapsed mutant consequence, thus a were and keratin used, Second, stretch that mechanical IFs. thickening IFs keratin showed showed here. of stretch observed observed mechanical compaction authors ones after and the these cells resemble different, normal that were First, responses study cellular this and some stress conditions in stretch mechanical lines the same Although cell 2004). the al., et using (Russell repeated cycles system to relaxation exposed and were stretch simplex adhesion bullosa epidermolysis cell–cell causes of degree some maintained. but being function desmosome ti neetn ont h ako euto fDPlasto leads DSP of reduction or lack the note to interesting is It h rdce oeo SI ncl–elahso htemerged that adhesion cell–cell in DSPII of role predicted The two the for phenotypes different the for explanation Another that mutation 14 keratin a expressing lines cell Keratinocyte SI n emsmlahso 2859 adhesion desmosomal and DSPII DSP tde eela odcesdlevels decreased to lead here studied Journal of Cell Science 2860 h uieitsiewspbihd(uiryadLechler, and report (Sumigray a published of review, epithelium was the under intestine in was DSP murine for paper function the non-adhesion our a While detailing structure function. tissue therefore and and isoforms adhesion DSP disease desmosomal the to cardio-cutaneous which contribute by or mechanisms differing disease which demonstrating skin mutations non-syndromic isoform-specific that two cause partial. suggests of only tissue, mechanism is molecular DSPI this of in lack the expressed for still DSPII the of although is that compensation skin, DSPII the observation of enough, affects The be 100% still function. to mutation seems nonsense heart DSP DSPI-specific normal of for amount of This however, defect dosage variants. a splice causing to by DSP owing epidermis, cells. adhesion palmoplantar control inter-keratinocyte particular NT reduced in or skin, siI the in affect not but 4, Fig. marked of in as indicative example arrow for the structures cells, by are siI/II observed the stretched to possibilities have in weight half-desmosomes direct We both in lends junctions possibility. data identify. that EM observed second our of to monolayers, likely number siI/II and in is harder siII decrease are it the becoming that to believe junctions contributing therefore failed we of apart, number Although rudimentary the pulled in more there increase increase being junctions), the an electron-dense by be less the indicated smaller, could (as of proportion cells to the siI/II in and This owing siII cells. in desmosomes control cell–cell Additionally, defective and the explain adhesion. to cells mechanism obvious siI an defective suggests with structurally and compared fewer the have and/or both to desmosomes why appear 1997) explain cells siI/II al., would and This et siII 1999). al., (Kowalczyk et previously (Kowalczyk PG PKPs membrane with plasma complex As the interactions desmosomal to to through the cadherins components. desmosomal failure organise the to recruiting a desmosomal by crucial to is other DSP owing published, desmosomes in when the decrease explain assembled a cells recruit to cause of directly stretched possibilities number could DSPII the in mechanistic of depletion two particularly siI First, the this. are cells, with There comparison control in available. cells NT siI/II and and siII both in (Armstrong structures contacts 1999). al., cell–cell et Whittock fewer patients 1999; well of al., et skin as the in to desmosomes deficiencies in structural the shown leading including observe. been haploinsufficiency, we have DSP possibly that with ours bundles to affected, filament results the Similar be of collapse and subsequently retraction and underdeveloped rudimentary might these more the to the attachment IDPs of determine IF structural cause in IDPs. the DSPII to electron-dense of be have levels less to lower thought likely the to are 1999), cells al., is et siI/II appear (North DSPII IDP the that of that as extent desmosomes Given well in in of increase as deficiencies. an to number cells), DSPII leads cells) the (siI/II (siII for DSPII isoforms lacking specifically role cells both of key observed haploinsufficiency the defects demonstrate (sII adhesion keratinocytes. DSPII in cells) The of adhesion desmosomal amount siI/II reduced cells. a and expressing siI/II cells in and specifically in adhesion siII cell–cell in reduction both a demonstrating assay dispase a twsas paetta hr eefwrdesmosome fewer were there that apparent also was It that revealed analysis ultrastructural EM Our with confirmed independently was adhesion in role DSPII This hssuyhspoie oe nihsit h functional the into insights novel provided has study This mutations haploinsufficiency DSP that suggest results Our ora fCl cec 2 (12) 125 Science Cell of Journal auatrrsisrcin.Poen eeimblsdot nitrocellulose onto gels immobilised procedures. Mini standard were the using to Tris-acetate immunoblotted according gels Proteins and proteins, SDS-PAGE 3–8% membrane other 10% instructions. all hand-cast for Novex system or manufacturer’s Mini-PROTEAN NuPAGE DSP, BioRad for the on blotting using western resolved 0.001% when glycerol, (Invitrogen) (v/v) and 20% SDS, M minutes (w/v) 1.44 5 4% and 6.8, Blue pH Bromophenol Tris-HCl (w/v) M 0.125 using later el eepae thg est n rw vrih orahconfluency. reach to overnight grown and density high at plated were Cells secondary Immunocytochemistry IgG pig -guinea UK). pan-cytokeratin (Paisley, or Invitrogen anti-mouse and from Fluor conjugated purchased Alexa UK) (red) were Denmark). antibodies 568- (Poole, (Glostrup, and Dako Sigma (green) from 488- purchased from was purchased antibody polyclonal was antibody monoclonal vinculin monoclonal the UK), mouse against (Cambridge, DSG3 Abcam the and from and purchased PKP3 PKP2 were The and antibodies DSC2. PKP1 against DSC3, antibody PG, along polyclonal Sciences, against Germany) rabbit antibodies Life (Heidelberg, monoclonal Progen of mouse from the (Faculty purchased with guinea Garrod was DSPI-specific ‘GP’ antibody David available The polyclonal 1987). from al., pig are et gift (Parrish UK) against immunocytochemistry generous Manchester, antibody of a University monoclonal and mouse DSPII, 11-5F blot and the DSPI of western exception the for with commercially used antibodies All Cxb7 The 2010). al., Antibodies junction. et (Bishop RNA elsewhere being described c.3861–c.5659 duplexes as siRNA DSPII-specific used knockdown two was siRNA with the siRNA strategy, siII was across site 2010). isoforms trans-splice al., DSP designed a of et using knockdown (Cabral performed siRNA described was siI/II previously and as cultured siI performed was protocols. line cell standard keratinocyte using human immortalised spontaneously HaCaT siRNA-mediated The transient and culture Cell Methods and Materials ecie lehr He ta. 02.Clswr rw oahg est na in as density performed high a was to assay grown dispase were Cells a 2002). attachment, al., cell–cell et (Huen of elsewhere strength described the test To 18%) was to assay 0 Dispase stress to hours. up severe 4 range to amplitude more up Hz, of 5 analysis), described to duration EM up as a (frequency for immunocytochemistry the monolayer (unstretched for the for hours to prepared 0 applied (including then 14%. between to and Additionally time, 11 hours, of from above. stretch ranging 2 periods amplitude mechanical and different of station cyclic for control) elongation loading to an stretched the and subject were Hz over rubber were Cells 2 placed of Cells a frequency on was posts. contain a density plate cell faced with which Each high planar a (Flexcell) well. a 6 subject 35-mm pronectin to containing each to with grown in were coated used membrane to cells plates was strain HaCaT 6-well plates, static stress. BioFlex or culture mechanical cyclic to flexible-bottomed apply monolayer to on pressure computer- cultured vacuum a cells uses NC), Hillsborough, that (Flexcell, bioreactor System regulated Tension FX-4000 Flexcell The assay adhesion Flexcell DSP DSPI Total of blot. cells. measurements western control densitometry each NT the in of samples of level The sum DSPI DSPII. the the and from levels of protein calculated change DSPII were fold and levels DSPI a non-targeting graph, as a fold DSPI/DSPII presented with the a transfected are For as cells cells). presented of (NT the are level pool and protein to siRNA corresponding sample, normalised the individual the of each were to change for according levels (vinculin) MD) protein control Bethesda, loading NIH, blots These J, western recommendations. (Image of program manufacturer’s measurements analysis densitometry image from an using calculated were levels blots Protein western of quantification Densitometric formation junction promote to density high at (Zeiss, (7 seeded were browser keratinocytes HaCaT image and LSM blotting were Western minutes using images 5 Zeiss Illustrator. processed Immunfluorescence (Carl and microscope for ng/ml. Photoshop and confocal 100 Adobe scanning and methanol:acetone UK) at laser Germany) 510 Hertfordshire, stain cold LSM nuclear DAPI Zeiss Ltd, ice a procedures. a with standard as 1:1 acquired to used according in was performed fixed was immunocytochemistry were Cells 02,wihi ossetwt n uprstedt nthis in data the supports and with consistent manuscript. is which 2012), 6 10 5 el e elo -elds)adttlpoenwsetatd2 hours 24 extracted was protein total and dish) 6-well a of well per cells t ts o ace ar a sdt opr h en ftwo of means the compare to used was pairs matched for -test b mratehnl yae eebie for boiled were Lysates -mercaptoethanol. DSP knockdown Journal of Cell Science hlbese . en,F,Bruye F., Senni, L., Chalabreysse, alcn,G . oki,P,Bur . i,M,Vsoki,V,Dgnti,L and L. Degenstein, V., Vasioukhin, M., Yin, C., Bauer, P., Kouklis, I., G. Gallicano, P. A. South, and P. D. Kelsell, P. J., D. D. Abrams, L., Kelsell, C. Cole, H., and Wan, M., D. R. Cabral, Nitoiu, A., M. F. Brooke, M. Jonkman, and C. M. Bolling, ihp .L,Bri,A . esr,D,Brdrf . azmsua . Garbe, E., Hatzimasoura, V., Borgdorff, D., Fessart, M., A. Bergin, Leigh, A., L., R. C. Eady, Bishop, J., K. Green, E., P. Purkis, E., K. McKenna, K., D. Armstrong, ttsia nlsswr are u sn h w-ald ardStudent’s paired two-tailed, the using out * carried were analyses Statistical analysis Statistical Ca containing PBS in once washed and dish 60-mm ns,B . ils .A n re,K J. K. Green, and A. L. Nilles, D., B. Angst, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.084152/-/DC1 online available material London Supplementary the and 6 Barts after the release for from PMC in grant Deposited months. studentship. a MRC by a and funded Charity was study This Claire thank Funding manuscript. to the like of reading also critical with would for help Scott for We Ann Facility and imaging. Microscopy Dentistry) siRNA, Advanced immunofluorescent of siI/II Blizard (Institute the the with of Wan Wheeler particularly Hong assistance thank technical to for like would authors The Acknowledgements 60 at monolayer the them over embedded Araldite cured were incubating partly cells with The and filled Araldite. then capsules with were ‘BEEM’ infiltrated They inverting and series. by oxide ethanol 1% propylene graded in in a post-fixed through cleared glutaraldehyde, dehydrated 4% and buffered tetroxide phosphate osmium in fixed were Specimens microscopy Electron ncpe rd n tie ihuay ctt n edctae hywere They citrate. lead with collected and images acetate and were microscope 2K uranyl electron highest ‘Morada’ 1230 with Olympus was JEM an stained J.E.O.L. density a and in mounted cell cut, examined grids were the nm) copper where (60–80 sections on Ultrathin areas stained microscopy. and electron Specific cut for were targeted Blue. microscopy light Toluidine for mm) with (0.5 sections semi-thin membrane, rnfre oa1 ltb otiigPSadsbetdt ehnclsrs by microscope. stress fragments dissecting monolayer were mechanical of a to number Monolayers under subjected The counted tube). detached. and the was PBS of monolayer containing inversions rapid the tube (10–20 ml agitation until 15 dispase a to mg/ml transferred 5 in incubated el ntemnlyrwt orkokonefcec,wr icutdfor discounted were efficiency, knockdown with poor associated presumably with mean, monolayer analysis. the statistical keratinocyte the density from a in on deviation showed that standard showed cells outliers falling that the one cells siI/II), siRNA-treated than points and expressed the (siII greater For density and test points. desmosome calculated test in was cytoplasm of decrease per density a DM number DM of the ratio per a total counts DMs as of these the number From of total only. to the total cytoplasm counting a by relative produce achieved to was negative coherent negative density DM EM A 0.55 of is each scored. Estimation square to (1 were applied points software. was test plaques 144 image over opposing Photoshop lattice negatives Adobe square recognisable The single using displaying examined. computer were DMs personal sample Only a per into fields scanned arbitrary were 10 and magnification P omauedsooedniy lcrnmcorpswr ae t5,000 at taken were micrographs electron density, desmosome measure To emsmsadctseea linkage. cytoskeletal and desmosomes E. Fuchs, mutations. human desmoplakin-dominant to of secondary isoform novel P. Bouvagnet, a DSPIa, of characterization desmoplakin. and Identification (2010). disease. and desmosomes x.Dermatol. Exp. p16(INK4A). H. inhibits D. signaling Beach, and Hedgehog J. -independent Koh, and R., M. Stampfer, C., J. keratoderma. palmoplantar E. of A. subtype Hughes, and M. I. etitdt taiidepithelia. stratified to restricted # .5 ** 0.05, P 19) empai srqie al ndvlpetfrasml of assembly for development in early required is Desmoplakin (1998). # elTsu Res. Tissue Cell .1 *** 0.01, 21) e yooiootasnrm:Craa/ao syndrome Carvajal/Naxos syndrome: hypo/oligodontia new A (2011). 18 658-668. , P 6 19) alisfiinyo empai assastriate a causes desmoplakin of Haploinsufficiency (1999). .Pathol. J. # ˚ Kdgtlcamera. digital 2K 0.001. o 8hus fe utn wytesilicone the away cutting After hours. 48 for C 341 m .Cl Sci. Cell J. wide M r,P,Am,B,Olgir . oi,A and A. Bozio, C., Ollagnier, B., Aime, P., `re, 121-129. , 20) knadhat n iio dangereuse. liaison une heart: and Skin (2009). 226 u.Ml Genet. Mol. Hum. .Cl Biol. Cell J. 158-171. , 5 19) empai Iepeso snot is expression II Desmoplakin (1990). 2 iesfrmaueetpurposes). measurement for pixels 220 97 247-257. , .Dn.Res. Dent. J. 21) rmr cilium-dependent Primary (2010). 143 21) elcl connectivity: Cell-cell (2012). 2009-2022. , 2+ 8 143-148. , n Mg and o.Cell Mol. 90 58-64. , 2+ eoebeing before 40 533-547. , t -test. 6 htok .V,Aho,G . opn-eesa,P . rta,M . Keane, J., M. Gratian, J., P. Dopping-Hepenstal, H., G. Ashton, V., N. Whittock, a,H,Suh .P n at .R. I. Hart, and P. A. South, H., Wan, oacy,A . azed . onlee,E . op .S,Brwrt .E., J. Borgwardt, S., D. Kopp, A., E. Bornslaeger, M., Hatzfeld, P., Dhaliwal, A. L., Kowalczyk, H. Palka, E., J. Borgwardt, A., E. Bornslaeger, P., A. Kowalczyk, V., E. Amargo, J., L. Bannon, X., Chen, M., L. Godsel, K., J. Park, C., A. Huen, a,H,DpigHpntl .J,Gain .J,Soe .G,Zu . Purkis, G., Zhu, E. G., M. Fuchs, Stone, and J., M. L. Gratian, J., Degenstein, P. Dopping-Hepenstal, C., H., Bauer, Wan, E., Bowers, V., Vasioukhin, re,K .adSmsn .L. C. Simpson, and J. K. Green, zmu . ogt,E . idr . yue,O,Nsi . asrl,H,Sahin, H., Kayserili, K., Nisli, O., Uyguner, A., Dindar, E., E. Norgett, A., Uzumcu, osl .M,Dbs,A . asZbk .E,Aag,E . lsnr .L., J. Klessner, V., E. Amargo, E., A. Bass-Zubek, D., A. Dubash, M., L. Godsel, uiry .D n ehe,T. Lechler, and D. K. Sumigray, acaGa,E,Lmad,R,Good,M . ilro,J . cnie,M D., M. Schneider, T., J. Willerson, J., M. Giocondo, R., Lombardi, E., Garcia-Gras, ot,A P. A. South, alcn,G . ae,C n uh,E. Fuchs, and C. Bauer, I., G. Gallicano, ot,A . hde,M . lre .P,Brse,W .adGro,D R. D. Garrod, and G. W. Bardsley, P., J. Clarke, A., M. Chidgey, J., A. North, kro,C . llad .G n kro,D. Skerrow, and G. D. Clelland, J., B. C. E. Skerrow, Lane, and J. James, D., P. Andrews, I. D., A. Russell, Magee, and A. D. Rees, C., Hobson, J., E. Penn, J. W. Nelson, and O. M. R. Weller, Pasdar, and C., R. D. H. Garrod, Cordingley, V., P. A., Steart, P., E. E. Parrish, Bornslaeger, J., Hyam, G., W. Bardsley, J., A. North, hu . tat . etn .E,Rdiuz . ol .adGliao .I. G. Gallicano, and B. Hoel, G., Rodriguez, E., L. Dettin, A., Stuart, X., Zhou, ogt,E . uk,T . oes . ur,C . eg,I .adKelsell, and M. I. Leigh, S., C. Munro, B., Bowers, W., T. Lucke, E., E. Norgett, i,T n re,K J. K. Green, and T. Yin, .M,Ed,R .adMGah .A. J. haploinsufficiency. McGrath, desmoplakin and from A. resulting R. Eady, M., F. ntae hog oneuaino empai yRAinterference. RNA by 313 desmoplakin of downregulation through initiated lkpii- id odsolknadehne t erimn odesmosomes. to recruitment J. its K. enhances disease. Green, and cutaneous and for desmoplakin Implications A. to binds Settler, desmosomal plakophilin-1 M., clusters C. and J. Corcoran, plakoglobin K. Green, to and binds F. desmoplakin complexes. M. cadherin-plakoglobin of Denning, al. M., domain C. et terminal Corcoran, P. S., D. A. Kelsell, M., actin- I. with strength. adhesive Leigh, 1017. synergistically intercellular regulate K., function to contacts A. dependent attachments Mongiu, filament-membrane Y., Intermediate T. Hudson, soitdwt otatn etrain fdsooe n h eai filament keratin the and desmosomes is of mutations perturbations 1 network. al. desmoglein contrasting et and S. with R. desmoplakin Buxton, associated from K., D. arising Armstrong, keratoderma F., Keane, palmoplantar P., A. South, E., P. .Ivs.Dermatol. Invest. J. sfr asseryostcrimoah n er alr naNaxos-like a formation. sheet in epidermal in failure essential heart is Desmoplakin and al. cardiomyopathy et M. onset I. early Leigh, syndrome. J., causes N. I Severs, isoform E., Dupont, E., S. eoeigt emsmlpau sebyvaRhoA. J. via assembly K. plaque Green, desmosomal and to X. remodeling Chen, P., R. Hobbs, o elahso rkrtnognzto nteitsia epithelium. intestinal the in organization keratin 23 or adhesion cell not inln yncerpaolbnrcptltspeoyeo rhtmgncright arrhythmogenic of phenotype recapitulates cardiomyopathy. plakoglobin ventricular nuclear by J. A. signaling Marian, and S. D. Khoury, Dermatol. xr-mroi coemrvasteiprac fti rti nebyncheart, embryonic in vasculature. protein this and of skin importance neuroepithelium, the reveals ectoderm extra-embryonic niesadlci-idn ie uigdfeetaini omlhmneiems a epidermis: human normal study. in ultrastructural differentiation quantitative during sites lectin-binding and antigens cells. kidney canine 13-16. Madin-Darby of glycoproteins desmosome major R. D. Garrod, tumours. intracranial of and diagnosis I. the in A. antibody monoclonal Magee, plaque. desmosomal J., the K. of Green, map Molecular M., (1999). Hatzfeld, B., show Trinnaman, and desmosomes epidermis. same nasal the bovine in in USA distributions occur graded isoforms reciprocally desmocollin Distinct (1996). 20) empai srqie o irvsua uefraini culture. in formation tube microvascular for Sci. required is Desmoplakin (2004). tit ampatrkrtdraadwol arascae ihanvlinsertion novel a with associated hair desmoplakin. woolly in mutation and keratoderma palmoplantar striate P. D. nue rfudrmdligo eai iaet n eljntosi epidermolysis in junctions keratinocytes. cell and simplex filaments bullosa keratin of remodelling profound induces analysis. Biochemical desmoplakin I. of contact. regulation cell-cell Biol. spatial upon and stabilization temporal and cells: organization epithelial kidney canine Darby ei.Cl e.Biol. Dev. Cell Semin. 792-799. , 2336-2344. , 117 93 106 20) al et rmcrimoah nafml ihatsmldominant autosomal with family a in cardiomyopathy from death Early (2006). 7701-7705. , 3129-3140. , 677-685. , r .Dermatol. J. Br. 29 20) lkpii :a motn tblzro desmosomes. of stabilizer important an 1: Plakophilin (2004). .Md Genet. Med. J. 161-167. , SI n emsmlahso 2861 adhesion desmosomal and DSPII 127 15 2499-2515. , 20) euaino emsm sebyadadhesion. and assembly desmosome of Regulation (2004). 665-677. , .Ivs.Dermatol. Invest. J. 150 43 .Ci.Invest. Clin. J. e5. , 878-891. , 18) ieiso emsm sebyi Madin- in assembly desmosome of Kinetics (1988). .Cl Sci. Cell J. 20) emsms e esetvso classic. a on perspectives new Desmosomes: (2007). 21) empai otosmcoil eghbut length microvilli controls Desmoplakin (2012). .Cl Biol. Cell J. .Cl Sci. Cell J. 20) upeso fcnnclWnt/beta-catenin canonical of Suppression (2006). .Bo.Chem. Biol. J. Development 20) nrae eaioyeproliferation keratinocyte Increased (2007). 20) ecigdsolknfnto in function desmoplakin Rescuing (2001). 117 19) tit ampatrkeratoderma palmoplantar Striate (1999). 21) lkpii ope actomyosin couples 2 Plakophilin (2010). 116 92 5233-5243. , 139 126 2012-2021. , .Ivs.Dermatol. Invest. J. 667-677. , 773-784. , 1651-1654. , 274 18) hne odesmosomal to Changes (1989). .Cl Sci. Cell J. 128 19) h eddmi of domain head The (1999). o.Bo.Cell Biol. Mol. 18145-18148. , 20) oso desmoplakin of Loss (2006). a.Cl Biol. Cell Nat. 18) h sebyo the of assembly The (1989). 929-941. , 20) ehnclstress Mechanical (2004). 18) Antidesmosomal (1987). .Pathol. J. .Cl Biol. Cell J. rc al cd Sci. Acad. Natl. Proc. 112 19) h amino- The (1997). 4325-4336. , 113 ESLett. FEBS o.Bo.Cell Biol. Mol. 21 20) Striate (2004). x.Cl Res. Cell Exp. 3 153 1076-1085. , 2844-2859. , 940-946. , 159 ln Exp. Clin. 265-273. , (2002). (2001). 1005- , .Cell J. .Cell J. 247 ,