Journal of Cell Science ulam Jacquemet Guillaume through pathway sites IQGAP1–filamin-A–RacGAP1 activation an integrin at deactivated is Rac1 Article Research oriae ciainaddatvto fRc,adacrigya accordingly and requires Rac1, migration of deactivation cell and Productive activation healingcoordinated 2007). al., wound et suppressed Tscharntke Pankov and 2009; 2007; al., 2005) et al., (Bass of et matrices loss fibrillar as such on defects, directionality migration cell and major in of re-organisation results Disruption regulates signalling 2006). cytoskeletal Rac1 Ridley, dynamically 2003; promoting therefore (Ridley, protrusion and by membrane 1998), migration al., cell al., et et Morgan Price 2007; Schwartz, the and 2007; of Pozo (Del activity Rac1 the GTPase small modulates al., et adhesion (Askari ligands Integrin-mediated their for 2009). affinity high a have integrins and activated extended or are a primed whereas have a ligands, and their conformation for by bent affinity low a of in regulated exist including integrins assembly is Inactive affinity. ligand-binding phenotype, function extracellular the modulates Integrin that actin cell switch for conformational 2002). the control platforms (Hynes, and that migration as (ECM) and complexes serve matrix invasion signalling extracellular and tumour physical the cytoskeleton, as a between provide such receptors link processes Integrin transmigration. to pathogenesisleukocyte healing. disease contributing of wound regulation the by and to central required also surveillance is process Migration immune physiological embryogenesis, fundamental a for is migration Cell Introduction migration. cell Further filamin-A, directional coordinate by thereby partner. followed and words: engagement, spatially binding Key integrin activity these whereby its with constrain model IQGAP1 Consistent to a Rac1 fibronectin. suggest novel deactivates directional findings on recruitment, disrupted These RacGAP1 and spreading a matrices. protrusion and during membrane extracellular IQGAP1 unconstrained as fibrillar activity triggered on active RacGAP1 Rac1 RacGAP1 migration or elevated and IQGAP1 cell IQGAP1 triggered filamin-A, identified of expression to expression recruited RacGAP1 analysis, reduced findings, is of biochemical RacGAP1 suppression that that and demonstrated and active analyses to pull-downs immunocytochemistry recruited and is and filamin-A Rac1 that immunoprecipitation immunocytochemistry, complex and a and immunoprecipitation of Using part IQGAP1 GTPase-activating fibronectin. be to IQ-motif-containing on shown spreading and were cell IQGAP1 filamin-A during suggests activity Rac1 complexes link dysregulated with integrin-associated that understood. membrane candidates poorly engagement as activated are dynamic activity (IQGAP1) integrin- off 1 the Integrin of control switching protein for Rac1, that pathogenesis. analysis responsible pathways GTPase are the that proteomic disease small mechanisms Although the migration. Here, the and defined, directional well of reasonably for physiology are deactivation required activation are normal and Rac1 mediated that activation both events cyclical reorganization cytoskeletal to the and via protrusion contribution controls, fundamental spatially ligands a extracellular makes migration Cell Summary 10.1242/jcs.121988 doi: 4121–4135 126, ß Science Cell of Journal 2013 June 12 Accepted ( correspondence for *Author 3 2 1 hitpe .Chen S. Christopher dnug acrRsac KCnr,Isiueo eeisadMlclrMdcn,Uiest fEibrh dnug H X,UK 2XR, EH4 Edinburgh Edinburgh, UK USA of 9PT, 19104, University M13 PA Medicine, Manchester Philadelphia, Molecular Manchester, Pennsylvania, and of of Genetics University University of Sciences, Bioengineering, Institute Life of Centre, of Department UK Faculty Research Research, Cancer Cell-Matrix Edinburgh for Centre Trust Wellcome 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. La QA1 nern irto,Rc,RacGAP1 Rac1, Migration, Integrin, IQGAP1, FLNa, [email protected] 3 arc .Caswell T. Patrick , 1 akR Morgan R. Mark , b nernt a1 iN-eitdkokono ihrflmnAo QA1idcdhigh, induced IQGAP1 or filamin-A either of knockdown siRNA-mediated Rac1. to integrin 1 nvivo in 1 ) 1 dmByron Adam , n atnJ Humphries J. Martin and Lue al., et (Liu 2 oahnD Humphries D. Jonathan , nterglto fRc ciiyuo nernactivation. Results integrin upon activity Rac1 of and migration. regulation cell the MgcRacGAP during in f novel activity as a protrusive reveal findings restrict known These to order (also in RacGAP1 CYK4) protein activating activity Rac1 constrain to integrins a FLNa Specifically, elucidated. Ra integrin-dependent hypothe The Rac1. and (FL integrin (IQGA analyses 1 filamin-A network protein 2011), identify activating al., GTPase et to Schiller used 2011; al., were et Kuo 2009; al., et (Humphries complexes integrin-associated (FN)-induced, fibronectin resolved. partially only integrin still whereby is mechanism activity 2003; Rac1 the coordinates However, activation Negishi, 2005). and al., in et involved (Katoh Nishiya be regulation to reported Rac1 been integrin-dependent have GTPase (GAPs) and proteins (GEFs) activating factors exchange nucleotide guanine of range ciiy aafo he rtoi nlsso FN-induced, of analyses proteomic three from data which activity, by mechanisms new identify To of downstream activity engagement Rac1 FN–integrin suppress IQGAP1 and FLNa b nern assetoercaayi fidvda filamin-A, individual of analysis spectrometric Mass integrin. 1 nti td,wihbid npbihdpoemcaaye of analyses proteomic published on builds which study, this In 1, * 1atvto a etdadtemechanism the and tested was activation c1 nto o LaIGP complex FLNa–IQGAP1 a for unction i htFN n QA1modulate IQGAP1 and FLNa that sis 1 oi .Choi K. Colin , dIGP r erie oactive to recruited are IQGAP1 nd i h erimn fteGTPase- the of recruitment the via 1 spttv ik between links putative as P1) a n IQ-motif-containing and Na) b nernrgltsRac1 regulates integrin 1 3 , b integrin, 1 4121 b 1 Journal of Cell Science QA1ascae ihrdrcl ridrcl,a h leading the at and activity. indirectly, FLNa Rac1 that or regulate suggest in to directly data edge paxillin either These protein 2C). associate, (Fig. complex IQGAP1 and FN adhesion on FLNa the plated with cells assessed. and was other 2B,C) subcellular each (Fig. IQGAP1 co- with co-localisation and substantial not the demonstrated IQGAP1 FLNa did To analyses, of 2A). 2009), (Fig. distribution immunoprecipitation al., IQGAP1 or et FLNa complement either (Humphries with in Arf6 immunoprecipitate enriched GTPase, protein a and small links that CD98hc, FLNa complexes contrast adhesion between FN-induced By Rac1 co-immunoprecipitation 2A). association (Fig. constrain Reciprocal an IQGAP1 to cells. demonstrated same the cooperate in experiments of part IQGAP1 complex were they and protein whether assessed FLNa initially we if activity, test integrin of To sites at complex might a activation form IQGAP1 IQGAP1 activity and and FLNa Rac1 FLNa IQGAP1 engagement. integrin sustained and following that activity Rac1 and FLNa hypothesised constrain to of cooperate elevated we suppression in 1D,E), as (Fig. and resulted and 1G), FLNa expression (Fig. of Rac1 kinetics the of As to 1D,E). recruitment correlated (Fig. IQGAP1 and inactivation 1G), (Fig. Rac1 to FN on IQGAP1 with spread and cells as FLNa time with of increased recruitment The 1G). (Fig. with complexes FLNa of with precipitate to recruitment the IQGAP1 in tested we engagement and 2005), ligand al., for et requirement Nakajima the 2001; with al., co-immunoprecipitate et to (Calderwood reported to integrin FLNa been both enriched have As 1A). specifically IQGAP1 (Fig. and were engagement FN IQGAP1 upon complexes and adhesion FLNa that analyses Proteomic suggested competency. ligand-binding and state activation activity FLNa Rac1 both engagement. suppressing FN that in human of role indicate during downstream important data in an activity These play expression 1F). IQGAP1 Rac1 and (Fig. enhanced IQGAP1 FN contrast, in on or spreading resulted By FLNa cells Similarly, 2009). either in 1D,E). osteosarcoma (Fig. resulted al., of activation expression Rac1 et silencing IQGAP1 sustained or 1D,E) Humphries and FLNa (Fig. enhanced minutes 2007; either 45 of at al., suppression MEFs observed on et activity spreading of during (Bass peak activity by Non-targeting-siRNA-treated a Rac1 with measured of FN, wave were transient GTP-Rac1 pull-down. on a exhibited of plated different levels using were effector and knockdown 1C) oligos, siRNA-mediated (Fig. targeting to cells and subjected 1B) osteosarcoma FN, (Fig. U2OS (MEFs) fibroblasts human embryonic IQGAP1 mouse and activation, FLNa studies. that three activity. hypothesis all Rac1 integrin-modulated the in to contribute tested identified we confidently Therefore, were IQGAP1 and components the between links putative of Analysis connecting network. PPI protein–protein with FN-induced, integrin-associated hypothetical integrated a generate were to databases, 2011) (PPI) interaction al., et al., et Schiller Kuo 2011; 2009; al., et (Humphries complexes integrin-associated 4122 nern neg ofrainlrglto htdtrie their determines that regulation conformational undergo Integrins Rac1 to IQGAP1 and FLNa of contribution the assess To ora fCl cec 2 (18) 126 Science Cell of Journal b nernt a1rvae LaadIGP as IQGAP1 and FLNa revealed Rac1 to integrin 1 b b nerni el nsseso,btwr on in found were but suspension, in cells in integrin 1 nernwe el dee n pedo FN on spread and adhered cells when integrin 1 b nern LaadIGP i o co- not did IQGAP1 and FLNa integrin. 1 b b nernadRc Fg A.Bt FLNa Both 1A). (Fig. Rac1 and integrin 1 nerncreae ihteinactivation the with correlated integrin 1 b nernt another to integrin 1 b 1integrin b 1 rnfri eetr(i.3) ie htFN n QA1co- IQGAP1 and FLNa that Given 3A). (Fig. receptor transferrin teryahso tutrs(i.2) hc r ie fdynamic of sites 2009). are al., which et of 2C), (Machacek sites activation (Fig. lamellipodia, Rac1 structures are to instead adhesion which but early adhesions, 2010), at al., focal et FLNa–IQGAP1 (Askari to activation the localise integrin that not fact Rac1 the does dysregulated with prevent complex consistent and is activity IQGAP1 This Rac1 and signalling. integrin reduce FLNa to that containing serves complex postulate that the a by to of followed Rac1 recruitment us of dynamic activation led the triggers findings initially engagement these together, active to complex Taken the recruit to necessary are IQGAP1 active with co-purification reduce to b and either 3B) anti-active (Fig. of patches to coated reduce proteins Suppression substantially both to of IQGAP1. sufficient recruitment or the was expression FLNa IQGAP1 to inactivation or of necessary FLNa integrin depleted were IQGAP1 cells of or active with FLNa to regions either complex of the if to sites recruit test to not To immobilise 3B). enriched but (Fig. and activation to recruited integrin specifically and IQGAP1–GFP were fabricated Both FLNa–RFP conformation. integrin constrain were spatially anti- surfaces conformation-specific complex. FLNa–IQGAP1 the Micropatterned of localisation the controls active activation with antibodies co-purify anti-integrin activating and on plated are cells when localise rti rvosyrpre orglt a1atvt (Humphries activity Rac1 regulate a RCC2–GFP, to and reported GFP previously 4A–C). protein (Fig. (MS) identified binding were spectrometry proteins mass transiently associated by IQGAP1 co-purifying cells and alone RCC2–GFP and GFP in Rac1–GFP, or performed IQGAP1–GFP, FLNa FLNa–GFP, were expressing new pull-downs Rac1 GFP identify we modulate activity partners, Therefore, GTPase To could additional 1999). of IQGAP1 modulators. recruitment al., the and et through FLNa activation (Ho proteins that these activity however, hypothesised 1999); GAP Rac1 al., to intrinsic et directly Ohta bind lack 1996; to al., reported et been (Kuroda have activity IQGAP1 Rac1 and suppress FLNa to RacGAP1 recruits IQGAP1 niois(i.3) La QA1adtln rti that co- protein 2003), al., a et talin, (Tadokoro active integrin with and affinity purified IQGAP1 high with FLNa, associates 3A). (Fig. formation, antibodies anti- activation-state-specific complex using the immunoprecipitated FLNa–IQGAP1 regulates and activation IQGAP1. integrin and FLNa that of distribution suggests subcellular cells finding of lamellipodia This the in observed affinity, active was with IQGAP1 low and a FLNa of induces of high that conformation a inactive antibody induces monoclonal that anti-human of (a conformation antibody 12G10 activated immobilised monoclonal affinity, on spread anti-human to IQGAP1– allowed and mouse were FLNa–RFP 2E) expressing (Fig. cells human GFP U2OS expressing and MEFs on 2D) integrins cells. (Fig. of conformation of the surface dictate to (Byron the used states were activation 2009) al., integrin et different induce activation Anti- that integrin complex. FLNa–IQGAP1 of antibodies the role of the formation the studied in we 1G), (Fig. engagement nern(i.3) hs aaidct htbt Laand FLNa both that indicate data These 3C). (Fig. integrin 1 oaayefrhrterltosi ewe nernactivation integrin between relationship the further analyse To with co-purified IQGAP1 and FLNa As b nern u o inactive not but integrins 1 b nernbtntwt inactive with not but integrin 1 b b b nern.Etnieco-localisation Extensive integrin). 1 nern etse hte integrin whether tested we integrin, 1 nernatbde n therefore and antibodies integrin 1 b nern saswr performed were assays integrin, 1 b nern r44( mouse (a 4B4 or integrin) 1 b nern Fg 2D,E). (Fig. integrins 1 b b nernuo FN upon integrin 1 nernantibody- integrin 1 b 1integrinwas b integrin–GFP 1 b nernor integrin 1 b b b integrin. 1 integrin 1 integrin 1 Journal of Cell Science F a1atvto eesi 2Scls nce onfrFN rIGP a esrdatr1hu fsraigo N( FN on spreading of ( hour (* blotting s.e.m. 1 represent western after bars by measured Error integrin. analysed was oligonucle and IQGAP1 control #1 K20 or (siFLNa with suspension FLNa antibody treated in for integrin were kept down cells (C) knocked siCTRL cells cells, of U2OS to U2OS recruitment that IQGAP1 and in to normalised (B) levels p was MEFs activation activity in is level activation activation, Rac1 Rac1 Rac1 area and Rac1 (D,E) (F) (D–F). Node FN approach in pull-down effector on 2011). IQGAP1 an spreading using al., and measured cell was during et FLNa activity measured Rac1 (Schiller of and IQGAP1, al. role of or FLNa binders the et targeting siRNA direct study Schiller or Reported (siCTRL) To pr and/or 2009). (B–F) two 2011) al., between organised. al., et interaction manually (Humphries et reported al. were (Kuo between et a al. links Humphries represents putative et by (line) selected identified Kuo highlight edge proteins edges by each the identified and of Mate also name) count the gene was (see spectral network with normalised protein PPI (labelled particular literature-curated protein a a a onto whether mapped represents indicates were (circle) 2009) node al., Each et (Humphries details). complexes for adhesion FN-induced in identified Proteins Rac1. i.1 LaadIGP upesitgi-eitdRc activation. Rac1 integrin-mediated suppress IQGAP1 and FLNa 1. Fig. b nerndrn elsraigo N o ahtm on,total point, time each For FN. on spreading cell during integrin 1 P , .5 ** 0.05; b nernadRc.T lo la iulsto ftecneto between connection the visualisation of clear a allow To Rac1. and integrin 1 P , .1 *** 0.01; n 5 ) h ieiso LaadIGP erimn to recruitment IQGAP1 and FLNa of kinetics The 4). P , .0) U rirr nt B muolt P immunoprecipitation. IP, immunoblot; IB, unit; arbitrary AU, 0.005). A h ewr fF-nue deincmlxsta connect that complexes adhesion FN-induced of network The (A) La QA1adRcA1rglt a14123 Rac1 regulate RacGAP1 and IQGAP1 FLNa, b nernwsimnpeiiae rmHFcl yae sn h pan- the using lysates cell HFF from immunoprecipitated was integrin 1 b nernwr omlsdt h mutof amount the to normalised were integrin 1 b b nernadRc r ipae,adred and displayed, are Rac1 and integrin 1 nernadRc,ndso hsnetwork this of nodes Rac1, and integrin 1 n 5 n 5 ) G uniiaino Laand FLNa of Quantification (G) 4). ;sIGP #1 siIQGAP1 6; b 1integrinto tis oecolour Node oteins. il n Methods and rials ootoa othe to roportional n 5 4). Eswas MEFs otide b b 1 1- Journal of Cell Science eetdi h iepl-on splmnaymtra Table material the were against (supplementary validation proteins specificity 1371 and pull-downs normalisation total, Following five S1). In the controls. as in used detected were 2009), al., et complex. ( a form FN IQGAP1 and FLNa 2. Fig. 4124 T el xrsigFN–F,pae nF o or.(,)Sub (D,E) hours. 2 for FN cr on were plated images FLNa–GFP, mean) expressing cells the 3T3 from differences the of intensity (product PDM hours. 2 for D rninl xrsigFN–F n i and FLNa–RFP expressing transiently (D) nuedfeetitgi ciainsae 1G0 cie B,inactiv 4B4, active; (12G10, 20 states activation integrin different induce m n m(B,E);25 5 ) B olclsto fFN n QA1wsdtrie yfluorescence by determined was IQGAP1 and FLNa of Co-localisation (B) 3). 2 enrdintensity) red mean ora fCl cec 2 (18) 126 Science Cell of Journal m m(D). 6 genintensity (green 2Scls()tasetyexpressing transiently (E) cells U2OS n A oascaino LaadIGP a sesdb immuno by assessed was IQGAP1 and FLNa of Co-association (A) 2 engenitniy.()C-oaiaino La IQGAP1 FLNa, of Co-localisation (C) intensity). green mean ellrlclsto fFN a FLNa of localisation cellular ) mgsaerpeettv falclspae nat-nernantibo anti-integrin on plated cells all of representative are Images e). ae ycluaigaddslyn th displaying and calculating by eated QA1GPadFN–F,pae nanti- on plated FLNa–RFP, and IQGAP1–GFP irsoyi 2Sclsepesn IQ expressing cells U2OS in microscopy aeilTbeS) QA1GP(upeetr material (supplementary IQGAP1–GFP S2), were (supplementary Table proteins FLNa–GFP 408 to material and enriched 122 specifically as 141, identified controls, RCC2–GFP and GFP dIGP a nlsdi human in analysed was IQGAP1 nd n ailnwsdtrie yfursec irsoyin microscopy fluorescence by determined was paxillin and rcptto rmHFcl yae olwn pedn on spreading following lysates cell HFF from precipitation D au o ahpxl hr PDM where pixel, each for value PDM e A1GPadFN–F,pae nFN on plated FLNa–RFP, and GAP1–GFP b nernmncoa nioisthat antibodies monoclonal integrin 1 b -nernGPepesn MEFs 1-integrin–GFP-expressing is cl as 10 bars: Scale dies. 5 (red m m(C); Journal of Cell Science xrsigFN–F rIGP–F n nce onfrete Lao QA1wr ltdo 21 ace.()Tercuteto Laand FLNa of recruitment The (C) patches. 12G10 on plated 10 were bar: IQGAP1 Scale or immunoprecipitation. FLNa IP, either immunoblot; for down active knocked to and IQGAP1 anti- IQGAP1–GFP on or plated FLNa–GFP were expressing IQGAP1–GFP and FLNa–RFP expressing b eseiial nihdi h QA1GPadRac1–GFP and IQGAP1–GFP GAPs, the to in Rac1 found protein enriched were two relative srGAP2, specifically approach, and of be CYK4) this or measure (MgcRacGAP Using a RacGAP1 4B). as (Fig. using counts abundance clustered regulator hierarchically spectral ‘GTPase were term normalised proteins the these with and used annotated was activity’, analysis proteins Ontology identify Gene Rac1, to of regulation the in roles IQGAP1– the direct. that not suggest is association might FLNa the not observations material These (supplementary but in S1). pull-downs detected enriched Table FLNa–GFP was weakly the in IQGAP1 be enriched and to pull-downs found IQGAP1–GFP was the FLNa in in S2–S4). Interestingly, 1993) 1997) Tables al., material al., (supplementary et pull-down et Rac1–GFP (Chuang (Bashour the RhoGDI Cdc42 and and pull-down; calmodulin Rac1 al., IQGAP1–GFP pull-down; 1999), et FLNa–GFP al., (Griffiths et the R-Ras (Ho in and 2011) identified al., were et For 2011) 4C). (Gay (Fig. B identified refilin were known proteins example, FLNa–IQGAP1–Rac1 many bait approach, the this the of of partners validation binding of a As 4C). view (Fig. complex network-based S4), to a Table databases PPI material construct with integrated (supplementary were datasets Rac1–GFP These respectively. and S3) Table integrins. active to recruited are IQGAP1 and FLNa 3. Fig. -nernatbde 9G,atv;mb3 ncie n nitasernrcpo nioy(K9 sangtv oto ( control negative a as (OKT9) antibody anti-transferrin-receptor and inactive) mAb13, active; (9EG7, antibodies 1-integrin ohglgtpoen eetdb Sta a aeputative have may that MS by detected proteins highlight To b nern a sesdb muorcptto sn E7fo 2Scls nwihete Lao QA1wskokddw.IB, down. knocked was IQGAP1 or FLNa either which in cells, U2OS from 9EG7 using immunoprecipitation by assessed was integrins 1 m m. (A) b -nernatbd-otdpths(21,atv;44 ncie,adUO el transiently cells U2OS and inactive), 4B4, active; (12G10, patches antibody-coated 1-integrin b nernwsimnpeiiae rmHFcl yae sn ciainsaeseii anti- activation-state-specific using lysates cell HFF from immunoprecipitated was integrin 1 La QA1adRcA1rglt a14125 Rac1 regulate RacGAP1 and IQGAP1 FLNa, aGP ncl yl euain(iohm ta. 03] both 2003)], of al., et role (Minoshima reported regulation the cycle cell with in [consistent RacGAP1 the nuclear are although mostly RacGAP1– were proteins Furthermore, and FLAG RacGAP1 5B). two endogenous (Fig. of these localisation directly subcellular that interact suggesting between to maximum nm) (theoretical likely 30–40 assays cells within of proximity that ligation distance close indicated in RacGAP1 proximity were co- endogenous both and addition, by IQGAP1 IQGAP1 validated endogenous In with further RacGAP1 5A). and (Fig. endogenous 4D), of IQGAP1– (Fig. FLNa–GFP, immunoprecipitation Rac1–GFP of and pull-downs GFP GFP of between immunoblotting the which interaction activity. by Rac1 mechanism potential suppress possible might the a complex FLNa–IQGAP1 as 2006). on RacGAP1 al., and et focused IQGAP1 (Ohta such therefore previously though GAPs reported even We Rac1 been pull-down known FLNa–GFP have no the interactions Surprisingly, in 4C). identified of (Fig. position were function central network a a this an had as RacGAP1 in network using that a revealed the IQGAP1, within connectivity, of their nodes and analysis clusters FLNa all that addition, algorithm in Rac1, In identified 4B). connecting (Fig. robustly sub-network repeats was biological RacGAP1 three only but pull-downs, aGP–QA1ascainwsspotdby supported was association RacGAP1–IQGAP1 A n 5 ) B 2Sclstransiently cells U2OS (B) 4). Journal of Cell Science rgee nices nRc ciiyfloigintegrin–FN following activity Rac1 in increase an expression RacGAP1 triggered of knockdown addition, RacGAP1 In periphery. cell of these the to together, RacGAP1 recruits loss complex FLNa–IQGAP1 All the that 5C). the indicate data (Fig. triggered ruffles membrane expression in from localisation staining FLNa RacGAP1 or membrane of IQGAP1 suppression of Importantly, 5C). loss (Fig. the cells RacGAP1-knockdown by and confirmed specific was was The RacGAP1 5C). endogenous (Fig. of localisation ruffles membrane membrane in IQGAP1 proteins. GFP-tagged with co-localisation the i performed of substantial pull-downs displayed each RacGAP1–FLAG GFP of and of positions RacGAP1 analysis endogenous the blot western represent cl Asterisks by that tested Rac1–GFP. algorithm was or an IQGAP1–GFP using IQGAP1–GFP organised with FLNa–GFP, automatically RacGAP1 GFP, were of network expressing Co-purification this a transiently (D) of onto cells connectivity. Nodes their mapped GAPs. L Rac1 of were pull-down. known function respective pull-downs highlight each a Rac1–GFP borders in as protein node and each red IQGAP1–GFP FLNa of and abundance FLNa–GFP, connecting proteins, relative sub-network the bait the F illustrating three the of charts the GFP, pie analysis of as in spectrometric representation displayed spectrometry Organic mass are Nodes mass (C) by network. by asterisks. PPI identified literature-based identified proteins with activity’ highlighted The regulator are Rac1. ‘GTPase GAPs and term Rac1 IQGAP1 Ontology Known Gene pull-downs. the RCC2–GFP with and proteins. annotated IQGAP1–GFP Rac1-associated proteins and of IQGAP1- clustering FLNa-, Hierarchical of (B) analysis MS 4. Fig. 4126 ora fCl cec 2 (18) 126 Science Cell of Journal A okfo sdt dniynwFN,IGP n a1bnigpartners. binding Rac1 and IQGAP1 FLNa, new identify to used Work-flow (A) Neggmn Fg G,ada h FLNa–IQGAP1 the as and 1G), (Fig. engagement FN aGP n osri a1activity. Rac1 FLNa– constrain and recruit a RacGAP1 to functions of activation, integrin formation of sites the at Rac1/2/3 complex, whereby IQGAP1 Toure mechanism toward a 2012; with activity consistent al., GAP et Since functional (Bastos 5E). (Fig. exhibits integrins active RacGAP1 with only co-purifies RacGAP1 using immunoprecipitation anti- Furthermore, activation-state-specific 5D). (Fig. engagement sFN n QA1c-uiidwith co-purified IQGAP1 and FLNa As ´ b nernatbde eeldthat revealed antibodies integrin 1 ta. 98,teefnig are findings these 1998), al., et b re oe represent nodes arger 1integrinupon LNa–GFP, sesnodes usters 293T n , Journal of Cell Science oto.()Tekntc fFN n aGP erimn oIGP eeasse yimnpeiiaini 2Sclsdrn elsraigo N IB FN. on spreading cell during cells U2OS in 20 immunoprecipitation by bars: assessed Scale were assay. 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(Fig. FN increased on IQGAP1 to spread RacGAP1 and and FLNa adhered of FLNa–I recruitment were The cells c when the RacGAP1 only and FLNa 5F). fo of (Fig. requirement periphery, the cell assembly the to tested RacGAP1 recruit we to found was complex IQGAP (PLA ImageJ using cell per spots proximity positive of by number assessed the counting was by RacGAP1 quantified endogenous were PLAs and hour. IQGAP1 1 endogenous for between FN on association plated Direct RacGAP1 cells (B) U2OS activity. in FN. Rac1 (PLA) on suppress assay plated ligation to cells IQGAP1 U2OS to in recruited immunoprecipitation is RacGAP1 5. Fig. n 5 n 5 ) ro asrpeetsem (*** s.e.m. represent bars Error 5). 0 L IQGAP1/IgG PLA 70; n 5 GP–aGP complex QGAP1–RacGAP1 iadeggmn nthe in engagement ligand r 9.Arw ihih h oiiesosa h elprpey C uclua oaino aGP edgnu or (endogenous RacGAP1 of location Subcellular (C) periphery. cell the at spots positive the highlight Arrows 79). -rcpttdwt IQGAP1 with o-precipitated b -nernatbde 9G,atv;mb3 ncie n nitasernrcpo nioy(K9 sanegative a as (OKT9) antibody anti-transferrin-receptor and inactive) mAb13, active; (9EG7, antibodies 1-integrin P , .0) E h erimn fRcA1to RacGAP1 of recruitment The (E) 0.005). A oprfcto fedgnu QA1adRcA1wsasse by assessed was RacGAP1 and IQGAP1 endogenous of Co-purification (A) La QA1adRcA1rglt a14127 Rac1 regulate RacGAP1 and IQGAP1 FLNa, nF,t upesRc activity. Rac1 suppress to FN, on complex FLNa–IQGAP1–RacGAP1 at a assembles that demonstrate data nciain(i.1,)adwt LaadIQGAP1 and Rac1 with FLNa correlating with 5F), (Fig. and to FN 1D,E) recruitment on (Fig. spread inactivation cells as m B;25 (B); m b nernwsasse yimnpeiiainfo F cell HFF from immunoprecipitation by assessed was integrin 1 m b (C). m nernatvto ie,drn elspreading cell during sites, activation integrin 1 b nern(i.1) ae oehr these together, Taken 1G). (Fig. integrin 1 1/ , Journal of Cell Science sn um1,adtevlct fec oewscluae.Frec odto,tetm rjcin()admtlt a G ftecl ulnswr p ( were siCTRL outlines in cell period recording the the of over (G) outline map cell by motility connected the and nodes of 400 (F) retrac motility into membrane projection ( mean segmented of time siRacGAP1 areas the were represent the outlines as (blue) condition, cell calculated values motility point, each were negative time dynamics whereas For protrusion, each membrane calculated. membrane For of was seconds. areas represent node 180 (red) each every values frame motility of Positive 1 velocity at plat the minutes and and siRacGAP1 420 QuimP11, or for siIQGAP1 using imaged siFLNa, were siCTRL, Cells with transfected hours. were 4 Lifeact-mEGFP expressing for transiently cells U2OS (F-H) Methods. and Materials T el,siCTRL cells, 3T3 ltdo Nfr1hu.Rc ciiywsqatfe ymauigteaeaednrlftm fteRih rb costewoecl lwlftm,hi lifetime, (low cells), cell U2OS whole in the #2 across and probe #1 Raichu (siRacGAP1 the RacGAP1 of siCTRL or lifetime cells, cells) donor average U2OS (U2OS the in FLIM/FRET measuring #2 using by siIQGAP1 a activity) quantified cells; #1 was low 3T3 (siFLNa activity lifetime, in FLNa Rac1 high targeting 3 hour. siRNA and 1 or #1 for (siCTRL) (siIQGAP1 oligonucleotide FN IQGAP1 control on cells), with plated U2OS pre-treated in (D,E), dynamics. cells #2 protrusion 3T3 siFLNa membrane and cells; and (A–C) 3T3 activity cells U2OS Rac1 in regulate probe RacGAP1 Rac1-Raichu and the IQGAP1 FLNa, 6. Fig. in (FLIM), microscopy lifetime Rac1- fluorescence a using by monitored was probe findings effector Rac1 these Raichu of an validate state To using activation 5D). by the Fig. further, FN 1; on (Fig. modulate spreading approach to pull-down cell demonstrated during were activity RacGAP1 Rac1 and IQGAP1 FLNa, cell directional migration and activity, dynamics Rac1 protrusion regulate membrane RacGAP1 and IQGAP1 FLNa, 4128 ora fCl cec 2 (18) 126 Science Cell of Journal n 5 9 el.Errbr ersn ...(*** s.e.m. represent bars Error cells. 19) n 5 5 iQA1#1 siIQGAP1 25, n 5 7 iQA1#3 siIQGAP1 27, P , n .0) cl as 20 bars: Scale 0.005). 5 2 iLa#1 siFLNa 22, n 5 3 iQA1#2 siIQGAP1 73, n 5 0 iLa#3 siFLNa 30, eutdi oe ieieo h a1Rih rb,i both expression in cells, probe, RacGAP1 Rac1-Raichu RacGAP1 control-treated the or of targeting lifetime IQGAP1 with lower FLNa, in oligos comparison resulted of siRNA In suppression IQGAP1 individual and 6B,D). FLNa using (Fig. two targeting performed or oligos were and siRNA possible IQGAP1 experiments out individual rule FLNa, these three To 2002). for effects, al., down et off-target (Itoh knocked 6A,C,E) (Fig. previously RacGAP1 cells live m (A,F). m n 5 3 iLa#2 siFLNa 63, n 5 1.(,)FE fiinywscluae sdsrbdi the in described as calculated was efficiency FRET (C,E) 31). AE ciainsaeo a1wsmntrdusing monitored was Rac1 of state Activation (A–E) n 5 3 iaGP #1 siRacGAP1 63, n 5 4,sFN ( siFLNa 34), n n 5 5 7,sIGP ( siIQGAP1 27), 8 iaGP #2 siRacGAP1 58, in H h overall The (H) tion. hactivity; gh n 5 do FN on ed d# in #3 nd 2 and 22) n lotted. 5 edges 47; Journal of Cell Science i.5) n eosrt htFN,IGP and IQGAP1 during FLNa, activity Rac1 1D,E; that (Fig. suppressing in demonstrate assays cellspreadingonFN. involved are pull-down and RacGAP1 effector obtained 5D), results Fig. with using consistent are activation previously, Rac1 data higher These indicating 6A,C,E). lines, (Fig. cell mouse and human ciiy(i.6– n upeetr aeilMve 1–4) Movies material cells. supplementary 2010). control with and compared al., 6F–H FLNa, (Fig. et protrusive of membrane activity enhanced suppression (Tyson expression activity, RacGAP1 or QuimP11 Rac1 IQGAP1 increased using were with outlines and Consistent 6H) 6G) (Fig. cell (Fig. measured condition, velocity quantified membrane each and 6F) For (Fig. tracked and 1–4). U2OS Movies in dynamics (supplementary down material Lifeact-mEGFP with FLNa, knocked actin transfected we was microscopy, transiently expression cells assess live-cell RacGAP1 activity, by or cellular To IQGAP1 activity Rac1 regulating protrusive in migration. regulate molecules membrane and these to of protrusion role cooperate the investigated IQGAP1 RacGAP1 FLNa, al., whereby et mechanism Wu and a 2005; identified al., et Having (Pankov 2009). migration cell directional during a1hsacnrlrl nrgltn elmmrn protrusion membrane cell regulating in role central a has Rac1 La QA1adRcA1rglt a14129 Rac1 regulate RacGAP1 and IQGAP1 FLNa, yrgltn ohcl ietoaiyadcl speed. cell and directionality cell IQGAP1 migration both cell FLNa, regulating regulating in by that role fundamental indicate a play data RacGAP1 IQGAP1 cell and These regulating in 7E,F). and cooperate (Fig. proteins cellmigration FLNa these increase that or both directionality suggesting decrease speed, al., further of et not Pankov did suppression 2007; expression the al., FN speed et Combined and on (Bass 7A) plated 1D–F) 2005). (Fig. cell cells (Fig. CDMs in or activity 6A–C) activity with in (Fig. protrusive Rac1 consistent 7C,E) in (Fig. is of increase increase migration consequent elevation persistent migration concomitant observed of the directional loss a The 7D,F). in (Fig. by decrease cells,accompanied U2OS a and FLNa, MEFs of both triggered in suppression (Pankov expression, siRNA-mediated RacGAP1 7C,E) or However, (Fig. IQGAP1 2005). CDMs al., a on et in manner migrated persistent cells directionally Non-targeting-siRNA-treated in assessed. (CDMs) together and matrices cell-derived or was FLNa on migration individually and and 7E,F), either 7A–D), (Fig. (Fig. down MEFs cells knocked also U2OS were in IQGAP1 proteins down these expression knocked RacGAP1 was whether and IQGAP1 FLNa, investigated migration. cell regulate next we protrusion, ie htFN,IGP n aGP upesmembrane suppress RacGAP1 and IQGAP1 FLNa, that Given cl a:20 bar: Scale ...(*** s.e.m. ( analysed were (F) speed cell and (E) directionality cell condition, For each hours. 16 for recorded migration their hours and 4 for CDM on plated were siFLNa/siIQGAP1 or siIQGAP1 siFLNa, siCTRL, with transfected ( analysed were (D) speed cell and (C) cell directionality and (B), created were 0.5) below directionality with cells tracks highlight red tracks; cell (40 plots spider condition, each For areas. inset delineate boxes Red on CDMs. cells the of morphology the demonstrating fields Representative (A) manually tracked. and hours 16 for recorded migration and their hours 4 for CDMs on plated were siRacGAP1 or siIQGAP1 siFLNa, siCTRL, with transfected cells U2OS (A–D) migration. cell directional regulate RacGAP1 and IQGAP1 FLNa, 7. Fig. n n 5 5 ) ro asrepresent bars Error 3). ) EF MEFs (E,F) 3). P , m 0.005). m. Journal of Cell Science r ato ope htfrsdwsra of downstream forms that IQGAP1 complex and FLNa a (1) of findings: part major Rac1 are following coordinates the that on based mechanism a novel of GAPs downstream a and activity GEFs identified of Rac1 level have suppress protein to We and RacGAP1 activation recruit Hypothetical IQGAP1 edge. and its Discussion FLNa around 3). gradient and red (2 a nodes. recruited by their are represented of recruit IQGAP1 is the size and level via the FLNa likely activation by most and Rac1 represented activity, maximum, 4). Rac1 a and in to (3 increase increase activity an levels triggers engagement activity active Integrin–ECM Rac1 to activation. (1). RacGAP1 Rac1 and regulating IQGAP1 events signalling FLNa, mediated of Recruitment 8. Fig. 4130 fRc ciiycudocrtruhasmlrmechanism similar a through occur could activity potentiation Rac1 IQGAP1-mediated that of proposed was activity of it GTPase Thus, binding 1999). intrinsic that Cdc42 suggested been inhibits regulates vitro has IQGAP1 IQGAP1 It to which al., unclear. et Cdc42 by Ho is 1996; mechanism al., activity et precise Rac1 (Hart the GAP a so or GEF 1999), a either to as this directly act contrast that show in we activity. and cues, Rac1 the IQGAP1- suppresses activity, extracellular describe mechanism in soluble to Rac1 plays with report of stimulation engagement first the modulation cell–ECM is dependent that This role 2009). al., essential al., et et David activity 2006; Hu by Sacks, 2011; and triggered Rac1 (Brown response cytokines IQGAP1-dependent the or and factors on growth of primarily positively focused studies al., have both et modulation date, Jeong 2012; GTPases al., To et small Casteel 2007). 1997; al., of et (Bashour activity negatively the regulate 8). (Fig. migration model cell a directional activity coordinates Rac1 suggest thereby over and and control IQGAP1 data tight and FLNa, maintains recruitment, by our protrusion RacGAP1 followed Thus, engagement, membrane integrin recruits migration. whereby disrupts FLNa, cell of activity IQGAP1 RacGAP1 Knockdown directional (4) Rac1 and (3) activity; suppress Rac1 IQGAP1 suppress engagement; IQGAP1 to RacGAP1 and FN-integrin FLNa following (2) activation; QA1cnan niatv aGPdmi n osnot does and domain RasGAP inactive an contains IQGAP1 to function can IQGAP1 that indicates evidence Accumulating rlnigCc2atvto Hr ta. 96 oe al., et Ho 1996; al., et (Hart activation Cdc42 prolonging , ora fCl cec 2 (18) 126 Science Cell of Journal b nernatvto.Ti ocuinis conclusion This activation. integrin 1 b integrin 1 b nernrgltsRc activity. Rac1 regulates integrin 1 in eeqiieydpneto ohmcoevrnetland environmental micro the both that on likely is dependent will it exquisitely activity GTPase IQGAP1, be coordinates precisely. IQGAP1 via which behaviour by Rac1 inhibition, mechanisms cell both promote and orchestrate can stimuli activation to extracellular to different edge, that the tempting Given leading effectors and to is the regulators scaffolding GTPase due to GTPase it small ‘small both a is Therefore, recruiting as that act platform’, IQGAP1 could effectors proteins. is IQGAP1 ours, by that other speculate from downstream activity of and 2004; GTPase studies, recruitment their these al., of from 2008). et modulation al., and theme et (Noritake recurring Sakurai-Yageta A 2011; 2007) S3) 2010; al., al., et Table et Pelikan-Conchaudron al., (Awasthi material Rap1 and small (supplementary et al., 2009) et is other al., Kuroda there et regulate Jeong 1997; (Hu al., RhoA, and Arf6 et supplementary and (Bashour recruit 1996), Rac1 Cdc42 and can IQGAP1- including to IQGAP1 GTPases 4B in addition (Fig. that In involved evidence RhoA S3). be Table could of material which domain-containing activation 3, homology mediated member putative pleckstrin Proteomic G a well-established 2011). namely family identified a al., GEF, proteins et 2012), RhoA IQGAP1-associated (Guilluy al., of activity promoted analyses Rac1 et IQGAP1 of that (Casteel antagonist reported to activation study recent RacGAP1 suppressing RhoA a IQGAP1 recruit activity, with subsequently Consistent Rac1 sites hydrolysis. and at GTP activation Rac1 promote active integrin immobilise might of 1996), that al., IQGAP1 et reported that (Kuroda have by suggesting GTP-Rac1 to others recruited preferentially is model, binds that our IQGAP1 Rac1 with of identified Consistent regulator have IQGAP1. we negative and that the time, activity as first Rac1 RacGAP1 the suppress for also report, can we IQGAP1 Here, 2006). Sacks, and (Brown ceerpeetn yl fsqeta integrin- sequential of cycle a representing Scheme eto GEF a of ment re Journal of Cell Science eetr,sc sgot atrrcposo syndecans, or IQGAP1. to differential receptors effectors other and the regulators factor GTPase the of dictate these of growth recruitment how modifications downstream post-translational identify as signals and/or to such intracellular and receptors, of time, and/or discrete convergence at locations these or simultaneously if determine IQGAP1 subcellular to to recruited be are will challenge molecules next The context. cellular eeietfe sIGP-soitdpoen (e.g. proteins proteins junction IQGAP1-associated cell–cell Lehtonen and as to 2012; 2004) identified al., al., known et were et Noritake than is 2005; (Krishnan functions al., IQGAP1 junctions et of context, cell–cell range at this broader localise of In a reports anticipated. has previously These RacGAP1 2012). that suggest al., functions RacGAP1 centralspindlin-independent et and cytokinesis- (Ratheesh at RacGAP1 junctions for role interphase cell–cell an identified interphase. also during has edge work leading Recent cell the at activity investigations. The Rac1 in regulating future role of S1). a subject play the could be Table here will division described material cell pathway the (supplementary cohesin that possibility Rac1– and by pull-downs and identified IQGAP1–GFP 1995) were FLNa–GFP, GFP 2001) al., the al., in et et kinesin-like spectrometry (Hauf mass (Lee 2005), 11) SA-2) al., and and KIF23) SA-1 et 2 (subunits and (Yu (isoforms (KIF11 calmodulin 2012; septins 2010), al., proteins regulators al., as et cytokinesis et such Mondal (Estey addition, components 1998; In spindle al., 2011). et and al., Eng et 2008; division Padmanabhan cell for al., important et be to Oceguera-Yanez reported (Canman IQGAP1 been FLNa, 2003; also Interestingly, have 2005). al., Rac1 Fang, and et and Zhao Minoshima 2005; al., 2001; cytokinesis et during al., role et regulatory a (Hirose play to previously described uiidadlclsdwt activated with localised and purified junctions. cell–cell at RacGAP1 recruiting also in may involved IQGAP1 be Therefore, S3). Table material (supplementary nieyt edrc n n ftenx hlegslaigfrom leading challenges next the of is one integrin and to FLNa direct of be recruitment in to the Therefore, unlikely that talin. predict inactivate with we context to along recruited this is integrins therefore key FLNa activated that to and a demonstrated specifically 2003), data talin, our al., with However, integrin. et to compete binding (Tadokoro to FLNa 2006). activator thought al., et is (Kiema overlapping region site integrin a binding in talin 2004), the al., to et with The Travis reported 2011; been al., surprising. et the has Ehrlicher to FLNa predict is integrins. bind would the integrin inactive directly association binds restrict integrin–FLNa addition, active FLNa the to that In to of integrins model protrusions. FLNa current primed uncontrolled of specifically unligated of IQGAP1 recruitment of and formation FLNa sub-set 2007). the al., that the et possible (Galbraith target is sites adhesion therefore it unligated for and Therefore, lamellipodia, probe polymerisation to in actin ‘primed’ by that, be activated structures reported get adhesion been can al., early has integrin et at It (Askari lamellipodia, 2C). activation to (Fig. focal integrin instead classical 2004); of but to al., sites 2010), localised et are IQGAP1 Travis which 2006; or adhesions, Suzuki, FLNa Nakajima and neither 2006; to Takahashi however, al., recruited 2005; et be Kiema al., 2001; to et al., reported et previously (Calderwood been integrin have FLNa and u aadmntae htRcA1pasa motn oein role important an plays RacGAP1 that demonstrated data Our been has complex, centralspindlin the of part as RacGAP1, u aadmntae htFN n QA1seiial co- specifically IQGAP1 and FLNa that demonstrated data Our b nernti Clewo ta. 2001; al., et (Calderwood tail integrin 1 b nern ohIQGAP1 Both integrin. 1 b integrin 1 b -catenin) b 1 La QA1adRcA1rglt a14131 Rac1 regulate RacGAP1 and IQGAP1 FLNa, otlcl eu FS isr)ad2 /lmueinterferon mouse U/ml 20 and Biosera) (FCS; serum calf foetal (vol/vol) 10% with supplemented Sigma-Aldrich) (DMEM; medium Eagle’s modified 33 and Wild-type culture Cell Methods and Materials MMsplmne ih1%(o/o)FSa 37 at in cultured FCS (vol/vol) were 10% cells osteosarcoma with supplemented U2OS DMEM human and cells kidney embryonic 293T ihrn irtr eet(adsar ta. 09 ra or 2009) al., et (Baldassarre defect a in migratory migrate to no previously ability of reported the either speed have in migration. the Others loss manner. increased a cell directionally-persistent only triggered not also disrupted but proteins migration these tightly expression of be any RacGAP1 Silencing must or activity IQGAP1 is Rac1 it temporally. that and behaviour spatially both surprising cellular coordinated in and plays integrin not Rac1 polymerisation perhaps that upon role during actin mechanosensation, fundamental activity activity regulating the Rac1 multiple Given integrin-mediated constraining processes. Rac1 of that these importance during fact limit the highlights the or to analyses, activation, exist spectrometric mass we GFP–FLNa mechanisms with Although FilGAP 2006). our of al., association (Ehrlicher et in the Ohta can detect strain 2010; to al., integrin unable mechanical FLNa et were and Nieves by that 2011; FilGAP, upon modulated al., activity, et previously is Rac1 IQGAP1 association of reported suppressor this been alternative and an has recruit FLNa it are activation, IQGAP1 containing and FLNa complex how determine integrins. to activated be to recruited will study this yais n h euaoymcaim fti network. the structure, this of the unravel mechanisms regulatory to the help binding and will FLNa point, dynamics, starting and of IQGAP1 a using analysis as network, partners proteomic this systems-level of regulate that components likely to the is information It transduce migration. next they their cell network, The how this of and spatially. components other inter-connection, identify behaviour to be cell will network, challenge control the regulatory from these to broader signals envision much soluble microenvironment and We a matrix-associated of migration. multiple nodes integrating cell the controls as that components activity Rac1 whereby to mechanism restricted. one spatially be be can may events activation protrusion integrin cell complex of FLNa–IQGAP1 sites the dysregulated and of to integrin become recruitment of The would cycle control. the lack events and break protrusion to switch activation, off protrusion. Rac1 an initial of the absence of sites the promotion adhesion In and transient activation these new Rac1 at making in activation results by Integrin environment adhesions. its protrusion cell–ECM explores expansion a cell their during a Indeed, our restrict activity. event, to with Rac1 order suppressing consistent in locally is by protrusions recruited This are formed RacGAP1 2005). newly and (Bass to IQGAP1 al., FLNa, cells et that these model concomitant Pankov proposed in the 2007; measured CDM and These al., dynamics activity on et Rac1 protrusion observed FN. high in of defects by increase Migration mode 2010) 2D explained on 2009). to be different al., compared al., could matrices et the cells fibrillar (Petrie et on by FN utilise for explained cells (Leung that be migration migration knockdown could cell discrepancies FLNa of following speed decreased ˚ u xeiet eosrtdta upeso fFLNa, of suppression that demonstrated experiments Our a to recruited was RacGAP1 that discovered we While ncnlso,w eotasgaln aha linking pathway signalling a report we conclusion, In ,5 vlvl CO (vol/vol) 5% C, b -nernGPepesn Eswr anandi Dulbecco’s in maintained were MEFs 1-integrin–GFP-expressing 2 ua oeknfbolss(Fs,mue33cls human cells, 3T3 mouse (HFFs), fibroblasts foreskin Human . ˚ ,5 vlvl CO (vol/vol) 5% C, c SgaAdih at (Sigma-Aldrich) b 1integrin 2 . Journal of Cell Science nvriyo abr,Mrug emn) n iec-EF fo Roland (from Lifeact-mEGFP Pharmacology, and of Germany), Institute Marburg, Wedlich-So Grosse, Marburg, Robert of (from Hospital, University Women’s FLNa– IQGAP1–eGFP and Japan), Brigham MA), Nagoya, Nakamura, Boston, (from Fumihiko Medicine, (from domain of FLNa–GFP CRIB School and GST-PAK-1 RFP University gifts: Nagoya as Kaibuchi, obtained Kozo were plasmids following The prahwt S-A ed.Clswr eu tre n anandin pull-down maintained effector and 10 starved an on plating serum using before were lysates hour Cells 1 from for beads. purified suspension GST-PAK affinity with was approach Rac1 Active assay activity Rac1 NaCl, mM 150 6.8, CSK pH in Pipes, lysed and mM PBS 10 cold MgCl X-100, mM with 3 washed Triton sucrose, hours, mM 2 (wt/vol) 150 for [0.5% FN on buffer plated were Cells experiment. in every Immunoprecipitation for RacGAP1 blotting down western Levels knock (J-008650-08). by to #2 verified and used down were (J-008650-06) oligos knock knockdown #1 siRNA of to siRacGAP1 were The used lines lines. was cell (J-058520- cell human Dharmacon) #3 human (L-012579-00, and knock and in (J-058520-20) pool to #1 FLNa smart (mouse siFLNa J-004694-06) used FLNa were #1 ON- oligos The lines IQGAP1, siRNA siIQGAP1 cell 18). mouse The The were in (human J-040589-10). 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Thermo Santa Santa Biotechnology), Cruz (PA5-22265; (C20; GFP Santa Biotechnology), RacGAP1 CD98hc (D-3; Cruz and IQGAP1 Santa anti-human anti-human (H-109; (BD mouse goat IQGAP1 Sigma-Aldrich) anti-human Rac1 were rabbit (1G6; human Biotechnology), used and Sigma-Aldrich), antibodies RacGAP1 mouse (AC-40; Polyclonal international), actin human human MBL Biosciences), and (K0100-3; mouse IQGAP1 Cultures), mouse mouse European Cell BD Other OKT9; 349; of and (clone Biotechnology. 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Red Reagents Detection 2 mgswr olce sn Coolsnap a using collected were Images . 6 .Pitvstn a sdt allow to used was visiting Point ). 4 Had05 vlvl rtnX-100). Triton (vol/vol) 0.5% and OH 6 180 6 m N .0Pa Apochromat Plan 1.40 /NA ,5 m, m ˚ /lacri cd(Sigma- acid ascorbic g/ml ro ouse. to prior C m /lDaeI(oh)at (Roche) I DNase g/ml ˚ m na xoet200M Axiovert an on C atcesz;Waters) size; particle m 6 N .5H Plan HC 0.15 /NA 5 100*(1– 6 75 t da m / m, t d ) La QA1adRcA1rglt a14133 Rac1 regulate RacGAP1 and IQGAP1 FLNa, lsee ntebsso nete ero orlto sn lse . (C 3.0 Java Cluster using using visualised and correlation 2004) al., Pearson et uncentred Hoon (de hierarchically of 1.50) were version basis Library, Proteins Clustering the in regulators. GTPase on provided activity’ as clustered regulator selected ‘GTPase are term were Ontology Gene (GO:0030695) proteins the with annotated identified Proteins 2009). all of S1–S4. Tables Details material supplementary 26836–26850. numbers ta. 07,FN n QA1bnesadRc euaos(hsstudy). (this regulators Rac1 and binders (Zaidel-Bar IQGAP1 proteins and FLNa adhesion-associated 2007), al., integrin Interaction et and Protein of 2009) al., the et databases (Wu in 2011) literature-curated (June reported a network onto sapiens PPIs Homo mapped platform of Analysis were Network consisting Proteins 2011). interactome al., human et performed merged (Smoot was 2.8.1) analysis (version network Cytoscape PPI using 2004). (Saldanha, 1.1.6r2) (version TreeView ta. 03,GPwsue stengtv oto.Dt eecnetdusing converted were PRIDE Data the in control. (Vizcaı deposited negative and (http://www.ebi.ac.uk/pride) 2009) the al., et as database (Barsnes (Mollinari used 2.5.5) Rac1 (version was with Converter GFP interact Rac1–GFP PRIDE to the 2003), reported For al., previously recruited. been et specifically has be RCC2 controls to since over considered dataset, the twofold were in than RCC2) Proteins more and count). enriched (GFP spectral and datasets (normalised sample IQGAP1–GFP protein entire and that the given in FLNa–GFP of a observed weight of spectra molecular of count the number spectral to total unweighted the an the to normalised using in protein calculated resulted was abundance datasets. criteria protein all acceptance for 0.1% at These of of rate assignment peptides. discovery and false level validated protein protein estimated the unique, at probability two at 99% probability least least 90% least at at level, of peptide identification of the analysed threshold a further were using Data Scaffold were Software). in Proteome validated 2003) Data 3.00.03; Beavis, (version respectively. and Scaffold (Craig within 2007.01.01.1) Da, from (version and implemented 0.5 Tandem precursor X! and engine for and search Da the variable tolerances doubly using 0.4 Mass only a were considered. and ions were as used, fragment ions allowed were precursor values charged was triply mass cleavage methionine missed precursor one to of Monoisotopic up 2.2.03; permitted. with oxidation considered, (version were peptides and tryptic server Only as modification. modification set Mascot was cysteine fixed in-house of Carbamidomethylation an a 1999). al., to et (Perkins submitted Science) Matrix were Searches origin. ald nqa variance). unequal tailed, upeetr aeilaalbeoln at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.121988/-/DC1 online available PhD material months. Trust Supplementary 6 number Wellcome after [grant a release for by Trust PMC supported Wellcome in was Deposited the G.J. studentship. by M.J.H.]; to supported 092015 was work This Funding redaction the with manuscript. assisted P.T.C. the M.J.H. and of patches. P.T.C. entire the A.B., antibody-coated supervised M.R.M., protrusion M.J.H. project. the experiments. and RacGAP1 provided the migration with analysis. assisted C.S.C. MS cell the and with the assisted manuscript. C.K.C. J.D.H. for and the A.B. data wrote experiments. and analysis the experiments collected the Selley of M.R.M. most Julian performed G.J. and analyses contributtions Author spectrometric mass support. bioinformatic for the Manchester) of with (University Manchester) help of (University Knight for David and Warwood Stacey thank for We (University UK) Carisey Manchester, Alexandre Manchester, thank We of antibody. JB1A the (Univer for Wilkins Canada) John and constructs Marbur of University Hospital, Pharmacology, Germa Wedlich-So Women’s Martinsried, Roland Biochemistry, and Japan), (Brigham (Nagoy Nagoya, Kaibuchi Nakamura Kozo MA), Fumihiko Boston, thank We Acknowledgements Student’s the cases other all in 30, than legends. higher figure the in and asterisk appropriate, an when performed was analysis Statistical analysis Statistical eeOtlg nlsswspromduigDVD(eso .)(un tal., et (Huang 6.7) (version DAVID using performed was analysis Ontology Gene he ilgclrpiae eepromdfrec F uldw.Relative pull-down. GFP each for performed were replicates biological Three Z tsswr efre hntesml iewas size sample the when performed were -tests y n oetGos Isiueof (Institute Grosse Robert and ny) ,Mrug emn)frproviding for Germany) Marburg, g, epwt h irsoyanalyses. microscopy the with help iyo aioa inpg MB, Winnipeg, Manitoba, of sity lnr(a lnkIsiueof Institute Planck (Max ¨ldner nvriySho fMedicine, of School University a ´ oe l,21)udraccession under 2010) al., et no t ts a sd(nard two- (unpaired, used was -test P ausaeidctdby indicated are values Journal of Cell Science ioe . aahm,T,Iaoo . oaa .adKtmr,T. Kitamura, and T. Nosaka, I., Iwamoto, T., Kawashima, K., M. Hirose, J. Peters, and C. I. Waizenegger, S., Hauf, at .J,Clo,M . oz,B n oai,P. Polakis, and B. Souza, G., M. Callow, J., M. Hart, ulu,C,Gri-aa .adBrig,K. Burridge, and R. Garcia-Mata, C., Guilluy, hag .H,Bh,B .adBkc,G M. G. Bokoch, and P. B. Bohl, H., T. Chuang, rfih,G . rnl . le,J . r n atr .L. M. Matter, and 3rd S., J. Allen, R. 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