Journal of Cell Science Fs.Fsdfe rmfclcmlxsb en agr( larger being by complexes focal al., from 2 adhesions et differ focal into Zamir FAs mature are (FAs). 2001; complexes complexes original Geiger, focal the (FAK) new while and advances, formed kinase lamellipodium Zamir the adhesion 2012; As 1999). focal al., and et (Lawson integrins, comprising h oa ope sasal( small cell. a migrating a is of complex lamellipodium, focal or and Focal edge, receptors The 1999). leading between al., the contact et at Adhesome initial ECM Zamir (Zaidel-Bar 2001; after 2007). Geiger, types assemble and three complexes al., Zamir into 2004; et al., classified et generally Zaidel-Bar the are with 2012; structures numerous, Zaidel-Bar, (Geiger 150 are over and numbering complex sites ‘adhesome’ the attachment comprising components molecular matrix The 2001). Geiger, of Burridge and of 2007; Zamir 1988; regions Yamada, al., et and along (Berrier components attachment (ECM) cell–substrate interaction matrix receptor extracellular by mediated with is cells mammalian of Adhesion Introduction of assembly the to rise gives functional lung and compositional the unique in possess words: environment also Key but mechanical arrangement, reorganization the distinctive attendant in a an that show found with suggest only absent, is largely not We adhesions, are FAs mature/fibrillar . properties. cFAs These of depleted, AEC. marker is in following The and a expression FAs Unusually, rates periphery. tensin, unconventional cell (cFAs). turnover whereas which the the in cell pFAs, paxillin at AEC both from rapid the FAs primary absent of show in by In normally cFAs centrally pFAs. generated is and those Moreover, located and cFAs to bundles. ring cFAs both similar keratin FA to forces as another localizes traction micropatterned well plectin and exert and as and (pFAs), glass photobleaching array periphery on after actin cell cultured recovery aster-like when the fluorescence an cells circles at adhesions epithelial with concentric located alveolar focal two associate primary FAs termed in cFAs cultured of FAs of complexes ring subset /paxillin/talin-rich protein A one of cytoplasmic . pattern substrates: the of unique to assembly a matrix induces extracellular display the substrate (AEC) indirectly, the albeit to connect, which attachment (FAs), cell upon clustering Receptor Summary 10.1242/jcs.128975 doi: 3746–3755 126, ß Science Cell of Journal 2013 May 13 Accepted ( correspondence for *Author 3 2 cells 1 epithelial lung Eisenberg L. primary Jessica in forces traction adhesions exert focal localized centrally Plectin-containing, 3746 usqety A rnlct etieal n eri tensin recruit and 2001). 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Susan , 1 ia Mrksich Milan , eerhArticle Research 1,2 , Journal of Cell Science 20 eecluae eaiet h eesi idtp AC h rp hw vrg ltdniisfor densities blot plectin average of shows levels graph the The and rAEC. densitometrically, scanned wild-type were in human-spe Immunoblots or levels A/C. KD) the (DG to or rat-specific DG relative KD), plectin, calculated (Plec against were plectin antibodies rat-specific using encoding immunoblotted viruses were with shRNAs infected rAEC and rAEC wild-type uninfected, ls fFspeeti E htbeksm fterlsof rules the of some Results break that AEC in unique a FAs. present describe traditional results FAs Our edge. of forces cell class traction the at exert FAs and fluorescence to comparable by (FRAP) assessed photobleaching as after pFAs recovery than properties dynamic more AIsann.Tebxdaesi h vrasaesona ihrmgiiain ntergtclm zo) mgso el xiiigdul Aring FA ( double percentage exhibiting mean cells the of as inc Images presented and (zoom). are column condition; column Data third per the right FAs. counted ( in the of cells presented. shown rings in are total are magnifications double images rings) exhibiting higher of (w/o mAEC at columns not and shown left do rAEC), are two that overlays the those the of and in overlays The rings) areas indicated. boxed as The tensin, staining. or DAPI plectin talin, cells. paxillin, epithelial vinculin, alveolar against in organization FA 1. Fig. two other These (cFAs). the cell while the (pFAs) of a periphery region circle central cell One found the the 1C). towards near Fig. occurs experiments; locates We separate FAs circles three of in concentric 1A,B). less (Fig. two or in eight for talin FAs coverslips display (25.1 rAEC glass and of on subpopulation paxillin maintained (rAEC) vinculin, AEC immunostained we AEC, rat in organized primary are FAs FAs how of determine arrangement To unique display AEC Primary m nteoely 10 overlay; the in m 6 03 fcells, of 10.3% m nzoom. in m n 5 4 oa el one ngop of groups in counted cells total 444 n . 0 el muotie ihF rtisi w rtresprt xeiet.* experiments. separate three or two in proteins FA with immunostained cells 200 ( A C , B ecnaeo AC lcK AC ACmitie vrih nlwclimmdu lwCa (low medium calcium low in overnight maintained rAEC rAEC, KD Plec rAEC, of Percentage ) , E rmr ACmitie ngascvrlp eeimnsandwt obntoso antibodies of combinations with immunostained were coverslips glass on maintained rAEC Primary ) lci sas opnn fFso aiu irbat,epithelial 2011). fibroblasts, various al., of of FAs et mediator of component (Takawira key a also FAs a is Plectin in is present indicated mechanosignaling data AEC better previous to our protein which proteins cytolinker the plectin, additional for stained for We adhesions. screened these characterize We 1A,B). (Fig. talin to S1A). Fig. 1.5-fold, material supplementary increases 1C; to (Fig. pattern cells media media ring normal of number double calcium the FA 37.6 reduced By junctions, the in cell–cell 1A,B). exhibiting (Fig. of overnight cells assembly entire cells decrease the the the over of distribution cell culturing interface the random at cell–substrate a locate show interior, FAs others some In while rAEC, 1A,B). edges of (Fig. population cells with edge, single remaining free in one the pronounced least more at being with pattern cells in the observed are FAs of rings nrE,bt FsadcA oti ailn iclnand vinculin paxillin, contain cFAs and pFAs both rAEC, In 6 .%( 2.1% 6 n .. fclsdslyn A ntedul igptenotof out pattern ring double the in FAs displaying cells of s.d.) 5 eta A xr rcinfre3747 force traction exert FAs Central $ 4 el)we oprdt el utrdin cultured cells to compared when cells) 247 ifrn ape e odto ( condition per samples different 3 P , 0.05; # P , .1 ( 0.01. 6 D ...Saebars: Scale s.d.). xrcsof Extracts ) uenuclear lude ii control cific n DG and (w/ s 2+ Journal of Cell Science dein nohrcl ye Zmre l,19;Zmre al., et Zamir 1999; fibrillar al., as et region cell (Zamir same types the cell to other localize in rAEC adhesions traditional in not cFAs are The rAEC in ring adhesions inner fibrillar the in cFAs The to reduced is FAs of rings double shRNA the plectin 11.7 exhibiting with with infected KD) infected rAEC (Plec AEC of shRNAs. cells control percentage in of or the pattern number KD) However, ring (DG the dystroglycan double in al., encoding a difference adenovirus et in no (Takawira FAs is hours 48 displaying There after formation. 1D). (Fig. rAEC their in 2011) plectin for achieved of we to necessary shRNA, knockdown us plectin led of is infection AEC adenoviral primary plectin Using of cFAs whether the in investigate plectin dependent of plectin enrichment is The formation ring double 2009; of FA al., formation AEC. the primary et that to arc indicate particular (Hamill is data or previously cFAs These spots plectin-rich 1991). while us al., rich periphery, by et protein cell Riddelle detailed hemidesmosome the as at in structures FAs found small is cell to these plectin proteins of localizes both two talin in plectin the Instead, lines, and of S1A). Fig. co-localization talin material no (supplementary with and observed cells keratinocytes) and filamentous epithelial antibodies epidermal bladder diffusely the rat human stained (804G, appears HEK, also lines with We cell primarily epithelial S1). colocalized other Fig. plectin material possibly, (supplementary cells cytoskeleton or, FAs these S1A). some distributed Fig. in material in (supplementary talin edge MLE Indeed, cell with in the co-localization plectin to close whereas limited located FA periphery, shows the similar at cells not 12 a located but FAs cell RLE-6TN random display the in In co-distribute within poorly cells S1A). talin However, and Fig. RLE-6TN plectin cells, S1A). material nor Fig. (supplementary 12 arrangement material MLE these supplementary double in cFAs neither possesses 1C; in co-localizing (Fig. mAEC plectin and of cells talin with subset RLE- in FAs, and A of FAs 12 rings (MLE respectively). analyzed alveoli cells lung We from 6TN and derived cells. mouse cells immortalized as epithelial epithelial well rat as lung (mAEC) AEC all mouse primary to common is 1B). from (Fig. absent periphery is plectin cell while the to rings, at with not some cells FAs although in observe FAs, as 1B). degree we interior same (Fig. in rings, the pFAs talin FA with from double co-localization absent co- without plectin mostly it rAEC where is in cFAs, but Similarly, 2002; in al., talin, matrix enriched et with plectin Tsuruta epithelial localizes found 1992; in al., We 2009; et 1998). found Seifert al., Wiche, 2010; et also al., et (Bhattacharya is Burgstaller hemidesmosomes and termed cells junctions endothelial and 3748 ieo A n84 el splmnaymtra i.S1A). Fig. material or (supplementary distribution cells the 804G plectin in in change Conversely, FAs any, interior of of if S1A). size loss little, a Fig. in as results the well material knock-down in as edge increase (supplementary cell similar the a down FAs at in FAs Knocking large results of cells S1B). numbers versus RLE-6TN rAEC the Fig. KD in Plec material plectin in is FAs (supplementary there larger latter Additionally, of controls number the 1E). the (Fig. whether in rings increase of double an regardless FA FAs lack cells, internal or control fewer possess with in periphery, than cell observed the to localize primarily enx eemndi h omto fcA npiayrAEC primary in cFAs of formation the if determined next We 6 .%( 4.5% ora fCl cec 2 (16) 126 Science Cell of Journal n 5 5 el;Fg C.TeFsi lcK rAEC KD Plec in FAs The 1C). Fig. cells; 355 , 65% oad h el deo lcK AC(i.1) Control and with 1E). cells shown). (Fig. FAs (not in rAEC rings large organization FA KD tensin the double on Plec the in impact without of no present marker. edge has rAEC also shRNA adhesion of cells is cFAs the fibrillar tensin and towards pFAs a Moreover, both 1E). tensin, to (Fig. localizes for tensin adhesions, stained Remarkably, fibrillar traditional were are they rAEC whether assess To 2000). de lowt oatrlk eta ci ra Fg A.mAEC 2A). (Fig. array actin central aster-like cell the no at with FAs large also the edge, to localize Plec mainly In bundles 2A). actin (Fig. rAEC, detected KD is organization aster-like no throughout but of FAs cell, connecting rings the filaments few outer a encircling with and bundles periphery, actin cell inner thick the display the rings FA connect without bundles rAEC of FAs. any, center at terminate if the bundles These Few, at array. occurs aster-like cFAs. or bundles with starburst actin a associated in thick cell is of the actin and set of pattern, periphery second set A cell One ring pFAs. the 2A). double (Fig. encircles fashion bundles FA unusual al., actin highly the et a in Geiger displaying arrange bundles 2001; rAEC Bershadsky, In and 2009). Geiger Chrzanowska- and 1996; (Burridge Wodnicka, cytoskeleton actin the actin organize influence FAs rAEC in ring organization inner cytoskeletal the in cFAs The 09.Ti ehdpoie a ocnrlsz,curvature, size, al., control et to Ostuni way advantage 1998; a al., provides substrates took et method (Chen arrayed This cues We 2009). create geometric areas defined contractility. adhesive to fibronectin with cell-sized technique single and/or uniform, of micropatterning influenced composed is shape a rAEC of cell in organization by cytoskeletal actin distinct RLE-6TN of interior cytoplasm S2B). co- cell Fig. interior material phospho- the (supplementary the spanning against cells fibers antibodies within actin contrast, stain phospho-myosin In is for 2D). there but (Fig. periphery, staining cell the phosphorylated at bundles show little actin also thick observe actin the Additionally, in aster-like myosin 2D). inner (Fig. We the indicating cell contractile lacking arrangement, are the cells actin arrangements myosin. at aster-like actin bundles the both as actin total well the as both periphery and with myosin antibodies phosphorylated phosphorylated with rAEC staining 2C). by against (Fig. contractility rAEC of KD levels Plec different than thicker edge cell significantly the exhibit at Additionally, also bundles 2C). rings actin (Fig. without rings rAEC with rAEC around rAEC wild-type bundles wild-type than in that actin periphery edge thicker found cell also cell significantly the We we the display rings. at rings Therefore, without bundles without and actin rings. with the rAEC with of rAEC cell that thickness cells the the at observe those bundles measured we actin actin versus of rAEC plots, regions central periphery to thicker these display in compared from rings drawn rings line without Also increase the with lines rings. dramatic of rAEC without plot white in the The intensity intensity 2A). shows the fluorescence Fig. fluorescence clearly in on scan images scans stained based line actin through 2B; single (Fig. performed not, do measurements which first rAEC those with we rings double possess which rAEC rat S2A). primary Fig. their material do (supplementary cells as HEK not arrangement and 804G RLE-6TN, actin 12, MLE and whereas counterparts, FA same the exhibit enx se hte h omto facArn n the and ring cFA a of formation the whether asked next We display organizations actin different the if determined we Next, in arrangements actin the in differences the quantify to order In Journal of Cell Science i.2. Fig. e etpg o legend. for page next See eta A xr rcinfre3749 force traction exert FAs Central Journal of Cell Science atr u fttlclscutdprcniin(average FA condition ring per double counted the cells containing total separate cells of three of out or percentage pattern two the in are patterns Data different experiments. four on counted proteins seas i.1 o unpatterned). for substrates 1B unpatterned Fig. with also compared (see substrates micropatterned on plating as staining for ( prepared indicated. and substrates micropatterned fibronectin-coated, hlodnt akteatnctseeo.Teiae ntergtcolumn right ( the actin. in for images stain The phalloidin cytoskeleton. the actin show the Alexa-Fluor-647-conjugated mark an to with phalloidin stained total- simultaneously and and phospho-myosin myosin, against antibodies with immunofluorescence n tie oso iclnadatn cl as 20 bars: Scale actin. and vinculin 10 show to stained and ci tiigsmlrt hs hw nA ausaeteaeaethickness of average the images are using Values cells A. in seven s.d.; shown least those to at ( similar across staining the A. places actin measuring in by four images determined in was actin fluorescence bundles the actin actin in outer the lines of white thickness the average by column indicated the position in the magnifications ( higher (zoom). at right The shown the actin. are at overlays for the marker in vinculin a areas against as boxed antibody phalloidin an Rhodamine-conjugated using with immunostaining together for prepared were rAEC # the in shown ( are nuclei. images of of staining columns DAPI left include two and the AEC. column of in third 2009; overlays organization G cytoskeletal al., and actin influence A,D,E rings In et double FA (Bhattacharya 2. surface Fig. cell the to involved is IFs and linking proteins (IF) in keratin filament in intermediate involved binds are Plectin rAEC in ring organization inner cytoskeleton the in cFAs The seeded We of area al., 2004). adhesive et an al., had James substrates et shape micropatterned 1999; each McBeath shaped al., where differently et 2010; several Dike Brock onto al., 1997; rAECs 2007; et apoptosis, al., Kilian al., et properties attachment, Chen 2008; these et 2003; as how (Bhadriraju al., assess et differentiation such to and processes cell migration a cellular of influence polarity and tension 3750 ( 54.1 of average An utrn L 2 L-T,84 n E el ntestar the S2C). actin and on Fig. FAs material of cells (supplementary organization an HEK S2C). such and induce Fig. to 804G fails material micropatterns RLE-6TN, (supplementary 12, rAEC actin MLE micropatterned central in Culturing the the star as from being array emanating latter onto bundles aster-like the the with plated for cFAs, site mAEC and termination pFAs that both exhibit found substrates 2G). (Fig. also we substrates what unpatterned to We on similar maintained edge, cells cell the in near observed bundles actin and (22.3 FAs micropattern large a such onto n plated rAEC and in KD ring 2.4-fold Plec double decreased FA is arrangements the cytoskeleton with actin cells aster-like of number cells corresponding the using the rAEC, studies to wild-type subsequent Compared micropatterns. our star focused on we maintained ring, FA inner of the organization 2E). the 2F). (Fig. studied, (Fig. shape pattern substrates cell each mirrors unpatterned on cFAs on cells in rAEC Moreover, from 2.1- a increase arrangement, FAs aster-like fold of actin central organization corresponding concentric the and double a display micropatterns) P n 5 , . ie htw a oifuneo elsaeo h omto of formation the on shape cell of influence no saw we that Given m 9 oa el one)(i.2,) hs el ipa mostly display cells These 2F,G). 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Plec Only and pFAs, cFAs the 3A). and between associate (Fig. observed closely cFAs and cFAs area of cell al., central with keratin rings et the to Takawira IF restricted exhibiting are 2011; bundles the rAEC al., of In et (Eisenberg 2011). distribution rAEC plectin- the of the if cytoskeleton influence determined next cFAs 2010; we al., Thus, 1998). containing 2006). et Wiche, al., Burgstaller IF et 2009; 1996; for Windoffer al., important al., being et FAs (Bhattacharya et for organization precedence Svitkina is 2001; 2012; there al., Additionally, et al., Gonzales 1999; et al., et Karashima Geerts 2010; al., et Burgstaller o worns r rsne.Saebr:20 bars: Scale do that presented. right those are and the rings) (w/rings) on rings (w/o images FA not double in the exhibiting areas in cells of boxed magnifications Images The higher (zoom). the nuclei. at in of shown shown staining are are DAPI images overlays of include columns and left for column two stained the third also of and Overlays substrates keratin. and micropatterned talin star ( on indicated. maintained as were keratin rAEC and talin double for the immunostained to rAEC. relative in organize FAs of filaments rings intermediate . 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Journal of Cell Science rn yai rpris(ai ta. 2000). al., but little et exhibit tensin, (Zamir they properties types, marker dynamic cells other no adhesion of or periphery fibrillar the at the FAs the unlike Like contain fibrillar character. unique pFAs and exhibit AEC cFAs, Moreover, primary FAs 1999). are of al., pFAs They and et complexes, the Zamir structures even types. 2004; adhesion focal al., cell et of (Zaidel-Bar traditional cultured class adhesions new other from a in cFAs distinct to FAs belong of to from composition appear 1999). protein apart al., and them et location Zamir sets which unusual 2004; adhesions al., the fibrillar et Indeed, classical (Zaidel-Bar vinculin not proteins paxillin, are these our adhesion lack they plectin, latter Thus, contain by the talin. also of supported and marker they is a However, tensin, This devices. contain they adhesions. that fibrillar finding to to relationship relationship from a al., 2011). et al., Ozawa distinct et 2006; bear al., Tsuruta et 2010; talin, are (Litjens and not may do paxillin hemidesmosomes FAs vinculin, while contain they they of these since ring hemidesmosomes However, that inner the FA hemidesmosomes. within suggests unusual plectin plectin highly of of this cFAs loss presence possessing a cytolinker The cells and large of organization. cFAs, the number the the that to reduces demonstrate only of localizes region we ( central plectin addition, the protein AEC within In one primary concentric cell. and two periphery the of in the FAs at subset of one pattern circles, unusual, a highly novel, that a display indicate results Our Discussion 2009). cell al., other in et for forces (Na reported in 6C) results (Fig. decrease with types consistent a rAEC, observe KD we Plec the surprisingly, not and, rAEC 3752 h eiula oaino h Fsas niae potential a indicates also cFAs the of location perinuclear The ora fCl cec 2 (16) 126 Science Cell of Journal , 25%) ifrn rmFslctdtwrstecl neiri te cell other in interior cell the the towards exquisitely on located them FAs make dependent that from properties different is contractile exhibit the they formation Indeed, with latter. their association Moreover, their with the aster-like cytoskeleton. consistent actin Nonetheless, rAEC, perinuclear by contractility. cells. myosin-mediated the and the actin and on dependent of cFAs is both array polarity of aspect or the formation in changes curvature by form to influenced ratio, seems directly AEC not in is pFAs and and inherently cFAs of arrangement unique the otatlt,wihi unatr Aasml n localization The 2008; and al., et assembly Pathak FA 2003; al., and alters et tension (Chen turn reported cellular in previously the arrangements which changes been aster-like size/shape contractility, has cell actin it of the since distortion that shape and cell FAs of of independent 1992; rings al., double et the (Seifert 1998). cells Wiche, actin 1996; these the al., but both in et pattern, of Svitkina cytoskeletons ring organization keratin a perinuclear into the and formation control cFA also regulate may plectin only cells that in suggest not link in data decrease to these might decrease ability cytoskeletons, a its keratin significant given and pFAs, Indeed, actin a rings. the and pFA in and cFAs cFA results the of displaying most plectin rings Although of pFAs. display expression and not cFAs do both periphery of cell rAEC rings the Moreover, to exhibiting extending cells zone, respectively. found in perinuclear are the rings bundles in keratin inner cFAs few and with and associate cFAs also and outer are bundles pFAs keratin There the between pFAs edge. connections up the cell filament making actin the while any, along array, if parallel central few, filaments aster-like actin unusual connect highly a into Fsas otiuesgiiatyt h rcinfre exerted forces traction the to significantly contribute also cFAs ewr eysrrsdb h nsa blt fACt form to AEC of ability unusual the by surprised very were We organized bundles actin anchor to serve cFAs Remarkably, 10 * type; o ahtm on.( point. values time recovery each fluorescence for cell. average per bleached the FAs are six Data to two with seven type, to ring per five double cells for quantified the were with data rAEC FRAP in pattern. cFAs in faster occurs the ( of signal. recovery fluorescence the monitor following to seconds photobleaching 10 every cells the collected of were Images images). pre-bleach cell box whole white in with designated images with zoomed indicated in areas arrows (bleached FRAP were to GFP–paxillin well subjected expressing as pattern cells FA RLE-6TN ring as double the without and with photobleaching. after exhibit fluorescence recovery ring following double pFAs the than turnover within faster cFAs The 5. 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Sprague-Dawley approved male Northwestern were pathogen-free, studies of animal Committee for culture and Protocols isolation cell epithelial Alveolar Methods and Materials o eti xeiet,floigratcmn,rtclswr netdwith and infected plectin were DG, rat cells against (shRNAs) rat RNAs reattachment, hairpin short following encoding adenoviruses experiments, certain For infections Adenoviral 5 at conditions D and of drugs cytochalasin amount deionized The and target. were: double time cytoskeletal minimal respective in used the the dissolved for disrupt optimized was to was required which drugs drug chosen Y-27632 the except of DMSO, Each water. in mg/ml PA). 1 Grove, of (West immunofluorescence isothiocyanate Laboratories for fluorescein ImmunoResearch Jackson (HRP)-conjugated antibodies as from well secondary Goldman purchased peroxidase as were Rhodamine-labeled D. blotting Horseradish and Alexa-Fluor-647-conjugated western Robert (FITC)- OR). for rabbit Dr antibodies (Eugene, and A secondary from Probes University). 4 gift Rhodamine- (Northwestern Molecular and Chew a University). 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(FBS) Eagle’s F-12 mg/ml serum 100 lung Mixture and bovine penicillin Nutrient (mouse fetal units/ml in 10% RLE- 12 University). containing maintained MLE (Northwestern Aldrich) Ridge were and Karen cells Dr negative) from 6TN T-antigen gifts were epithelial, cells epithelial) lung (rat RLE-6TN culture cell Immortalized coated also and MA) according 2011; 10 Waltham, sulfo-SANPAH al., Scientific; with with et (Thermo activated (Eisenberg instructions were gels manufacturer’s kPa The to 5 1998). about Pelham, a measuring with and substrates Wang value generate stiffness to bis-acrylamide modulus acrylamide/0.2% Young’s 7.5% of ratio a using 0mnts ooaoea 5 at nocodazole minutes, 30 MOfr3 iue o3husa appropriate. as hours 3 to minutes 30 for DMSO 10 at PF-573228 and l hmclihbtr eefo im-lrc n aeu taconcentration a at up made and Sigma-Aldrich from were inhibitors chemical All b m m D 4DG/D)wsotie rmAcmIc Cmrde A.A MA). (Cambridge, Inc. 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(Invitrogen). per different samples three three Express cell least or after at and TrypLE to cell on prior Live performed cells using multiple NY). scanning for Melville, captured resonant Inc.; detachment were temperature laser Instruments room (Nikon A1R at software images Nikon Elements a Nikon gel deformation. 60 using and substrate a During performed with for microscope was markers above. confocal fiducial 1.1 imaging as described with cell serve Live to impregnated as Probes) were prepared (Molecular cultured beads substrates cells substrates the RLE-6TN gel and polymerization, rAEC polyacrylamide on performed on was microscopy force analysis Traction and microscopy force Traction after 37 recovery at GFP–paxillin-expressing fluorescence captured from and were images dynamics AEC Live paxillin experiments. of (FRAP) imaging photobleaching were live-cell vector expression for GFP-paxillin the used containing adenovirus with infected dynamics rAEC FA of imaging Live-cell 1:1 with extracted and fixed were filaments. actin cells and keratin, proteins or of at adhesion plectin focal minutes mixture staining 5 X- optimal including Triton for for stains achieve 0.3% utilized PBS with For to was permeabilization in PBS of used in formaldehyde minutes were 100 5 3.7% by protocols using followed temperature fixation method room fixation Two on A immunostaining isolation. for staining. after prepared and 3–5 media culture day from removed were Substrates the at as from NIH; presented Immunofluorescence lysates are Health, using and combined blots of were three were Institutes isolations of Immunoblots cell minimum (National means independent 2011). a previously ImageJ three al., from least as Results using et MD). 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G. Whitesides, X., Jiang, P., LeDuc, C., C. Ho, E., Chang, A., Brock, rsne sbxadwikr lt aeuigGaha rs software Prism are GraphPad data was using force density made Traction signal CA). plots Pa. Jolla, integrated 300 La whiskers Software; the than and GraphPad 3.0, rAEC, greater box (Version KD forces as Plec traction presented and for outer rAEC determined four from and analysis inner within FAs four of quantitative outer least and number forces for At inner cell. equal used the given an were that a indicates in to FAs 1 forces attributable traction to similar close forces exerting ratio of are FAs force inner a sum of Therefore, the number FAs. given to outer a in to relative variations attributable cell-to-cell determined forces slight of was for sum compensate the to force, order traction In overall cell. given a within edge) etakDsRbr .Glmn ae ig,CrsohrS. Christopher Ridge, Karen Goldman, D. Robert Drs thank We Acknowledgements two-tailed, the or as analyses identified comparison was Tukey’s Student’s with paired either testing with explored ANOVA were one-way conditions experimental between differences Significant analysis Statistical htahra . ozlz .M,Dbae .J,Teo .E,Glmn .D., R. Goldman, E., H. Trejo, J., P. Debiase, M., A. Gonzalez, R., S. Bhattacharya, C. Chen, and J. Tan, D., Pirone, S., Ruiz, Alom M., Yang, K., Bhadriraju, M. K. Yamada, and L. A. Berrier, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.128975/-/DC1 online available material Supplementary release for PMC months. Trust in 12 Community Deposited after Chicago M.M.]. the to C2006-00997 at Consortium number Funds Biomedical [grant Searle the Chicago from number the support [grant to and with Institute RO1HL092963 M.M.]; Cancer number to and National U54CA151880 [grant the J.L.E.]; postdoctoral G.R.S.B.]; Health to and of affiliate J.C.R.J. 12POST8610002 Institutes Midwest number National the J.L.E.]; [grant Association to program T32HL076139 Heart training number American [grant Heart, National Institute the the from Blood grant and training a Lung by supported was work This Funding experimental in advice. participated scientific J.C.R.J. provided J.L.E., and and and constructs. design J.L.E. G.R.S.B. plasmid work. M.M., generated experimental S.B.H. K.G.B., out data. manuscript. carried analyzed the D.T. K.G.B. wrote and and K.G.B. experiments J.L.E., designed J.C.R.J. Lurie and J.L.E. H. by contributions Robert Author supported the Facility Center. to Cancer Imaging Comprehensive in Institutional awarded Cell the Facility the at NCI-CCSG-P30-CA060553 performed B University by was Microscopy Core outlined cells, Committee. Northwestern Use Division guidelines antibodies, and Care Pulmonary proper of Animal the gifts with AEC of by their compliance Isolation respectively. performed for analyses, data Hamill was with help J. and Kevin reagents and Turner ircnatprinting. microcontact E. D. ea nernadpetnmdae deinstrength. adhesion mediates plectin and integrin C. J. beta3 Jones, and W. F. Flitney, 565-573. ciaino OKb hAi euae ycl dein hp,adcytoskeletal and shape, adhesion, cell by regulated is RhoA tension. by ROCK of Activation 20) emti eemnnso ietoa elmtlt eeldusing revealed motility cell directional of determinants Geometric (2003). x.Cl Res. Cell Exp. t P ts sn rpPdPimsfwr.Asgiiatdifference significant A software. Prism GraphPad using -test , . 0.05. 5 m Langmuir 313 nadfo eleg)adotr(ihn5 (within outer and edge) cell from inward m 3616-3623. , 20) erimn fvmni otecl ufc by surface cell the to of Recruitment (2009). 19 20) elmti adhesion. Cell-matrix (2007). 1611-1617. , n 5 – el e ye o traction For type. per cells 7–9 .Cl Sci. Cell J. .Cl.Physiol. Cell. J. 122 1390-1400. , m fcell of m (2007). 213 , Journal of Cell Science ozls . ese,B,Tuua . oda,R . on .J,Hopkinson, J., K. Yoon, D., R. Goldman, D., Tsuruta, B., Weksler, M., Gonzales, egr . pt,J .adBrhdk,A D. A. Bershadsky, and P. J. Spatz, B., Geiger, R. Zaidel-Bar, and T. Geiger, egr .adBrhdk,A. Bershadsky, and B. Geiger, ers . oto . ivr,M . caped .Q,Pri,P . Wheeler, E., P. Purkis, Q., R. Schaapveld, G., M. Nievers, L., Fontao, D., Geerts, E. D. Ingber, and M. G. Whitesides, S., Huang, M., Mrksich, S., C. Chen, cet,R,Prn,D . esn .M,Barrj,K n hn .S. C. Chen, and K. Bhadriraju, M., C. Nelson, M. M., Post, D. Pirone, and R., McBeath, K. A. Tanswell, M., Liu, iebr,J . ai . e,X,Epns,H . uigr .S,Tkwr,D., Takawira, S., G. Budinger, D., H. Espinosa, X., Wei, A., Safi, L., J. Eisenberg, C. J. Jones, and M. Wilson, K., Ridge, S., E. Budinger, K., D. Lane, Ingber, J., and P. DeBiase, M. G. Whitesides, E., Ostuni, L., J. Alonso, S., C. Chen, E. D. Ingber, and M. G. Whitesides, S., Huang, M., Mrksich, S., C. Chen, ijn,S . ePrd,J .adSnebr,A. Sonnenberg, and M. J. Pereda, de H., S. Litjens, ie .E,Ce,C . rsc,M,Te,J,Wieie,G .adIngber, and M. G. Whitesides, J., Tien, M., Mrksich, S., C. Chen, E., L. Dike, C. Turner, and G. Nuckolls, T., Kelly, K., Fath, K., Burridge, eat .R,Co,C . ilr .S,Sa,L,Go . ezg .adChen, and E. Betzig, L., Gao, L., Shao, S., J. Miller, K., C. Choi, R., W. Legant, M. G. Whitesides, and L. J. C. Wilbur, J. J., Jones, R. and P. Jackman, DeBiase, B., S. Hopkinson, J., K. Hamill, urde .adCraosaWdik,M. Chrzanowska-Wodnicka, and K. Burridge, G. Wiche, and L. Winter, M., Gregor, G., Burgstaller, M., C. Baker, O., Z. Burgess, E., S. Chiarella, J., P. DeBiase, D., Urich, R., G. Budinger, asn . i,S . ru . hn .L,Clewo,D .adSchlaepfer, and A. D. Calderwood, L., X. Chen, S., Uryu, T., S. Lim, C., Lawson, M. Mrksich, and C. Liu, H., Hu, D., E. Goluch, J., James, aahm,T,Tuua . aaa . si,N,Oo . ahkw,K., M. Mrksich, Hashikawa, and T. F., B. Lahn, Ono, B., Bugarija, N., A., K. Ishii, Kilian, T., Hamada, D., Tsuruta, T., Karashima, A., D. Dean, C., R. Geiger, E., Lecuona, B., S. Hopkinson, K., Lane, C., J. Jones, soitdmti deini nohla cells. C. endothelial J. in Jones, adhesion and matrix W. associated F. Flitney, B., S. oa adhesions. focal adhesome. integrin oa contacts. focal lh6ea opetnpeet lci soito ihFatnbtde o interfere not A. does but Sonnenberg, binding. F-actin and filament with M. intermediate association plectin with I. prevents Leigh, plectin B., to alpha6beta4 E. Lane, N., G. arxdpsto yavoa pteilcells. epithelial alveolar by C. J. deposition Jones, matrix and B. S. Hopkinson, function. and position, shape, cell of Prog. control for surfaces Micropatterned elsae yokltltnin n hArglt tmcl ieg commitment. lineage cell stem regulate RhoA and Cell Dev. tension, cytoskeletal shape, Cell cells. lung in transduction hemidesmosomes. of breakdown and formation ifrnito uigagoeei sn irptendsubstrates. micropatterned Anim. Biol. using Dev. angiogenesis during differentiation E. D. cells. alveolar I-like type by Cytochem. assembly fiber (Laminin-6) Laminin-311 assembly. adhesion focal of control Commun. global Res. provides shape Cell the death. and and life cell matrix of control extracellular Geometric the between junctions cytoskeleton. transmembrane adhesions: oainlmmnsaotfcladhesions. focal about moments rotational S. C. Rac1 of modulation activities. integrin-mediated cofilin beta4 and through polarity keratinocyte maintains n signaling. and dystroglycan. C. requires 672. lung J. the Jones, in and kinase M. AMP G. Mutlu, S., Soberanes, motility. D. 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