Teeatoscnrbtdeulyt hswork this to equally contributed authors *These India. 560065, ltleHuet-Calderwood activation Clotilde kindlin integrin determines and complex localization ILK adhesion the isoform to binding in Differences ARTICLE RESEARCH 4308 2014 July 2 Accepted 2014; April 28 Received ` 3 2 that protein 1 adaptor events cytoskeletal signaling integrin the the intracellular of of tail cytoplasmic by by binding the to triggered talin the or be in can ECM culminate states) (the to active ligand- activation to binding high Integrin inactive with from 2004a). conformation conversion (Calderwood, ‘active’ organization, for an affinity affinity low in binding relatively tissue Integrins or with physiological state ligands response. ‘inactive’ of ECM an immune migration, in found range the be can and cell a hemostasis govern Calderwood, angiogenesis, including integrins and such, (Harburger processes As (ECM) matrix 2009). and cells extracellular adhesion between signaling the transmembrane bi-directional mediate heterodimeric that receptors essential are Integrins INTRODUCTION Kindlin activation, kinase, Integrin Integrin-linked Integrin, adhesion, Focal WORDS: KEY not but kindlin-2, and kindlin-2 rescue not, can does GFP-tagged kindlin-3 kindlin-3, respectively); whereas FAs FERMT3, to known localizes we and (also -3 Here, FERMT2 and examined. kindlin-2 as been between differences not functional three generally report differences have the functional their the isoforms and for of elucidated, between basis fully each be signaling. molecular to the yet of and has adhesion, function cell importance activation in accepted kindlins integrin mammalian widely proper the for Despite required (FA) proteins adhesion focal FERM-domain-containing essential are Kindlins ABSTRACT 2sboanbtpol ota fknln3 sn point-mutated a Using kindlin-3. of that integrin-linked to poorly FA kindlin-2 the but the that subdomain to drives F2 strongly find binds We complex subdomain (ILK)–PINCH–parvin activation. kinase F2 integrin the and kindlin-2 that targeting show uncharacterized we kindlins, relatively chimeric Using fibroblasts. knockdown n ifrnilIKbnigcnrbtst idi sfr specificity. isoform function complex. kindlin kindlin-2 to ILK normal contributes binding the for ILK crucial to differential and is integrin binding binding require kindlin-2-mediated ILK-complex targeting Thus, efficient FA that and activation establish we kindlin-2, iu .Boggon J. Titus uhrfrcrepnec ([email protected]) correspondence for Author nttt o tmCl ilg n eeeaieMdcn,Bnaoe Karnataka USA. Bangalore, 06520, Medicine, CT Regenerative and Haven, Biology New USA. Cell University, Stem 06520, for Yale CT Institute Biology, Haven, Cell New of University, Department Yale Pharmacology, of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,40–31doi:10.1242/jcs.155879 4308–4321 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 1 n ai .Calderwood A. David and a 5 b nernatvto eet nkindlin-2- in defects activation integrin 1 1, ,Nn .Brahme N. Nina *, b uui (Calderwood subunit 1,2, 2, ` ,NktKumar Nikit *, n inln Clewo ta. 03 Karako 2013; al., et regulation (Calderwood integrin applies normal signaling for and crucial and are the that senses and and tail for cell cytoplasmic the points Harburger which anchorage at 2012; as force. sites mechanical serve Zaidel-Bar, and and cytoskeleton signaling and 2009) and scaffolding Calderwood, (Geiger cytoskeletal, of array proteins assemblies large multi-protein 2009; a to complex Calderwood, contain cluster These and can (Harburger 2012). receptors (FAs) Wehrle-Haller, adhesions integrin or activated focal molecules, Once form 2012). ECM al., et by Ye 2010; engaged al., et Shattil 2013; al., et ncdw fknln1(sa ta. 08,adls fkindlin-3 of of activation loss or impaired and 2008), knockout in al., following results Shi et reported (Ussar 2011; are kindlin-1 al., of defects cells et knockdown Similar Pluskota depleted, 2007). 2008; al., Ussar is al., et 2007; et kindlin-2 (Montanez al., 2009; spreading when et cell al., Shi example, et 2008; in For defects 2008; Malinin al., exhibit 2008). et 2008; al., al., Moser al., 2009; et et et al., Ma (Dowling et 2009; cells Moser knockdown experiments al., and et overexpression knockout Harburger of by studies processes. by and integrin-mediated three supported and all is activation This implicating integrin strongly in phenotypes, kindlins knockout or disease (LADIII) 2009; al., defects III et immune by the Moser bleeding. characterized severe 2009; type is and al., which of 2009), et al., deficiency FERMT3) Malinin et loss Svensson 2009; as al., adhesion Finally, et known (Kuijpers leukocyte 2008). basement (also al., causes the kindlin-3 from et detachment hematopoietic-specific (Montanez cell peri- to the membrane at due kindlin-2 development stage murine expressed Ussar halts ubiquitously implantation FERMT2) 2003; the as al., of known et Knockout (also (Siegel 2008). in colitis al., manifesting ulcerative et syndrome, and Kindler blistering to (also kindlin-1 skin leads epithelial FERMT1) mainly the as of known Loss 2008). al., et Larjava n idi- ohtre oFsi deetcls kindlin-1 cells, adherent in FAs to to target preferentially targets both tissue kindlin-1 whereas kindlin-2 and example, subcellular For and integrin observed. in in been the have differences to localization kindlin-3 tails, bind 2008). al., cytoplasmic isoforms non-functional et three subunit Ussar 2009; all for al., al., although et et Moser Furthermore, 2009; or Lai-Cheong instance, al., 2010; et Malinin al., skin of 2008; For et loss (Bialkowska the for cells functions. compensate hematopoietic fully in to isoform-specific also unable kindlin-1 they three is adhesion, have all kindlin-2 cell although endogenous of to However, regulators 2008). key appear al., are et isoforms Moser kindlin 2009; al., et idisaeafml fpoen htbn oteintegrin the to bind that proteins of family a are Kindlins marditgi ciaini omnfaueo kindlin of feature common a is activation integrin Impaired 2 m .Stiegler L. Amy , b and 1 a b v 1 nernatvto,F omto and formation FA activation, integrin 3 rkl Raghavan Srikala , b -ihahsosin adhesions 6-rich b 1, b and 2 b nern (Moser integrins 3 b s ta. 2010; al., et ¨se -ncotcells 1-knockout 1,3 , b

Journal of Cell Science bn .,ern aii,mei)dmi opsdo four of composed domain moesin) FERM atypical radixin, an of ezrin, up 4.1, made to are remain (band Kindlins signaling determined. and fully activation be integrin affect kindlins is which function and localization isoform molecular understood. The in not 2006). differences generally al., the et to for Ussar localize 2010; basis not al., does et kindlin-3 (Bialkowska and FAs 2012), al., et (Bandyopadhyay ARTICLE RESEARCH ope ifr ewe hs w idi sfrs We isoforms. ILK-KD–parvin-CH2 kindlin the IPP to two the site to these kindlin-binding binding the between that localized found differs We reverse complex fibroblasts. to in unable was knockdown expression localize of kindlin-3 not suppression addition, the did kindlin-3 In noticeably, FAs. observed Most to -2. kindlin-3 initially and overexpressed kindlin-1 of We and localization the partners. in differences their binding and striking subdomains kindlin relevant key identify functionally to means a as functions 2008). integrin antibodies al., on et effects signaling. exerts Tucker and ILK 2009; ligand-mimetic how reporter activation about al., known et ligands, is Honda little conformation-specific However, soluble 2004; al., of et and (Friedrich binding al., the antibodies et activation by Sakai integrin 2009; assessed inhibit al., integrin-mediated to et undertake reported Kogata to 2013; required Wickstro and al., is 2003; and et al., architecture proteins (Elad et FA signaling Wang other FA of 2009; or directly range maintain al., interacts a tri- ILK al., et parvin, with et this and Stanchi indirectly PINCH (Fukuda and to 2007; function addition 2013) al., In ILK 2008). al., et to et central Gkretsi Stiegler is 2003; complex 2009; IPP al., al., protein et parvin et Chiswell protein 2010; Fukuda al., domain et 2008; its (Chiswell (CH) (through domain) homology kinase PINCH its pseudokinase calponin (through protein the domain a and LIM protein. be the ARD) adaptor catalytic to an with as key partners thought primarily lacks ILK functioning generally 2009) domain al., is et kinase (Fukuda ILK The (ILK-KD) and domain 2009). kinase residues, al., C-terminal (Chiswell a et (ILK-ARD) by (Fukuda followed domain 2008) repeat al., ankyrin composed et was is N-terminal and ILK 1996), an al., 2003). integrin integrin of et for al., (Hannigan normal partner screen et two-hybrid binding and yeast Sakai a as 2004; development identified al., for originally et essential (Friedrich protein signaling is ILK FA 2013). al., a et (Calderwood proteins kindlin-binding difficult. known action of made 2012a; kindlin mechanism has kindlin isolated Xu signaling of the al., 2011; or inability of activation al., The characterization integrin et et 2012). support Perera al., to 2011; (Bouaouina et subdomains al., Yates membrane- et domains 2013; of al., Liu et because 2009; these part al., et in in Harburger least at sites the domains, 2008). requires binding PH al., also and et adhesions, F1 Ussar to F0, localization 2008; that and show al., al., function, studies et mutagenesis kindlin Moser and et deletion 2008; subdomain (Harburger al., However, proteins. et activation Ma adaptor site integrin 2009; binding or a talin-mediated contains scaffold enhance subdomain as F3 integrin the for act this, domains with kindlins enzymatic Consistent recognizable of that lack The suggests 2013; 2009). al., al., et (PH) et (Calderwood homology subdomain Goult F2 pleckstrin the a within with nested F3) domain and F2 F1, (F0, subdomains eie nern nernlne iae(L)i n ftefew the of one is (ILK) kinase integrin-linked integrin, Besides by mechanisms the 2013), al., et (Ye progress recent Despite nti td,w netgtdteioomseiiiyo kindlin of specificity isoform the investigated we study, this In b tis n hsbnigi eurdfrknln to kindlins for required is binding this and -tails, me l,21) oal,ls fIKhsbeen has ILK of loss Notably, 2010). al., et ¨m a 5 b nernatvto rgee ykindlin-2 by triggered activation integrin 1 nvitro in and b nvivo in tisi a in -tails as ciain n rvdsisgtit o idi n ILK and kindlin how into signaling. drives insight integrin integrin to ILK provides kindlin-mediated contribute that and and and shows localization activation, targeting work subcellular our FA kindlin Overall, that supports kindlin showed activation. we and binding integrin complex, preserved, ILK ILK-complex the was strong binding in structure defective domain mutants inhibited the which strongly kindlin in that chimeras overall kindlin-2–kindlin-3 mutation using F2 point By kindlin binding. a ILK the identified to site and ILK-binding domain, the mapped complex, domain fF agtn yqatfigbidytepretg fclsin cells of percentage extent the blindly the quantifying assessed by We targeting 1A). FA (Fig. readily of FAs, level to contrast, expression were target clear of not In did -2 1A). regardless and (Fig. cytoplasmic and replating always after was h GFP–kindlin-1 GFP–kindlin-3 24 at 2009; 2009), FAs al., in al., et detected Goult et As 2012a; al., microscopy. Harburger et fluorescence (Bouaouina to and by reported fibronectin assessed kindlins previously on was plated mammalian cells localization CHO three their in -3 and overexpressed the -2 each kindlin-1, of were GFP-tagged N-terminally ability FAs, in accumulate the compare to cells To target CHO kindlin-3, in not FAs but integrin-rich -2, and kindlin-1 GFP-tagged RESULTS l he idisbudwild-type with 2007). bound al., coated kindlins et beads (Lad three tails All with cytoplasmic expressing integrin incubated cells His-tagged CHO purified were from kindlins recombinant Lysates GFP-tagged to tails. 1B). binding cytoplasmic (Fig. isoform FAs integrin kindlin to compared targeting kindlin therefore for We crucial is binding Integrin binds Kindlin-3 -2 and GFP–kindlin-1 to for cells. CHO in required that FAs demonstrating at the and accumulation is 1B), assays 2008), (Fig. our al., FAs of integrin-binding et specificity in the Ussar accumulate confirming and to 2008; failed GFP–kindlin-1-W612A al., GFP–kindlin-2-W615A mutants et defective Montanez integrin-binding 2009; (Harburger not al., expected could As et we FAs. to However, than targeting respectively). GFP–kindlin-3 better observe 86%, targeted and most consistently (99% in kindlin-2 FAs kindlin-1 to and targeted -2 cells, and vinculin- expressing GFP–kindlin-1 with 1B). colocalizes (Fig. FAs clearly rich protein GFP-tagged the which a pcfca twsrdcdt akrud( background to reduced was it as specific was in accumulate to failed also kindlin-3 2008), al., a et (Moser h osre rpohni h 3sboano h kindlins that the observed of subdomain we F3 Notably, the 1E,F). in (Fig. tryptophan conserved in motif the NPxY membrane-distal the mutating point for crucial partners several indicating or one 1D), FAs. to due (Fig. to binding be localization might in not that differences isoforms to did kindlin to between GFP–kindlin-3 differences targeted for intrinsic needed GFP–kindlin-2 whereas be cells, FAs, 2010) could (EA.hy926) and al., (CMK) cofactor megakaryocytic endothelial-like et in both cell-specific (Bialkowska However, hematopoietic targeting. a endothelial kindlin-3 1C). to and (Fig. therefore 2006) restricted cells, target al., normally et did is (Ussar GFP–kindlin-2 expression whereas Kindlin-3 fibrinogen, on infcnl esknln3ta idi- Fg F,suggesting 1F), (Fig. kindlin-2 than kindlin-3 less significantly IIb h nblt fknln3t cuuaei A sntspecific not is FAs in accumulate to kindlin-3 of inability The a b 5 -ihFsin FAs 3-rich b -eitdahsosa,cnitn iherirreports earlier with consistent as, adhesions 1-mediated ora fCl cec 21)17 3842 doi:10.1242/jcs.155879 4308–4321 127, (2014) Science Cell of Journal b nerntisls elta te isoforms other than well less tails integrin 1 a IIb b -nernepesn H el spread cells CHO 3-integrin-expressing b -al Fg E.Binding 1E). (Fig. 1-tails b -al ulddown pulled 1-tails a I)lvl by levels IIb) b nernor integrin 1 4309 b 1- b 1

Journal of Cell Science EERHARTICLE RESEARCH idi- Fg BC.Hne ecnld httedifference the of that 4310 that conclude like we binding, Hence, integrin 2B,C). kindlin-2, (Fig. diminished of kindlin-3 that had K3332 to K2223 comparable chimera whereas binding, the FA integrin Notably, enhanced 2B). bound of (Fig. had and masses specifically molecular tail in lack expected integrin well the of expressed were the kindlins cells, chimeric CHO The between whether kindlin-3. integrin subdomain subdomain and F3 reduced kindlin-2 integrin-binding by test the the explained swapped digits, we be which binding, To could the kindlin-3 four from of with 2A). localization using subdomain, isoform (Fig. F3 kindlins the or originates F2 chimeric indicating F1, F0, number named kindlin the We representing material S1). supplementary boundaries FERM in regions indicated Fig. (subdomain predicted are the conservation isoforms swapping map kindlin and kindlin To by between 2012). we chimeras subdomains 2011; targeting, al., FA al., generated et in et therefore differences Yates Perera isoform-specific 2008; 2008; for al., responsible al., et 2011; al., et Moser et 2008; Liu Ussar 2009; al., al., al., et et et (Bouaouina Harburger Montanez targeting 2009; FA al., impairs et domain kindlin Goult the F3 2012a; of or mutation PH or F1, deletion that F0, subdomain shown F3 have others the and only in We are differences to isoforms due kindlin of partially targeting FA in Differences to target to kindlin-3 of inability the to FAs. contribute could this that b 1 ugs htaohrknlnsboanmksimportant makes subdomain kindlin data its targeting. these another and FA together, cells to Taken contributions of 2D). that 37% (Fig. only weak in suggest was FAs differences. FAs to in isoform targeted intensity K3332 F3 the 2C), kindlin- the to (Fig. of levels that 2 comparable part at suggesting integrin that binding only (78%) despite Likewise, indicates cells for FAs. most to accounts 2E) (Fig. in target subdomain targets to targeting still kindlins of K2223 of quantification abilities varied However, the to contributes (mean o 22 n 1.25 and K2223 for hnwl-yeGPknln2[Atreigidcso 1.75 of indices targeting [FA GFP–kindlin-2 wild-type than agtdsgiiatyls el( well less GFP–K3332 significantly and GFP–K2223 mediumtargeted both method, targeting. this indicated strong by very assessed indicated 2 When 4 and targeting GFP–kindlin-2), targeting, strong of indicated that low (like 3 GFP–kindlin-1), of indicated that (like indicated targeting 1 0 of the targeting, score categorize a to Briefly, no adhesions. system at scoring signal GFP blinded exhibiting of a cells intensity used of percentage we the targeting, scoring FA to addition kindlin-3 Conversely, In to 2D). compared 2D,E). (Fig. when (Fig. gain-of-function modest kindlin-2 a FA had to diminished K3332 exhibited compared K2223 when that targeting found to we due chimeras, tagged is integrin subdomains. binding F3 in their of kindlin-3 properties and kindlin-2 between hnw xmndsbellrlclzto fteeGFP- these of localization subcellular examined we When et.Saebr:10 bars: Scale test). (C) FAs.experiments. to target WA) mutants indicated by and kindlin-3-W597A, kindlins and GFP-tagged kindlin-2-W615A the (kindlin-1-W612A, for ( which stained cells in and transfected interval) later of confidence h Percentage 24 (B) fixed vinculin. fibronectin, endogenous on plated were localization kindlins subcellular binding. different integrin have and isoforms Kindlin 1. Fig. rmCOcl yae ihprfe recombinant purified with lysates kindlins cell GFP-tagged CHO of from Pulldown and (E) fibronectin vinculin. cells endogenous on for EA.hy926 plated stained or were CMK kindlins (D) GFP-tagged vinculin. overexpressing endogenous fixed for fibrinogen, stained on and plated were kindlins GFP-tagged overexpressing al a uniidadepesda ecnaeo input of percentage a as expressed (mean and quantified recombinant was purified tails to binding GFP–kindlin-3- (K3WA) and W597A (K3) GFP–kindlin-3 GFP– (K2WA), GFP–kindlin-1 (K2), (F) kindlin-2-W615A GFP–kindlin-2 lysate. input (K1WA), of GFP–kindlin-1-W612A 3% (K1), indicates lane 3% The staining. ( 6 b YA nerntis allaigwsasse yCoaseBlue Coomassie by assessed was loading Tail tails. integrin 1Y/A) ora fCl cec 21)17 3842 doi:10.1242/jcs.155879 4308–4321 127, (2014) Science Cell of Journal s.e.m., 6 s.e.m.; n 5 ).Teerslsso htteF subdomain F3 the that show results These 4)]. 6 n 5 a .5frK32vru 3.0 versus K3332 for 0.25 IIb ) * 4). m b m. -nernepesn H cells CHO 3-integrin-expressing n P A H el vrxrsigGFP-tagged overexpressing cells CHO (A) 5 , 6–8 el rmtreindependent three from cells 160–280 .5 ** 0.05, P , P , .5 ardStudent’s paired 0.05, .1 *** 0.01, P 6 , a a .0 (Student’s 0.001 .0frkindlin-2 for 0.00 IIb, IIb, b b 1or and 1 6 b 95% 1Y795A b 6 t 1Y/A -test) 0.25 t -

Journal of Cell Science EERHARTICLE RESEARCH lotcmltl,ad oeipraty nrdcn the generated targeting introducing (K3323) FA kindlin-3 importantly, into abrogate kindlin-2 more into of to subdomain and, subdomain F2PH sufficient 3A). completely, F2PH (Fig. was (F2PH) almost kindlin-3 domain (K2232) PH the kindlin-2 inserted introducing only its swapping plus by Strikingly, domain chimeras generated F2 motif we the this, of FA-targeting subdomain test major F2PH To uncharacterized kindlin-2. a relatively that the within suggest resides results preceding The subdomain independently F3 targeting, the FA from dictates kindlin subdomain F2PH between The targeting FA therefore the are in We regions differences 3B,C). F2F3 isoforms. of (Fig. their drivers within respectively differences major the -2, well as that and FAs conclude and to (K3311 targeted kindlin-1 K1122) kindlin-1 and as from (K3322 F2F3 kindlin-2 to a or the target K2211) bearing to of chimeras those failed Notably, K2233) were whereas and shown). FAs, and (K1133 more F2F3 not well kindlin-3 a the (data expressed bearing mass produced -3 F2F3 molecular and swapping 3A) expected from -2 (Fig. resulting kindlin-1, and (F2F3) chimeras between six kindlin-2 subdomains All effect. affect F3 the dramatic both partially swapping and whether swaps tested F2 next we F3 targeting, FA that kindlin-3 shown FA Having in isoforms differences kindlin drive between subdomains targeting F2F3 the in Differences mato agtn,a idi- otiigteknln3PH kindlin-3 the little had containing this kindlin-2 it 3H,I, as Fig. swapped in targeting, shown we on As targeting impact -3. is domain and FA PH kindlin-2 the isoform-specific between whether their test in for To implicated 2011). been responsible al., has et it (Liu and targeting kindlins, FA to unique feature a to is target to isolated unable the shown). was 2008), not F3 kindlin-2 al., (data et of and FAs Ma domain 2009; F1 Goult F2PH al., 2012a; et F0, GFP-tagged al., Harburger FA et 2009; of al., (Bouaouina drives importance et localization kindlin FA the for of describing subdomains region data with F2PH consistent even However, published the fashion. kindlin-2, isoform-specific Therefore, an that kindlin-3. as in like targeting show well well, results as less tails almost our integrin FAs binds it to F2PH integrin though targets bound 3F,G). the (Fig. K3323 K3323 respectively and kindlin-3, Thus did and K2232 with kindlin-2 it to as targeting, similarly binding, tails tracked FA integrin controlled alter kindlin-2 F2PH not the and Although targeting subdomain. FA kindlin-1 showing cells the of here between percentage even the but 3E), in differences (Fig. FAs small to efficiently targeted K2212 and 2.29 n of [FA GFP–kindlin-2 index wild-type 3D,E). than targeting (Fig. GFP–K3323 well less that levels slightly showed only kindlin-2 intensity targeted FA-targeting wild-type of scoring almost Blind at targeting FA 5 rmtreidpnetexperiments. independent three from hc h F-agdknln n hmrstre oFAs. to target chimeras and kindlins GFP-tagged bars: the Scale which fibronectin. on plated cells 10 CHO in vinculin, endogenous (Student’s to lysates cell CHO from chimeras kindlin a GFP-tagged nomenclature. of chimera Binding and (B,C) kindlins chimeric binding. FA of integrin isoform-specific representation to in contributes differences subdomain through F3 targeting kindlin The 2. Fig. uniid()adnraie osatn aeillaig(mean loading material starting to n normalized and (C) quantified h Hdmi meddi rdce EMF subdomain F2 FERM predicted a in embedded domain PH The 7; IIb, 5 ) h %ln niae %o nu yae * lysate. input of 3% indicates lane 3% The 5). m P .()Pretg ftasetdcls( cells transfected of Percentage (E) m. b , ora fCl cec 21)17 3842 doi:10.1242/jcs.155879 4308–4321 127, (2014) Science Cell of Journal and 1 .5 ardStudent’s paired 0.05, t ts) D oaiaino F-agdknln,and kindlins, GFP-tagged of Localization (D) -test). b 75 ( Y795A 1 b 6 YA a sesdb immunoblotting, by assessed was 1Y/A) .8vru 2.86 versus 0.18 t ts).A xetd ohK1121 both expected, As -test)]. 6 5 ofdneitra)in interval) confidence 95% 6 .4(mean 0.14 P , A Schematic (A) .5 ** 0.05, n P 5 , 6 6 cells 160 6 0.01 s.e.m., s.e.m.; 4311

Journal of Cell Science EERHARTICLE RESEARCH soit ihknlni es w-yrdadco- 4312 (ILK-KD). and domain kinase C-terminal two-hybrid a and yeast ARD) to shown in Montanez 2002; previously 2008). al., al., protein kindlin et et FA (Mackinnon assays essential particular with immunoprecipitation of an is is not ILK associate it was 4A). protein, as (Fig. FA GST–K2-F2PH interest another by This vinculin, down 4A). lysates. as pulled (Fig. kindlin-binding cell specific ILK CHO is known endogenous with interaction of including pulldown assays revealed pulldown proteins, proteins, in candidate bait as are Testing from it that (K2-F2PH) used kindlin-2 F2PH and of recombinant kindlin-2 the domain purified F2PH the we GST-tagged targeting, for FA in partners involved potentially binding identify To domain kinase PH the ILK with the of interacts of directly F2PH outside Kindlin-2 interactions to effect. this points mediating and in that domain targeting determinant show isoform-specific to FA strong first a the of bears is still whereas work subdomain (K3-K2PH) our F2PH FAs together, the domain Taken to PH target. to kindlin-2 targeted failed the efficiently containing still kindlin-3 (K2-K3PH) domain L scmoe fa -emnlakrnrpa oan(ILK- domain repeat ankyrin N-terminal an of composed is ILK nvivo In .coli E. ,the w-yrddt Mcinne l,20;Qdt ta. 02 and 2012) al., the et with Qadota yeast 2002; not with al., consistent et and (Mackinnon is complex data This two-hybrid 4D). ILK-KD (Fig. the alone domain with parvin-CH2 down domain K2-F2PH pulled The only GFP–K2-F2PH. expressing with cells incubated GST– of and lysates GST, beads glutathione–Sepharose alone. were on proteins immobilized His–parvin-CH2 parvin-CH2 plus we GST–ILK-KD bound produce or parvin-CH2, parvin-CH2 kindlin cannot of absence whether we the Although tested in ILK-KD 4C). recombinant (Fig. soluble complex ILK-KD interferometry the dissociation apparent complex. bio-layer 4.4 an estimated of ILK-ARD Using constant we the Sciences) GST. Life not Pall bound (BLItz, but complex GST–K2-F2PH complex 4B, Neither ILK-KD Fig. the in a shown bound down As pull complex. to PINCH1-LIM1 GST–K2-F2PH used ILK-KD– we purified direct whether is test interaction and ILK 2009). the al., in et domain 2012). kindlin-interacting Yang 2009; the al., identify Wu, et To Fukuda 2008; and al., al., in et et (Chiswell Qin Chiswell partners 2010; ILK-KD binding 2013; respective or their al., with purification ILK-ARD et CH2 of PINCH, (Ghatak C-terminal of the Production parvin with domain of associates LIM domain domain N-terminal kinase the the whereas with associates ARD ora fCl cec 21)17 3842 doi:10.1242/jcs.155879 4308–4321 127, (2014) Science Cell of Journal he needn xeiet.Saebr:10 bars: Scale experiments. independent three agdknln n hmrstre oFAs. to target chimeras and kindlins tagged to kindlins chimeric of FAs. Binding to (F,G) target chimeras and kindlins n GFP-tagged the which fibronectin. ( in on cells transfected plated of cells Percentage CHO (E) in chimeras vinculin kindlin endogenous GFP-tagged and of localization FAs. Subcellular to (D) target chimeras and kindlins n GFP-tagged the which in nCOclspae nfboetn I ecnaeof Percentage (I) ( fibronectin. cells on transfected vinculin plated endogenous cells and CHO chimeras in PH kindlin GFP-tagged of ( fibronectin. ( on cells transfected plated of cells Percentage CHO (C) in GFP-kindlins vinculin of endogenous localization and Subcellular (B) chimeras. kindlin isoform-specific drives subdomain differences. F2 targeting FA kindlin The 3. Fig. yae ** lysate. input of percentage a (mean as expressed and quantified b 5 5 YA nerntiswsasse yimmunoblotting, by assessed was tails integrin 1Y/A) 6–8 rmtreidpnetexperiments. independent three from 160–482 experiments. independent three from 123–162 6 0.8 6 s.e.m.; P a , pri-H ope n nILK-ARD– an and complex -parvin-CH2 m .1(Student’s 0.01 ewe h idi- 2Hdmi and domain F2PH kindlin-2 the between M n 5 6 ) h %ln niae %o input of 3% indicates lane 3% The 5). 5 ofdneitra)i hc h GFP- the which in interval) confidence 95% A ceai ersnaino the of representation Schematic (A) t ts) H uclua localization Subcellular (H) -test). 6 6 a 5 ofdneinterval) confidence 95% 5 ofdneinterval) confidence 95% IIb, .coli E. b and 1 n 5 2–0 from 120–400 m eursco- requires m. b Y795A 1

Journal of Cell Science EERHARTICLE RESEARCH 32 hmr on rfrnilyt h iia IPP K2232 minimal non-targeting the the and to FA-targeting kindlin-3 the preferentially and whereas kindlin-2 bound 5E,F), ILK-KD, (Fig. chimera the with K3323 with 2013). saw al., complexed we et ILK, As (Stiegler full-length minimal His–PINCH1-LIM1 a and GST-tagged using His–parvin-CH2 of obtained were complex ILK by results determined IPP Hence, Similar is region. and 5D). F2PH isoforms (Fig. between the complex chimera differs efficiently kindlin more K2232 ILK-KD to binding chimera non-targeting we the K3323 the when FA-targeting than swap-chimeras, the Notably, down F2PH 5C,D). pulled (Fig. the kindlins examined less full- GFP-tagged with significantly obtained were down length results pulled Similar complex 5B). complex ILK-KD (Fig. ILK-KD GFP–K3-F2PH the the bound assays. 5A), pulldown subdomains (Fig. in F2PH complex both GST–ILK-KD Although the to compared binding and cells their CHO in from (K3-F2PH) were GFP–kindlin-3-F2PH kindlin-3-F2PH and we recombinant Unfortunately, soluble -3. coli purify and to kindlin-2 the explain unable compared of we could isoforms, binding binding kindlin ILK-KD of ILK targeting in FA in differences differences properties whether ILK-binding evaluate their in To differ -3 and Kindlin-2 domain. kinase direct ILK a the mediates region with F2PH interaction kindlin-2 the that suggests strongly D. and B C A, parvin for with shown complex with in are Pr coated GST–ILK-KD GraphPad 3%) Biosensors or in or domain, regression GST parvin-CH2 nonlinear (5% BLItz GST-tagged by Inputs the to staining. curve GST, immunoblotting. a control, immobilized Blue to to by a Coomassie fitted lysates assessed and as by cell subtracted was CHO parvin-CH2 assessed was from of was alone GFP–kindlin-2-F2PH GST or GST–kindlin-2-F2PH of to Binding Binding complex, or (D) temperature. GST solu ILK-KD–parvin-CH2 room purified immobilized the at of to measured of binding was Direct domain concentrations GST–kindlin-2-F2PH (B) parvin-CH2 increasing staining. + S of Ponceau ILK-KD Binding by or (C) assessed domain was loading domain. PINCH-LIM1 protein kinase + GST ILK ARD (IB). immunoblotting to by directly assessed was binds GST–kindlin-2-F2PH F2PH Kindlin-2 4. Fig. eteeoeepesdGPknln2FP (K2-F2PH) GFP–kindlin-2-F2PH expressed therefore We . A idn fedgnu L n iclnfo H ellstst moiie S or GST immobilized to lysates cell CHO from vinculin and ILK endogenous of Binding (A) E. hmr on eyltl.Tu,w aeietfe kindlin- a and targeting identified FA have binding. both ILK we dictates that Thus, determinant little. isoform-specific very bound chimera 9 u o nK1 in not K1 in but (present domain 499 PH the ( preceding 2012) linker PH amino-acid al., defined et structurally ( (Yates the the domain only domain lacking PH separated deletion the that smaller including another residues subdomains kindlin-1 all different F2 lacking two split kindlin tested one in then implicated constructs: we is 2011), deletion domain al., PH et kindlin (Qu the function subdomains As F2F3 6A). C-terminal As with (Fig. the 2012). interact did whereas not al., complex, did et ILK-KD subdomains Yates the GFP– F0F1 2009; N-terminal generated al., the previously et expected, (Goult expressing fragments cells kindlin-1 the from incubated kindlin, lysates and within beads with on site immobilized ILK-binding was the complex GST–ILK-KD map precisely more To is binding region ILK F2PH for kindlin the required within residue leucine conserved A Fg B.Asqec lgmn fti rvosyuncharacterized linker previously this this GFP– of alignment lacking the sequence GFP–kindlin-2 A 6B). down not (Fig. pulled but alone, complex linker GST–ILK-KD kindlin-2 the as 6A). (Fig. binding, dispensable was itself domain PH the whereas nknln2 hslne sas eesr n ufcetfrILK for sufficient and necessary also is linker this kindlin-2, In ora fCl cec 21)17 3842 doi:10.1242/jcs.155879 4308–4321 127, (2014) Science Cell of Journal D 2–9)wsrqie o L binding, ILK for required was 326–495) D 6–9) efudta h 38- the that found We 363–499). D 2–9)and 326–495) l ILK- ble D 4313 363– ism. H2

Journal of Cell Science EERHARTICLE RESEARCH 4314 in GFP–kindlin-2 the residues within mutation additional L357A mutating a Introducing region. between this by interaction ILK the and map kindlin-2 a to kindlin-2mammalian attempted full-length not and therefore or did We kindlins linker kindlin-2) kindlin-2 6D). human human (Fig. the three in to binding (S351V all ILK site in inhibit this serine at a substitution is valine residue This to 2012). reported the been binding has for key UNC-112) be ILK-KD in 6B). (D381 (Fig. UNC-112 the otholog bind the kindlin of bind not region this did in residue to acid region aspartic an Interestingly, N-terminal sufficient the were whereas complex, region, and conserved species between more C-terminal conserved The 6C). region (Fig. middle isoforms a revealed linker .elegans C. L rhlgPT4(aoae al., et (Qadota PAT-4 ortholog ILK , 5aioais nldn the including acids, amino 25 .elegans C. ehrs ed,adc-rcpttn noeosIKwas with cells, co-immunoprecipitated ILK ILK endogenous HEK293T immunoblotting. co-precipitating by in and detected beads, overexpressed M2-coated anti-FLAG Sepharose using we lysate were cell L334 from immunoprecipitated on kindlins depends tagged nonetheless FLAG- ILK experiments. co-immunoprecipitation kindlin-3 of low series the a performed for whether test is seen To it Notably, (L334). binding kindlin-3 6C). 6E), (Fig. in (Fig. species conserved and also isoforms complex across conserved is IPP ILK. full-length with minimal interaction for importance to its underscoring abrogated binding mutation L357A the Moreover, GFP–kindlin-2 6D). (Fig. binding ILK of loss dramatic a in resulted GFP–kindlin-2 full-length within or linker ossetwt t motnei L idn,knln2L357 kindlin-2 binding, ILK in importance its with Consistent ora fCl cec 21)17 3842 doi:10.1242/jcs.155879 4308–4321 127, (2014) Science Cell of Journal oca tiig inputs staining; S Ponceau by assessed was proteins GST 3)aeas shown. also are (3%) (Student’s ** mean ( (F) in quantified and (E) immunoblotting by assessed was IPP minimal or GST immobilized using lysates cell CHO from kindlins GFP-tagged of mean ( (D) quantified and (C) immunoblotting by assessed was CH2 parvin- + GST–ILK-KD or GST immobilized by chimeras -3 and and -2 GFP–kindlin-1, of A n uniidi B ( (B) mean in quantified and (A) immunoblotting by assessed was parvin-CH2 + KD GST–ILK- or GST immobilized by lysates cell CHO from F2PH GFP–kindlin-3- or 2-F2PH GFP–kindlin- of Pulldown binding. (A,B) ILK in differ kindlin- 3 and Kindlin-2 5. Fig. P , .1 *** 0.01, 6 6 6 ....* s.e.m.). ....(,)Pulldown (E,F) s.e.m.). ....(,)Pulldown (C,D) s.e.m.). t ts) odn of Loading -test). P , n § P 0.001 n , 5 3; 0.05, 5; n 5 3;

Journal of Cell Science EERHARTICLE RESEARCH N-1/idi aiiae N-1/idi neato with interaction UNC-112/kindlin PAT-3/ facilitates than UNC-112/kindlin difference. this better for responsible ILK is F2PH binds the same kindlin-2 and the does involves and but kindlin-3 interaction kindlin-2 pulldown residue, the both leucine and in cells Thus, conserved in 6F,G). observed ILK (Fig. bind previously ILK kindlin-3 purified chimeras had with K3323 we assays gain-of-function what 6F,G). and recapitulated (Fig. the with K2232 residue co-immunoprecipitation ILK alanine loss-of-function endogenous an of ILK co-immunoprecipitation with Moreover, kindlin-3) inhibited in leucine L334 conserved strongly the kindlin-2, of replacement in cases, (L357 both In 6F,G). (Fig. FLAG–kindlin- with 3 extent) lesser much a (to and FLAG–kindlin-2 thsbe eotdta in that reported been has It b nern(aoae l,21) eteeoecompared therefore We 2012). al., et (Qadota integrin .elegans C. A-/L idn to binding PAT-4/ILK , h idn fwl-yeadL5Amtn idi- to kindlin-2 mutant L357A and wild-type of binding the a togyipie Fg A,idctn htIKbnigis binding ILK that indicating GFP–kindlin-2-L357A 7A), of (Fig. targeting impaired FA FA strongly kindlin-2 cells. was in GFP–kindlin- CHO defective in involved ILK-binding 2-L357A is the expressed binding we ILK targeting, whether evaluate targeting FA To kindlin-2 aids binding ILK h idi- 3ceryihbtdbnigto binding within inhibited mutation clearly W615A the F3 2008), kindlin-2 al., Ma et 2009; the Montanez al., et 2008; (Harburger al., reported et previously As tails. integrin osntngtvl mattemmainkindlin-2–integrin effect mammalian no the binding had impact ILK interaction. of negatively binding disruption not that ILK conclude does therefore abrogates We 6H). that (Fig. mutation L357A ora fCl cec 21)17 3842 doi:10.1242/jcs.155879 4308–4321 127, (2014) Science Cell of Journal % %ipt B immunoblot. IB, input; 3% 3%, lysates. cell HEK293T from chimeras and mutants isoforms, kindlin FLAG-tagged and ILK endogenous of Co- (IP) (F) immunoprecipitation (E). IPP minimal or immobilized GST to or (D) parvin-CH2 + GST-ILK-KD or GST immobilized to truncations/mutants kindlin-2 GFP-kindlin-2, and of binding of analysis Immunoblot are (D,E) L357 highlighted. kindlin-2 human and UNC-112 D382 shaded. are (right) PH domain inserted and (left) subdomain the elegans and kindlins human region three linker the in F2 the of amino sequence the acid of Alignment S (C) Ponceau staining. by assessed was loading protein GST immunoblotting. assessed by was parvin-CH2 + GST– ILK-KD or GST immobilized to cell lysates CHO from truncations and/or deletions kindlin-2 or GFP–kindlin-1 binding. for residue crucial leucine a of identification ILK– and the interaction of kindlin-2 mapping Fine 6. Fig. idn oprfe nerntails (mean integrin purified to binding mutants kindlin-2 or GFP–kindlin-2 (Student’s (mean experiment each in kindlin-2 co- FLAG– ILK with of immunoprecipitated amount the to normalized are Results kindlins. tagged FLAG- with immunoprecipitated co- of ILK amount endogenous the of Quantification (G) 6 6 s.e.m., s.e.m., rhlgUC12 h F2 The UNC-112. ortholog t ts) H uniiainof Quantification (H) -test). n n § § b 5). *** 3). al,btthe but tails, 1 AB idn of Binding (A,B) P , 0.001 4315 C. b -

Journal of Cell Science n akdtecnrlylctdfbilrahso eni ILK in seen adhesion fibrillar located centrally the lacked and EERHARTICLE RESEARCH 4316 and kindlin-2 ILK GFP-tagged in cells, FAs CHO to in targeted observed K3323 As cells. (ILK ILK-knockout ILK- several in generated K3323 of We localization cells. the assessed deficient chimera we effect, K3323 targeting Furthermore, is binding the FAs. effect. ILK gain-of-targeting that into the to illustrating for 7A), mutation important (Fig. kindlins targeting L357A its of abolished the targeting introducing for important idi- i o Fg C.Hwvr 32 agtn was targeting K3323 However, 7C). (Fig. not did kindlin-3 ILK newborn immortalized infecting by lines clonal el pedadfre iclnrc A hnpae on plated when ILK that al., FAs noticed et (Elad we vinculin-rich reports other 2013), with formed agreement levels in However, and kindlin-2 fibronectin. endogenous spread ILK, and cells of PINCH1 by absence diminished led proteins the also also ILK-interacting this In 2008), the 7B). al., et (Fukuda (Fig. of Immunoblotting Wang reported 2007; loss al., previously et to recombinase. Gkretsi as 2003; and, al., et adeno-Cre knockout ILK confirmed with fibroblasts safrhrts fteiprac fIKi h gain-of- the in ILK of importance the of test further a As , 0 Fg B.Mrhlgcly ILK-null Morphologically, 7B). (Fig. 50% 2 / 2 el a otyprpea FAs peripheral mostly had cells fl/fl irbat,weesGFP- whereas fibroblasts, fl/fl a -parvin mouse 2 / fl/fl 2 ) ihti,atog noeosknln2wsawy eetdin detected always Consistent ILK was in level. kindlin-2 FAs reduced endogenous much although a this, at with FA albeit ILK-independent fibroblasts, aids an in in binding manner accumulate ILK can although GFP–kindlin-2 Thus, targeting, 7D). (Fig. GFP–kindlin-2 bandi w te needn ILK independent other two in obtained Fg C.W lontcdta,i otatwt htwe ILK what of with absence contrast some ILK the in in displayed targeting in GFP–kindlin-2-L357A that, FA cells, weak noticed FAs CHO also in to observed exhibited We targeting GFP–kindlin-2 7C). clear, (Fig. that but observation weak, surprising initially nieyls nILK in lost entirely fe ltn u a eetdi A 1hps-ltn Fg 7E). (Fig. ILK post-plating in h levels 21 kindlin-2 FAs of in reduction detected The was but plating after idn srsosbefrtegi-ffnto ofre by conferred gain-of-function of kindlin-3. the presence into for F2PH the kindlin-2 responsible introducing in ILK that is establish even data our binding targeting together, Taken 7C). FA (Fig. FLAG–ILK in defective L357A K3323 remained point-mutated the whereas FLAG–ILK, expressing agtn fGPK33wsrsoe nILK in restored was GFP–K3323 of targeting ntecus forsuiso ILK of studies our of course the In ora fCl cec 21)17 3842 doi:10.1242/jcs.155879 4308–4321 127, (2014) Science Cell of Journal fl/fl el,i a adydtcal nILK in detectable hardly was it cells, irnci.Saebr:10 bars: Scale fibronectin. n ILK and ILK in chimeras GFP-tagged and of kindlins localization ILK Subcellular in (C) vinculin fibroblasts. and PINCH kindlin- parvin, ILK, endogenous 2, fibronectin. of analysis on (IB) plated Immunoblot cells (B) CHO in mutants chimeras kindlins, and GFP-tagged and vinculin to endogenous kindlin-2 FAs. for to required target is efficiently binding ILK 7. Fig. ltdo irnci.We pcfe,ILK specified, When fibronectin. on plated idi- n iclnb muoloecnei ILK in immunofluorescence by endogenous vinculin of and Detection kindlin-2 (E) fibronectin. on plated idis uat n hmrsi ILK in chimeras and GFP-tagged mutants and kindlins, vinculin endogenous of Subcellular localization (D) were immunofluorescence. ILK by and visualized Vinculin FLAG–ILK. with co-transfected 2 / 2 2 / 2 el Fg C.Smlrrslswere results Similar 7C). (Fig. cells 2 el,4had2 fe ltn on plating after h 21 and h 4 cells, / 2 el,smlrt hto wild-type of that to similar cells, 2 A uclua oaiainof localization Subcellular (A) 2 m / 2 2 m. fl/fl / / 2 2 ie.Ipraty FA Importantly, lines. n ILK and el Fg B might 7B) (Fig. cells el,w aethe made we cells, 2 fl/fl 2 / 2 2 / n ILK and 2 / 2 2 2 el yco- by cells fibroblasts / / fibroblasts 2 2 el were cells el h 4 cells 2 / 2 fl/fl

Journal of Cell Science euto fedgnu idi- eespriskindlin-3 permits levels kindlin-2 endogenous of Reduction of FAs the but in found localization was kindlin-2 kindlin-2 in endogenous ILK some reduction that this fact of the some for account ARTICLE RESEARCH utemr,GPknln2L5Aas agt oFsin FAs kindlin-3. to of targets targeting also FA GFP–kindlin-2-L357A of facilitates we reduction Furthermore, time, GFP– Thus, kindlin-2 first 8C). the expected, (Fig. for endogenous targeting but, As GFP–kindlin-3 cells these clear levels. in saw spread FAs kindlin-2 to partially targeted endogenous cells kindlin-2 this in low localization on GFP–kindlin be with assess 8B). Focusing to also (Fig. us adhesions shown). could allowed kindlin-2 cells population not partial in these of in (data the levels staining adhesions exhibited detected to low within localization kindlin-2 retain ILK cells that Weak weak they that some noted very suggesting we with population However, spreading cell targeting FA cells. assess knockdown to knockdown kindlin-2 difficult of fibroblasts it loss in making 2008), spreading knockdown al., cell et impairs (Montanez Our reported previously FAs. GFP– as permit in would only exhibited 2013) in localization kindlin-2 al., endogenous et kindlin-3 down (Brahme we knocking fibroblasts does, whether NIH3T3 kindlin-3 test than efficiently to more aimed ILK binds kindlin-2 As FAs to targeting icmtne,knln2cntre oFsi nILK- an in FAs certain to in adhesions, target to can manner. independent kindlin-2 kindlin-2 of circumstances, localization efficient 2 / 2 el ugssta,atog L sipratfor important is ILK although that, suggests cells , 5 eiulknln2(i.8) Unfortunately, 8A). (Fig. kindlin-2 residual 15% idn otiuet h Alclzto fkindlins. ILK of integrin localization and and FA ILK integrin the both to that GFP–kindlin-2- both suggest contribute data in binding double-mutant These competing 8C). impaired the (Fig. of is binding of not which absence not targeting L357A,W615A did is we the FA however, binding cells; observe GFP–kindlin-2-W615A in kindlin-2-knockdown ILK surprise, in targeted our targeting also that To FA kindlin-2. confirming endogenous for 8C) (Fig. essential cells these idi- n 3rsu of been compared rescue therefore also We -3 have 2013). and al., activation kindlin-2 et activation, in (Calderwood integrin FAs. in described roles to differences key targeting play kindlin isoform-specific kindlins differential three ILK all the in Although to differences isoform-specific kindlin contribute in that binding binding reveal ILK and for targeting role FA a demonstrate data preceding The kindlin-2-mediated activation for integrin important is binding ILK ncdw I33fbolss(rhee l,21) The assessed 2012b; used al., We widely 2013). et 2004b). a (Bouaouina Calderwood, activation provides al., integrin ligand of soluble et readout bind functional to (Brahme integrins of fibroblasts ability NIH3T3 knockdown idn faslbeboiyae rgeto h endogenous the of fragment biotinylated soluble a measures of which assay, binding flow-cytometry-based well-established a a n‘ciainidx hti omlzdfor normalized is that index’ calculated ‘activation we an conditions, experimental across activation compare ees nareetwt ulse aa(otnze l,2008), al., et (Montanez data published with agreement In levels. 5 b iadfboetn(N-1 Buoiae l,21b.To 2012b). al., et (Bouaouina (FN9-11) fibronectin ligand 1 ora fCl cec 21)17 3842 doi:10.1242/jcs.155879 4308–4321 127, (2014) Science Cell of Journal a 5 a b 5 10 (Student’s *** * mean represent graphs Bar cells. transfected in assessed was levels and activation integrin and kindlins or GFP-tagged GFP with transfected were fibroblasts kindlin-2-knockdown or control Scramble (F–H) b to normalized and cytometry flow by measured index, activation Integrin (E) flow cytometry. by measured levels surface (D) h. 5 for fibronectin on plated cells shRNA K2 in vinculin endogenous of and kindlins, GFP- tagged or GFP of localization Subcellular (C) imaging. for selected were cells knockdown kindlin-2- Spread fibroblasts. NIH-3T3 kindlin-2-knockdown and control scrambled in vinculin and kindlin-2 endogenous of staining Immunofluorescence (B) cells. NIH3T3 shRNA) (K2 kindlin-2-knockdown and SC) (scrambled, control parental, in vinculin and of kindlin-2 analysis (IB) Immunoblot (A) activation. integrin for required is interaction Kindlin-2-ILK 8. Fig. ciaini kindlin-2- in activation 1 P b tiig( staining 1 , P nernatvto using activation integrin 1 m , .5 ** 0.05; m. .0;n,ntsignificant not ns, 0.001; b t ts) cl bars: Scale -test). P nernsurface integrin 1 n , 5 0.01; 4). 6 b s.e.m.; 1 b b a integrin 1 surface 1 5 n b a § 1 5 4317 b b 3; 1 1

Journal of Cell Science eosre infcn eraei surface in decrease significant a observed we ARTICLE RESEARCH 4318 these and binding with screen exhibit hybrid ILK isoforms function. and and kindlin localization integrin on (3) impact in and differences its exert differences activation; to kindlin-2 integrin marked for to on crucial integrin, binding is (2) role ILK–parvin only subdomain; and F2PH a not integrin through its both complex, within ILK to located the to region binding also linker but subdomain, requires F3 kindlin-2 its FAs through optimal (1) and at that: ILK-knockout accumulation report in for we studies cells responsible ILK– integrin functional kindlin-2-knockdown the the and subdomain of in interaction kindlin and analysis kindlin biochemical kindlins the combining localization, sites Here, of isoform-specific of understood. kindlin mapping adhesion poorly roles chimeric remained control at the have that activation underlying retention to these interactions signaling mechanisms for and/or recruitment integrin the important kindlin recruitment in likely However, and is roles processes. structures binding, key adhesive ligand integrin-rich play by of so also integrin do downstream might trigger Kindlins they to 2013). and 2013) talin isoform-specific al., clustering have with et also (Calderwood cooperate activation kindlins Kindlins implicated but is kindlins functions. activation mammalian three integrin the (Calderwood of in proteins Each 2013). regulatory al., integrin et essential are Kindlins DISCUSSION H ln antspotknlnbnigad oehrwith together and, binding kindlin Parvin- support complex. mammalian cannot ILK-KD–parvin-CH2 direct alone the show CH2 now to We binding 2008). al., kindlin al., et et (Mackinnon Montanez cells 2002; mammalian in co-immunoprecipitation in this decrease for significant allowing a Even see 8D). (Fig. we cells reduction, shRNA kindlin-2 F–idi xrsina eceppltoswr l ae to gated all in were differences populations to due rescue not as GFP– is expression This with 8F). GFP–kindlin of (Fig. not overexpression cells did whereas GFP–kindlin-3 activation, kindlin-2-knockdown integrin restored the Importantly, kindlin-2 8E). of (Fig. cells reconstitution kindlin-2-knockdown in activation nbet usiuefrknln2i uprigatvto of activation supporting in into kindlin-2 takes for index fibroblast substitute activation to the unable and in levels changes any GFP account same the have htdfeecsi L idn otiuet functional to contribute binding ILK kindlin-3. is and binding kindlin-2 in between ILK differences that differences suggest show and activation, that data integrin kindlin-2-mediated our not for together, important did Taken GFP–K3323-L357A 8H). levels whereas (Fig. to GFP-kindlin-2, activation with integrin restored seen also ILK-binding chimera the kindlin Furthermore, K3323 activation. integrin interaction kindlin-2–ILK for activation. the important that integrin is evidence kindlin-mediated first the and for provides ILK This kindlin-2 both necessary to W615A binding are that and indicating integrin 8H), L357A (Fig. increased not the did GFP–kindlin-2 mutants whereas contrast, In activation, binding on ILK. integrin relies not levels levels, surface but integrin that integrins on surface showing kindlin-2 8G), of kindlin-2- effect (Fig. integrin the not did GFP–kindlin-2 reconstituted rescued GFP–kindlin-2-W615A kindlins. whereas GFP–kindlin-2-L357A we point-mutated with and activation, cells knockdown integrin mediated nIKknlnitrcinwsfrtietfe nayattwo- yeast a in identified first was interaction ILK–kindlin An ots hte L idn sipratfrkindlin-2- for important is binding ILK whether test To a a 5 IIb b 1. b nern Fiesne l,21;Y tal., et Ye 2011; al., et (Feigelson integrins 3 .elegans C. b ufc xrsin hs idi- is kindlin-3 Thus, expression. surface 1 rtis n a envldtdby validated been has and proteins, b nern in integrins 1 b integrin 1 b 1 fIKi ILK in ILK of ihK33i o bevdILK observed not is K3323 with h 3sboanadi nfetdb L idn.Tu,a least at by Thus, binding. determined ILK is by unaffected binding is and integrin in subdomain that F3 differences the find isoform-specific we binding, observe integrin we although PAT-3/ However, in to that proposed binding been 112/kindlin has it and elegans integrin targeting, triggers both FA for and binding FAs. important activation in also ILK retention are how interactions kindlin Kindlin–integrin or unclear for activation ILK- is and integrin data kindlin- interactions kindlin-mediated It our known phenotypes. with ILK–kindlin Thus, keeping deficient in of shown). are activation importance (not integrin integrins the Consistent cell-surface establishing 2008). al., active an et have (Friedrich Tucker fibroblasts in is ILK-knockout 2009; activation our al., ILK integrin this, et with normal Honda and 2004; for al., activation required et be integrin a to is of known Kindlin activation. regulator integrin that for suggest well-established important also we is cells, integrin binding the kindlin-2-knockdown ILK rescue in to defect chimeras kindlin activation some is and kindlins site mutant integrin-binding of their absence that the providing FAs, in intact. to Indeed, target subdomain. integrin- kindlins can the F3 ILK-binding-defective to kindlin-2, the endogenous due competing likely in is site cells binding ILK-knockout localization FA in ILK residual kindlin-2 cells, the and of some required in absolutely least not is at binding Nonetheless, kindlins. mutant L357A hmrcknln,adwt L-idn eetv mutants, defective ILK-binding with FA and in significance kindlins, with studies functional FA-targeting chimeric its Our activation. for integrin and evidence localization first ILK-binding the which provide altered unclear specificity. isoform still for with is responsible it are in kindlins Therefore, residues not shown). kindlin but yield not -2, (data and not properties kindlin-1 did in conserved targeting kindlin-3, residues studies linker mutational F2 initial the Our specific. ILK are kindlin-3 weak, an interactions although by that, ILK ILK demonstrates to linker for mutation binding kindlin-3 L334A required F2-PH of is inhibition the that and kindlin-2. kindlin-3) in binding, in complex to L334 residue and than leucine kindlin-2 in conserved kindlin-3 (L357 consistently a to identified binding ILK, as We ILK well as endogenous less constructs, demonstrated ILK with of ILK–parvin-CH2, variety a for co-immunoprecipitation to with -3 assays and inability pulldown kindlin-2 Our but of us affinity well. prevented the kindlin-3 less comparing of binds fragments three recombinant kindlin-3 all soluble produce although ILK, Notably, bind kindlin-2. of kindlins subdomains PH F2 split the a N-terminal and at kindlin–ILK-KD the between occurs direct linker binding a uncharacterized complex is previously ILK–parvin there kindlin, that On suggests interaction. Qadota this 2002; 2012), al., al., et (Mackinnon et data two-hybrid yeast published obn L.Hwvr diinleiec uprsthe (1) supports targeting: is kindlin-2 FA evidence expressed ILK localization in exogenously in impaired and additional endogenous interactions FA of However, kindlin–ILK targeting testing kindlin the of ILK. of from ability importance bind recapitulate the us ILK diminishing retaining when to mutants kindlin-binding-defective preventing or observe we localizing no identified, phenotypes such in yet, been whether ILK As have for FAs. mutants role at a kindlins support strongly ae nteiaiiyo idi-,ILK-binding-defective kindlin-3, of inability the on Based nadto ocaatrzn h idi–L neato,we interaction, kindlin–ILK the characterizing to addition In ora fCl cec 21)17 3842 doi:10.1242/jcs.155879 4308–4321 127, (2014) Science Cell of Journal A-/L idn oUC12knlnrgltsUNC- regulates UNC-112/kindlin to binding PAT-4/ILK 2 / 2 2 / 2 el etrsF agtn fwl-yebtnot but wild-type of targeting FA restores cells irbat,()tegi ffnto observed function of gain the (2) fibroblasts, b 2 nern(aoae l,2012). al., et (Qadota integrin / 2 el,ad()re-expression (3) and cells, , 5 reduction 75% C.

Journal of Cell Science idi- nitgi ciaini irbat ihtebroad the with fibroblasts in activation integrin activation. or integrin on on recruit impact kindlin-3 to might ILK of ILK also ability might that the action into scaffolding suggesting play This in proteins. 2012) FA increase multiple al., stabilize an of targeting et from FA to (Stiegler contributes distinct also ILK mechanism binding. integrin a integrin and through localization FA activation kindlin-2 regulates ILK mammals, in ARTICLE RESEARCH ecie Bam ta. 03 i ta. 03 aaaae al., et previously Nakazawa 2013; as al., et grown Seiji Liu were (from 2013; al., cells Japan) et CMK University, (Brahme and described Osaka EA.hy926 NIH3T3, Tadakoro, HEK293T, CHO, transfection and culture Cell al., 2013). et al., (Bandyopadhyay et described hairpin Brahme previously and short 2012; as Sigma produced kindlin-2 from library-based was purchased and lentivirus TRC were SHC002) plasmids sequencing. (TRCN000019185) no. DNA (sh)RNA by (catalog pET-32- verified into scramble subcloned were lentiviral and ILK-KD inserts PCR by GST-tagged All by tag. TT. pET32 generated bacterial introduced GST was modified N-terminal 175–452) a For were an (residues or with pGEX-4T3 mutations (pET32-TT) (Stratagene). into vector cloned Point were mutagenesis kindlins 552–663. expression, site-directed 255–551, F3 F2PH 1–254, QuikChange F0F1 346–478, kindlin-3, 278– 571–680; F2PH PH F3 1–277, F0F1 369–501, kindlin-2, PH PCR 568–677; 570, vector F2PH F3 overlap 1–275, pEGFP-C1 366–498, F0F1 splice kindlin-1, PH the boundaries: 276–567, by acid into amino generated the following the subcloned were from using chimeras and purchased Kindlin was PCR (Invitrogen). 3677274) by (encoding clone amplified DNA domain as IMAGE were form ATTC, 1–663; PINCH1-LIM1 2013) short al., acids kindlin-3 et the Stiegler human amino 2012; and The al., described. et ILK-ARD, Stiegler previously 2008; and al., His-FLAG-tagged et ILK or (Chiswell His-tagged domain, al., GST-tagged, et CH2 (amino the (Harburger 1–680) kindlin-1 and acids (amino 2009) human kindlin-2 human GFP-tagged and 1–677) acids N-terminally encoding Vectors Constructs were antibodies secondary purchased. (Li-Cor) IRDye-conjugated and (Proteintech (Invitrogen) PINCH1 Signaling), and (Cell Group) ILK kindlin-2 (Rockland), AbCam), GFP and (Sigma), (ProteinTech vinculin complex against antibodies Primary ILK–kindlin Antibodies the METHODS studies. AND future the MATERIALS of which subject and the be The accumulation, in will adhesions at FA isoforms. participates pathways kindlin integrin kindlin influences kindlin-mediated signaling regulates and between ILK activation which differences by mechanisms functional contribute binding ILK in to activation. differences that integrin reveal for we required Furthermore, are time interactions first kindlin–ILK the for that establish and targeting, FA kindlin-2 for important integrins. hematopoietic regulate with to interactions complex require IPP not might the kindlin-3 2008), al., kindlin-3-knockout et platelets (Tucker ILK-knockout of blood-cell-specific and phenotypes 2008) al., Alternatively, et in (Moser activation. difference integrin the trigger considering efficiently that to to binds ILK requires addition kindlin-3 kindlin-3 with in that that be interactions might find hematopoietic-specific it we additional ILK, to and specifically 2008) but platelet weakly al., of et activation for (Tucker important al., also integrins et is Moser ILK 2009; as al., However, et 2008). for (Moser cells required hematopoietic in is activation kindlin-3 that evidence nsmay ehv hw htIKcmlxbnigis binding ILK-complex that shown have we summary, In of effect of lack the reconcile to how unclear currently is It a pri Cl inln) n Alexa-Fluor-conjugated and Signaling), (Cell -parvin b 1, b and 2 b integrin 3 b a -parvin -Parvin -ncdw n oto I-T el eegnrtda previously as 2013). generated al., were et cells (Brahme NIH-3T3 described control MW Kindlin- and Polyethylenimine, Lonzabio). (Amaxa, 2-knockdown (Linear nucleofected PEI or Inc.), with Polysciences, 25,000, transfected were Cells 2013). od4 aaomleyecnann .%Tio -0 o 0min, 30 NH for mM with X-100 50 h Triton permeabilized with 24 blocked 0.1% and At and containing FBS. fixed quenched paraformaldehyde 0.1% simultaneously containing 4% were medium cold cells in plating, plated fibrinogen were after When BSA. cells 2% used, with was blocked and (Sigma), coated fibrinogen coverslips plasma glass on plated 10 were with cells nucleofected targeting or FA Transfected of scoring and Immunofluorescence to according from performed were gift GFP- lines experiments guidelines. h, cell (a approved animal clonal 48 10 All promoter than After established. more CT). same and were sorted repeated Haven, single-cell the were New cells through co-expressing University, under expressing adenovirus Yale immortalized GFP Yang, an flox/flox and Xiaoyong and with ILK infected Recombinase 2003) new-born then Cre a al., were of Cells et skin passaging. the (Terpstra from mouse isolated were Fibroblasts fibroblasts ILK-knockout of Generation odn a eiidb tiigterlvn oto fteglwith gel the of membrane nitrocellulose portion the S. Bead staining relevant Ponceau control. by the with or internal stain an staining ‘input’ protein to the Blue by relative of Coomassie verified binding intensity of of was band percentage quantification the a loading a on as For as expressed (based or was input (Li-Cor). interest lane) total of the Lite protein of bound Studio the percentage analyzed of Image intensity and immunoblotted, the (Li-Cor) or binding, System conditions, Imaging ImageJ were Infrared reducing proteins using Odyssey Bound in an 2007). on previously al., SDS-PAGE imaged et as by Lad lysates 2013; cell fractionated al., CHO et from (Brahme performed described were assays Pulldown Tobacco assays the Protein-binding with removed was tag kept GST were protease. the virus Proteins or Etch Healthcare). beads on (GE immobilized beads was glutathione–Sepharose GST–kindlin-2-F2PH on 2013). in produced al., was parvin-CH2 et 2007; previously al., Stiegler et as in 2012; Lad produced purified 2009; al., al., and et et Harburger produced Stiegler 2008; al., were et complex (Chiswell described IPP minimal and GST–kindlin-1, GST, GST– tails, cytoplasmic integrin His-tagged Recombinant purification and production Protein i betv.Iae ipae nfgrsaeivre ohglgtFAs. highlight to inverted are figures in displayed a Images with objective. were equipped microscope Cells oil TI (Invitrogen). Nikon reagent a anti-fade using methods Gold visualized immunofluorescence ProLong standard in using mounted stained and and PBS, in X-100 vrgdfo tlattreeprmnst eeaeaF targeting FA a generate then to were experiments construct three each for least Scores index. at targeting. (like strong from targeting very GFP–kindlin- averaged medium for for that for 4 (like 2 and targeting targeting, strong 2), low for 3 for arbitrary score GFP–kindlin-1), 1 (in The for targeting, that experiment. score no each representative in for a construct 0 from each so was little for experiment blindly varied given given The confidence construct was a blindly. given units) 95% in assessed a cell also for the to was targeting per cell with FA FAs cells of in three proportions intensity 1000 signal least overall as GFP to at of 120 shown Intensity in of interval. are tested scoring non-dividing Data was reflect results construct well-spread, construct. final Each were 40 and as condition. fluorescence experiments, least -3 each cytoplasmic in and At moderate-to-low scored GFP–kindlin-2 with experiments. cells using all transfected blindly, in scored controls was proteins tagged h ecnaeo F-oiieclssoigF agtn fGFP- of targeting FA showing cells GFP-positive of percentage The a pri-H,adteGTIKADPNH-I1complex GST–ILK-ARD–PINCH1-LIM1 the and -parvin-CH2, ora fCl cec 21)17 3842 doi:10.1242/jcs.155879 4308–4321 127, (2014) Science Cell of Journal m /lbvn lsafboetn(im)o 10 or (Sigma) fibronectin plasma bovine g/ml .coli E. oet el n S–L-Di ope with complex in GST–ILK-KD and cells Rosetta .coli E. e L1cls ohwr purified were Both cells. 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Journal of Cell Science 150 nest MI fF91 binding, FN9-11 of (MFI) intensity EERHARTICLE RESEARCH 4320 N., N. Brahme, N., Bate, C., Huet-Calderwood, T., B. Goult, M., Bouaouina, X., Zhang, L., and Izem, K., A. Sossey-Alaoui, K., D. Bledzka, Q., Y. Calderwood, Ma, K., Bialkowska, S., Kim, G., Rothschild, A., Bandyopadhyay, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.155879/-/DC1 online available material Supplementary material Supplementary release for PMC in months. Deposited 12 RSG-12-053-01]. after numbers American number [grant the [grant Health by Society of and Cancer Institutes R01GM088240]; National and the R01GM068600 by T32GM007223, supported was work This the Funding wrote and data and analyzed expertise experiments, and/or designed reagents analyze paper. D.A.C. and essential paper. experiments provided the perform T.J.B. helped edited and results A.L.S. S.R. and analyzed A.L.S., N.K. experiments, data. paper. performed the and wrote designed and N.N.B. and C.H.C. contributions Author interests. competing no declare authors The interests Competing endogenous of state activation The activation integrin of Analysis npeec fihbtr and inhibitor, of presence in elt eelsdi ufrX( MNaVO mM (1 X buffer in lysed were pellets idn otasetdcls yoer aawr nlzduigFlowJo using analyzed were data software. Cytometry cells. transfected to binding rpPdPimi h olna ersin‘qiiru idn – in binding calculated ‘equilibrium was regression constant nonlinear dissociation mode. the binding’ The in specific GST. signal Prism the to GraphPad against from referenced binding and ranging instrument from series BLItz the dilution with in temperature buffer ILK- assay The in Sciences). 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FERM a kindlin D. the Calderwood, in and loop R. lipid-binding D. Critchley, L., I. Barsukov, endothelial the cell, non-hematopoietic a in cell. functional F. and E. expressed Plow, and is T. Kindlin-3 Byzova, J., Qin, N., Malinin, keratinocytes. in S. Raghavan, oimnpeiiainwspromda olw.HK9Tcell HEK293T follows. as performed was Co-immunoprecipitation iaedmi esrmnswr efre sflos GST– follows. as performed were measurements domain Kinase 8 follows. as performed was assay protein-binding purified The 5 IIb m ( a b F .Bnigrsosst S–idi eemaue troom at measured were GST–kindlin to responses Binding M. pri-H ope,or complex, -parvin-CH2 .Bo.Chem. Biol. J. nerncoactivation. integrin 3 2 F o )/ F integrin 21) ucinldfeecsbtenknln1adkindlin-2 and kindlin-1 between differences Functional (2012). .Cl Sci. Cell J. 285 where , 18640-18649. , .Bo.Chem. Biol. J. 125 F F 2172-2184. , a integrin stegoercma fluorescence mean geometric the is pri-H ln sngtv control, negative as alone -parvin-CH2 a 5 F b stenraie F fCD29 of MFI normalized the is o nern a sesdb flow by assessed was integrins 1 287 steMIo N-1binding FN9-11 of MFI the is 6979-6990. , 21) h nernco-activator integrin The (2010). 4 0m a,4 mM 40 NaF, mM 50 , 21a.Aconserved A (2012a). , m 0.5– gof b 1 rerc,E . i,E,Sna . ok . isoe .S,Mca,C A., C. MacRae, S., D. Milstone, S., Cook, S., Sinha, E., Liu, B., E. Friedrich, R., Pasvolsky, E., Manevich-Mendelson, V., Grabovsky, W., S. Feigelson, Spatz, C., Grashoff, V., Hirschfeld-Warneken, I., Patla, L. T., E. Volberg, Feldman, N., and Elad, J. Golden, S., Kim, P., A. Vreede, J., J. Dowling, ujes .W,vnd ivr . eemn .A,d or . ol .T,van T., A. Tool, M., Boer, de A., M. Weterman, E., Vijver, de van W., T. Kuijpers, hsel .P,Sige,A . aii,Z,Nlbtk,E,Bgo,T .and J. T. Boggon, E., Nalibotski, Z., Razinia, L., A. Stiegler, P., B. Chiswell, A. D. 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