Journal of Cell Science n erdcini n eimpoie htteoiia oki rpryattributed. properly is Attribution work Commons original the Creative that the provided of 2009; medium distribution terms any use, the in unrestricted reproduction under al., permits and distributed which article (http://creativecommons.org/licenses/by/3.0), Access et License Open an is Arp2/3 (Ismail This and which Rac1 in targeted Fibroblasts GTPase The directly 2009). 2004). Rho Kirschner, be and al., the Lebensohn can et by of 2010; Steffen complex tips activated al., 2001; the WAVE et al., at pentameric et (Chen accumulates that (Hahne complex 2010), Scita, lamellipodia WAVE Gautreau, and the and Stradal 2001; Derivery called al., now Scar/WAVE et by al., (Stradal 2006), 2008) al., et proteins the et associated (Miyoshi Lai between and 2007; network Mullins, interface actin and the Iwasa growing 2006; at and activated membrane is plasma 2012). the complex al., filaments, et by Arp2/3 Vinzenz actin 2010). generated 2011; of The Svitkina, junctions and of Sixt, branch (Yang types by complex and networks Arp2/3 part different in Renkawitz of connected to 2002; composed directly al., are cells et Lamellipodia epithelial (Small and ranging leukocytes types, fibroblasts cell various of from migration the initiate Lamellipodia for essential Introduction is signaling Rac that migration. demonstrate chemotactic that data suggest in also polarity our findings of Our maintenance Together, words: formation. or Key filopodium assembly. establishment or of spreading the adhesion removal for to although genetic not crucial focal – but by Rac are migration, unaffected to how GTPases cell largely explaining Rac efficient contribute potentially were of for veils adhesions, intensity loss and cytoplasmic can focal their protrusion of notable in established as lamellipodium advancement – components a well and the different and by as essential filopodia of followed adhesions migration mobility formed not filopodia the focal cell spread, of increased of to random extension deficiency size able the Rac and and However, by were closure number achieved cells wound The was by Rac-deficient them. not in cells the defects, but Rac-deficient between these reduction to member, these Rac3. in strong subfamily Spreading and Despite adhesion Rac showed adhesions. Cdc42. Rac2 gradient. nascent either focal of or Rac of chemotactic of levels expression RhoG in a by initiation detectable by deficient to restored lack also and were sensitivity Cells that but structures space Rac1 RhoG. subfamily, these new for but or Rac deficient lamellipodia, of the Cdc42 apparent genetically of exploration of lines GTPases devoid both cell were Rho to fibroblast cells by viable relevant stimulated describe considered potently we be protrusion is Here migration, can cytoplasm two-dimensional formation In of Lamellipodium lamellipodia. and sheets substratum. ruffles thin membrane of these protrusion by of accompanied commonly is migration Cell Summary 10.1242/jcs.118232 doi: 4572–4588 126, ß Science Cell of Journal 2013 July 9 Accepted ( correspondence for *Author 7 6 5 4 3 2 1 formation not adhesion is Steffen focal Anika but and migration spreading cell for for required crucial is function Rac 4572 a Faix Jan ahi .Ladwein I. Kathrin imdclIsiue RC nvriyo oehgn K20 oehgn Denmark Germany Copenhagen, Hannover, DK-2200 D-30625 Copenhagen, 1, of Carl-Neuberg-Strasse Germany University Austria School, Hannover, BRIC, Vienna, Medical D-30625 Institute, A-1030 Hannover 1, Biomedical 3, Chemistry, Carl-Neuberg-Strasse Bohr-Gasse Biophysical School, Dr. for Medical Sciences, Institute Hannover of Toxicology, Academy for Austrian Mu Institute Germany Biotechnology, of Braunschweig, Molecular University D-38124 of Germany Biology, 7, Institute Bonn, Cell Inhoffenstrasse D-53115 Molecular (HZI), 13, for Research Strasse Institute Infection Karlrobert-Kreiten for Bonn, Centre of Helmholtz University Genetics, of Institute 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. 6 hrsaE .Stradal B. E. Theresia , ci,Rc,Mgain dein aelpda iooi,Ceoai,CAAX Chemotaxis, Filopodia, Lamellipodia, Adhesion, Migration, Rac1, Actin, 1 aksLadwein Markus , [email protected] 2 .Mri Holleboom Margit J. , ) 2 2,3 eriA Dimchev A. Georgi , odBrakebusch Cord , ntr clspaz5 -84 Mu D-48149 5, Schlossplatz ¨nster, 1,2 lra Schur Florian , 1 7 neHein Anke , n lmn Rottner Klemens and Nihv eetyidctdmlil,rdnatpathways redundant multiple, indicated by proteins recently Rho of have depleted RNAi fibroblasts (Nobes primary structures of these performance trigger Aspenstro also 1995; Rac- Hall, can the and Cdc42 as major al., such or proteins et RhoG Rho (Ridley other drives related 1995), ruffles Hall, induce and membrane to Nobes which known and 1992; are lamellipodia Rac1 (Aspenstro of of as such formation cytoskeleton proteins Rac most actin Although 2004). Rottner, the of and of Ladwein reorganizations overexpression 2008; Ridley, 2008), and (Heasman 2012). mammals al., et remained complex Wu 2012; efficiency al., Arp2/3 of migration et (Suraneni impact to on controversial the complex WAVE suppression However, al., protrusion. lamellipodium of WAVE lamellipodial et for importance Steffen 2008), of signaling Higgs, 2004; and the Nicholson-Dykstra al., depleted 2006; confirming et al., form et cells to (Innocenti Steffen fail 2004; RNAi 2012) do by al., components et as (Suraneni ArpC3 lamellipodia, subunit Arp2/ the complex ES- for or 3 deficient 2012) genetically al., cells et fibroblastoid (Wu cell-derived suppressed stably is expression complex 4 h aiyo h Tae opie 0mmesin members 20 comprises GTPases Rho of family The .Vco Small Victor J. , ntr Germany ¨nster, 1 iaSchwenkmezger Lisa , 4 aetSchwarz Janett , 1,2, me l,20) nlsso migratory of Analyses 2004). al., et ¨m * 1 tfnArens Stefan , eerhArticle Research 5 afGerhard Ralf , me al., et ¨m Rac1 3 , 5 , . Journal of Cell Science eragmnsb yasn a Tae.Frinstance, cytoskeletal For induce GTPases. also unclear Rac could remained bypassing it RhoG by 2003), or Negishi, rearrangements Cdc42 and both 2005) (Katoh al., Rac, whether et Nishimura on RhoG RhoG 2005; al., signaling and et and (Baird connections of Cdc42 of proteins biochemical convergence downstream Although Rac potential 2009). established Cdc42, al., et of (Monypenny downstream operating utemr,i ol o efral xlddta d4 can Cdc42 Rac. that of excluded independent formally formation 2008). be lamellipodium al., to drive not 2006), et reported could with (Meller was independent it (Ridley, complex nucleotide complex Furthermore, WAVE and proposed the weak WAVE rather to be been GTPase the this of of previously binding interaction had direct RhoG-GTP a although eeini hs ouain,n niiulcoe n thus and clones individual no primary populations, gene Rac1 in efficient permanent these reasonably observed of in in spite In deletion also deletion 2009). Rac1 al., et were tamoxifen-mediated (Liu mice after formation isolated be fiber site fibroblasts to adhesion stress migration, Rac1 al., in et defects and concluded (Guo modest assembly recently, al. fiber only and More stress 2006). and et spreading adhesion found focal Guo cell for essential formation, colleagues efficiency, adhesion migration and focal on effects Vidali 2006). al., moderate al., et although et (Vidali (Guo fusion Interestingly, HIV–TAT adenovirus cell-permeable through a or delivered 2006) recombinase Cre by odtoa a1allsi irbat eepeiul deleted previously were fibroblasts in alleles Rac1 Conditional a ucini rtuinadmgain4573 migration and protrusion in function Rac Rac1 2 / 2 ellnswr eeoe,peldn faithful precluding developed, were lines cell el analyzed. cells EH and (E–H) Rac1 Rac1 s.e.m.; means as expressed are and experiments independent three from collected were Data stimulations. factor growth of Quantification (M) (H,L). or HGF (G,K) EGF (F,J), PDGF with stimulated or (E,I), starved either were (I–L) stimulation. factor Growth (E–L) of staining Phalloidin (C,D) panel). (lower antibody tubulin and panel) (upper antibody Rac1 of blot Western (B) indicated. as genotypes for clones individual and cells of Genotyping (A) ruffles. and form lamellipodia to unable are MEFs Rac1-deficient 1. Fig. 2 2 fl/fl / / 2 2 n n individual and 5 Es(D). MEFs Rac1/3 with clones oa ubrof number total Rac1 Rac1 Rac1 2 fl/fl fl/fl / 2 Rac1 C and (C) Rac1 MEFs MEFs 6 fl/fl 2 / 2 Journal of Cell Science aelpda swspeiul soitdwt a1ls of Arp2/3-mediated of loss inhibition Rac1 lacking and previous cells 2006) with al., these associated with et in previously (Vidali Furthermore, variance blebbing function 2009). was noticeable al., at as observe et lamellipodia, 1C,D), Liu not (Fig. 2006; did al., we types et comparable (Guo displaying cell appeared observations pattern both instead, fiber points. stress in attachment overall architecture peripheral the between Otherwise, bundles starfish-like actin and prominent fibroblasts, a elongated of slightly typical the developed morphology lost polarized confirming The had frequently 2006). cells 1D), al., and et (Fig. Rac1-deficient Vidali of 2006; structures appeared al., majority et these MEFs (Guo observations of previous Rac1-deficient cell devoid whereas the completely 1C), at (Fig. lamellipodia and spontaneous periphery their Rac1-expressing fibronectin formed expected, and fibroblasts As on cytoskeleton. control seeded MEFs actin the were Rac1-deficient for stained counterparts structures, floxed regulation actin the parental, in GTPases specific Rac of of function crucial the confirm in To formation ruffle and lamellipodia fibroblasts S1E), for essential is Fig. Rac1 proliferation. material for essential propagation (supplementary absolutely slower not is MEFs much Rac1 that control a showing at than albeit rate years, over continuously individual phenotypes, variability studied clonal relative potential and the minimize levels on #22 to #17, order of based #13, In protein #3, 1A,B). termed selected, (Fig. and #24 rates, were and growth of clones comparability data) experiments, Rac1-deficient subsequent For five unchanged S1D). (unpublished Fig. showed material (supplementary transcript cells 1B, Ridley, (Fig. Rac1-deficient and RhoG (Heasman Interestingly, Rac1 Rac2 to identity the 2008). thus and to 72% belongs bearing system. RhoG lines family, that Rac1 cell determined Rac cell was fibroblastoid it for Rac-free analysis, These sequence western virtually By case S1A–C). first, by the Fig. the constitute undetectable material was remained (unpublished supplementary protein counts as threshold Rac3 blotting, exceeded and hardly data), in levels clones absolute below), (see these analyses further for selected clones in deleted was Rac1 permanent of Generation Results derive could study phenotypes removal. our in Rac1 for incomplete variability developed from that single lines possibility the cell the at Rac-deficient avoid deletion The gene Rac1 level. of cell consequences the of analysis 4574 Blse l,20;Cret ta. 05 n a2epeso is Although expression increased 1989). indicated Rac2 al., analyses and et microarray (Didsbury 2005) development cells al., hematopoietic brain et to Corbetta of confined 2003; stages al., specific et Rac3 to (Bolis S1A). restricted Fig. Rac1 see material western is recognizes (supplementary by clones expression further that well confirmed antibody of equally also and Rac3 an selection was and employing isolation a protein 1B), (Fig. Rac1 for after blotting of was dozen; Loss obtained 3 a 1A). exon Fig. than clones in alleles. (more all floxed deletion expansion and respective in excised the isolated detected of harboring were presence alleles clones the Rac1 Individual for genotyped recombinase. and Cre by (MEFs) anandsprtl n oldpirt ahexperiment. each to prior all pooled and Surprisingly, separately maintained ora fCl cec 2 (20) 126 Science Cell of Journal Rac1 Rac1 2 / 2 Rac1 fl/fl lnswr bet divide to able were clones os mroi fibroblasts embryonic mouse 2 / 2 irbatcl lines cell fibroblast Rac3 Rac1 RAin mRNA 2 / 2 lnswere clones Rac1 2 / 2 n d4 opeeyfie ognrt a1dfcetcells Rac1-deficient generate of to (0% RhoG failed lamellipodia (Aspenstro active displaying completely Rac3-L61, studies Cdc42 and previous Rac2-L61 and to unlike However, similar ( 2004). Rac1 and of lamellipodium that 2G) to triggered Fig. comparable 2C,E) efficiencies (Fig. with Cdc42 formation, and RhoG S4A,C), Fig. 2G). material Fig. (supplementary see quantifications Rac1-L61 for to S4B,D; identical effects indicating the Rac3 had or S3), of Rac2 active injection Fig. constitutively Furthermore, upon protein. 3; wild-type effects GTP-loading comparable and Rac GEF-mediated had 2 potential Rac1 Movies wild-type material as (supplementary Moreover, well Rac1. as variant by active (Rac1-V12) constitutively alternative, activation an harboring to protein Rac1 receptive lamellipodial dormant readily a of machinery presence the confirmed data Fig. These material S3). supplementary lamellipodia and of 1 induction Movie material abrupt (supplementary caused protein constitutively Rac1-L61 of active Microinjection see images S2). overview Fig. (for material re-expression supplementary Rac1 ectopic by function eragmns eepoe ramnswt aiu growth various specific with treatments under factors. employed extents we rearrangements, certain separable. to mechanistically are blebs interdependent, yet and conditions, filopodia be lamellipodia, Filopodium indicating types, may below), 2012). protrusion (see distinct observed al., that commonly et also was (Bergert formation formation lamellipodium eihr Fg DFG htehbtdtednmc fbona-fide of dynamics the exhibited that 2D,F,G) their (Fig. at periphery extensions projected frequently fibroblasts Rac1-deficient S4). Fig. material supplementary 2D,F,G; Fig. nitnusal rmta fclslcigedgnu Rac1 endogenous lacking cells of 2B,B (Fig. that in from 2A,A lamellipodia indistinguishable (Fig. induced fibroblasts a Rac1-L61, of control expression Rac1, 1992), of al., active characterization et initial constitutively (Ridley the fibroblasts in in described function As Rac1 in type. proteins Cdc42 cell Rac of same distinct the forms the by of active induction comparison direct as lamellipodium a of well allowed efficiency as also 3 approach This or RhoG. 2 and constitutively and Rac1, GTPase, expressed Rac of ectopically a variants we of active events, absence secondary the Rac- to to in solely not formation due lamellipodium is of cells absence deficient the that confirm complex To WAVE and the formation with lamellipodium interact restore proteins Rac All omto tmltd o xml,i epnet extracellular lamellipodium to fibronectin. response as growth- as in such well in example, data matrices proteins for as These Rac stimulated, ruffling 1M). for formation Fig. role membrane in essential factor-induced quantification an suggest see cells (1710 strongly total, factors growth in different the analyzed upon showed of ruffling one dorsal cells any of with capable to control treatment cell failed Rac1-deficient Rac1 We single respectively. a of treatment, detect EGF 35% and PDGF treatment and after HGF ruffles 33% after highest ruffle whereas was dorsal cells (68%), of control frequency Rac1 In The Rac1- in data). formation in 1J–L). abolished (Fig. (unpublished entirely fibroblasts was ruffles deficient formation peripheral ruffle dorsal ruffles few dorsal contrast, prominent but of 1F–H) formation the (Fig. with addition HGF and ncnrlcls a2 a3(upeetr aeilFig. material (supplementary Rac3 Rac2, cells, control In oepoeRcfntosi tmltdatncytoskeletal actin stimulated in functions Rac explore To sd rmtelc faprn aelpdalk structures, lamellipodia-like apparent of lack the from Aside Rac1 9 ,idctn ulrsoaino a1gn osof loss gene Rac1 of restoration full indicating ), fl/fl el epne ihnmntst DF EGF PDGF, to minutes within responded cells 9 .Ti hntp a virtually was phenotype This ). Rac1 2 / 2 el ihlamellipodia; with cells me al., et ¨m . 90%, Journal of Cell Science Nisuis(tfe ta. 06 oe ta. 2007; genetic al., or more et suppression confirmed complex Gomez and Arp2/3 2006; stable 2008), al., using early Higgs, recently in et and proposed (Steffen as of Nicholson-Dykstra lamellipodia, formation studies of the devoid RNAi with cells consistent in are filopodia Movies observations material These (supplementary as 1–3). well kinking as and substrate up-lifting the frequent to parallel protrusion including filopodia, i.2. Fig. e etpg o legend. for page next See h ontemlmlioilmciey oivsiaethis investigate To machinery. with also interacting data lamellipodial of Our capable downstream pathway. equally are Rac the proteins the Rac of that upstream suggest RhoG acting with formation, Cdc42 lamellipodium and in proteins Rac different of al., et 2012). (Suraneni al., ArpC3 et subunit Wu complex 2012; Arp2/3 the of deletion oehr hs eut togyagefrardnatfunction redundant a for argue strongly results these Together, a ucini rtuinadmgain4575 migration and protrusion in function Rac Journal of Cell Science nrae ahrta erae eeso cieRo a and Rac Rac endogenous Rho, of absence active virtual The of 2011). al., levels et (Khan decreased strongly Cdc42 than have (Philips, rather macrophages activation increased essential GGTase-I-deficient and an Indeed, Rho-GTPase positioning being in 2011). membrane solely showed beyond I) for functions recently prerequisite (GGTase have macrophages to I prenylation type and geranylgeranyltransferase fibroblasts of deletion rtoyial lae npr atrs) B-agdRo-1 a used was RhoG-V12 was MBP-tagged confirmation. GST–RhoG-L61 (asterisk), as The As part panel). proteins. in (upper recombinant cleaved Rac3 of proteolytically and input as Rac2 shows controls Rac1, panel and to lower and GTPases binding RhoG Rho Sra-1 not expressed shows but recombinantly indicated Rac3, of and down Rac2 Pull Rac1, Cdc42. with interacts complex WAVE (H) o t amtyaino ytie18 eetyipiae in actin implicated Rac1-mediated pre-requisite recently (Navarro-Le and a 178, partitioning remodeling be cysteine membrane C-terminal to plasma on 2005). concluded proper palmitoylation Isberg, also its and was for (Wong Rac1 of nucleus 2003), prenylation the al., its inducing et to and membrane (Shao passage the cysteine from GTPase modified the releasing the thereby of upstream directly n r xrse smeans as experiments expressed independent ‘no three are as from and collected scored neither were were Data case cells (purple). in detectable, protrusion’ and were the orange), filopodia present, (light nor were assessed lamellipodia the lamellipodia was for no filopodia scored case of first In presence were (blue). Cells lamellipodia active evaluated. of constitutively were presence of indicated versions as myc-tagged GTPases with (L61) transfected or (–) GTPase h bec flmlioi nDadEa poe oA ,CadE. and C Rho B, different A, of to expression opposed by as caused GTPases. E phenotypes and of D Quantification in (G) lamellipodia (A of anti-myc absence and the (A,B,C,D) phalloidin with stained and fixed (A–B Rac1-L61 myc-tagged i.2 a1 a2adRc etr aelpdaaditrc ihthe (A,A with Cdc42. interact and and RhoG lamellipodia not restore but Rac3 complex, and WAVE Rac2 Rac1, 2. Fig. effector YopT their the instance, for For 2002). essential lipid al., pathogenic et from (Hori considered Solski polyisoprene function 1991; biological al., been correct the et thus of long and C-termini by association the has membrane of modified GTPases Prenylation Rac1. is Rho of case C-terminal that in common, geranylgeranyl the motif harbor GTPases CAAX Rho-family and Ras- for reorganization essential actin not to is signaling Rac1 of prenylation Post-translational WAVE. to connect to inability proteins Rac their ectopic induce of or to because endogenous fail of probably absence the RhoG in and lamellipodia Cdc42 Cdc42 either conclusion, contrast, with In Sra-1 In RhoG. of 2H). or interactions specific (Fig. WAVE detect Sra-1 to the of failed analyzed. we binding with been by interacted evidenced yet Rac3 complex as not complex and WAVE has Rac2 of but proteins Rac1, independent 2008), Rac Recombinant and other al., weakly with best et association at (Meller RhoG loading to 1998), nucleotide bind al., et to (Kobayashi shown Rac1 active identified and of was partner Sra-1 binding 2007). direct a Suetsugu, (Stradal as and Takenawa protrusion 2004; WAVE lamellipodium and al., for et pentameric Nap1 essential Abi1/2, The is HSPC300, Sra-1/PIR121 complex. WAVE1/2, individual WAVE comprising of complex, the interaction the with tested GTPases we biochemically, pathway 4576 osiuieyatv h Tae in GTPases Rho active constitutively 9 ,C,C Rac1 9 ,E,E ora fCl cec 2 (20) 126 Science Cell of Journal fl/fl 9 and ) and Yersinia Rac1 Rac1 2 2 ´ 9 / 6 2 ,Ro-6 (C–D RhoG-L61 ), / iae l,21) oee,genetic However, 2012). al., et rida tan sacsen rtaeta cleaves that protease cysteine a is strains 2 (B,B s.e.m.; Eswtotoeepeso fayRho any of overexpression without MEFs 9 ,D,D Rac1 n 5 9 oa ubro el analyzed. cells of number total ,F,F fl/fl 9 and (A–F Eswr rnfce with transfected were MEFs ) 9 n d4-6 (E–F Cdc42-L61 and ) Rac1 9 xrsinof Expression ) 2 / 2 9 cells. ,B 9 ,C 9 Rac1 ,D 9 .Note ). fl/fl 9 ), fiinyo ci eoeigb coi a1hroigor harboring Rac1 ectopic Surprisingly, (CLLL). by box CAAX the remodeling C-terminal the of lacking actin comparison allowed of lines cells efficiency permanent our in GTPases hs aasgetdta ysnatvt sntcuilfor crucial not is activity myosin that suggested data These atyb ciae ntectslpirt aelpda targeting, studies. lamellipodial future to in least prior addressed at cytosol be might the will complex in activated WAVE be the partly association whether membrane and plasma 2011), residual (Philips, to contribute may Rac1 in Rac1- of Rac1- association D membrane blotting. plasma western compromised indicating by analyzed D and fraction, membrane hrceie ylmlioi ihirglr rydedges frayed morphologically the irregular, of was half with activity Approximately lamellipodia 3C). Rac (Fig. weakest by of the reconstitution characterized and 3B), of (Fig. networks form actin concave- by lamellipodial represented was shaped response the Rac expressing prominent less cells slightly same the in on ob eue oeta ieodi a1dfcetcells Rac1-deficient were in fivefold rates than Migration more reduced (33.4 4). be Movie to found material supplementary also otiue opeoyi ucm.I umr,ti analysis significantly this Rac1 summary, endogenous In of outcome. phenotypic presence to the contributes Rac1- that the reveals and Rac1-L61 expressing ectopically o nuigatnplmrzto nlmlioi.Fnly Rac1- Finally, lamellipodia. in D polymerization actin inducing essential not for are palmitoylation actin as to such post- modifications subsequent translational signaling the probably efficient and Rac1 for of prenylation remodeling, helpful although that established eghRc.Drc oprsno oto and control of comparison Direct Rac1. length ovxlmlioi Fg A ihsot de,fre n36% in large, formed with edges, smooth cells of with by 3A) represented (Fig. lamellipodia was convex phenotype 3A– strongest lamellipodium Fig. in The in shown of C. robustly, as categories penetrance defects three into classified quite The was modest induction efficiency. ruffles revealed remodeling ( quantification actin membrane sequence thorough and CLLL although the lamellipodia lacking formed Rac1 expressing cells omoi-Imdae otatlt.T u upie ROCK surprise, both, of speed our and migration increased To signaling Y27632 of by involve contractility. inhibition target could the major myosin-II-mediated inhibited Rac a to we (ROCK), of Rho-kinase this, absence effector address the RhoA To in contraction. wound whether myosin-II-based examined a we into so these eliminated, migration in entirely migration not However, was conditions 4D). Fig. respectively; fibroblasts, might this induced how Wounds investigated migration. of cell monolayers we of cell in efficiency GTPases, the with Rac coincide the in of formation lamellipodium in absence phenotype strong the to Owing in fibroblasts impaired Rac1-deficient dramatically is capacity Migration enin seen the fe naeaeo 05huswith hours achieved 10.5 was of closure average an whereas after S5) Fig. material (supplementary uclua oaino h y-agdRc ainsused, variants Rac1 myc-tagged the the of compare transfected biochemically location To lamellipodia. subcellular WAVE/Arp2/3 for with consistent essential shown), being (not tips their at complex AX owa xetteplbscrgo -emnlt CLLL to N-terminal region polybasic the extent what To 3E), CAAX. (Fig. fraction membrane the in reduced clearly was CAAX CAAX-expressing Rac1 D Rac1 AXvratsoe h atrpeoye hc a not was which phenotype, latter the showed variant CAAX m /orad6.5 and m/hour fl/fl Rac1 2 el xrsigcntttvl cieRc,btwsnot was but Rac1, active constitutively expressing cells / 2 Rac1 2 el,t 54 to cells, / 2 el rnfce ihcntttvl cie full- active, constitutively with transfected cells fl/fl Rac1 Rac1 el eesprtdit yoo and cytosol into separated were cells 2 m m 2 /orad12 and m/hour / /orfor m/hour 2 / 2 el a aelpdawt WAVE with lamellipodia had cells el aldt ls fe 0hours 20 after close to failed cells D AXvrin(i.3) A 3D). (Fig. version CAAX Rac1 Rac1 fl/fl Rac1 m /or respectively. m/hour, 2 el Fg AB see 4A,B, (Fig. cells / 2 fl/fl D el expressing cells AXvariant CAAX Rac1 and D 2 Rac1 Rac1 / CAAX) Rac1 2 cells 2 2 fl/fl / / 2 2 Journal of Cell Science A A (A anti-myc (see and outline (A,B,C) cell convex (C with lamellipodia associated irregular lamellipodia regular follows: as contours D osrcsa niae eeasse o h rsneo ovx ocv rirglrlmlioi.Dt r rmtreidpneteprmnsan experiments independent three from are Data lamellipodia. irregular or concave convex, of presence the means for assessed were indicated as constructs yoo C n ebae()fatos ape eepoe ihatbde sidctd nimcpoe ebaefatosaeadtoal sho additionally are fractions membrane Anti-myc-probed indicated. as antibodies with probed panels). were (bottom Samples enhancement fractions. contrast (M) membrane and (C) cytosol h eiulmtlt bevdi h bec fRcadthat and Rac Since of function. require absence not does Rac the migration in of increase observed ROCK-inhibitor-mediated motility residual the Rac1- 3. Fig. hs el ttewudeg,i atclr ic irto was migration since particular, in of edge, cytoskeleton wound actin the the at investigate cells these to sought we lamellipodia, AXfr aelpdawt itntpeoye.Frqatfcto frbsns flmlioimfrain aeoiswr sindaccordin assigned were categories formation, lamellipodium of robustness of quantification For phenotypes. distinct with lamellipodia form CAAX 0 ,B 0 ,C 6 0 cl asi C in bars Scale . s.e.m.; D AXi aal frbs u o ulrsu flmlioimformation. lamellipodium of rescue full not but robust of capable is CAAX n 5 oa ubro el nlzd (E) analyzed. cells of number total 9 0 9 .Clswr rnfce ihmcRc-6 ntson e loFg )admyc-Rac1-L61- and 2) Fig. also see shown, (not myc-Rac1-L61 with ,B transfected were Cells ). Rac1 frA,A (for 9 ,C 9 oietf rnfce el.Elreet fteatnctseeo ttecl eihr frgosbxdi – r hw in shown are A–C in boxed regions of periphery cell the at cytoskeleton actin the of Enlargements cells. transfected identify to ) 2 / 2 9 ,B,B irbat akdspontaneous lacked fibroblasts 9 C n C and ,C) 0 (A Rac1 0 ,B 0 .()Qatfcto flmlioilphenotype. lamellipodial of Quantification (D) ). fl/fl and Rac1 2 / 2 el rnfce ihmctge a1cntut,a niae,wr eaae into separated were indicated, as constructs, Rac1 myc-tagged with transfected cells togyrdcdbtntaoihd oepoewhether explore To abolished. not but reduced strongly a rmnn b tiiga h iso aelpdafre at formed lamellipodia 4G,G of (Fig. tips edge the wound at which the cells, staining control Abi to prominent opposed As had F- complex. for WAVE cells the stained and we actin migration, for lamellipodia employed cells 0 ,rglrlmlioi soitdwt ocv elotie(B outline cell concave with associated lamellipodia regular ), a ucini rtuinadmgain4577 migration and protrusion in function Rac Rac1 fl/fl A and (A) Rac1 Rac1 fl/fl 9 ,G 2 and / 2 0 I,tecl eihr of periphery cell the ,I), D BC el xrsigmyc-Rac1-L61- expressing cells (B,C) AXadsandwt phalloidin with stained and CAAX Rac1 2 / 2 Esepesn Rac1 expressing MEFs xrse as expressed d 0 n small, and ) Rac1 Rac1 otheir to g nwith wn 2 2 / / 2 2 Journal of Cell Science on rai eodo aelpdabthsnmru filopodia. numerous has but lamellipodia of devoid is area wound ihpalii (E phalloidin with nrae h on lsr pe nbt eltpst h aeetn.Errbr indicate bars Error extent. same the to types cell both in speed closure wound the increases ahcl yetetdwt h iaeihbtrY73 vrtm.Dt eecletdfo w needn experiments; independent two from collected were Data time. over Y27632 inhibitor kinase represent bars Rho Error with analyzed. treated experiments; type independent cell three each from collected were Data S5). rvoscnlsos(iaie l,20) coi xrsinof with expression line ectopic in In 2006), Rac1 al., 5A). dominant-negative (Fig. et defects (Vidali migratory conclusions previous by caused migration mostly healing wound was in reduction the that suggesting velocity, aneac fplrt rbt.T itnus ewe these or between initiation distinguish migration assays, random To with In assays. both. additional problems performed we or possibilities, and polarity derive defects of could maintenance migration migration wound-healing from in both reduction strong chemotaxis for The essential is signaling a Rac suggests Abi in which conditions. least these at area, dot-like under – – operating wound part migration, for of the mechanism 4H,H filopodia-driven into except (Fig. projected tips unstained, frequently filopodium essentially at accumulations was cells time. over type cell each MEFs. for Rac1-deficient closure in wound reduced Average strongly (B) is cells. capacity Migration 4. Fig. 4578 tml.T e pceoai,w sd25 eu ncombination in serum 2.5% used we chemotaxis, up set To stimuli. be 5A). solely (Fig. functions cannot Rac Rac1-N17 with interference by by exerted explained effects that establishing etw xlrdtemgaincpct oad chemotactic towards capacity migration the explored we Next Rac1 ora fCl cec 2 (20) 126 Science Cell of Journal 2 / 2 9 ,F el hwdafufl euto fmigration of reduction fourfold a showed cells 9 ,G 9 ,H 9 n niAi(E anti-Abi and ) 6 ...()Wudcoigsedof speed closing Wound (D) s.e.m. Rac1 2 / 2 el lmntdmigration, eliminated cells 0 ,F 0 ,G 0 ,H 0 .(,,,)paecnrs,(,)mre images. merged (I,J) contrast, phase (E,F,G,H) ). 9 ,H 0 J.Filopodia ,J). n 5 Rac1 oa ubro oisaaye.Errbr indicate bars Error analyzed. movies of number total Rac1 Rac1 2 / 2 fl/fl fl/fl el r o bet ls h on fe 0hus(e upeetr aeilFig. material supplementary (see hours 20 after wound the close to able not are cells (E–E and A eetdfae rmwudhaigmve fcnrl( control of movies healing wound from frames Selected (A) 0 Rac1 ,G–G el omgaetwrsceoatcstimuli. chemotactic towards these migrate of ability to of cells lack independent additional, an by accompanied ne,temda fwihapoce eo(setal no (essentially zero approached in which migration) of directional median migration the forward the the towards by index, reflected frequency also path is difference of This increase gradient. an lacked entirely almost ihhptct rwhfco,a nuto frfln in ruffling of induction as factor, growth hepatocyte with a Tae aepeiul enipiae nboth loss Since in 2009). al., et spreading implicated Liu 2006; cell al., et of been Vidali regulation 2006; al., previously the et (Guo and have formation lamellipodium GTPases spreading cell for Rac required not is Rac as defect, additional the given prominent directionality- 5D), a (Fig. a plots for revealed for Rose bias by 1). data identified or (Fig. a was arguing migration when healing of factor Indeed, direction wound 5B,C), defects. (Fig. in additional growth than dependent, migration reduced this assay drastically random this with more in even deletion robust Rac was upon velocity most migration Interestingly, was cells ugs httemgaindfcsosre in observed defects migration the that suggest 2 0 / I and ,I) 2 el iho ihu 262tetet oeta h iaeinhibition kinase Rho that Note treatment. Y27632 without or with cells 6 Rac1 ...(–)Telaigfotof front leading The (E–J) s.e.m. 2 / 2 el (F–F cells 0 ,H–H Rac1 6 0 J or fe onigwr stained were wounding after hours 4 ,J) 2 ...()Aeaewudcouefor closure wound Average (C) s.e.m. / 2 n 5 el.Teedt strongly data These cells. oa ubro movies of number total Rac1 2 / 2 Rac1 Rac1 el aigthe facing cells fl/fl Rac1 2 and ) / 2 2 el are cells / Rac1 Rac1 2 cells 2 fl/fl / 2 Journal of Cell Science a ucini rtuinadmgain4579 migration and protrusion in function Rac Rac1 nlzd B hmtci irto of migration Chemotactic (B) analyzed. three from are experiments, sets independent Data respectively). 0.000, 0.090, 0.380, (medians Rac1-N17 dominant-negative expressing Rac1 rmtegot atrsuc r ooe e.()Rose (D) red. colored 10 are of source plots factor away growth paths the migration from the black, towards colored paths are Migration HGF-gradient paths. cell individual all Rac1 ein,1t,2t,7t n 0hpretlsaddots and points. percentiles data 90th individual and show 75th show 25th, plots 10th, whiskers medians, and Box respectively). 0.070, experiments, independent three n from respectively). are 0.040, sets and Data 0.310 (medians HGF ng/ml 100 irto of migration MEFs. are Rac1-deficient chemotaxis in and abrogated migration Random 5. Fig. E owr irto ne FI of (FMI) index migration chemotaxis. Forward during (E) paths migration of direction 5 oa ubro el nlzd C rjcoyposof plots Trajectory (C) analyzed. cells of number total 2 fl/fl 2 / / 2 2 and el uigceoai mdas030and 0.370 (medians chemotaxis during cells and serum 2.5% of gradient a towards cells ˚ emnssoigtefeunyo given a of frequency the showing segments Rac1 Rac1 2 fl/fl / 2 , el nteceoai sa show assay chemotaxis the in cells Rac1 n 2 5 / 2 oa ubro cells of number total and Rac1 A Random (A) Rac1 Rac1 2 / 2 fl/fl cells fl/fl and and Journal of Cell Science nlzdatrdfeettms gi,Fatnsann revealed in staining lamellipodia F-actin of absence Again, virtual times. the different after analyzed ihs oadesti oedrcl,cnrladknockout and control (25 fibronectin directly, on spread more to tissue-culture allowed this were on fibroblasts address spreading or To to dishes. adhesion only with indicated clonal cells problems during Rac-deficient minor observations of Initial maintenance Rac and spreading. of effects expansion on the function explore to of sought loss we directed migration, in reduction cell strong random a or with well correlated lamellipodia of ofre yimnsann o b Fg 6D (Fig. Abi for immunostaining by confirmed 4580 oto el a rmnn aelpdaatr2 or on 6C (Fig. hours tips points, 24 their 6A,A time earlier (Fig. at at after observed proteins also lamellipodia was Abi formation Lamellipodium with prominent 6C), (Fig. had fibronectin cells control ocnrtoso irnci (5 fibronectin Indeed, of 6H). concentrations (Fig. lamellipodia low and with coated coverslips Rac on efficiency spreading quantifying when of absence the in defects no revealed spreading of initiation after times different at areas cell of aii Fg I rgltn(i.6J), (Fig. gelatin or 6I) (Fig. laminin ora fCl cec 2 (20) 126 Science Cell of Journal 9 ,B,B 9 ,abi esfeunl.eakby quantification frequently.Remarkably, less albeit ), m /l i.6)a ela on as well as 6G) Fig. g/ml; Rac1 Rac1 2 / 2 2 / 2 el lal covered clearly cells 9 ,E el Fg 6D–F), (Fig. cells 9 ,F 9 .I contrast, In ). m /l and g/ml) 9 ). ic pedn of spreading filopodium Since and adhesion focal for formation required not is Rac rmgainefcec a efntoal uncoupled. functionally pronounced. be can efficiency migration less or formation lamellipodium much unclear, and spreading currently that show experiments are was these discrepancies the these of for ArpC3 reasons removal the Although subunit genetic al., upon complex et observed (Wu Arp2/3 defect expression the complex although Arp2/3 2012), reduced those with with variance at cells also are Vidali data from 2006; Our 2009). al., al., et Liu et 2006; frequently are al., (Guo et a previously had spreading lamellipodia function with in implicated Rac engagement thus been although of receptor, and integrin independent specific spreading, function for Rac-GTPase first dispensable the for that show, data These controls. time, than area cell larger even an omto fnsetahsosi rqetydsrbdt occur process. to this described during frequently is over adhesions turned nascent of and focal Formation how assembled wondered are we controls, adhesions with compared decreased than Rac1 2 / 2 irbat perdicesdrather increased appeared fibroblasts 4hus(C,C hours 24 n5 on 0mnts(B,B minutes 60 (A,A minutes 15 (A anti-Abi and (A,B,C,D,E,F) phalloidin (D–F B in prominent the tip, at lamellipodium Abi of localization that 25 on (A–C cells. spreading (A–F for required spreading. not cell is Rac1 6. Fig. and matrices. extracellular or concentrations fibronectin different on area the spreading of Quantification (G–J) cells. el analyzed. cells s.e.m.; the indicate bars Error experiments. independent three are from datasets All Materials Methods. the and in detailed gelatin as 0.2% (J) or (I) laminin ml 25 9 m n C and Rac1 /lfboetn() 25 (H), fibronectin g/ml 9 9 m 9 eesandwith stained were ) muoloecneof Immunofluorescence ) and ) m /lfboetn(G), fibronectin g/ml /lfboetn Note fibronectin. g/ml 9 2 sasn in absent is , / n 2 9 Rac1 5 ,B Eswr plated were MEFs 9 oa ubrof number total 9 ,F,F ,C Rac1 9 9 2 ,E,E ,D,D 9 ,D / 0 2 fe plating after ) 9 fl/fl 0 ,E cells 9 and ) Rac1 ), Rac1 9 ,F 9 ) fl/fl 2 m / 2 g/ Journal of Cell Science rti iclna ifrn ie fe pedn.In spreading. after times different at vinculin protein irbat,teoealdsrbto ffclahsosappeared adhesions focal of distribution overall the fibroblasts, al., these et in lamellipodia. formation (Choi adhesion without explore front spreading to cells protruding curious were the we so to 2008), close lamellipodia, below Rac1 fl/fl and Rac1 2 / 2 el eesandfrtefcladhesion focal the for stained were cells Rac1 2 / 2 vrg esplrzdthan polarized less average pedn a opee(4hus,temjrdifference After major 7A–F). the (Fig. hours), cells and (24 control control complete Rac1 in between was that spreading with comparable ewe h w eltps with differences types, cell morphological two from the derived between pattern adhesion their a ucini rtuinadmgain4581 migration and protrusion in function Rac Rac1 Rac1 epesn oto el.These cells. control -expressing 2 Rac1 / Rac1 of movies phase-contrast of frames Selected filopodia. employing Rac1 oaiain(elwarw nF). (G,H) in arrows (yellow polarization of lack indicating nucleus, the from equidistant appeared ttebs fflpdain filopodia of base the formed at are adhesions Focal (I–K) filopodia. to point arrows lamellipodia, to point Arrowheads spreading. during acquired (H) hl oedslyfcladhesions focal display C), some in while arrowheads (yellow cell the under distributed evenly focal adhesions show cells time Some earlier points. at than cells between variable more focal is of adhesions distribution the After hours, plating. 24 after (C,F) hours and 24 (B,E) minutes 60 (A,D), tahetpit in points attachment peripheral most hours spreading, 24 of after controls As to C). opposed in arrowheads (blue points attachment peripheral at mainly tiigof staining formation. filopodium for and essential adhesion not focal is Rac1 7. Fig. n e o2 minutes. 20 to red minutes and 16 to corresponds green minutes, 12 after localization GFP-VASP to corresponds Blue points. time different of three merge from a images in GFP-VASP shown is time adhesions over focal of Maturation (L) frames. (green) and GFP–VASP (gray) contrast phase merged shows (K) of filopodia. base protruding the at formed focal adhesions in VASP of accumulation show GFP–VASP, expressing cells of frames (J) channel epifluorescence green and (I) phase-contrast Selected cells. 2 irbat ihrgr to regard with fibroblasts 2 fl/fl 2 / Rac1 / 2 2 G and (G) DF el 5minutes 15 cells (D–F) irbat en on being fibroblasts Rac1 2 AF Vinculin (A–F) / 2 Essra by spread MEFs fl/fl Rac1 AC and (A–C) Rac1 2 Rac1 / 2 2 Rac1 cells 2 / 2 / 2 cells 2 / 2 Journal of Cell Science mgsof images oa deinfrainsotyatriiito fsraigof spreading of initiation after as shortly 6). formation 7G), (Fig. (Fig. adhesion immunolabelings lamellipodia Focal anti-Abi and flat phalloidin and from expected ruffles of formation 2012). through al., et Wu and 2012; Nicholson-Dykstra al., 2007; et al., Suraneni et 2008; formation Gomez Higgs, filopodium 2006; in complex al., Arp2/3 et the (Steffen of function with consistent of axis, lack signaling a Rac–WAVE cytosol activation filopodium the Arp2/3 to alternative of the and an pathways as involve spreading S6; in not do Rac-independent Fig. probably complex formation Hence, material (supplementary 6). Arp2/3 1998) Movie the Insall, and sequester (N-WASP- (Machesky N-WASP to of domain WWCA) C-terminal the overexpressing Rac1 oa deinitniy()adfclahso ie()wr acltd 0clswr nlzdfrec eltype. cell each for analyzed were cells 30 calculated. were (F) size adhesion focal and (E) intensity adhesion focal (A threshold binarized yaiso pedn of the spreading as Importantly, between. of operated in cytoplasm dynamics filopodia of protrusion These the 7H; for 5). (Fig. struts became contact Movie substrate which material upon supplementary filopodia, seconds 20–60 protrusive within apparent employing by efficiently irsoy n ogtt orlt opooia changes that Time- revealed formation. morphological adhesion movies correlate early lapse with to spreading sought accompanying and microscopy, process. this for essential 1999a), al., not et are Rottner GTPases nascent 1995; Rac in Hall, and involved (Nobes certainly assembly although adhesion that revealed clearly data in parameters adhesion focal of Analysis 8. Fig. 4582 rtuiebhvo fflpdawsfeunl olwdby followed frequently was filopodia exploratory, of of the absence however, behavior the Interestingly, protrusive confirming 7J). (Fig. struts, squeezing cytoplasm, lamellipodia filopodial VASP of regions between Importantly, peripheral the forward 7I). to (Fig. target filopodia to failed protrusive presence multiple, the confirmed of supplementary movies of also Phase-contrast see 7). accumulation VASP Movie 7J; material (Fig. strong adhesions marker focal showed in phase-contrast adhesion EGFP–VASP microscopy using imaging fluorescence and Sequential and 1999b). protrusion al., et the (Rottner of microscopy ncnrs,sraigo a1cnrlclswsmsl achieved mostly was cells control Rac1 of spreading contrast, In enx nlzdsraigb hs-otatvideo phase-contrast by spreading analyzed next We 2 / 2 Rac1 ora fCl cec 2 (20) 126 Science Cell of Journal el a lofloe yfursec video fluorescence by followed also was cells fl/fl (A,A 9 ,B 9 and ) 9 ssont ersn aaetato.Fo hs mgs ubro dein e el() ubro dein e elae (D), area cell per adhesions of number (C), cell per adhesions of number images, these From extraction. data represent to shown is ) Rac1 Rac1 Rac1 2 / 2 2 (B,B / 2 2 / 2 el a nfetdwhen unaffected was cells 9 el tie ihvnui niois(,) mgswr hehle sdtie nMtrasadMtos a Methods; and Materials in detailed as thresholded were Images (A,B). antibodies vinculin with stained cells ) el pedrpdyand rapidly spread cells Rac1 2 / 2 MEFs. AF seseto oa deinprmtr rmvnui tiig.Representative stainings. vinculin from parameters adhesion focal of Assessment (A–F) o fetv eltasoainadmigration. essential and reorganizations translocation cytoskeletal cell critical effective in the for in activity to contrast Rac delay cells strong of in these no function was by this consequence, covered However, area observed. a substratum was of As reduction lamellipodia. or adhesions spreading nascent as seed well and that spreading equally evidence cell direct mediate provide can data of filopodia our Together, dynamics S7). (supplementary Fig. same VASP material for observed the as formation essentially adhesion nascent showed paxillin fluorescent oetices fahso raprcl iho without or with cell in per area area cell increased when adhesion to the insignificant of Moreover, normalization area. appeared increase cell (supplementary to increase modest relative fashion this intensity Rac vinculin significant S8), expressing upon statistically increased Fig. total was a material Although cell size types. in per cell and staining deletion both In vinculin 8E) in 8D). (Fig. of equal Fig. intensity roughly 6; intensity (Fig. were adhesion Rac 8F) (Fig. of focal to absence average due the mostly addition, in was difference areas this cell but increased 8C), (Fig. cell per numbers iooi Nmtoae l,2008). 7). in al., rich Movie fibroblasts et material CAR (Nemethova in supplementary observed filopodia also 7K; were (Fig. adhesions Similar 1995) Hall, (Nobes previously described and complexes focal of Cdc42-induced reminiscent base, the their at formation adhesion VASP-associated ttsial ninfcn splmnaymtra i.S8C,D). Fig. material (supplementary insignificant statistically eeietfe sn mg hehlig(i.8A,A (Fig. thresholding image using Interestingly, assembly identified adhesion fiber antibodies vinculin stress with were that stained focal and adhesions Focal adhesion concluded 2006). focal al., colleagues different et both (Guo and for crucial of Guo is MEFs, Rac1 turnover primary gene adhesion in Rac1 as Cre-recombinase-mediated deletion focal acute of well Using components. parameters as quantitative assessed patterns components we adhesion focal Next, of and mobility number increases adhesion but focal size, affect not does deficiency Rac Rac1 2 / 2 el ipae nrae adhesion increased displayed cells Rac1 Rac1 2 / 2 2 / 2 el expressing cells el a also was cells 9 ,B,B 9 ). Journal of Cell Science i.9. Fig. e etpg o legend. for page next See a ucini rtuinadmgain4583 migration and protrusion in function Rac Journal of Cell Science qaincefcet r ie nsplmnaymtra al S3. figure, Table the material in supplementary displayed in are given component are each coefficients for equation fits curve of equations rcin.DFHso itdcre faeae aafo hc aftmsof times half immobile which from and data mobile ( averaged of of recovery (sum curves fitted fraction show are total D,F,H fractions the fraction). Mobile of after fraction. percentages and immobile as before IF, shown points fraction, time mobile acquired MF, for bleaching. s.e.m. with means arithmetic loecneitniiso GPtge yi CD,pxli EF and (E,F) paxillin in (C,D), (G,H) normalized zyxin VASP of EGFP-tagged curves of Recovery intensities Yellow (C–H) fluorescence indicated. areas. and points bleached seconds) time mark (0 the quadrilaterals bleaching at after recovery immediately fluorescence panel), during left adhesions FRAP, focal in (pre accumulation before EGFP–zyxin show experiments bleaching of i.9 unvro oa deincomponents. adhesion in focal EGFP–zyxin of Turnover 9. Fig. 4584 bevtos(u ta. 06 iaie l,20) hs results These 2006). al., previous et Vidali corroborating 2006; growth-factor- al., fibroblasts, et and in (Guo cell observations the ruffling formation our at dorsal lamellipodium Rac3 central in mediated be and to spontaneous Rac1 protein Rac2 revealed results to detect Rac1 our expected, to As residual level. unable protein 2006; were of can al., and we presence lines, et populations, cell was knockout (Guo the permanent Rac1 proliferation our exclude In fibroblasts, previous for 2006). in al., mind essential et gene Vidali in be Rac1 to bearing directed the concluded note, eliminate and Of to random attempts spreading. adhesion, and of protrusion, requirement migration the for of for function analysis possible, molecular made Rac1 detailed a clones time, knockout developed first Rac1 the we viable of generation and rearrangements, the individual Importantly, actin cells. morphology, fibroblastoid dynamic Rac1-deficient fibroblast in and functions Rac1 migration understand to order In Discussion stabilization. their focal or of structures association these promoting within Rac constituents Rottner from adhesion 1995; derive may Hall, 1999a) and al., (Nobes et GTPase active of upon induced microinjection phenotype assembly material and complex focal supplementary the stabilization S7–S9), 7–9; Figs. initiation, although (Figs turnover site that component the adhesion suggest adhesion in data for fractions these dispensable immobile Collectively, being greater Rac. towards of (FRAP) shift presence a photobleaching revealed after data recovery components fluorescence all examined, to the common for However, turnover fraction. in increase exchanging an slowly revealed and VASP, and paxillin fractions for exchanging of differentially that two with suggested compared fits and types, Curve paxillin cell zyxin. both both in of complex behavior more recovery was VASP the addition, material In supplementary supplementary S9B). 9G,H; Fig. (Fig. 9E,F; Rac VASP (Fig. and S9A) of paxillin Fig. material absence components, distinct for adhesion the found was focal increase in similar a Interestingly, increased 9C,D). (Fig. substantially focal in was zyxin of fraction adhesions mobile the respectively), seconds, 15 and 01.Itrsigy lhuhtetroe fzxnwsvery was zyxin ( al., Rac1 of of et absence and turnover Wolfenson presence the focal both 2006; the in within expected similar al., although over et turn as Interestingly, to (Lele 2011). known 8), seconds is within Zyxin Movie adhesions 2001). al., material et types (Rottner cell supplementary both in 9A,B; expression (Fig. transient upon incorporated zyxin EGFP-tagged readily adhesions. focal individual in turnover protein ial,w etdwehrRcdfcec a osqecson consequences had deficiency Rac whether tested we Finally, t 1/2 ora fCl cec 2 (20) 126 Science Cell of Journal eecalculated. were ) Rac1 Rac1 fl/fl fl/fl A and (A) bu)and (blue) N Rac1 5 ubro nlzdmve;respective movies; analyzed of number Rac1 2 / 2 2 / B Es ersnaieframes Representative MEFs. (B) 2 el oag) ,, show C,E,G (orange). cells AD RPaayi of analysis FRAP (A–D) t 1/2 fruhy14 roughly of eurmn fCA-rcsigfrpoe Rho-GTPase Rac1- as active controversy, constitutively this proper on a light of for shed actin expression can Rac-induced experiments CAAX-processing proposed Our on function. the of questioning effects 2005), minor requirement al., et exhibited (Michaelson Icmt remodeling them or of increased deletions genetic strongly with Rce1 fibroblasts Moreover, but 2011). GTPases, al., et not active (Khan did of macrophages in levels this GGTase-I challenged decrease of have removal instance, enzymes For different view. these of deletion genetic ufigi h rsneo eiulRcatvt,adi thus is We remodeling. and actin activity, to Rac signaling residual Rac of bypassing lacking induce of presence lines only incapable the can cell RhoG in that permanent speculate ruffling using we on activity, Based here Rac observed experiments. GTP-loading-specific detectable presented our polymerization be in results could confirmed no actin as complex the 2008), WAVE al., the as et with (Meller RhoG to elusive, have of signaling interaction might remained populations addition, machinery cell In study, 2006). this un-recombined in with Rac- contaminated proposed been recent employed driving was be clonal From in since migration not However, RhoG 2008). Cdc42. and al., et of ruffling (Meller and function membrane RhoG potential independent a the unlike with experiments, interact Rac1. complex, to of observed independently were WAVE nor variants lamellipodia Rac RhoG drive all to neither Consistently, able formation, as efficient were lamellipodium as Cdc42 were inducing Rac3 GTPases. and in examined Rho Rac2 also Rac1 although other 2003; We that by show 2004). al., could formation al., We et in lamellipodium et Rogers Rac of Steffen 2003; by 2004; regulation al., al., played et through et role (Kunda machinery Innocenti polymerization complex key actin WAVE the the the by to signals explained relaying likely most are n h soitdps-rnltoa oiiain fRho of these modifications to ascribed post-translational functions crucial Icmt associated (Navarro-Le the GTPases by of the and spite (Dai isoprenylcysteine In and 1997), 1998). methyltransferase) the al., al., et of et carboxyl (Boyartchuk methylation (isoprenylcysteine-directed by Rce1 1) carboxyl protease followed enzyme prenyl-CAAX-specific by GGTase-I, the actin converting of by by (Ras C-terminus hence AAX geranylgeranylated the of and first at removal sequence targeting is CLLL Rac1 positioning membrane The events. considered plasma subcellular been postprenylation remodeling long for have and with Rac1 prenylation of essential C-terminus instance, conjunction the of For processing in modifications activation. post-translational and GTPases of requirements of the carefully and Lebensohn 2009; contrast, al., In 2009). et al., Kirschner, (Ismail 2005). or complex remodeling al., et WAVE the actin et lamellipodium Snapper through Czuchra Arp2/3-complex-dependent 2001; 2002; in triggers al., al., Rac-GTP not et et Benesch vesicle but (Lommel 2001; in formation instance endocytosis, for N-WASP filopodium involved and is through activators. which complex trafficking 1999), complex al., Arp2/3 Arp2/3 lamellipodium et the (Rohatgi with drive activates GTPase to of Cdc42-GTP each specificity able the with of is consistent Rac, interaction Cdc42 of absence that the in formation exclude also can togyrdcdi h ebaefato,i stmtn to tempting is it as fraction, efficiently membrane the less Rac1- in Since reduced albeit Rac1. strongly length full lamellipodia, active constitutively reconstituted cells u ellnssol lopoeisrmna ndsetn more dissecting in instrumental prove also should lines cell Our ´ iae l,21) oercn tde with studies recent some 2012), al., et rida Rac1 D Rac1 lee Vdl tal., et (Vidali alleles AXin CAAX nl el could cells -null D AXwas CAAX Rac1 2 / 2 Journal of Cell Science omto n togyrdcdmgaincpct,agigfor arguing capacity, migration reduced strongly and formation and processing how temporally clarify and spatially correlated. are to complex WAVE have and Rac will of activation previously work than positioning Future less complex be thought. WAVE might correct association for membrane relevant plasma Rac1 that speculate eeerdcino irto pnRcrmvl a the focal form was changes to morphological removal, and of efficiently analysis Rac spread careful to However, upon cells adhesions. migration these of of capability reduction severe with or polarity. propagation of or chemotactic maintenance transduction a and signal separable follow establishment arises with and defect this additional, sense problems whether to from explore will an cells experiments of Future caused ability signal. the deficiency in defect Rac migration, reduced 2012). al., et the (Suraneni light recently cells observed ArpC3-deficient migration in of cell in random especially in phenotype tested, causative of lack be the to is of cell remains observed cells cell the the rates at Rac-deficient migration activation Arp2/3 at of of lack the activation complex periphery not complex or Arp2/3 Whether WAVE of sites. these lack at a of probably thus accumulation and periphery, lack continuous with coincided expression of investigated. Rac of be removal and case, to WAVE- the remains Whatever additional, Rac/WAVE/ Rac from the of or functions of Arp2/3-independent axis inhibition signaling efficient complex Rac-deficient more Arp2/3 of control phenotype from of penetrant derives 50% more cells to the only whether In reduced So 2012). were cells. al., rates et migration (Wu cells, RNAi these complex Arp2/3 fibroblasts in by observed suppressed also stably recently was lines cell Rac-deficient in of speed and matrices. will behavior three-dimensional it migration work, in assay future fibroblasts In to signaling. exciting Rac from be separable contractility fashion myosin-II-based is a migration exaggerated in of by type this suppressed addition, clearly In signaling. studied Hall, Rac as efficient, requires surfaces, and here, that two-dimensional conclude on (Nobes as We migration signaling. stimulation mesenchymal Rac migration, of RhoA irrespective counteracts for 1999), previously mostly observed on induced surfaces as contractility that two-dimensional indicate al., mutually data et our be (Sanz-Moreno However, migration to 2008). of since reported modes -independent previously counterintuitive, different to were be and antagonistic signaling might Rac Rac-dependent this Surprisingly, and glance, Rho both body. first increased At decreased cell contraction migration. to of the signaling RhoA instead of of inhibition translocation seemingly mostly however, bundles, for contractile appeared of between employed formation cytoplasm which with and together filopodia migratory migration, them, of protrusion effective residual by accompanied cells reasonably displayed Rac1-deficient permanent Rac1 mediate also our systems residual Nevertheless, to experimental that performance. earlier sufficient speculate these in We were present 2009). activities studies al., protein RNAi in et Rac1 observed be defects (Monypenny of to migration efficiency reported mild with the case consistent latter Again, the 2006). in al., depletion, Our et observed motility. than (Vidali phenotype cell migration previously dramatic efficient more for a showed lamellipodia cells of requirement the a1dlto opeeyaoihdrfl n lamellipodium and ruffle abolished completely deletion Rac1 qal upiig osdrn h bec flmlioi and lamellipodia of absence the considering surprising, Equally directed and random on effects strong of Irrespective observed as filopodia involving migration of mode ineffective An , 0,i reasonably is 90%, Rac1 2 / 2 hs tutrsfi ocnrbt fetvl orno or random to effectively contribute to Although Rac1-deficient fail striking filopodia. of protrusive structures most multiple, cytoskeleton of The these formation actin GTPases. the peripheral is cell Rac and cells the efficient RhoG on of by for depends feature data lamellipodia signaling strictly first Our of Rac Cdc42 induction system. the of Moreover, cell here importance migration. Rac1-deficient the present viable underline we and cells, permanent through fibroblastoid formation differentiated adhesion nascent to stabilization. a component contributes suggests adhesion cause This Rac mobility. to component components deficiency that adhesion all in Rac increase For a revealed marked phenotype. experiments revealed FRAP detectable components of tested, clearly adhesion assessment but Rac, focal of modest individual adhesions absence focal of the of in turnover sizes unaffected and virtually as numbers well were as Although formation adhesion spreading. and cell filopodia is both activity for Rac essential that remain establish not that unequivocally reasons they for determined, 2006), be al., to disagree et (Guo data study these previous Although one with adhesions. nascent of formation seed to the appeared which of prominent shafts by substratum-attached mediated the filopodia, is spreading that revealed seeding cell after a-eiin ellnswl ehlflfrdsetn the dissecting diverse for in GTPases helpful Rho of be processes. subfamily permanent, cellular relevant will to undefined this Our of lines contribute yet components. functions might cell adhesions additional, and focal Rac-deficient has signaling, of chemotactic Rac stabilization in migration, spreading. functions cell mediate from to sufficient Apart are they migration, directed mroi irbat rprdfo mroi a (E)14.5 day embryonic from prepared fibroblasts embryonic lae 1-1cl yae nlssbfe 1[0m rsHl 0m NaCl, mM 50 Tris-HCl, 800 mM [50 with L1 incubated buffer were lysis resin in lysates to cell bound B16-F1 pmol) cleared (200 proteins Recombinant down Pull snap MgCl mM were 5 matrix NaCl, the mM to 150 bound Tris-HCl, mM Proteins 50 (GE (NEB). in glutathione–Sepharose resin frozen using amylose procedures, and standard Healthcare) in to overexpressed according were Healthcare) proteins recombinant All supplementary proteins in Recombinant previously listed described are study as this transfected in used and S1. plasmids Table maintained The material 2004). 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L-glutamine, glucose sodium g/l mM 4.5 2 DMEM, (Sigma), in maintained were MEFs transfection and culture Cell 2001). and (DeChiara, (fl) previously floxed described the as for MEFs Genotyping from isolated was DNA Genomic Genotyping Rac1 of Generation Methods and Materials Tngcie l,19)adslce ih5 with selected and 1998) al., et (Taniguchi sdsrbdpeiul Crse ta. 2006). al., et (Chrostek previously described as V0lreTtasuigrtoiu.T eeaecoe ooyosydltdfor gene, deleted homozygously Rac1 clones generate To the retrovirus. transducing T large SV40 niios(iiCmlt,ET re oh) H75splmne ih10% (Ridley with previously described supplemented 1992). as 7.5 al., purified pH et were microinjection Roche), for free, Proteins EDTA glycerol. Complete, (Mini inhibitors niiulygoigcl lnswr sltdadaaye ygntpn and genotyping by analyzed and isolated were blotting. clones western cell growing Individually nsmay sn eedsuto ehdlg in methodology disruption gene using summary, In fl/fl a ucini rtuinadmgain4585 migration and protrusion in function Rac irbati el eegnrtdb motlzto fpiaymouse primary of immortalization by generated were cells fibroblastoid Rac1 Rac1 fl/fl fl/fl and Eswr rninl rnfce ihpCre-Pac with transfected transiently were MEFs Rac1 Rac1 2 / 2 MEFs 2 / 2 koku C)allswsperformed was alleles PCR) (knockout m /lprmcnfr2weeks. 2 for puromycin g/ml .coli E. 2 L1D3pyS(GE BL21(DE3)pLysS MDT protease DTT, mM 1 , Rac1 fl/fl mro with embryos m g Journal of Cell Science otae(iirnSses a used. VisiView random by was driven Systems) for (Photometrics) (Visitron phase- camera for software below Coolsnap-HQ2 VIS-LED light a a DG4 and also a illumination, imaging, stage, epifluorescence contrast see for automated an Instrument) (Zeiss, with (Sutter equipped source Observer shutter experiments) FRAP Axio and Devices). and (Molecular migration inverted for (Photometrics) software lamp an Metamorph camera by halogen Alternatively, driven a Coolsnap-HQ2 Corporate) illumination, (Uniblitz a drivers epifluorescence an for imaging, with equipped (Visitron) phase-contrast 100TV lamp Axiovert Zeiss 120 inverted HXP an on objectives oil Apochromat nlsso pedn ra,clswr tie ihpalii n randomly and phalloidin with stained 40 were a using the cells acquired For were levels. areas, fields contrast chosen and spreading brightness of adjust to analysis used were MetaMorph and ImageJ processing and analysis Data (see observer Axio inverted an on mounted 37 6- was a a plate with in the equipped (Lai subconfluently above) hours, seeded previously 6 were described cells After as assays, plate. out migration well carried random were For plates 2009). of al., 6-well assays et or by healing 12- monitored Wound into fibronectin- medium. was seeded previously onto microscopy cells cells suspensions described with of cell equilibrated pipetting Spreading as coverslips after 2001). coated movies performed al., of et was acquisition Rottner immediate 1999a; microinjection al., and et (Rottner imaging cell FRAP and Live microinjection microscopy, video Time-lapse 40 using captured were Images acquisition Image PBS in diluted was FN coatings, For urea. 5 M 2 in 25 mg/ml to 1 matrix at respective dissolved the was of Roche) dilution a 100 preparing a by pre- placing out and were carried coverslips was coating 4E–J, (HCl)-washed cases, Fig. other in acid shown 500 onto cells with For seeded incubated follows. were as prepared MEFs coverslips analyses, immunofluorescence For by separated Immunofluorescence were nuclei and cells Intact broken. 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CB, with or 60 permeabilized with PBS and for in washed minutes BSA CB were 20 or MEFs for PBS CB stainings, in or other 100 PBS (PFA) all in paraformaldehyde 4% PFA For with 4% with minutes. fixed with 20 pre-extracted and for minute 1999b), 1 CB al., for in et CB in (Rottner X-100 (CB) Triton 0.3% buffer cytoskeleton with washed lcl35,1 rtnX10 H75adpoes niios(iiComplete, 4 (Mini at inhibitors hour protease 1 and for 7.5 pH Roche)] X-100, free, Triton EDTA 1% 3350, glycol etr ltigacrigt tnadpoeue.Atbde,raet and by reagents analyzed Antibodies, were S2. 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Stradal, A., Steffen, S., Lommel, S., Benesch, ar,D,Fn,Q n ein,R A. R. Cerione, and Q. Feng, D., Baird, Aspenstro Deutsche References the by for at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.118232/-/DC1 PMC online available part in material Supplementary Deposited T.E.B.S.]. in to SFB629 supported release. K.R., and immediate to FOR629 SFB621 was programs and within [grants work Forschungsgemeinschaft This Funding J.F. reagents; generated provided C.B. R.G. antibody; and Rac2 and J.S. the performed T.E.B.S. generated T.E.B.S., J.V.S., J.M.H. data; F.S., and analyzed L.S., K.I.L. A.H., K.R. G.D., S.A., M.L., manuscript; L.S., A.S., the A.H., experiments; wrote G.A.D., and experiments M.L., the A.S., conceived K.R. and A.S. (UKE Himmel contributions Author Mirko analysis. FRAP to on Braunschweig, advice and valuable (HZI for analyses Geffers Germany) Hamburg, microarray Robert to for grateful Germany) technical are excellent for We Landsberg Gerd assistance. and Denker Brigitte thank We Acknowledgements eoeyfrrpdyadsol xhnigfatoswr acltdusing calculated were fractions exchanging slowly and equations rapidly for recovery aksmts Sgalt1.) aawr rcse sn irsf xe 14, 14.0.0. Excel Illustrator Microsoft Adobe Adobe using and Software), processed 11.0.2 (Systat 12.0 were Photoshop SigmaPlot Data (http://rsbweb.nih.gov/ij/), 12.0). 1.43u (SigmaPlot ImageJ test sum rank o RPaayi,itniyvle ftebcgon usd ftecl were cell the of outside background the of values intensity analysis, FRAP For was images all of intensity maximum the quantification, adhesion focal For ttsia nlsswspromduigtennprmti Mann–Whitney non-parametric the using performed was analysis Statistical nue eil oeetdpnso -APadivle c,WP n Grb2. and WIP, Nck, involves and Chem. N-WASP Biol. on J. depends movement vesicle K. induced Rottner, and J. Wehland, d4-a inln cascade. signaling Cdc42-Rac system. a filament actin the of organization the on m . rnsn .adSrs J. Saras, and A. Fransson, P., ¨m, t 1 = 2 ~{ t 1 277 = 2 b 1 ~{ y 37771-37776. , n0 ln 5 o h is iepitatrbecig aawr itdin fitted were Data bleaching. after point time first the for 0 : b 1 and 5 n0 ln : .I h aeo ailnadVS,hl ie of times half VASP, and paxillin of case the In 5. t 1 20) hshtdlnstl45bpopae(PIP2)- 4,5-biphosphate Phosphatidylinositol (2002). = ur Biol. Curr. 2 ~{ 20) h ol2apaPxpoenmediates protein Cool-2/alpha-Pix The (2005). d 1 n0 ln 20) h Tae aedvreeffects diverse have GTPases Rho (2004). 15 : ,respectively. 5, 1-10. , ice.J. Biochem. Rac1 377 fl/fl 327-337. , cells. Journal of Cell Science u,F,Dbda . ag . ilas .A n hn,Y. Zheng, and A. D. Williams, L., Yang, M., Debidda, F., Guo, oe,T . ua,K,Mdio,R . hmz,Y,Lisn .J and J. P. Leibson, Y., Shimizu, B., R. Medeiros, K., Kumar, S., T. 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