col nvriyo imnhm imnhmB52T UK. 2TT, B15 Birmingham Birmingham, of University School, lao Wilson Eleanor focal regulates and complex disassembly GIT–PIX adhesion the with interacts RhoJ ARTICLE RESEARCH eevd1 uut21;Acpe 9Arl2014 April 29 Accepted 2013; August 10 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This ` 1 forms GDP-bound inactive and GTP-bound Rho active dynamic 2011). between are cycles Manser, its and cytoskeleton (Sit orchestrate movement actin cell which enable the to switches of rearrangement molecular regulation pre- the GTPases, matrix. and a Cell the extracellular to cytoskeleton the organs. is the with Central in distant contacts of it of to remodelling modulation crucial where the tumours coordinated on of cancer being depends metastasis as movement the as such for and well diseases requisite developmental of as numerous pathogenesis to processes, fundamental physiological is motility Cell INTRODUCTION vessel Angiogenesis PIX, tumour tumour GIT, adhesion, Focal and RhoJ, mediating WORDS: motility KEY cell in regulating molecular RhoJ the in into formation. RhoJ for insight of new role give function studies a These angiogenesis. and identifying vessel tumour GIT1 diminished density, and both growth tumour reduced of showed mice regulated knockdown complex or with to focal GIT2, recruitment GIT–PIX formation, in their the tube for colocalised Functionally, endothelial other all adhesions. each RhoJ on focal and depended ARHGEF7) and adhesions as GIT2, GIT1, known GIT1. this of (also and domain homology experiments, Spa two-hybrid the and on yeast depended daRhoJ using between confirmed of was interaction regulator GIT1 An a disassembly. complex, adhesion whereas adhesion GIT–PIX focal the time, focal with Furthermore, disassembly co-precipitated it. decreased daRhoJ (daRhoJ) adhesion regulate mutant active focal an of to expression increased knockdown RhoJ found (siRNA)-mediated to of RNA aimed interfering was timelapse small study Using disassembly; RhoJ. tube RhoJ This of endothelial function numbers. microscopy molecular invasion, the adhesion delineate motility, focal further cell tumour and and cells regulates formation endothelial in which expressed GTPase cells, Rho a is RhoJ ABSTRACT Teeatoscnrbtdeulyt hswork. this to UK. equally 2TT, contributed B15 authors Birmingham *These Birmingham, of University Biosciences, ee .Noy J. Peter uhrfrcrepnec ([email protected]) correspondence for Author colo muiyadIfcin nttt o imdclRsac,TeMedical The Research, Biomedical for Institute Infection, and Immunity of School 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,33–01doi:10.1242/jcs.140434 3039–3051 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. hJblnst h d4 ufml fRoGPss and GTPases, Rho of subfamily Cdc42 the to belongs RhoJ b PXpeooyn hJkokon RhoJ-knockout knockdown. RhoJ phenocopying -PIX 1 nraBacon Andrea , 1, ,KtryaLeszczynska Katarzyna *, 1 ohaZ Rappoport Z. Joshua , 2 colof School 1, ,NtleS Poulter S. Natalie *, b -PIX 2 onK Heath K. John , r Ya ta. 01.Fntoal,Ro a ensonto shown vivo been has vitro RhoJ factor al., formation Functionally, lumen transcription et and 2011). tubulogenesis the motility, Takase endothelial al., by regulate 2011; induced et and al., (Yuan 2011) et Erg al., Kaur endothelial et 2011; in Yuan al., expressed 2012; be et al., to et (Fukushima Vignal found cells 2003; was al., and it et Aspenstro Nishizuka Subsequently endocytosis 2003; 2000). 2003; al., et early al., Toledo de et RhoJ cytoskeleton, 2004; for (Abe actin roles differentiation suggested the adipocyte studies early modulating 2000 and in in 2000) identified al., first was et It (Vignal 2004). Wennerberg, and (Burridge 99 iCsr ta. 00 rmn ta. 98 unre al., et Turner proteins, 1998; (PIX) al., factor et exchange Premont Pak-interacting two al., 2000; (GIT) and al., 1999), et et (Bagrodia Cesare target two Di GIT2/CAT-2/PKL are 1999; and There kinase-interacting 2000). GIT1/CAT-1 al., et proteins, receptor Zhao 2006; al., et Kuo G-protein-coupled 2010; Nayal to al., 2011; functions et al., (Feng it et disassembly adhesions, and 2008; focal maturation Hansen, their Within and regulate 2006). (Frank Berk, integrator and signal and Hoefen scaffold a 2010). as al., acts et (Parsons motility focal cell of to disassembly crucial and are assembly adhesions coordinated transmembrane the are and which proteins, integrins, the through to matrix cytoskeleton extracellular actin localise intracellular 2011). both al., the to et connect (Kaur found adhesions numbers been These their regulate has to RhoJ and adhesions 2011). focal al., to increased et Yuan and 2011; increased (Kaur Cdc42 al., chain light et and myosin with phosphorylated and Rac RhoA associated active active of levels of in is knockdown levels increase RhoJ decreased turn This that causes observations 2011). in with consistent al., is this contractility et Kaur (Kaur and 2013; contractility al., 2011), actinomyosin et (Ho al., motility in et impairment an is 2013). with (siRNA) al., associated RNA interfering et small the (Ho using expression melanoma regulating RhoJ malignant Reducing in of spread identified RhoJ metastatic for the been role in a suggesting has cells, melanoma RhoJ of invasion and for motility role a Recently, A ofclahsostruhisbnigt I.I addition, In PIX. to binding kinase its the of through Zhao recruitment adhesions 2004; the focal in al., results to et turn PAK in their (Premont which Matafora 2000), homology proteins in al., Spa 2000; PIX et their with al., involved through (SHD) associate et proteins likely domains and GIT Cesare 2000) 2001). is al., (Di al., et et localisation which Zhao and 1999; activity trafficking al., are et ARF-GAP (Di Turner paxillin proteins have 2000; have of al., GIT binding proteins their et PIX partners. through Cesare and adhesions GIT interacting focal Both to and recruited 1997). al., domains et Manser multiple Oh 1998; and al., 1998; Cool-2) et al., (Bagrodia and et Cool-1) and ARHGEF6 ARHGEF7 as as known known (also PIX h I–I ope sa lgmrcpoenasml that assembly protein oligomeric an is complex GIT–PIX The Kme l,21;Tks ta. 02 une l,2011). al., et Yuan 2012; al., et Takase 2014; al., et (Kim Ku ta. 01 une l,21)advascularisation and 2011) al., et Yuan 2011; al., et (Kaur 2 rnec Edelmann Francesca , 2 o Bicknell Roy , 1 n itraL Heath L. Victoria and 1 itraA Salisbury A. Victoria , b PX(also -PIX me al., et ¨m 3039 1, ` a in in 1 - ,

Journal of Cell Science nrgltn uoragoeei n nmdaigfocal GIT–PIX mediating the with in association and its complex. angiogenesis through RhoJ dynamics tumour for adhesion role a regulating demonstrates compared study in tumours the This controls. reduced subcutaneous wild-type RhoJ of with of vascularisation Knockout and discovered. growth adhesions. for was focal role formation to tube other a and each RhoJ, GIT2 of GIT1, Like recruitment RhoJ RhoJ, the active GIT1. potentiated of that all SHD identified the with also experiments. interacted pulldown experiments in RhoJ two-hybrid complex of Yeast GIT–PIX form active the the with and interacted disassembly adhesion focal regulate ehns ywihRo ouae oa deindynamics adhesion focal modulates RhoJ which by 2000). mechanism al., et 2002; Zhao al., 2001; adhesions et the al., (Manabe focal promoting et protrusions that to West and indicate complex motility guanine-nucleotide- cellular studies PAK–PIX–GIT increases of the Manser Rac number of 1998; A al., localisation and 1998). et al., (Bagrodia Cdc42 domains et (GEF) contain factor exchange proteins PIX ARTICLE RESEARCH sebyaddssebytmst ecluae;teassembly the 3040 calculated; be enable longer to to no times measured were disassembly also they and was when assembly time intensity to the fluorescence appearance be The first visible. to their each considered from in from and cells shown taken tracked in adhesions is manually focal images were of of times condition imaging duration series minutes, The representative 1A. 90 Fig. A of period minutes. a 2 is over phenotype every tracked adhesion cell then focal the the were high at where evident, a cells wound, Motile scratch siRNA at the scratched. of was plated a edge monolayer duplex, the either then RhoJ-specific Paxillin– and a with density shown). or treated not duplex (data were control adhesions siRNA- HUVECs focal its that RFP-transduced of not confirm did confirmed numbers paxillin–RFP expressing to affect paxillin–RFP- was that of and order adhesions, numbers it focal positive increased In adhesion studies, knockdown focal RhoJ 2011). these mediated studying al., for for et this suitability used and (Berginski previously 2010). protein dynamics al., been adhesion et focal has (Mattheyses well-characterised fusion surface a cell is the to Paxillin close visualisation reflection for structures internal suited of 2004; is total which to microscopy, al., subjected (TIRF) fluorescence and et and with paxillin transduced RFP-tagged (Debreceni were sequestering human (HUVECs) an adhesions, cells Rac focal endothelial track RhoJ vein to or order umbilical In of Cdc42 2002). Hall, and of mutant Schmidt GEF GEFs of dominant-negative binding result inhibiting to a likely promiscuous reduce be would the specifically GTPases in Rho GTPase; related to multiple Rho to used proteins this was of To mutant levels than RhoJ. rather dominant-negative of knockdown activity siRNA a activity, and endothelial RhoJ expression in reduced increased their adhesions investigate for focal track and manipulated of to mutant disassembly cells performed affects and adhesions were GTP-bound RhoJ assembly how experiments the focal determine dynamics, active precisely adhesion more fewer numbers, focal an to order adhesion in In of motility. focal results expression increased (daRhoJ) and the leads motility motility RhoJ whereas of cell level reduced both the in regulate reduction to A to number. shown adhesion focal been and previously had disassembly RhoJ adhesion focal regulates RhoJ RESULTS angiogenesis in role its determine and h ups fti td a ocaatrs h molecular the characterise to was study this of purpose The b PX I1adGT nendothelial in GIT2 and GIT1 -PIX, nvivo in hJwsfudto found was RhoJ . b -PIX ihteGPcnrl n ltigtedt sahistogram a adhesions as focal of data majority the the with left plotting the to and shift a control, in resulted GFP compared a the times in disassembly with resulted in adhesion GFP–daRhoJ that reduction of focal assembly significant Expression than statistically faster 1E). rather RhoJ, (Fig. disassembly in as changed of was was resulted monitored it expression again were RhoJ and reducing scratch turnover active to a of contrast of introduction edge In the above. at described cells Focal paxillin– (GFP–daRhoJ). in daRhoJ both GFP-tagged adhesions or express GFP to either and transduced RFP were 1D). HUVECs to (Fig. adhesions, control minutes loading 20 a as than tubulin with more blot confirmed were western knockdown by taking RhoJ of Levels adhesions 1C). (Fig. focal focal disassemble higher with of right of the to that number shift numbers a shows the in results intervals RhoJ with 1B). of knockdown 10-minute (Fig. histogram, siRNA during RhoJ a disassembling as of adhesions data knockdown the after Displaying longer times, assembly significantly the not but were times, disassembly times, the disassembly that in and found assembly was resulted it into down knockdown broken was RhoJ this shown). When not that (data times duration found adhesion the focal was longer of significantly disappearance It to peak adhesion. and the fluorescence focal from peak being the time to disassembly appearance the from time the being time iasml,addRo asn erae ubr wn to owing slower numbers disassembly. to decreased rapid knockdown owing causing more RhoJ adhesions daRhoJ focal 2011): and regulates of al., disassembly, numbers RhoJ et increased that (Kaur causing is numbers observations and adhesion previous disassembly, focal our adhesion with focal demonstrate promotes consistent data activity These RhoJ 1G). the blots and (Fig. that Western GFP proteins both 1F). of fusion (Fig. expression GFP–daRhoJ substantial minutes was 20 there that than showed less in disassembling rtisGT 1 etds .%cvrg)and adhesion coverage) focal 4.8% the peptides, were (11 spectrometry. proteins GIT1 mass candidate interacting proteins using and candidate identified lysate Two were HUVEC proteins with (GST)- interacting incubated S-transferase daRhoJ glutathione tagged were recombinant experiments using Pulldown partners. of performed interacting role performed its the were determine for experiments to basis disassembly, molecular adhesion focal the in characterise RhoJ better to complex order GIT–PIX In the with interacts RhoJ re od hsaGL N-idn oanfso with fusion domain This DNA-binding constructed. GAL4 was In (dn)RhoJ a system. dominant-negative this this of in and RhoJ both daRhoJ do with or to interact either to two- order whether able yeast were investigate complex, proteins to GIT–PIX these performed the with was characterise RhoJ hybrid further To of 2A). interaction (Fig. alone the GFP revealed with not blotting but daRhoJ, Western beads. GFP-trap GFP–daRhoJ, that with or GFP incubated either and expressing HUVECs from prepared RhoJ. for explored partner also interacting was GIT2 potential GIT1, a to as similarity its while than Given HUVECs, level shown). in higher not GIT1 a of at those expressed than al., was higher et be Nayal to found 2011; and al., and GIT1 GIT2 et 2000). to Kuo al., particular et 2010; adhesion Zhao focal of al., 2006; in et complex were GIT–PIX (Feng the proteins disassembly of role These the given coverage). interest 6.9% peptides, olo tteefc ficesdRo ciiyo focal on activity RhoJ increased of effect the at look To nodrt ofr hs neatos ellrlstswere lysates cellular interactions, these confirm to order In b PX I1adGT l oimnpeiiae ihGFP– with co-immunoprecipitated all GIT2 and GIT1 -PIX, ora fCl cec 21)17 0935 doi:10.1242/jcs.140434 3039–3051 127, (2014) Science Cell of Journal b PX epciey eeso I2mN were mRNA GIT2 of Levels respectively. -PIX, a a PXaesrcual related structurally are -PIX PXi hscl ye(data type cell this in -PIX b PX(53 -PIX b -PIX

Journal of Cell Science *** EERHARTICLE RESEARCH a oepesdi es ihGT or GIT1 with yeast in conditi co-expressed per was adhesions 165 of total a experiments, load independent a three from as cells tubulin ti (four with disassembly cells (mean blotting and 12 chart western assembly of bar adhesion by each focal a confirmed from and as was adhesions GFP–daRhoJ, displayed 10–15 expression or and from GFP RhoJ either above, in express as Reduction to measured (D) transduced were were times. intensi HUVECs disassembly Paxillin–RFP-expressing maximal different (E,F) the with control. from adhesions time focal the of were disappeari number times and disassembly 0 and time intensity at maximal appearing to (arrow) adhesion appearance focal first a from show time 10 images the bar: Representative were Scale tracked. times disappearance. were Assembly adhesions cells. minutes. focal endothelial 26 and in after microscopy disassembly TIRF by adhesion minutes focal 2 regulates RhoJ 1. Fig. eoeiaig sebyaddssebytmswr esrdfo 01 dein rmec f1 el treo orclsfo he independent three from cells four or (three cells (mean 11 chart bar of a each as from displayed adhesions and condition) 10–15 per from adhesions measured 155 of were total times a disassembly experiments, and Assembly imaging. before ahJwudas idti eino I2 hs aasuggest data that These likely GIT2. is of region it sequence this so bind a and also similar, with with would respectively, being daRhoJ SHDs SHD2, acids and have SHD1 amino for necessary GIT2 non-identical 87% being and and 90% as of GIT1 SHD identity Both the including 2B). RhoJ, with region (Fig. interaction a for identified necessary were these determine GIT1 of to of series created regions A were which GIT1 2B). of (Fig. that mutants not histidine truncation but showed lacking C-terminal GIT1 medium assays on with These growth interacted allow histidine. dnRhoJ, not lacking but plates daRhoJ, on strain GAL1- grow yeast the auxotrophic of to of this interaction transactivation enabling gene the close HIS3 in used, promoter-driven results strain domains yeast GAL4 the these In domain. activation P , .0;N,ntsgiiat(anWinytest). (Mann–Whitney significant not NS, 0.001; m .()Pxli–F-xrsigHVC eetasetdwt ihrcnrlsRA(ioto)o hJsRAdpe 8hours 48 duplex siRNA RhoJ or (siControl) siRNA control either with transfected were HUVECs Paxillin–RFP-expressing (B) m. 6 ... E n itga F.()Epeso fGPadGPdRo a ofre ihwsenbotn o GFP. for blotting western with confirmed was GFP–daRhoJ and GFP of Expression (G) (F). histogram a and (E) s.e.m.) b PXfsdt h GAL4 the to fused -PIX b PXto -PIX A UEsepesn ailnRPa cac dewr mgdevery imaged were edge scratch a at paxillin–RFP expressing HUVECs (A) hJwti nohla el loafce h ieo focal of performed size was vinculin the of were staining of GFP–daRhoJ affected immunofluorescence activity and or fixed GFP the also either that expressing cells observed HUVECs adhesions. endothelial we study within 2011), this al., RhoJ of et (Kaur course numbers the adhesion during focal regulating as well size As adhesion focal regulates RhoJ fGPdRo ihGT,GT and 2C). GIT2 colocalisation (Fig. GIT1, was with there GFP–daRhoJ and of S1A), GIT2, Fig. GIT1, material Endogenous (supplementary GIT2. and and/or RhoJ GIT1 with SHDs, association their through daRhoJ bind GIT2 and likely most and GIT1 that 6 b ....()Dt rmBwr lopotda itga hwn the showing histogram a as plotted also were B from Data (C) s.e.m.). PXi oimnpeiiae ihdRo hog its through daRhoJ with co-immunoprecipitated is -PIX ora fCl cec 21)17 0935 doi:10.1242/jcs.140434 3039–3051 127, (2014) Science Cell of Journal b PXalclclsdwt iclna oa adhesions focal at vinculin with colocalised all -PIX b PXa oa adhesions focal at -PIX 3041 ing yto ty mes on) ng

Journal of Cell Science EERHARTICLE RESEARCH 3042 Fg AC upeetr aeilFg S1B,C). Fig. experiments material three supplementary across 3A,C; compared (Fig. HUVECs area adhesion siRNA-transfected focal control in with reduction small a RhoJ-speci have consistently different In two 3B). (Fig. with compared GFP GFP–daRhoJ in expressing expressing increase HUVECs those three in significant from size statistically mean adhesion Data a focal the calculated. showed condition and experiments each cells replicate for three area 15 from least adhesion at selected focal for quantified randomly was adhesions staining vinculin focal of area The 3A). (Fig. by with probed interactions were for and Samples blot lysed beads. western were GFP-trap GFP–daRhoJ using or performed GFP pulldowns expressing with stably colocalises HUVECs and (A) interacts RhoJ 2. Fig. ie h hsclitrcin ewe I1adGT with GIT2 and GIT1 between interactions physical the adhesions Given focal at other each and GIT2 GIT2 GIT1, RhoJ, adhesions of focal to there. recruited phosphorylated being levels being and S3D) GIT2 Fig. GIT2 material reduced (supplementary reduced to of due phosphorylation of be small levels in level might was reduced The its lower reduction observed. consistently the resulted was to although it change and S2). lead rather 4C), not (Fig. not phosphorylation Fig. but did did material RhoJ protein, but of (supplementary (Y392), Knockdown been mobility phospho-GIT2 has reduced electrophoretic GIT1 FAK of and Src in of Inhibition levels residues 2006b). al., these et (Webb observed phosphorylation of threonine and phosphorylation serine to of likely because levels is increased this to its daRhoJ, expressing due HUVECs be in shift from a GFP–daRhoJ- GIT2 result also of is size in might There in degradation. daRhoJ from GIT2 protection with and total focal stabilisation interaction to tyrosine GIT2 as through of recruitment extent adhesions Increased be 4B). same (Fig. cells must the expressing be to to found (Brown also GIT2 were (FAK) elevated (Y392) phospho-GIT2 kinase its of Levels adhesion adhesions, 2005). for al., focal et and order of focal Src In levels by phosphorylated higher cells. to observed GFP-expressing GFP–daRhoJ,recruitment we control expressing alone GIT2 HUVECs with from GFP of compared lysates case expressing in protein the those GIT2 In with 4A). compared (Fig. cells expressing and GIT2 GIT1, needn experiments. 20 independent and bar: GIT2 Scale GIT1, area. for enlarged be stained GFP–daRhoJ to express and found to fixed transduced (SHD) were HUVECs domain (C) its homology binding. this map Spa for to the necessary plates performed with on were RhoJ, yeast GIT1 with of of interaction growth Truncations by (–His). indicated Histidine are lacking daRhoJ interactions of fusions Positive (DBD) dnRhoJ. candidate domain or DNA-binding of Gal4 fusions and yeast (AD) partners domain using interacting activation performed Gal4 were assays with transformed two-hybrid Yeast (B) control. binding rnfce ihtesRAcnrldpe,dpee pcfcfor specific were duplexes HUVECs duplex, adhesions. control RhoJ, focal siRNA to the with others affected transfected the components these of of recruitment each the of whether expression determine to down performed knocking were experiments RhoJ, and PIX I1adGT eepromdtgte.A asafter days immunofluorescence 2 then At and together. fixed performed were were cells both of GIT2 transfection, knockdowns HUVECs, and in GIT2 expressed GIT1 and are GIT1 both between fact redundancy the and and similarity the to Owing ossetwt hs bevtoswsteices nlvl of levels in increase the was observations these with Consistent b ora fCl cec 21)17 0935 doi:10.1242/jcs.140434 3039–3051 127, (2014) Science Cell of Journal PXo obnto fGT n I2duplexes. GIT2 and GIT1 of combination a or -PIX b PXfuda oa dein nGFP–daRhoJ- in adhesions focal at found -PIX b PXaeivle nrcutetof recruitment in involved are -PIX m .Teedt r ersnaieo three of representative are data These M. b PX I1adGT,uigGPa a as GFP using GIT2, and GIT1 -PIX, i iN ulxswr on to found were duplexes siRNA fic otat UEstransfected HUVECs contrast, b PX h o niae the indicates box The -PIX. b PX I1adGIT2. and GIT1 -PIX, b -

Journal of Cell Science EERHARTICLE RESEARCH aapit rmec xeietlgop nldn hs fthe of experiment, those including replicate group, each experimental for each for from Thus experiments, value points grey duplex. data different mean control average from the siRNA to the scaled generated In were cells. values data three grey of mean compare each other from to examined any were order total adhesions A immunofluorescence focal to S3A–D. 20 Fig. the material of specific of supplementary in Examples shown antibodies are complex. staining the with a in of staining resulted was member components It these of analysed. of for was any reduction vinculin down staining for knocking that positive their or found region GIT2 GIT1, by the RhoJ, in for identified value staining grey mean were the and adhesions vinculin, RhoJ, to Focal in specific vinculin GIT2. protein antibodies adhesion with focal the combination for performed was staining ( experiment each from values mean times focal the three mean performed are the plotted was and and This cells, condition. three each of for total calculated area a adhesion from cell per adhesions focal of 15 ImageJ using measured were GFP–daRhoJ-expressing HUVECs and 20 GFP- box bar: The Scale vinculin. area. for enlarged stained the and indicates fixed were GFP–daRhoJ or GFP express to transduced were HUVECs hours Similarly, antibodies. vinculin-specific 48 with after stained and duplexes siRNA RhoJ or (siControl) siRNA size. control adhesion with focal regulates RhoJ 3. Fig. * racluae e odto,potdaetema oa deinaesfrom areas adhesion adhesion focal ( focal mean experiments mean independent and the three cells are six plotted condition, or five per calculated of area total a from adhesions 106– of focal ImageJ 135 using measured were siRNA RhoJ or siControl with transfected deinaeswr bevdi aho h he experiments. three the of each in observed were areas adhesion P , .5(Student’s 0.05 t ts) C iial,fclahso ra rmHUVECs from areas adhesion focal Similarly, (C) -test). n 5 )(mean 3) m .()Fclahso ra from areas adhesion Focal (B) m. 6 ....Rdcin nfocal in Reductions s.e.m.). A UEswr transfected were HUVECs (A) n 5 b )(mean 3) PX I1or GIT1 -PIX, 6 s.e.m.). b -PIX eutdi eue I1 I2adRo tfcladhesions; focal RhoJ at less caused RhoJ together GIT2 and and and GIT1 GIT2 of a GIT1, knockdown likewise and only reduced GIT2 not in resulted GIT1, in resulted of also but adhesions RhoJ that focal to localisation down RhoJ in knocking decrease significant Thus, 5B). (Fig. together, GIT2 were and GIT1 results and RhoJ, similar of Knockdown Very S3E). (supplementary duplexes Fig. 5A. siRNA material of Fig. set then alternative in an were with shown obtained experiments from are Data independent and 100. three at combined set from negative was the adhesions which of experiment focal mean that the for to data scaled control were duplex, control negative xrc rmteEglrt–omSamsroacl hc is which cell membrane sarcoma basement Engelbreth–Holm–Swarm solubilised on the a seeded from are Matrigel, extract cells with endothelial coated and and assay, wells this Matrigel to GIT2, biochemical In a using assay. and assessed forming the also tube GIT1 was formation to RhoJ, tube in complex, Owing combination of GIT–PIX the role 2011). and the RhoJ al., in between interaction RhoJ al., et functional et for (Kaur Yuan role formation a tube 2011; and demonstrated migration had endothelial others regulating and we Previously b mlnaino ygni ei ugcrioaclsrsle in resulted cells carcinoma lung Lewis syngeneic is subcutaneous of RhoJ normal However, implantation development. that the embryonic indicating for at normally essential born not grew were and mice frequency RhoJ-knockout Mendelian exon. of second mice removal in and with the resulting crossed recombinase first were Cre genetrap expressing the exon. RhoJ constitutively second the the between for flanking sites homozygous inserted LoxP Mice had cassette which and trap which exons cells second gene stem embryonic a from derived contained RhoJ were of These role made. the was determine to order In angiogenesis tumour regulates RhoJ RhoJ, of combination the tube in down role positive Knocking a suggest plays data complex formation. These GIT–PIX to the 6). similar RhoJ, (Fig. cells, like knockdown that, of RhoJ networks with stable giving observed less formation, that and tube connected disrupted less the in to of resulted rise of indication GIT2 Knockdown an and 6B). GIT1 give (Fig. both to and were network image, the 12 each cells calculated of for after that connectivity loops software days, imaged of by and number processed 2 were the Matrigel, images After to The hours. on duplexes. 24 plated four and all harvested of siRNA 1989). combination or Kubota, duplex RhoJ, control and cell siRNA against Lawley with of transfected 1988; on stage were differentiation al., cells HUVECs dependent the et represent endothelial (Kubota to process of angiogenesis considered is a differentiation which and and structures, migration tube-like attachment, cells form endothelial the and induces to proteoglycans extract sulphate This 2003). heparin (Kalluri, laminin, entactin IV, collagen in rich aeuiga lentv e fdpee (supplementary duplexes were of observations set Similar alternative severe S4). Fig. more an expected. material and using be additive rather an made pathway, would where same pathways, the phenotype distinct in in together consistent acting than be would or proteins This 6). these similar RhoJ (Fig. with a GIT2 and out to GIT1 formation of knocking tube combination impair as to found degree was GIT2 and GIT1 PX I1adGT euaetubulogenesis regulate GIT2 and GIT1 -PIX, b b PXt ercutdt oa adhesions. focal to recruited be to -PIX PXwr ofre t4 or ywsenblotting western by hours 48 at confirmed were -PIX ora fCl cec 21)17 0935 doi:10.1242/jcs.140434 3039–3051 127, (2014) Science Cell of Journal b PX obnto fGT n I2o a or GIT2 and GIT1 of combination a -PIX, b PX Similarly -PIX. b PXidvdal rthe or individually -PIX nvivo in b PXaoeadin and alone -PIX b ncotmouse knockout a PXknockdown -PIX b PXand -PIX b -PIX, 3043

Journal of Cell Science EERHARTICLE RESEARCH 3044 PAK-mediated likely the by is potentiated GIT1 this is and interaction paxillin and This between GIT2. adhesions, interaction and/or the focal We through (Vicente- to mediated be 2012). other to disassembly each Wehrle-Haller, of adhesion recruitment 2011; RhoJ, with Horwitz, to found relative and Rac associated activated Manzanares of is ratio increased RhoA an not delineated, of has GIT–PIX disassembly activity been GIT–PIX-mediated GEF the of of the mechanism activity of precise of the the because increases recruitment turn Cdc42 of is the in and SHD This Rac RhoJ promote adhesions. the focal GTP-bound to through to complex interacts tumour GIT2 Active and in and 8. adhesions Fig. GIT1 role focal in a to depicted localised plays is RhoJ adhesions that show angiogenesis also tube endothelial for complex We required GIT–PIX adhesion is formation. complex focal the this regulating with that demonstrate in interaction and RhoJ its for through role disassembly a identified have We DISCUSSION RhoJ RhoJ- for role from angiogenesis. a tumour tumours demonstrate mediating data vessel in These in of reduced and 7C). analysis (Fig. was mice excised 7B), this knockout (Fig. that CD31 were sections indicated protein frozen tumours density endothelial on mice, performed the was of knockout staining was the vascularisation immunofluorescence tumour in whether vessels investigate affected and new To (Carmeliet proliferation the 2011). cell these Jain, angiogenesis, tumour of sustain on growth to rapid required dependent being The wild- highly 7A). in is (Fig. those tubulin weeks and tumours with 2 RhoJ compared GIT2, after Y392, tumours controls on type smaller with phosphorylated transfected GIT2 of were for formation HUVECs blotted (C) the and tubulin. lysed and were experiments. GFP cells independent GIT2, hours, three (pGIT2), 48 of Y392 After representative on duplexes. are phosphorylated siRNA data GIT2 RhoJ These for or blotted (siControl) either western siRNA and adhesions. and control vinculin focal GFP–daRhoJ for to or stained proteins GFP and partner expressing fixed of were recruitment and increases GFP–daRhoJ, expression GFP–daRhoJ 4. Fig. nvivo in b PXadteGTpoen l rmt the promote all proteins GIT the and -PIX oe o h oeo hJwti focal within RhoJ of role the for model A . b PX Although -PIX. b PX I1o I2 cl a:20 bar: Scale GIT2. or GIT1 -PIX, eitdihbto fmoi I rvosy ehv shown have we blebbistatin- Previously, II. by myosin induced of with cells inhibition contractility in our mediated adhesions focal actinomyosin in mirroring levels reduced increased investigated assembly, at it They finding RhoJ. than after of knockdown rather with observations found disassembly They of for 2011). knockdown adhesion role al., et a siRNA with the (Kuo identified consistent that turnover who in are adhesion al. focal variation findings et nascent Our context-specific Kuo are GIT2. of there and and work Thus, GIT1 cell-type- 2006). of al., be functions et increase (Frank to and study size likely this was times in adhesion turnover measured adhesion focal focal not on decrease effect the to epithelial al., although in motility, et found GIT1 Zhao not was 2001; but GIT2 al., cells of et knockdown West contrast, to In 2002; and 2000). al., motility complex cellular et of PAK–PIX–GIT (Manabe levels protrusions increased the in results of adhesions focal localisation knockdown the RhoJ 2011). that promoting al., observed et (Yuan who Cdc42 and al. Rac active et of consistent RhoJ levels Yuan reduced are of data down Our those recruited. knocking complex with GIT–PIX contrast, the reduced larger with of adhesions, In levels stable these more stable. smaller suggest in resulted would less expression would adhesion are size the adhesions increased of their The disassembly proteins. however, adhesion faster focal cells; of recruitment accelerated control an indicate the longer no in was size maximal than their reach to taken adhesions time larger these The with adhesions. for of larger of associated Expression assembly phosphorylation. the PAK promoted paxillin daRhoJ further the promote GIT– recruited, to the likely as is thus 2006), complex al., PIX et (Nayal paxillin of phosphorylation rvosyanme fgop aedmntae that demonstrated have groups of number a Previously ora fCl cec 21)17 0935 doi:10.1242/jcs.140434 3039–3051 127, (2014) Science Cell of Journal A UEswr rndcdt xrs F or GFP express to transduced were HUVECs (A) m .()Clua yae eepeae rmHUVECs from prepared were lysates Cellular (B) m. b PXseiial eue focal reduced specifically -PIX b PXi driving in -PIX b PXis -PIX b -PIX .

Journal of Cell Science fe 8ad7 or n lte o I1 I2 RhoJ, GIT2, GIT1, for blotted and hours 72 and 48 after EERHARTICLE RESEARCH en einncsayfrtebnigo ahJ eie RhoJ Besides daRhoJ. of binding the three as and for GIT of necessary the SHD region the a of identified being We two 2009). Rittinger, of and binds have (Schlenker GIT1 domains of studies SHD GIT-binding each structural which the in Recent complex heteropentameric 2001). a al., in et that Koh suggested 2001; al., of domains et similar C-terminus likewise 2004), the al., domains et in Premont coil 2003; coiled al., et their regulates (Paris through heterodimers RhoJ and that homodimers suggest GIT–PIX- would turnover. of adhesion modulation data focal and mediated (Parsons interaction Our its contractility so through 2010). regulate II contractility distribution, proteins al., and myosin a adhesion size maturation, et as focal is adhesion can There thus, focal adhesion too influence 4A). contractility; Fig. focal can actinomyosin and activity between and 2011 al., turnover relationship et both and (Kuo complex Thus, decreased adhesions 2011). in formation increased result focal al., associated expression an et fibre daRhoJ and and contractility (Kaur inhibition stress gels II myosin collagen actin light of in myosin contraction contractility phosphorylation, regulating and negatively activation RhoJ from chain RhoJ condition with between each cells, from link points endothelial a data is scaled the there all that of means the are Plotted 100. at set exper replicate was each which For siControl, Methods. (mean and the replicates Materials of the experimental mean to the according the of experiments to each independent scaled three from were ImageJ points using data cells three from cell per adhesions and GIT1/2 RhoJ, of RhoJ, recruitment siRNA, the RhoJ of (siControl), regulation Reciprocal 5. Fig. arxpoenpcooalhv ensont neatwt the with interact to shown been have all piccolo protein matrix h I–I ope smliei.GTpoen form proteins GIT multimeric. is complex GIT–PIX The b b PX hshlps C phospholipase -PIX, PX I1o I2 o aheprmn,tema ryvleo ihrGT,GT,Ro or RhoJ GIT2, GIT1, either of value grey mean the experiment, each For GIT2. or GIT1 -PIX, b PXfrstiesrte hndmr,resulting dimers, than rather trimers forms -PIX b b PXsRAo ohGT n I2(I12 iN ulxs fe 8hus UEswr ie n tie o icln and vinculin, for stained and fixed were HUVECs hours, 48 After duplexes. siRNA (GIT1/2) GIT2 and GIT1 both or siRNA -PIX PXeal t ooieiain(Kim homodimerisation its enable -PIX c A,MKadtepresynaptic the and MEK FAK, , 6 ....*** s.e.m.). b PXacmlto at accumulation -PIX P , b b .0 Mn–hte etcmaigec ftedt onst h ioto) B el eelysed were Cells (B) siControl). the to points data the of each comparing test (Mann–Whitney 0.001 PXo uui salaigcontrol. loading a as tubulin or -PIX PXproteins -PIX b PXt oa adhesions. focal to -PIX xrsin(i ta. 05 une l,21) oee,data However, and RhoJ 2011). FAK reduced piccolo, al., that et with suggested Yuan mutagenesis cells 2005; using endothelial and reduced generated al., MEK1 in et the a (Yin explain B-Raf as expression might active acts adhesions 2004; of GIT1 al., focal that levels et in finding Yin scaffold The 2003; 2000). al., ERK1/2 et al., Kim et 2003; Zhao al., et (Haendeler SHD xeiet ol ugs htdRo osntcmeewith pulldown compete not Our does SHD. daRhoJ the that of suggest regions would different experiments with interact all PIX I o I idn eas ahJwsal opulldown to able was daRhoJ because binding GIT for PIX nuainwt r n/rFKihbtr eue Y392 reduced inhibitors FAK most phosphorylation. and/or mass, daRhoJ-expressing threonine molecular and Src from serine increased with GIT2 increased an Incubation localisation to was The at due adhesion ran 2005). which likely focal cells al., GIT2, whose endothelial for residues et of tyrosine required three (Brown levels is of one increased phosphorylation of Y392, Expression in at 2006a). al., phosphorylated et paxillin results Webb as some 2013; such with al., daRhoJ et 2006b), proteins, (Jones al., interacting Vav2 et of and (Webb binding GIT1 the on modulating identified been on have depend mediates to activation. likely GIT and is type of which cell of SHD the nature the of the interactions, Thus displacement multiple through protein. might turnover GIT2 GIT-binding adhesion and/or another focal GIT1 to regulate is RhoJ It to active GIT2. act of or GIT1 binding with that interaction possible its through presumably PIX, utpestso eie henn n yoiephosphorylation tyrosine and threonine serine, of sites Multiple ora fCl cec 21)17 0935 doi:10.1242/jcs.140434 3039–3051 127, (2014) Science Cell of Journal A UEswr rnfce ihcnrlsiRNA control with transfected were HUVECs (A) b PXsann a niae)wscluae o 20 for calculated was indicated) (as staining -PIX mn,all iment, 3045 b b b - - -

Journal of Cell Science EERHARTICLE RESEARCH 3046 ori combination. in four RhoJ, individual the RhoJ, of RhoJ combination (siControl), siRNA control with siRNA, formation. transfected tube were impairs HUVECs similarly (A) four all of RhoJ, combination of Knockdown 6. Fig. ttsial infcn ifrnecmae ihtesCnrlduplex siControl the with [ compared difference significant statistically 200 bars: Scale after hours. imaged and 24 Matrigel and on 12 replated were cells the transfection, after iepit h eno hs ausfo he experimental per three view from ( of values plotted fields these is six of replicates to mean the five The experiment from point. each calculated time For was tubules. number the loop by mean the formed show to loops plugin of ImageJ number analyser angiogenesis the using formation P , ora fCl cec 21)17 0935 doi:10.1242/jcs.140434 3039–3051 127, (2014) Science Cell of Journal .5(Student’s 0.05 b PXsRAo I1adGT iN oehr(I12 ra or (GIT1/2) together siRNA GIT2 and GIT1 or siRNA -PIX t b ts),btteeaen ifrne between differences no are there but -test)], n PX I12kokon n ncdw fall of knockdown and knockdowns GIT1/2 -PIX, b 5 PX I1adGT ulxs t4 hours 48 At duplexes. GIT2 and GIT1 -PIX, )(mean 3) b PX ohGT n I2o a or GIT2 and GIT1 both -PIX, 6 m e) l ncdwsgv a give knockdowns All sem). .()Aayi ftetubule the of Analysis (B) m.

Journal of Cell Science EERHARTICLE RESEARCH soitdwt erae hAatvt n erae actinomyosin decreased is and This activity contractility. motility. RhoA and increased decreased Rac and with of disassembly associated activation adhesion and promote focal paxillin complex and with GIT–PIX Cdc42 interacts the GIT with domain (GIT). together homology GIT2 RhoJ Spa or the GIT1 with of interaction (SHD) its function. through RhoJ complex for GIT–PIX model A 8. Fig. 2007). al., et (Schmalzigaug of duct vascularisation impaired bile have the mice lining GIT1-knockout cells and Although cells, endothelial bronchi of to localised the expression not is (data widespread GIT1 whereas RT-PCR suggest GIT2, by analyses detectable Expression readily shown). more is GIT2 interacting although daRhoJ-expressing preferentially in be size increased might its find RhoJ GIT2. in not with that change did any suggesting We or cells, GIT2. GIT1 PAK, of activating phosphorylate also levels is to daRhoJ that it possible and (Manser is causing PAK it Cdc42 kinase and 1998), threonine al., active serine et the of of activation Co-expression increased S3). material (supplementary of Fig. HUVEC mass molecular daRhoJ-expressing increased from the GIT2 alter not did but phosphorylation mice. RhoJ-knockout in growth tumour Reduced 7. Fig. 100 * acnm el,atr2westetmuswr xie n ege (WT, weighed and excised were tumours the weeks 2 after cells, carcinoma P 5 efn htbt I1adGT r xrse nHUVECs, in expressed are GIT2 and GIT1 both that find We .11(anWinyts) B h xie uor eescindadvsessanduigat-D1wt ersnaieiae hw.Saebar Scale shown. images representative with anti-CD31 using stained vessels and sectioned were tumours excised The (B) test). (Mann–Whitney 0.0101 m .()Vse est a acltduigteAgoy otaeadpotd(mean plotted and Software Angiosys the using calculated was density Vessel (C) m. cieRo neat ihthe with interacts RhoJ Active b idtp W)adRo-ncot(O iewr uctnosyipatdwt 10 with implanted subcutaneously were mice (KO) RhoJ-knockout and (WT) Wild-type b PXcauses -PIX PX Active -PIX. n 5 ;Ro KO RhoJ 5; eurdt aiiaeti rcs.Ordt r iia othat to similar are variety a data that demonstrate Our they 2014); al., process. et (Kim this reported proliferationrecently facilitate is cell RhoJ that to suggest tumour would required data the our and sustain 2011), the Jain, al., and on to (Carmeliet dependent et vessels crucially (Masiero is of tumours vessels development of tumour growth rapid in The of upregulated 2013). one highly – signature for has angiogenesis genes tumour essential RhoJ 20 a Recently of not part cells. as endothelial identified is been by it expressed developing GTPases 2011), the related Rho closely in al., from compensation expressed to et due is presumably development (Kaur RhoJ vasculature and 2012), mouse al., vascularisation retinal neonatal et affect (Takase to reported been diminished a has RhoJ angiogenesis indicate in of tumour data in results These RhoJ density. for RhoJ vessel role reduced of and knockout growth tumour that observations our w I rtis oe o h I–I–A ope have complex PIX–GIT–PAK the for Roles al., these between et proteins. redundancy (Schmalzigaug GIT functional to phenotype knockout two due vascular GIT2 be might no the This 2009), for 2009). al., reported et been (Pang has lung developing the ciiyo inlitgaos uha h I–I complex. GIT–PIX and the as localisation such the integrators, controlling signal appropriately of and of Endothelial by activity 2011), balance Jain, act pathway. GIT–PIX the and likely by (Carmeliet same they the stimuli controlled anti-angiogenic tightly the and and is pro- RhoJ in activation or with together quiescence consistent acting are complex findings inhibition RhoJ, level down These knocking similar as a formation positively gives of tube complex of complex knockdown GIT–PIX Combined the GIT–PIX with formation. the RhoJ we tube RhoJ, study, endothelial this like impairing In regulates that, 2009). and al., shown adhesions, et have (Jones focal activity Upon from protrusive cellular away complex stability. paxillin–GIT1 GIT1 a vascular with the sequestering interacts for Robo4 promotes ligand ligand, a its that is binding Slit2 receptors 2012). al., in et roundabout (Liu haemorrhage expression either GIT1 cerebral down with increased been zebrafish vascular is also developing has van there the stability for 2007; because and role vascular A al., podosome suggested mediating 2009). in al., et et complex contractility Stockton cell Wang GIT–PIX 2004; 2003; al., et endothelial al., Amerongen function, Nieuw et regulating (Shikata barrier permeability in formation, identified been ossetwt t oei nohla ueformation tube endothelial in role its with Consistent ora fCl cec 21)17 0935 doi:10.1242/jcs.140434 3039–3051 127, (2014) Science Cell of Journal 6 n ....* s.e.m.). 5 ) A h uorwihsaepotd(mean plotted are weights tumour The (A) 7). P 5 .33(anWinytest). (Mann–Whitney 0.0303 b PXmtto rknocked or mutation -PIX nvivo in b lhuh knockout Although, . PXadGTalone. GIT and -PIX 6 s.e.m.). nvitro in 6 ei lung Lewis 3047 are , :

Journal of Cell Science I2sRA 5 siRNA, GIT2 AGAAACCUCUCACUUACGAG-3 esn H ta. 02.Telte eurdteatvto of activation the required latter damage The DNA 2012). affecting al., that by et but (Ho 2013), chemoresistance al., sensing modulated et did (Ho only also invasion not and that it have motility discovered their was affect it might lines, RhoJ cell signalling melanoma In RhoJ. RhoJ of inhibition mice, benefit. RhoJ-knockout therapeutic in that effective more suggesting are agents anti-cancer of ARTICLE RESEARCH rmtr a sda h otsri o h sas(et tal., 3048 et 5 (Heath siRNA, assays RhoJ the used: for were GAL1::HIS3 duplexes strain a siRNA host comprises following the which The as 2004). PJ69-4A, used strain was yeast promoter, The fusions. as GIT1,cloned domain. DNA-binding Gal4p the b with proteins fusion as expressed as box CAAX cloned C-terminal the lacking were each experiments, pWPXL- (T35N) from two-hybrid in dnRhoJ yeast or and (Q79L) For paxillin–RFP 2011). daRhoJ cassette. pWPXL GFP the pWPXL-GFP-daRhoJ, the into al., removing 2008) process subcloning the et al., or et by (Kaur (Parsons expression constructed pcDNA3.1-mRFP-N-paxillin described was GFP previously paxillin-RFP as for EMCV constructed an USA) (Addgene, with pWPXL or vector Cambridge, USA) expression Cambridge, mammalian Addgene, cassette; lentiviral IRES-EGFP (a combination in pWPI and USA) (Lentiviral with Cambridge, USA) psPAX2 Addgene, Cambridge, plasmid; were Addgene, (Envelope pMD2G production expression; mammalian lentivirus for for packaging used (Life plasmids The duplexes 546 siRNA and Plasmids Fluor PF inhibitor Alexa FAK were to used Inhibitors conjugated goat (1 UK) 573228 UK). Paisley, IgG Paisley, Technologies, Technologies, (Life anti-rat IgG 647 anti-mouse Technologies, Fluor polyclonal polyclonal Alexa goat (Life UK), to Fluor 488 Paisley, Alexa conjugated to Technologies, Fluor conjugated (Life IgG Alexa 546 anti-mouse polyclonal polyclonal donkey to goat to UK), UK), conjugated Paisley, conjugated Paisley, IgG Technologies, IgG (Life anti-mouse anti-rabbit 488 polyclonal used. Dako Fluor to goat conjugated Alexa anti-mouse (HRP; Healthcare), IgG was anti-rabbit polyclonal (GE peroxidase polyclonal HRP antibody donkey goat horseradish UK), Ely, follows: UK) to Cytomation, CD31 as conjugated Oxford, anti-mouse were immunoglobulin Biosciences, antibodies vessels Secondary tumour BD of For (MEC13.3; used. were GIT1 staining antibodies anti-human For USA) immunofluorescence Cruz, polyclonal used. Santa Biotechnology, rabbit were Cruz (Santa UK); (hVIN-1, antibodies [as vinculin Gillingham, 2011)] and polyclonal anti-human Sigma-Aldrich, al., monoclonal RhoJ blotting et mouse anti-rabbit immunofluorescence (Kaur western purified described Signaling UK); (Cell previously For GIT2 Hitchin, anti-human used. monoclonal Technology, rabbit rabbit anti-human UK); were and Livingstone, polyclonal UK); antibodies rabbit London, Technology, immunofluorescence, UK) Signaling UK, (Cell Research Hitchin, (Tyr392) Cancer anti-phospho-GIT2 3E1, monoclonal anti- (Clone monoclonal mouse UK); GFP Hitchin, anti-human Technology, polyclonal Signaling (Cell rabbit GIT1 anti- UK); anti-chicken monoclonal Gillingham, monoclonal UK) mouse (Sigma-Aldrich, mouse UK), tubulin (Gillingham, blotting, Cambridge, (Abcam, western Sigma-Aldrich RhoJ For human from stated. obtained otherwise unless were chemicals All Reagents METHODS AND MATERIALS RhoJ vasculature. tumour of tumour both the biology targeting and in the cells opportunities Understanding GIT–PIX therapeutic the provide involved. whether might discover also to is interest of complex be will it and PAK1, elcCe,Mnc,Germany). Munich, SelleckChem, PX I1aioais138adGT mn cd –5 hr each where 1–258 acids amino GIT1 and 1–378 acids amino GIT1 -PIX, eety oe eodagoeei aebe dniidfor identified been have angiogenesis beyond roles Recently, Eco m ,Slekhm uih emn) n aaii 5 nM, (50 Dasatinib and Germany); Munich, SelleckChem, M, Ifamnsit AT ocet a4 ciaindomain activation Gal4p create to pACT2 into fragments RI 9 Bam -CGAUGAAGUUGACAGGCGATT-3 HI- Eco Ifamnsit h lsi GT and pGBT9 plasmid the into fragments RI 9 Ergne,Suhmtn UK); Southampton, (Eurogentec, b PX(Millipore, -PIX 9 I1siRNA, GIT1 ; 9 - CCACUGUGUUUGACCACUAU-3 ntesplmnaydt eea follows: Life as 3 and were UK) data experiments for supplementary (Southampton, used the duplexes Eurogentec in Alternative both UK). (Paisley, from Technologies were siRNAs TAGACAAGATATTCATT-3 GGAATGACAATTT-3 5 0m a,2m NaVO mM 2 NaF, mM 10 c o 5mntspirt pniga 21,910 at spinning to prior minutes 15 for ice ofunei ei 9 otiig4m -ltmn,90 to L-glutamine, cultured mM 4 were containing HUVECs delivery; 199 consent. after obtained Media Trust were informed in cords NHS given Umbilical confluence Care had experiments. Health all mothers Women’s for between Birmingham used 6 were from and (HUVECs) 1 cells passage endothelial vein umbilical Human culture Cell l ellstswr rprduigRoasylssbfe 1 (v/v) (1% buffer MgCl lysis mM Rho-assay 30 N-octyl- using (w/v) prepared 1% were NP40, lysates cell All western and experiments blotting GFP-trap lysates, of Preparation Cre the express of 10 removal growth, tumour constitutively the induce To C57BL/6 in RhoJ. which resulting of to 2 1998) exon mice, bred flanked al., LoxP et were PGK-Cre were (Lallemand and of mice recombinase These with cassette. mice University the for C57BL/6 the crossed heterozygous were mice albino Project embryonic generate at mice of to into females injection Facility Mouse knockout by cells Mouse generated and stem Knockout were Transgenic mice USA) Chimeric the the ID Birmingham. Davis, by project from California, generated [RhoJtm1a(KOMP)Wtsi of procured cassette (University were driven CSD25401] Unit had JM8.N4 RhoJ-knockout promoter the C57BL/6N Services experimentation containing licensing. first cells Biomedical and stem and embryonic approval Birmingham feeder-independent maintenance Office the Home animal appropriate at UK), housed (Birmingham, were Mice tumour and assays mouse implantation RhoJ-knockout the of Generation t89weso g.Atr2wes uorms a eemndand determined at cut was sections mass serial and tumour compound OCT weeks, in 6 2 frozen were After tumours age. the mice then male of of weeks flank the 8–9 into at subcutaneously injected were cells carcinoma o etr ltigo ellsts yae eemxdwt sample 2 with experiments mixed GFP-trap were For SDS-PAGE. lysates to lysates, subjected cell and of buffer blotting western For 8 with supplemented Netherlands), The Amsterdam, (Corning, hog rnin rnfcinwt obnto ftepackaging, the of combination a HEK293T (Life with in with generated transfection (v/v) was lipofectamine 0.3% Lentivirus transient OptiMEM. of described RNAiMAX in through concentration nM final 10 as using a at siRNA duplexes at penicillin- with are UK) performed HUVECs Paisley, of Technologies, transduction Transfections were L-glutamine, 2011). for lentiviral al., duplexes Methods et mM serum. and (Kaur 4 bovine medium previously fetal transfection Eagle’s (v/v) with 10% modified siRNA and Dulbecco’s solution supplemented 1979). streptomycin in al., Co, Heidelberg, et cultured (Promocell, Culture (DMEM) (Maciag fibroblasts were Cell extract dermal The human brain Germany) and (PAA, bovine cells serum purified HEK293T and bovine UK) fetal Yeovil, (v/v) 10% heparin, a nuae nOtMM(ieTcnlge,Pily K with 36 UK) at Paisley, UK) Technologies, Gillingham, (Life (Sigma-Aldrich, OptiMEM plasmids of in polyethylenimine mixture The incubated above. listed was plasmids expression and envelope upeet:bvn ri xrc n eai sdsrbdaoe and transduced. above, be described to growth as HUVECs heparin endothelial the and with and incubated extract brain UK) bovine Gillingham, supplements: (Sigma-Aldrich, polybrene 0mntsa omtmeauepirt digt E23 el in harvested 0.45 cells was a HEK293T through virus passed to containing transfection, after adding Medium hours to medium. 48 prior growth temperature normal room their at minutes 10 9 9 I2 5 GIT2, ; -GGCAUUACAUCAUCCCACATT-3 m m. ora fCl cec 21)17 0935 doi:10.1242/jcs.140434 3039–3051 127, (2014) Science Cell of Journal 9 2 -CGUUGAUUAUGCAAGGCAATT-3 5 MNC,mmainpoes niio cocktail, inhibitor protease mammalian NaCl, mM 150 , 9 I1 5 GIT1, , b Dguoyaoie 5m EE H7.5, pH HEPES mM 25 -D-glucopyranoside, 3 .Clswr eupne nlssbfe on buffer lysis in resuspended were Cells ). 9 Lf ehoois ase,U) Control UK). Paisley, Technologies, (Life 9 -ACAUCUCCAUUGUCAAGCATT- 9 . 9 and ; b b g PXsRA 5 siRNA, -PIX PX 5 -PIX, o 0mntsa 4 at minutes 10 for m m 2 9 n hJ 5 RhoJ, and , oesrnefilter syringe pore 9 -CAACGACA- 6 ei lung Lewis m 9 /lfor g/ml -CAGA- m m 6 g/ml g/ml 10 ˚ C. 9 7 -

Journal of Cell Science UEswr ltdtedypirt h xeiet hywr lysed were they 25 experiment, buffer, the lysis to Rho-assay prior ml day 1 with the plated were HUVECs ARTICLE RESEARCH ei.Clsa h dewr eetdadmntrdfr15huiga (Nikon, using microscope 37 h inverted 1.5 Ti at for Eclipse UK) monitored Nikon and a Surrey, on selected system were TIRF edge Nikon (Sigma- the HUVEC red at for the to above phenol Cells described Prior in media. lacking as supplemented DMEM microscopy. made UK), with TIRF Gillingham, Aldrich, replaced was timelapse was by wound media monitored imaging, scratch were 20 a edge sterile scratch imaging, a were with day before cells next monolayer hours The imaging, 2–4 USA). before 1.5 Ashland, (No. and Corporation, day plates their MatTek MatTek microwell A for coverslip, mm uncoated indicated. 20 manipulated diameter, 35-mm as and onto replated RhoJ paxillin–RFP, of express expression HUVECs to turnover. adhesion transduced focal monitor were to used was microscopy TIRF microscopy TIRF NH antisera, minutes, mM anti-RhoJ 15 50 for with PBS than with in paraformaldehyde other (w/v) neutralised staining 4% with all fixed For were cells 2011). previously described al., as et is (Kaur staining immunofluorescence for protocol 30 The at days 5 staining to Immunofluorescence 3 amino-1,2,4- for incubated contained or were Plates containing also mM. either 3 histidine medium at synthetic lacking (3-AT) triazole to plates on Briefly, a histidine, spotted to 2004). diluted lacking and phase, al., with 0.5 stationary to et of medium OD600 (Heath selective transformed in described grown were previously was cultures as assays two-hybrid performed Yeast 1996) the 2002). were Woods, using and above) (Gietz al., (described method plasmids acetate lithium pACT2 described and et GBT9 of previously combinations (James gal4 as met2::GAL7-lacZ) were ura3- his3-200, leu2-3,112, trp1-901, conditions (MATa 52, PJ69-4A culture strain The 1991). and (Sherman, media Yeast two-hybrid Yeast Vco aoaois eebruh K.Imnsann was Immunostaining UK). 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Journal of Cell Science rn,S .adHne,S H. S. A. Hansen, R. and Cerione, R. and S. Frank, M. Antonyak, S., Yoo, D., Baird, Q., Y. M., Feng, Mann, Zheng, J., Andersen, and S., Dariozzi, B. C., Albertinazzi, Jia, S., Paris, K., A., Cesare, Zhu, Di F., Guo, Y., Gao, B., Debreceni, P. Fort, F., Comunale, G., Uze, J., Salamero, F., Senic-Matuglia, M., Toledo, de rn,S . dlti,M .adHne,S H. S. Hansen, and R. M. Adelstein, R., S. Frank, K. R. Jain, and P. Carmeliet, E. K. Wennerberg, C. Turner, and K. and Burridge, A. J. Cooper, S., J. Jamieson, A., M. L. Cary, S. C., Gomez, M. and Brown, M. K. Hahn, A., E. Vitriol, E., M. Berginski, Aspenstro it,R .adWos .A. R. Woods, and D. R. Gietz, uhrcontributions Author interests. competing no declare authors The Regulation. Immune interests for Competing Centre MRC the Transgenic of Birmingham part of is from University Facility support The Mouse with (AWM). Platform, Midlands Trials West Translational Infrastructure Advantage City and in Science Research used Birmingham Clinical microscope through Medicine TIRF obtained, The was mice. research PGK-Cre this the providing for UK) Birmingham, ARTICLE RESEARCH 3050 F., Mann, Y., Yoshida, M., Hirashima, H., Kataoka, M., Okada, Y., Fukushima, T., R. Premont, L., J. Guan, M., Hart, Z., Lenard, D., Bailey, S., A. R. Bagrodia, Cerione, and L. Aelst, Van A., K. Jordon, J., S. Taylor, S., Bagrodia, T. Endo, and T. Takenawa, H., Miki, M., Kato, T., Abe, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.140434/-/DC1 online available material Supplementary material for Supplementary PMC in Deposited Foundation. Heart British the release. by immediate funded was study This Funding manuscript. experimental the in wrote assisted V.L.H. J.Z.R. interpretation; and data A.B. J.K.H. and analyses; R.B., design image mice; with P.J.N. knockout F.E., assisted N.P., the V.A.S. K.L., generated experiments; E.W., the experiments; executed the V.L.H. of and conceived K.L. and E.W. V.L.H., o . rr,J,Kpda . er . ht,M .adGnsn .K. A. Ganesan, and A. M. White, H., Mehr, R., Kapadia, J., Aruri, S. H., M. Ho, Cyert, and S. Roy, L., S. Shaw, L., V. Heath, K., V. Sharma, C., Yan, M., Melaragno, Y., Saito, Y., Hojo, G., Yin, J., Haendeler, h irto n naieatvt fSctasomdcells. for Src-transformed signal of regulatory activity a 18806-18816. invasive as and serves migration protein the cool-1/beta-Pix the of Phosphorylation actin. I. of Curtis, reorganization de Rac-mediated signaling and R. Rac-mediated Longhi, mutant. and trio exchange negative dominant nucleotide a by guanine revealed of Mechanisms pathway. endocytic early the of regulator essential A. Blangy, and inln astei oto fcl shape. cell of control in cassette signaling angiogenesis. of and applications spreading cell regulate 116 and localization, linker, adhesion kinase paxillin focal phosphorylate protrusiveness. to its cooperate kinases stimulate FAK and living Src in (2005). dynamics spatiotemporal adhesion focal cells. of quantification resolution oi . ihd,K,Nsiaa .adUmr,A. Uemura, and ischemic mice. S. in in angiogenesis retinopathy Nishikawa, disoriented suppresses selectively K., adhesion signaling PlexinD1 Nishida, focal F., Cdc42-mediated Gomi, and spreading cell turnover. Rac1-regulated and p21-activated of family cool the on region regulatory important proteins. kinase-binding an to A. R. binds Cerione, and J. S. 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