fBooia hmsr,JhsHpisUiest colo eiie Baltimore, Medicine, of USA. School University 21205, Hopkins MD Johns Chemistry, Biological of cecs(CM) yt nvriy ay,Koo6680,Japan. 606-8507, Kyoto Sakyo, University, Kyoto (iCeMS), Sciences Teeatoscnrbtdeulyt hswork this to equally Japan. contributed 226-8501, authors Kanagawa *These Yokohama, Technology, of Institute nvriy ay,Koo6680,Japan. 606-8502, Kyoto Sakyo, University, h oeo h neato ftevnui rln-ihlne region linker Yamashita Hiroshi proline-rich vinculin the of vinexin interaction with the of role The ARTICLE RESEARCH eevd2 pi 03 cetd2 aur 2014 January 28 Accepted 2013; April 23 Received ` this transducing and stiffness 1 ECM sensing in 2000). stiffness al., role ECM et integral (Lo of cells fibroblast gradient of a migration rigid cell Furthermore, the stiffness a (Schedin directs 2011). ECM with progression Keely, increased cancer microenvironments, and and to tumor 2008), contributing in Engler al., microenvironment 2009; observed et al., been et Saha has (Discher stem 2006; become stiffness of al., ECM specification has such et by lineage directed cues, cell it is the physical cells Indeed, to addition, stiffness. respond ECM of cells In as that density migration. apparent proliferation, and cell increasingly regulate and variety to known the differentiation well including are components, cell al., cues, ECM et Parsons determining Chemical 2009; al., in et 2010). (Geiger fate important extracellular and behavior of are morphology, component key which a microenvironments, is (ECM) matrix Extracellular INTRODUCTION Vinculin Stiffness, Mechanotransduction, matrix, with Extracellular WORDS: vinculin KEY of region PRL the mechanotransduction. of the interaction results in vinexin the These Mutations migration. these that cell impair ECM. suggest of expression regulation soft impairs vinexin stiffness-dependent depletion vinexin on the of Furthermore, stiffness. depletion than ECM to the ECM responses or rigid region on PRL (FAs) adhesions focal rigid larger at A localizing vinculin, is stably substrates. vinculin cytoskeleton-associated of ECM fraction of the of level and stiffness the the increases sensing substrate vinexin in linker protein proline-rich involved PRL-region-binding the the are and that vinculin show of yet we region not study, (PRL) have this stiffness In progression, ECM elucidated. cancer the sense been affect that direct and mechanisms to molecular cells known the stem is of and specification microenvironment lineage extracellular aspect important the an is of stiffness (ECM) matrix extracellular Although ABSTRACT ua .Craig W. Susan uhrfrcrepnec ([email protected]) correspondence for Author iiino ple ieSine,Gaut colo giutr,Kyoto Agriculture, of School Graduate Sciences, Life Applied of Division 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,17–86doi:10.1242/jcs.133645 1875–1886 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. oa dein Fs,stso elEMahso,pa an play adhesion, cell–ECM of sites (FAs), adhesions Focal a ly rca oei esn C tfns n in and stiffness ECM sensing in role crucial a plays 4 rdaeSho fBocec n itcnlg,Tokyo Biotechnology, and Bioscience of School Graduate 3 cioHarada Ichiro , 1, ,Tkfm Ichikawa Takafumi *, a nsnigtesifeso h xrclua matrix extracellular the of stiffness the sensing in 2 nttt o nertdCell-Material Integrated for Institute 4 n oiuiKioka Noriyuki and 1, ,DiueMatsuyama Daisuke *, 3 Department 1, ` 06,tesgaigmcaim ywihclssneECM sense signals cells biochemical into which information this by al., transduce et mechanisms and Sawada stiffness 2009; signaling al., intracellular et the sensing Friedland higher 2009; in 2006), al., exert engage et FA Rio ECM to some (del suggested tension Although rigid ECM. been on soft have on cells al., proteins cells et 2010); than (Geiger tension al., accordingly intracellular et exert ECM Parsons they the that 2009; on tension ‘pulling’ the and FAs by adjusting through tension stiffness intracellular and ECM actomyosin-generated using talin sense including Cells proteins, stress vinculin. cytoplasmic actin various of transmembrane ends through the FAs, to fibers connected At are integrins, signals. receptors, ECM biochemical into information rti neatos tecigfrepligtevnui tail protein– vinculin to the of addition pulling 2002; simulation force in a al., stretching that, using a et shown study interactions, DeMali has protein recent A 2005; dynamics 2005). al., molecular al., et et proteins (Chen Takahashi PRL-region-binding vinculin the activated because region, change al., dynamic vinexin et PRL F-actin a Peng induces for the 2006; also affinity vinculin in al., of high et activation Cohen a The 2011; 2011). exhibits Ballestrem, which head change and form, conformational (Carisey the a ‘active’ its a induces of an reduces and in domain talin, to association tail resulting including the partners, for The 2004), affinity binding latter al., F-actin. other the with et for domain in Borgon affinity sites 2004; the actin-interaction decreased al., with the et associates masks (Bakolitsa and domain an In domain head 1995). tail the Craig, the and conformation, (Johnson between domains ‘inactive’ interactions tail fully intramolecular and head by vinculin not regulated is are FA-mediated actin, in details role crucial the a play understood. particularly however, in to vinculin, region) seems Thus, PRL mechanotransduction; region, 2010). the in al., PRL (or et the vinculin (Grashoff of cells domains living tail between observed and been head al., have the et strengths tension (Wolfenson various forces Vinculin and 2008). intracellular 2011), al., by et regulated Mierke vinculin 1998; are al., (Alenghat dynamics et where force Goldmann contractile 2000; areas al., intracellular et the in maintain to 2006). observed required al., is et (Mo tension Ziegler accumulates 2011; higher al., FA-mediated actin Indeed, in et the involved talin– (Peng be to to binds with mechanotransduction thought tail associates is vinculin Vinculin domain cytoskeleton. the head whereas complexes, vinculin the integrin to The head vinculin domain. the connects tail proline-rich that a region and (PRL) domain linker tail flexible C-terminal a domain, head terminal understood. well not are h fiiyo iclnfrisbnigprnr,icuigF- including partners, binding its for vinculin of affinity The N- an of composed protein FA cytoplasmic a is Vinculin 1 auiaKimura Yasuhisa , b n h r23cmlxpeeetal idto bind preferentially complex Arp2/3 the and h ta. 09,adteepeso fvnui is vinculin of expression the and 2009), al., et ¨hl 1 auis Ueda Kazumitsu , 1,2 , 1875

Journal of Cell Science 05.Tebnigo vinexin of binding The The 2005). 2003). al., et Wakabayashi vinexin Mitsushima 2002; 2004; al., vinexin of et al., localization 2010; Suwa et al., 2006b; Mitsushima et al., 2006a; Lalevee et acid 2001; al., retinoic al., et et flotillin, Mitsushima (Kimura WASF2), vinculin as and known receptor Abl, (also ERK, to including WAVE2 binds proteins, and Dlg5, cytoskeletal protein adaptor and an signaling as various functions Vinexin 2002). al., et einta nldsasri oooy(oo oan Vinexin domain. (SoHo) homology sorbin a of includes that variants region vinexin transcriptional and domains, Several (SH3) al., 3 2002). et vinexin known; al., (Kioka are vinculin protein vinexin et PRL-region-binding Kioka induce a 1999; as can identified domain have head 2010). Mofrad, the and (Golji from activation away domain ARTICLE RESEARCH eoe h yolsi rtisa ela rtista are that proteins as 1876 treatment well as CSK proteins (CSK). cytoplasmic buffer the treatment stabilization removes using of by cytoskeleton amount fraction, the cytoskeletal with on the stiffness in substrate included of vinculin effect the the mechanotransduction, of Because investigated in role we independently conditions. essential detected, an FAs experimental plays was cytoskeleton our at actin stiffness under localize stiffness different the area can ECM of vinculin among number, that substrates FAs the suggesting on vinculin-containing in grown of stiffnesses difference cells intensity substrate integrated significant tested or all No on vinculin 1A–D). comparably that (Fig. FAs By found at S1A). we on localized Fig. areas microscopy cell material immunofluorescence similar (supplementary showed using substrates and tested different similarly we spread the that Cells first substrates stiffness. gel all we of polyacrylamide on levels vinculin, on mouse various grown with (WT) To on cells substrates wild-type (MEF) behavior. in stiffness fibroblast vinculin ECM vinculin ECM embryonic of then localization affect of the stiffness, to effects examined ECM expected the sensing be examine in would involved stiffness is vinculin If localizing stably vinculin FAs of at fraction the increases ECM Rigid RESULTS with vinexin vinculin with region of PRL its association through the interaction reduces activity vinexin II and myosin FAs vinexin and at of vinculin localizes on stably dependent that is paxillin this ECM of Rigid fraction stiffness. vinexin the ECM of on decreases to depletion than responses the ECM or these region of rigid impairs PRL expression on the fraction larger of Mutation is of ECM. the FAs level soft at the addition, localizing sensing increases stably In ECM vinculin in rigid vinculin. that involved show cytoskeleton-associated are We stiffness. vinexin ECM protein the PRL-region-binding However, understood. 2005). NIH3T3 poorly al., in is et mechanotransduction FAs in Takahashi vinexin at 1999; of function actin al., et of (Kioka accumulation cells the promotes region species, among conserved is domain family SoHo including This a domains. have SH3 a which three of form and members SORBS1) the as family, protein known small also protein, (c-Cbl-associated CAP a oei esn C tfns n mechanotransduction. and stiffness ECM sensing in role rB2(r-idn rti ,as nw sSRS)and SORBS2) as known also 2, protein (Arg-binding ArgBP2 , nti td,w on httePLrgo fvnui n the and vinculin of region PRL the that found we study, this In we that protein FA an is SORBS3) as known (also Vinexin a a anradtselegans Caenorhabditis ae oehr hs eut ugs htvinculin that suggest results these together, Taken . a oaiea A ihu icln(aaah tal., et (Takahashi vinculin without FAs at localize can b eed nvnui xrsin whereas expression, vinculin on depends b scmoe ftreSchomology Src three of composed is a a otisa diinlN-terminal additional an contains u o vinexin not but , , Drosophila a xrsin Inhibition expression. n ua (Kioka human and a ly crucial a plays b otePRL the to , T2,wr lowse u yCKtetet n only and treatment, CSK by as known out (also washed FAK and also paxillin were proteins, FA PTK2), on other Fig. even material Two detected (supplementary S2). treatment were CSK signals washed after slight was substrates the only talin rigid on treatment; vinculin, to CSK stiffness contrast by ECM In out actin of talin. of effect the resistance the CSK to examined ECM bound We cytoskeleton. is rigid that that vinculin suggest FAs. of at results cytoskeleton fraction the These stiffness-dependent a increases 1G,H). in (Fig. increased CSK-resistant also of were intensity manner cell integrated the each and in cell vinculin FAs The per 1F). FAs of (Fig. of stiffness substrate area a number upon in dependent increased the was strongly that manner was that vinculin of CSK-resistant showed Quantification substrates contained on 1E). that signals out (Fig. rigid washed kPa) immunostaining 7.8 was on and vinculin the kPa most high (3.8 whereas substrates kPa), was of soft 43 FAs intensity and kPa The at (25 is immunostaining. vinculin that using by by vinculin the CSK-resistant followed visualized substrate Thus, be treatment can to 1997). the cytoskeleton al., CSK whereas actin on tightly et the to Ruhrberg remain cells, bound bind 1984; tightly but al., from that solubilized, et (Fey not proteins cytoskeleton are and the cytoskeleton proteins to cytoskeletal attached loosely moieo otsbtae Fg B.T 2B). (Fig. substrates 21% soft FAs. whereas on data substrates, immobile at the rigid on decreased curve-fitting FAs GFP–vinculin by at 29% substrates of fraction concomitantly that immobile rigid revealed fraction and in the of fluorescence on immobile shown Calculation of As the MEFs recovery measurements. increased of maximum fluorescence culture the growth the FA 2A, of to which Fig. contribution FAs, the decay peripheral minimize large or to were were to static, 2). analyses limited 1, relatively GFP–vinculin were are Movies FRAP studies material and FRAP supplementary expressing substrates, The 2A; (FRAP) rigid (Fig. by cells performed or FAs photobleaching then soft MEF at on after turnover cultured WT vinculin recovery the on analysis. investigated fluorescence next stiffness we substrate using Therefore, of 2011). effect al., et (Wolfenson actin that the suggests to binding also vinculin FAs. result of at This fraction cytoskeleton the substrates. increases rigid ECM detected or rigid were proteins soft were the on of paxillin amounts and either small BCAR1) only by as and out, known out washed (also contrast, p130CAS vinculin By washed 1I,J). talin, CSK-resistant (Fig. was most manner expected, stiffness-dependent vinculin a as Sheetz, in of increased and However, was Sawada portion treatment. fraction 1997; substantial CSK Otto, CSK-resistant of a and amount (Lee the 2002), significant cytosolic a then is Because vinculin and immunoblotting. by buffer examined treated were to CSK substrates rigid specific or with soft on are cultured cells resistance experiments, CSK on the that stiffness suggests substrates ECM vinculin. rigid result on This of even S2). effects detected Fig. were material signal (supplementary of amounts trace niaeta ii C nrae h rcino iclnstably results vinculin These of fraction FAs. 2C). the (Fig. at increases was localizing stiffness ECM recovery, rigid substrate that maximal the indicate the by of affected half not reaches intensity recovered ai saohrF rti htascae ihteactin the with associates that protein FA another is Talin nrclua eso eue iclntroe tFAs at turnover vinculin reduces tension Intracellular immunostaining from obtained results the corroborate To ora fCl cec 21)17 8518 doi:10.1242/jcs.133645 1875–1886 127, (2014) Science Cell of Journal 6 .%( 1.8% 6 ... fvnui a immobile was vinculin of s.e.m.) 6 1/2 .%o iclnwas vinculin of 1.7% h iewe the when time the ,

Journal of Cell Science EERHARTICLE RESEARCH uain eeitoue notePLrgo fvnui to vinculin P2 P843/844/846/847A; of mutant, region (P1 PRL cluster proline the each into three disrupt introduced 2010). species, Because were among al., region. mutations conserved et are PRL region (Grashoff PRL the including the cells in on clusters living focused proline tail in and we various head vinculin Indeed, Accordingly, the between of mechanotransduction. observed domains for been have region strengths the positioning PRL tension to important the the potentially binds to a of vinculin binds implying domain of tail cytoskeleton, head vinculin actin The the and 2010). complex, al., al., et integrin–talin (Geiger et accordingly Parsons exert they 2009; that tension the ECM the and adjusting on FAs by pulling through by tension stiffness intracellular ECM actomyosin-generated sense using FAs cells at that thought vinculin is of It vinexin localization with stable region for PRL required vinculin the of Interaction .elegans C. n ua Koae l,19) point 1999), al., et (Kioka human and a is h muto 1mtn nteCKislbefato was fraction amounts CSK-insoluble small only the whereas vinculin, in WT mutant of that P1 that to comparable showed of also removed amount were analysis mutants the Immunoblotting P3 treatment. and after CSK rigid P2 even after FAs most at whereas on detected treatment, that were these CSK vinculin vinculin revealed P1-mutant analysis and (supplementary whether vinculin of WT Immunofluorescence both S3C). coverslips) for resistance investigated Fig. glass necessary material CSK then (collagen-coated not the We substrates are affect FAs. region mutations PRL at proline the the localization that FAK in suggesting with S3B), colocalized clusters Fig. and and FAs material mutants at (supplementary vinculin localized the subcellular clearly All the vinculin examined. and WT was MEFs each into of introduced localization versions were GFP-tagged mutants S3A). these of Fig. material P872/873/875/876/ (supplementary mutant, 877A) P3 and P860/861/863/864A; mutant, ora fCl cec 21)17 8518 doi:10.1242/jcs.133645 1875–1886 127, (2014) Science Cell of Journal uniidb sn mgJ() h ausrpeetthe represent values The (J). mean was ImageJ vinculin using total by to quantified vinculin The CSK-resistant (I). of antibodies ratio indicated SDS-PAGE using to immunoblotting subjected CSK- and were of lysates volumes protein four CSK-resistant resistant or the lysates solubilize protein lysed to Total then PBS fraction. untreated, in left SDS or 1.5% buffer with CSK with stiffness type-I- of treated on levels were cultured various MEFs with WT substrates (I,J) collagen-coated kPa). (3.8 gel softest 1st the percentile. presenting 99th bars represent and the boundaries with box percentiles, plot, 25th–75th box the each cell For per F–H). and FAs (B–D, area vinculin-containing number, of the intensity displaying integrated are plots data box quantified as The presented condition. were each experiments for separate photographed three 10 from bars: cells Scale individual boxes. dashed areas by the indicated Insets of (A,E). images visibility higher-magnification their indicate images increase gray to the inverted was and were Vinculin immunostaining fixed treatment. using were CSK by cells (E–H) The visualized kPa). with 43 or or (A–D) kPa without 25 kPa, stiffness 7.8 of kPa, levels (3.8 various with substrates of collagen-coated levels various with substrates stiffness. on CSK-resistant FAs and at vinculin vinculin of Localization 1. Fig. 6 ...fo he needn experiments. independent three from s.e.m. AH TMF eeclue ntype-I- on cultured were MEFs WT (A–H) n 5 0 ** 30; P , .0 oprdwt the with compared 0.001 m .Thirty m. 1877

Journal of Cell Science EERHARTICLE RESEARCH Tvnui,btntta fteP uat(supplementary mutant P2 the of 1878 expected, that of As not CSK-resistance then but the and immunostained. promoted vinculin, substrates and substrates WT rigid rigid buffer or on CSK soft the rigid incubation on with confirm the on cultured expressing treated MEFs further were cells mutant vinculin-KO mutant (KO) To region, P2 PRL knockout P2 S4A). the of vinculin Fig. low vinculin importance in material was the observed mutant (supplementary were of P2 substrates vinculin fraction substrates, the soft immobile of on than WT fraction (17% substrates of immobile fraction rigid immobile on the the FRAP larger which the was in to vinculin vinculin On contrast WT In rigid 4). of vinculin. and analysis WT 3, to soft Movies comparable on into immobile, 21% material analysis substrates, mutant supplementary FRAP soft P2 2; (Fig. performed vinculin affect and substrates not EGFP-tagged MEFs S3F). does introduced Fig. WT mutation material we P2 (supplementary structure Next, the protein to that comparable overall vinculin, the implying of region the vinculin, PRL to the binds WT monodispersity in which cluster VASP, addition, the proline with In coprecipitated first or shown). was not mutant structure P2 data the secondary S3E; Fig. the material (supplementary affect retained mutation size- not P2 mutant the and did that this spectroscopic showed analyses dichroism whether chromatographic Circular exclusion examined are structure. we native clusters the First, substrates. proline-rich mutant. rigid on P2 third results vinculin of and These resistance CSK second CSK-insoluble the S3D). the for the in necessary Fig. detected that material were suggest mutants (supplementary P3 and fractions P2 the of Immobile (B,C) condition. each for T function and exponential fractions using single plotted a was ( on substrates experiments based boxes) independent (filled at three rigid (blue) from or mutant data boxes) P2 (open Normalized or soft (A) (black) on performed. WT FAs were EGFP–vinculin analyses of FRAP recovery and kPa) fluorescence (soft, on 43 stiffness cultured rigid, of were kPa; levels vinexin, 7.8 various for with region substrates. substrates interaction rigid the type-I-collagen-coated EGFP–vinculin-P2- lacks and or which soft (WT) (P2), on EGFP–vinculin FAs mutant expressing at stably vinculin MEFs of WT analysis FRAP 2. 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These of S1D). regulation vinculin vinexin Fig. that CSK-resistant material suggest of supplementary stiffness-dependence 4C,F; (Fig. the rescue not usrts iia oW Es ycnrs,tesubstrate in in vinculin the CSK-resistant vinculin of CSK-resistant contrast, amount of The By amount MEFs. the vinexin-KO MEFs. the affect not WT did to stiffness similar substrates, iei sapoenta ehv dniida PRL-binding a as identified have we that protein a is Vinexin ocrooaetefnto fvnxni h stiffness- the in vinexin of function the corroborate To 6 6 .%.Teerslssgetta vinexin that suggest results These 1.6%). vinexin Interestingly, 3C,D). (Fig. 1.5%) ora fCl cec 21)17 8518 doi:10.1242/jcs.133645 1875–1886 127, (2014) Science Cell of Journal b sesnilfrtesal oaiaino iclnat vinculin of localization stable the for essential is , a a sncsayfrtestiffness-dependent the for necessary is a nrae infcnl nrigid on significantly increased was a n vinexin and 6 .% nrgdsubstrates rigid on 1.4%) a and b b h el were cells The . 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Journal of Cell Science EERHARTICLE RESEARCH srqie o h tfns-eedn tblzto of stabilization stiffness-dependent together, the Taken for substrates. indicate interaction vinculin. rigid required vinculin–vinexin results the vinculin on that is These vinexin-mediated suggest FAs for 5). observations (Fig. these required at is mutant region stabilization P2 PRL the vinculin that the of vinexin and nrgdsbtae nrae olwn h eepeso of re-expression the for vinexin following either results fraction increased immobile the substrates substrates vinexin the rigid to that rigid on showed on contrast which performed into EGFP–vinculin, In then WT vinexin introduced coverslips). was re-expressing was analysis (collagen-coated MEFs mutant FRAP or and P2 MEFs vinculin vinexin-KO EGFP-tagged the ofrhrcnimterl fteitrcinbtenvinculin between interaction the of role the confirm further To a nvnxnK Es(i.3,) h eepeso of re-expression the 3C,D), (Fig. MEFs vinexin-KO in a a ntesifesdpnetrglto fvinculin, of regulation stiffness-dependent the in rvinexin or b i o fetteimbl fraction immobile the affect not did a or b , ysnI niio,t eueitaellrtnin then tension, vinexin against intracellular antibody coprecipitated clearly with reduce was an vinculin treatment, with to the Without vinexin. performed inhibitor, was II immunoprecipitation myosin a vinexin re-expressing MEFs tfns nteitrcino iclnwt vinexin with vinculin of untreated interaction vinexin from the with on coprecipitated promotes vinculin stiffness tension of was intracellular interaction that to that the suggests vinculin result amount coprecipitated This the samples. of of amount 55% the reduced blebbistatin effect the examine To vinculin–vinexin- 2010). the al., on et tension Parsons (Geiger intracellular ECMs 2009; of stiffer on al., tension et intracellular higher exert Cells interaction vinexin the with promotes vinculin ECM of rigid and tension Intracellular netgtd TMF eeclue nsf rrgdsubstrates, rigid or soft on cultured were MEFs WT investigated. eedanbsdo igeepnnilfnto o each T for and function fractions exponential Immobile single (A). curves a condition The on experiments. based independent drawn three were from data using plotted 10 on cultured were (WT) EGFP–vinculin of FAs. fraction at immobile vinculin the in increase stiffness-dependent Vinexin 3. Fig. omlzdfursec eoeyo GPvnui tFsi WT in FAs boxes, at (filled The EGFP–vinculin MEFs analysis. of FRAP recovery to fluorescence subjected normalized and dishes bottomed glass coated uvs() CD iei-OMF eepesn ok vinexin mock, re-expressing MEFs Vinexin-KO (C,D) (B). curves acltdfo itdcre D.Tevle ersn the represent values The (D). mean curves fitted from calculated vinexin eddot 10 onto seeded eoeyo GPvnui tFsi OMF eepesn mock re-expressing MEFs KO in boxes, FAs (open fluorescence at normalized EGFP–vinculin The of analysis. recovery FRAP to subjected and ucinfrec odto C.Imbl rcin n T and fractions Immobile exponential (C). single condition a each on for based function drawn were curves The experiments. triangles, ora fCl cec 21)17 8518 doi:10.1242/jcs.133645 1875–1886 127, (2014) Science Cell of Journal 6 s.e.m. b ihsal xrsino GPvnui W)were (WT) EGFP–vinculin of expression stable with n 5 a oprdwt h ots e 38kPa). (3.8 gel softest the with compared h 5h7t ecnie,wt h aspeetn the percentile. presenting 99th bars and the represent 1st with boundaries percentiles, box 25th–75th plot, box the each in For FAs cell. vinculin-containing each plots of box number as the presented displaying are data condition. quantified each The for separate photographed three were from experiments cells 10 individual bars: Thirty Scale (D–F) boxes. dashed areas by the indicated Insets of visibility. images their higher-magnification increase indicate to inverted images then The were immunostaining. by CSK-resistant visualized and was treatment vinculin Cells CSK (A–C) after stiffness. fixed of were levels various with type-I-collagen-coated substrates on cultured were (C,F) eepesn ok(,) vinexin (A,D), mock re-expressing vinculin. CSK-resistant of the amount in increase ECM-stiffness-dependent the Vinexin 4. Fig. 6 a lte sn aafo he independent three from data using plotted was 56) n 5 Fg AB.Itrsigy ramn with treatment Interestingly, 6A,B). (Fig. m a 8,vinexin 58), AB TadvnxnK Essal expressing stably MEFs vinexin-KO and WT (A,B) /ltp--olgncae ls otmddishes bottomed glass type-I-collagen-coated g/ml u o vinexin not but , n 5 a 8 rK Es(pnboxes, (open MEFs KO or 58) a a a u o vinexin not but , fle boxes, (filled eetetdwt blebbistatin, with treated were b sesnilfrteECM- the for essential is , n 1/2 5 0 * 30; a eecluae rmfitted from calculated were m h feto ECM of effect The . /ltype-I-collagen- g/ml n 5 P , 6 rvinexin or 56) a .5 ** 0.05, b BE rvinexin or (B,E) iei-OMEFs Vinexin-KO sesnilfor essential is , a n interaction, a 5 P m 1/2 , 2 was 52) m. a also was 0.001 were b 1879 (gray a or b

Journal of Cell Science h RPaayi n S eitneasy nwihvinexin which in assay, with resistance consistent CSK is a and analysis This FRAP shown). the not (data immunoprecipitation EERHARTICLE RESEARCH 1880 experiments. triplicate of representative anti-vinculin or are anti-vinexin Results using antibody. immunoblotting by by visualized 7.5, followed were pH deoxycholate), proteins Tris-HCl sodium anti-vinexin mM 0.5% the (50 and with for buffer NP-40 immunoprecipitation substrates lysis 1% kPa) with NaCl, (42 lysed mM rigid were 150 or cells kPa) The (2.7 hours. soft 12 on values cultured The were ImageJ. MEFs using by mean quantified that the was vinculin represent A of in amount vinexin The with (B) coprecipitated antibodies. anti- anti-vinculin immunoblotting with or by (IP) anti-vinexin visualized immunoprecipitation using were to proteins subjected Coprecipitated was antibody. lysate vinexin cell The h. 1 vinexin vinexin or with mock vinculin re-expressing of Coimmunoprecipitation 6. Fig. vinexin of effect the F-actin conformation, for its vinculin by of affinity regulated the is Because cytoskeleton. actin vinexin substrates with rigid interaction on vinexin- vinculin of a resistance in CSK the in vinculin increase by of The F-actin change for conformational vinculin a of inducing affinity the changes Vinexin that of interaction suggest the observations vinexin promotes with ECM these rigid vinculin and together, tension Taken intracellular ECM. rigid anti-vinexin vinexin the both when recognizes coprecipitated that was with comparable antibody a vinculin Interestingly, coprecipitated of 6C). (Fig. amount was substrates soft on vinculin cultured anti-vinexin- More with vinexin antibody. immunoprecipitated were specific lysates cell then h ofraino iclnwseaie yfluorescence by examined was vinculin of conformation the u o vinexin not but , a nclsclue nrgdsbtae hni cells in than substrates rigid on cultured cells in a dpnetmne asstepsiiiyta the that possibility the raises manner -dependent 6 ...fo he needn xeiet.()WT (C) experiments. independent three from s.e.m. b ly oei iclnsaiiainon stabilization vinculin in role a plays , a . a nacsteafnt fvnui o the for vinculin of affinity the enhances a eetetdwt 5 with treated were a pcfcatbd.Coprecipitated antibody. specific a and m /lbebsai for blebbistatin g/ml b a a sdfor used was . A OMEFs KO (A) a on a C 2. s (23.4 ECM fteitrcino iclnwt vinexin with vinculin of manner interaction the vinexin-dependent If a in of FAs localization at stable paxillin the regulates stiffness Substrate sda oiiecnrlta nue h ofrainlchange was conformational peptide the induces 3) that site control binding positive (vinculin a as VBS3 used talin from The purified 7A). (data and cells (Fig. similar in NIH3T3 expressed retains and were MEFs but shown), in vinexin not acids cytoskeleton conformation, active actin amino and the 156 an on probe inactive effects N-terminal in tail the is an lacks vinculin vinculin which the when in low Both is is conformation. efficiency occurs vinculin FRET FRET 2005). whereas al., when et (Chen conformation binding efficiently actin vinculin by in caused vinculin-tail changes are report a that can using which analysis probe, (FRET) FRET transfer energy resonance cRT ai y1.%cmae ihteehnlcnrl The control. ethanol vinexin the of with compared addition 15.7% (1.4 by 2006). ratio al., peptide (cFRET) et (Chen VBS3 vinculin F-actin, full-length with The together of and, activation 2004) the al., et induces (Izard head vinculin the of ailnimbl rcinwsrdcdo ii ECM fraction immobile rigid paxillin (32% on (24% the the ECM ECM reduced of 2), rigid soft (Fig. on amount was ECM FAs immobile at the rigid the fraction on 8A,B); vinculin, (Fig. increased to immobile (supplementary was contrast paxillin stiffness which In of of 8). fraction levels 7, performed various Movies was possibility, was analysis material with FRAP this GFP–paxillin substrates a paxillin. test and on protein, MEFs, To WT FA into another interaction. introduced be on this would focused the proteins, require we FA as to other such expected of events, regulation ECM-stiffness-dependent stiffness-dependent stiffness, ECM erae h msinrtob .%( vinexin 8.1% by expected, ratio emission the decreased otiuet h nraei h muto iclnco- vinculin form. of active indicate an results to amount vinexin vinculin these of of the change together, also binding in Taken can the that complex actin. increase ternary with the the sedimented of to Interestingly, formation contribute actin. the with Thus, shown). vinculin of vinexin dose-dependent of vinexin association binding a the the in that promote actin notion the with supporting co-sedimented manner, that vinexin vinculin 7E, of Fig. vinexin a performed in and also to shown vinculin We using As probe probe. assay P2-mutant whereas WT co-sedimentation the actin the mutation, for an of P2 observed as ratio the extent cFRET containing similar the probe decreased FRET VBS3 the of ratio ae nasnl xoeta ucinfrec condition T each and for fractions function Immobile drawn exponential (A). were single curves a The on experiments. based independent three from data OMF eepesn ok(pnboxes, in (open FAs mock at re-expressing (P2) MEFs EGFP–vinculin The KO analysis. of FRAP recovery to fluorescence subjected normalized and 10 dishes on bottomed cultured glass were coated (P2) mutant P2 vinexin-KO vinculin in FAs at vinexin mutant P2 the vinexin re-expressing of MEFs analysis FRAP 5. Fig. boxes, B.Tevle ersn h mean the represent values The (B). ora fCl cec 21)17 8518 doi:10.1242/jcs.133645 1875–1886 127, (2014) Science Cell of Journal aD tefwsc-eietdwt ci dt not (data actin with co-sedimented was itself N n a 6 5 rvinexin or 6 rvinexin or 46) .3s hno otEM(77s (27.7 ECM soft on than s) 0.93 6 aD .%.T 1.1%). aD i o infcnl fettecFRET the affect significantly not did N b (0.5 N ihsal xrsino h EGFP-tagged the of expression stable with m )dcesdtecretdFRET corrected the decreased M) b 6 a ga triangles, (gray 1/2 aD 1.2%, 1/2 m a nac h conformational the enhance can a )i h rsneo F-actin of presence the in M) nedicesdteamount the increased indeed N a lgtylwro rigid on lower slightly was or eecluae rmfte curves fitted from calculated were b . 6 6 OMF eepesn mock, re-expressing MEFs KO s.e.m. P ... a esta on than less was s.e.m.) , a .0,Fg BD.As 7B–D). Fig. 0.001, n sivle nsensing in involved is 5 n m 3 a lte using plotted was 53) 5 /ltype-I-collagen- g/ml 7,vinexin 47), shrci coli Escherichia 6 .7s.To s). 1.17 a a aD aD (filled can N, N.

Journal of Cell Science tFswseaie yuigMF tbyexpressing stably MEFs using vinculin by to paxillin examined of ratio stiffness was the ECM on FAs stiffness by ECM at hypothesis, altered of this test effect is To the interaction. FAs vinculin–vinexin the of through composition molecular ECM. rigid on vinculin– paxillin not of the a fraction (data that indicating suggest paxillin cells, results of vinexin- these regulation these together, the in Taken in shown). vinexin paxillin and vinculin of for MEFs of requirement fraction vinculin-KO overexpression immobile in the the fraction that immobile found paxillin the immobile on the vinculin; of b 30% fraction was paxillin immobile of fraction the for vinexin observed Interestingly, MEFs. vinexin-KO oprbet h TMF Fg CD.B otat the (34% contrast, high By was 8C,D). (Fig. paxillin MEFs of WT fraction the immobile to comparable EERHARTICLE RESEARCH otdcvrlp) h moiefato fpxli tFswas FAs (collagen- at substrates paxillin rigid of (23% fraction low on immobile performed The coverslips). was coated analysis introduced FRAP vinexin was re-expressing a GFP–paxillin MEFs or ECM, MEFs immobile rigid vinexin-KO lower into on the paxillin for of necessary fraction is vinexin whether investigate GST–vinexin- GST, purified Vinexin 7. Fig. lgtylwcRTCPrtoo 2ti rb,psil u oteefc fteP uaini h R ntedsac ewe F n F.()Actin (E) CFP. and YFP between distance the on PRL the in mutation (0.1 P2 probe the tail of WT effect Vinculin the probe. to tail due vinculin possibly the probe, using tail assay P2 co-sedimentation of ratio cFRET:CFP low slightly GST–vinexin- oiiecnrl h ensetawr omlzdt h msino rbsa 7 m D h ai fcRTsga oCPsga a acltd EtOH, calculated. 150,000 was at signal ultracentrifuged CFP to was staining. signal sample CBB cFRET the of then ratio F-actin, The pre-polymerized (D) nm. 476 at probes of mean emission the the to represent normalized values were The spectra ethanol. mean The control. positive et eivsiae h feto h xrsino vinexin of expression the of effect the investigated we Next, . h bv bevtosrietefrhrpsiiiyta the that possibility further the raise observations above The a 6 neato srqie o h eraei h immobile the in decrease the for required is interaction aD .% nteMF eepesn vinexin re-expressing MEFs the in 1.0%) eeicbtdwt r-oyeie -ci n hnsetawr esrd ai B3ppie(1.4 peptide VBS3 Talin measured. were spectra then and F-actin pre-polymerized with incubated were N aD a nueavnui ofrainlcag rma nciet natv form. active an to inactive an from change conformational vinculin a induce can N aD n i–iclnFE rb To 2mtn.(,)W B rP uat()vnui alpoe(0.1 probe tail vinculin (C) mutant P2 or (B) WT (B,C) mutant. P2 or WT probe FRET His–vinculin and N 6 .%i el eepesn vinexin re-expressing cells in 1.1% 6 s.e.m.; n 5 b ;ns,nnsgiiat * non-significant; n.s., 4; a a oefc,as effect, no had 6 i o affect not did .% nthe in 1.1%) a a n was and , or b and , a g o .Tespraatadple eesbetdt D-AEfloe by followed SDS-PAGE to subjected were pellet and supernatant The h. 1 for P , .5cmae ihcnrlsmls nardStudent’s unpaired samples; control with compared 0.05 m lestemlclrcmoiino A yrgltn the is that regulating manner a by in vinculin–vinexin- FAs FAs the in stiffness at on of proteins dependent stiffness ECM composition multiple that ECM of molecular immobilization suggest by the results significantly alters These altered MEFs. GFP– to not vinexin-KO grown mCherry–paxillin cells of was ratio with the compared vinculin contrast, ECM of By in rigid ECM. soft on ratio significantly on grown decreased The MEFs WT GFP–vinculin 8E). the (Fig. to mCherry–paxillin mCherry–paxillin and GFP–vinculin ewe otadrgdsbtae a bevd hs results These MEFs observed. no was vinexin-KO substrates; rigid substrates of soft rigid on migration and on the soft even migration in between WT slowly their difference to to migrated contrast significant In similar substrates MEFs MEFs. substrates, soft MEFs of vinexin-KO on migration WT MEFs, ECM the than that region 8F, regulates indicates substrates Fig. This wounded stiffness WT 9–12). rigid Movies the in using material on closed (supplementary substrates shown efficiently and rigid As faster more or significantly MEFs. soft migrated vinexin-KO on an performed assay or vinculin–vinexin we healing the stiffness, of ECM wound sensing role in physiological interaction the demonstrate To involves migration vinexin cell of regulation Stiffness-dependent )adGTo GST–vinexin- or GST and M) ora fCl cec 21)17 8518 doi:10.1242/jcs.133645 1875–1886 127, (2014) Science Cell of Journal A omsi rlin le(B)sann of staining (CBB) Blue Brilliant Coomassie (A) aD 0t 0.2 to (0 N a m )wt -ci a sda a as used was F-actin with M) interaction. m )wr nuae with incubated were M) m )ad0.5 and M) t ts.Nt the Note -test. m S or GST M nvitro in 1881

Journal of Cell Science EERHARTICLE RESEARCH ucin sasno o C tfns.Arcn eotthat report recent vinexin A vinculin stiffness. with 1882 ECM the interaction for the sensor that by a induced indicate as cell is functions of observations that stiffness-dependency activation the These impairs migration. vinexin on of cells vinexin depletion culturing with 2006; vinculin that al., of found interaction et a the also Cohen promotes We 2005; substrates at 2007). al., rigid vinculin al., et of et (Cohen cells Humphries fraction fibroblast immobile in that the FAs mutations strong increase and shown), showed vinculin not domains) activate prevent (data tail treatment that CSK and mutations to head resistance of the in presence between deficient the is interactions to be (which owing to T12 seems mutant autoinhibition The form vinculin vinculin. active active of in regulation the constitutively stiffness-dependent to change this form for conformational inactive important A the from vinculin. vinculin vinculin of the rigid behaviors of vinexin on dependent interaction with increased The region were ECM. PRL soft vinculin CSK-resistant with of compared that fraction, fraction ECM cytoskeletal show immobile the the we with and tightly Here, stiffness associates elucidated. ECM which vinculin, been sense 2011). that not Keely, and mechanisms have Schedin molecular 2008; al., the et progression However, Saha cancer 2006; affects the al., and directs et cells (Engler stiffness stem ECM of behavior. specification and factor lineage fate important an cell as determines attention that much attracted has stiffness ECM DISCUSSION migration cell of regulation vinexin. involves stiffness-dependent the that suggest n hsitrcinatvtsvinculin activates interaction this and , a a eesr o hs stiffness- these for necessary was nvitro in Furthermore, . a poigrsosso iclnadpxli aeas been Similar also 2011), have paxillin. al., et paxillin of (Wolfenson colleagues and that re- and Wolfenson vinculin The decreases by of reported paxillin. but responses of vinculin opposing that of decreases fraction but vinexin of vinculin expression of fraction the in process. performed vinexin, this be various understand to to further under needs to vinculin analysis future tension rigid of intracellular Detailed of on activation. binding conditions vinculin cells the to culturing induce leading by contrast, might induced By sites. substrates tension adhesion to intracellular localization recruit vinculin high resulting of tension, low to loss intracellular the ROCK abolishes sufficient model, in of completely is II inhibition this myosin Pharmacological substrate or and sites. In adhesion soft function. to (recruitment on vinculin vinculin mechanosensing tension of intracellular two-step model be a paxillin-positive can activation) observations from by These 2010). vinculin al., inhibition explained et in II of (Pasapera FAs Myosin sites disappearance adhesion to 2013). the al., vinculin et of induces (Carisey localization inhibiting cells by the osteosarcoma tension impairs intracellular activity of ROCK to Disruption that vinculin substrates of sites. suggested rigid localization the adhesion and have for necessary soft is reports tension both intracellular several on shown Interestingly, FAs have at comparably. we localizes study, vinculin this that In respectively. tension, intracellular supports also intracellular 2013) al., for finding. et requirement (Carisey our maturation the FA during bypass tension can vinculin activated u eerhrvae htrgdEMicessteimmobile the increases ECM rigid that revealed research Our high and low have substrates rigid and soft on grown Cells n10 on a ujce oFA nlss moiefatosadT and fractions Immobile analysis. FRAP to subjected mean OMF n2 kPa, 25 on MEFs KO . P;rgd 3ka n ujce oFA nlss Immobile analysis. FRAP to subjected T (soft, and and stiffness kPa) fractions of 43 levels rigid, various kPa; on with 7.8 cultured migration. substrates were cell collagen EGFP–paxillin I and expressing type paxillin stably on MEFs stiffness WT ECM (A,B) of Effects 8. Fig. eoeyo GPpxli tFso ot(pnboxes, (open boxes, soft (filled on FAs at EGFP–paxillin of recovery au fterltv lsdae t0mni % eut r shown are Results 0%. is min The 0 percentage. at mean a area the as closed as expressed relative open the MEFs initial of KO to value vinexin area and closed into WT relative migrated the in cells shows area and plot The removed area. for were open incubation inserts the After culture onto substrates. the seeded rigid hours, were or 24 Cells soft (F) on percentile. inserts bars 99th culture the and with 1st percentiles, the 25th–75th presenting the represent boundaries box needn xeiet W Eso . kPa, kPa, 3.8 25 on on MEFs (WT experiments independent eepoorpe o ahcniin n h ai fmCherry– ( of calculated ratio was the cells EGFP–vinculin 10 and to from condition, paxillin adhesions each focal for 30 of photographed total were A stiffness. with of substrates levels type-I-collagen-coated various mCherry– on and cultured EGFP–vinculin were both paxillin expressing stably MEFs KO boxes, (open n mock re-expressing MEFs KO n EGFP– in of FAs recovery at fluorescence paxillin normalized the from calculated ora fCl cec 21)17 8518 doi:10.1242/jcs.133645 1875–1886 127, (2014) Science Cell of Journal 5 5 rvinexin or 1) h ausrpeettemean the represent values The 111). vinexin 116), 6 m ...(,)VnxnK Esr-xrsigmc,vinexin mock, re-expressing MEFs Vinexin-KO (C,D) s.e.m. /ltp--olgncae ls otmddse and dishes bottomed glass type-I-collagen-coated g/ml n b 6 5 n ihsal xrsino GPpxli eecultured were EGFP–paxillin of expression stable with ...o h ubro ilsgvnblwfo three from below given fields of number the of s.e.m. 5 9 iei OMF n38kPa, 3.8 on MEFs KO vinexin 19; 1/2 a 0)sbtae.Tevle ersn the represent values The substrates. 108) eecluae rmtenraie fluorescence normalized the from calculated were a fle boxes, (filled nK Esas nrae h immobile the increases also MEFs KO in n 5 14). n 5 1)o vinexin or 112) 6 n ...()W rvinexin- or WT (E) s.e.m. 5 0.Frec o plot, box each For 30). n n 5 b 5 7 n vinexin and 17; ga triangles, (gray 9 TMEFs WT 19; n 1/2 5 9 rrigid or 89) were

Journal of Cell Science poiersosso iclnadpxli niaeta ECM The that al. vinexin indicate et as paxillin well Wolfenson and (as of results vinculin stiffness our of observations ECM, responses the soft intracellular on opposite and with than Because blebbistatin ECM consistent vinculin cells. rigid are by HeLa on of higher in tension are fraction paxillin tensions of immobile intracellular that the increasing of decreasing reduction treatment the with ARTICLE RESEARCH tfns.I diin h neato fvnui ihvinexin with vinculin of interaction the addition, In stiffness. a possibility. and this stretching examine studies Future for to proteins. necessary sensor FA other are a of accumulation as the non- for works that platform of and the vinculin X-100, fraction remaining Triton for some with this treatment that explanation after is even attractive stretching remains vinculin to an vinculin 2013), of FA sites by responsiveness adhesion al., core out to recruit et paxillin washed can and (Carisey were FAK vinculin paxillin p130CAS, active including and that proteins FAK Given 0.5% that treatment. and but CSK 0.25% buffer resistant was CSK containing vinculin our that to X-100, in showed We Triton used respectively. buffer X-100, on Triton CSK based the and are for Sheetz, FAK study experiments and sensors p130CAS, the Sawada in accumulating by as used buffer reported for the work both platform Interestingly, paxillin. to a and as seem Triton-X-100- and and the fraction (Sawada stretching in vinculin cytoskeletal included to are insoluble that not Molecules but including 2002). proteins, paxillin, Sheetz, FA and several FAK to binding p130CAS, its promote to 8). reported (Fig. was vinculin ECM to the paxillin of of stiffness ratio the by of that properties regulated found fraction molecular we immobile Indeed, the FAs. modulate the of rather (because but decreases), FAs paxillin entire stabilize merely iclnPLrgo ihvinexin with region PRL vinculin PRL-dependent analysis. future and in by redundancy examined p130CAS be Functional and should ECM latter. mechanosensing vinexin the for between of mechanosensor vinculin differences to dynamics a p130CAS of the as p130CAS binding the affects that functions of show also region They phosphorylation stiffness. PRL the stretch-induced that the plays in and (Janos p130CAS to role binds vinculin of a region PRL the in cluster Janos manuscript, species. between of half N-terminal deficits the vinexin lacks these that to variant in similar Interestingly, a dCAP, deficits by hearing. rescued in be dCAP and cannot resulting 2013). sensation organs, al., vibration chordotonal in et impairs ortholog (Bharadwaj the depletion vinexin structures During 2005). tension-sensing a al., of dCAP, et manuscript, Takahashi Drosophila this 1999; at of al., F-actin preparation of et accumulation (Kioka the increases FAs and vinculin This of absence vinculin. vinexin that of observation regulation ECM-stiffness-dependent vinexin- the in ae oehr hs bevtosidct oefrvinculin– for role a to indicate phenotype observations vinexin- these similar together, partially Taken a show importantly, mutants More vinexin mechanotransduction. that finding our with ee esoe htvinexin that showed we Here, been has cytoskeleton Triton-X-100-insoluble the Stretching nsmay ee edmntaeta h neato fthe of interaction the that demonstrate we here, summary, In current our revising of process the in were we While ˇ ike l,21) hsfrhrspot u conclusion our supports further This 2013). al., et tiak a a seii ucini ossetwt u previous our with consistent is function -specific neatosi ehntasuto hti conserved is that mechanotransduction in interactions a eotdt euaeteasml n function and assembly the regulate to reported was , ˇ ike l eotdta h eodproline second the that reported al. et tiak a b u not but , hs bevtosaeconsistent are observations These . a a n nrclua eso onot do tension intracellular and ) u not but , a u o vinexin not but , a sivle nsnigECM sensing in involved is b oaie oFsi the in FAs to localizes , b ly rca oein role crucial a plays , rspiavinculin Drosophila dCAP b sinvolved is , mutants. a is os niFKadat-10A eeprhsdfo BD Anti-vinexin- was from 1999). antibody al., antibody polyclonal et purchased (Kioka anti-vinexin Anti-GST previously described Rabbit as were CA). Healthcare. was GE Jose, from anti-p130CAS Technologies (San obtained Life and were Laboratories MO), Transduction antibodies anti-FAK respectively. Louis, TX), (Dallas, (St Biotechnology anti-GFP Mouse Cruz Santa Aldrich and and CA) Sigma (Carlsbad, from anti-paxillin purchased anti-vinculin, Mouse reagents and Antibodies METHODS AND MATERIALS -mnpoytiehxsln SgaAdih n a oee with covered with was treated and were that Aldrich) coverslips (Sigma glass and onto 3-aminopropyltrimethoxysilane persulfate dropped was ammonium acrylamide TEMED) previously 0.05% 0.1% 10% bisacrylamide, were reported 0.05–0.5% concentrations as monomer, (final reagent prepared acrylamide-mix the was substrate (Damljanovic gel substrates Polyacrylamide gel polyacrylamide of Preparation 37 at Technologies) Life Gibco, (FBS; serum 10% with bovine in supplemented grown (DMEM) fetal were medium Cells (Todaro through Eagle’s Japan. BRC method modified MEXT, RIKEN Dulbecco’s 3T3 of the Project standard by Bio-Resource a provided National were using the cells by 293T 2010) 1963). Green, al., and vinexin- et or (Kioka WT mice from KO generated were MEFs generation immortalized lentivirus Spontaneously and transfection culture, Cell (Takara vector pColdI tail into vinexin and subcloned Vinculin purification, were for CA]. Japan) 2005) Ohtsu, al., Clara, Bio, et Santa (Chen Kit (Stratagene), probes Mutagenesis Site-Directed Technologies QuikChange P877A) P861A, the [Agilent P876A, P1 (P860A, using P875A, by P873A, mutant vinculin generated (P872A, P2 were mutant The P3 P847A), and CA). P864A) P846A, P863A, View, P844A, Mountain (P843A, mutant Biosciences, Tsien) -blasti or 2005), pCDH-EF1-IRES-puro Y. (System vinexin into subcloned Roger and vector al., were Sabe), 1999) by Hisataka al., et et provided by (Kioka provided (kindly (Takahashi (kindly mCherry- paxillin fused (chick) or GFP- vinculin monomeric GFP-fused Monomeric construction Plasmid uiiidamshr otiig5 CO 5% containing atmosphere humidified olwdb nuainwt 1 with incubation and by lentiviruses, LTX with infected Lipofectamine were followed using pCDH Cells Technologies). cells and (Life 293T Reagent MA) PLUS into Cambridge, transfected were (Addgene, vectors psPAX2 pMD2.G, lentivirus, im lrc.Batcdnwsfo ae hraetcl(Tokyo, Pharmaceutical Kaken from was from obtained Japan). Blasticidin were blebbistatin Aldrich. and Sigma Puromycin Japan). 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Journal of Cell Science ice,D . ony .J n adta .W. P. M. Zandstra, Radmacher, and and J. J. Domke, D. Mooney, E., D. Discher, eai .A,Bro,C .adBrig,K. Burridge, and A. C. Barlow, A., K. DeMali, e i,A,PrzJmnz . i,R,Rc-uah,P,Fradz .M. J. Fernandez, P., Roca-Cusachs, R., Liu, R., Perez-Jimenez, A., Rio, del Damljanovic W. S. Craig, and M. D. Choudhury, H., Chen, A., Nazgiewicz, N., Heath, N., Nijenhuis, M., A. Greiner, R., Tsang, A., Carisey, oe,D . ushr . hn . upy .B n ri,S W. S. Craig, and B. D. Murphy, H., Chen, B., Kutscher, M., D. Cohen, W. S. Craig, and B. Choudhury, P., R. Johnson, H., W. Chen, M., S. D. Craig, Cohen, and N. Kioka, M., D. Choudhury, M., D. Cohen, H., Chen, T. Izard, and R. C. P. Ballestrem, Bois, and G., A. Carisey, Bricogne, C., Vonrhein, A., R. Borgon, hrda,R,Ry . haa . ia-okaoa . eany M., Delannoy, E., Sivan-Loukianova, T., Ohyama, M., Roy, R., Bharadwaj, aois,C,Chn .M,Bnso,L . okv .A,Cdel .W., G. Cadwell, A., A. Bobkov, A., L. Bankston, M., D. Cohen, C., Bakolitsa, e,E . a,K .adPna,S. Penman, and M. K. Wan, G., E. E. D. Fey, Discher, and L. H. Sweeney, S., Sen, J., A. Engler, optn interests Competing stage initial the during contribution work. her spectroscopic this for dichroism of Takahashi circular Honami Irie on thank Kazuhiro us We and advising analysis. Murakami for Kazuma Japan) thank University, also (Kyoto for We Japan) material. University, and valuable (Tohoku Science providing Maruoka of Masahiro Institute and (Nara Sabe Japan) Hisataka Kojima Technology, CA), Chojiro Diego, Japan), San University, California, of (Hokkaido (University Tsien Y. Roger thank We Acknowledgements ARTICLE RESEARCH lnht .J,Fby . si .Y,Glmn,W .adIge,D E. D. Ingber, and H. W. Goldmann, Y., K. Tsai, B., Fabry, J., F. Alenghat, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.133645/-/DC1 online available material Supplementary material Supplementary numbers [grant Scientists Young for Science of Promotion 10J03430,12J03633]. the the of for Bio-oriented Fellowships Society the Research Japan and from KAKENHI Institution; grant JSPS Advancement a Foundation; Research 2465809]; Technology Glass 20380186, Asahi the 24380185, by numbers part [grant in supported was work This Funding from contributions with manuscript the K.U. wrote the and T.I. contributed S.W.C. and S.W.C. provided H.Y. experiment. I.H. N.K., gel and tool. H.Y. Y.K. and analysis by experiments. purification input the protein additional performed on with D.M. expertise N.K., and H.Y. by T.I., designed T.I. and and conceived was project This contributions Author interests. competing no declare authors The oye im ihteaoi oc microscope. force atomic the with films polymer cells. stem control and combine forces and matrices, ope ovnui:culn ebaeporso omti adhesion. matrix to protrusion membrane Biol. coupling vinculin: to complex n hez .P. binding. M. vinculin Sheetz, and characterized to proteins assays. mechanotransduction matrix 39 cell for extracellular substrates of polyacrylamide conjugation micropatterned force- C. a in Ballestrem, proteins adhesion and focal core J. manner. of dependent release Spatz, and recruitment B., the regulates Derby, R., Kemkemer, ofrainlsic nvnui rvsfrainaddnmc fatalin- a of dynamics and formation adhesions. drives focal at vinculin complex vinculin in switch conformational by vinculin of activation suppress to cooperate talin. interfaces head-tail distinct Two cells. living in Biol. vinculin activated Cell of J. significance functional and distribution Spatial vinculin. activate to required is 40398. talin and filaments signalling. adhesion cell vinculin. human of structure Crystal rti euae sebyadfnto ftotninsnigsrcue in structures tension-sensing two of function L. and A. Drosophila. Kolodkin, assembly and F. regulates D. Eberl, protein M., Zlatic, E., T. 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