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License and Commons cited Creative properly same medium the is any to work in subject are reproduction original adaptation and the distribution (http://creativecommons.org/licenses/by-nc-sa/3.0), that use, Attribution provided non-commercial Commons License Creative unrestricted the permits Alike of terms which the Share under distributed article Non-Commercial Access Open an is This to into structures 2010). body al., cell functional et the (Schoumacher guide as vessels and blood invasive invadopodia membranes basement these use the perforate Consequently, can 2007). cells cancer (Linder, form in formed osteoclasts to Gimona, like 2009; and much ability al., are et that their (Buccione and that ECM 2008), the protrusions to remodel membrane and linked penetrate cells, -rich is cancer specialized such carcinomas, invadopodia, many Weinberg, particular of and capacity (Chaffer in sites invasive distant Enhanced at 2011). the extravasate of and (BM) blood membranes vessels and basement lymphatic breaking by into microvasculature intravasate cells invade locally, (ECM), cancer matrix tissue some extracellular surrounding remodel dissemination, to metastatic ability the of acquire During mortality. steps cancer-related initial of the cause major the is Metastasis Introduction process. this in involved insights scaffold provide a findings as These Met Gab1 . the identify words: of the from and Key and formation downstream specific promotes cortactin the invadopodia invasion and of and of cortactin cell cells domain mechanisms with Met-transformed and SH3 molecular in along biogenesis the the rosettes invadopodia invadopodia on invadopodia into to to dependent abrogates localizes localize a is Gab1 Gab1 Met Met-mediated as and interaction GRB2- cortactin, from kinase. that cortactin Gab1–cortactin protein, cortactin Both both regulatory show a Gab1. of actin Using that of we the uncoupling two region show activity. identify that cells P4/5 and demonstrate We the Met tumor we formation. Gab1 of approach, invadopodia integrity on for in structure–function for partner a dependent Gab1 essential By interaction are of is Gab1. Gab1 direct protein as within knockdown in formation scaffold (P4/5) and we the specific known invadopodia motifs formation, Here requires -rich invadopodia and invasion (also known. amplified, is promotes cell not fibroblasts Met factor, invadopodia is and is growth formation (Gab1)-null of formation kinase Met invadopodia hepatocyte invadopodia 1 formation Met, where tyrosine in for cancer, protein role cells receptor In associated-binding its the tumor the with prognosis, remodeling. cells poor gastric from matrix cancer podosomes and aggressive breast extracellular signals metastasis resemble of cancer in deregulated stimulation to structures that role Although linked show These crucial are potential. factor a invadopodia. growth invasive play called hepatocyte with and protrusions cells associated matrix-degrading normal strongly actin-rich some by form produced cells carcinoma Invasive Summary work this to equally ` contributed authors *These 4 3 2 1 Rajadurai V. Charles mediate invadopodia to a complex, through scaffold signals cortactin–Gab1 kinase tyrosine receptor Met 2940 o:10.1242/jcs.100834 doi: 2940–2953 125, Science Cell of Journal 2012 January 23 Accepted oiaNaujokas Monica uhrfrcrepnec ( correspondence for Author eateto nooy cilUiest,Montre University, McGill Montre Oncology, Montre University, of University, McGill Department McGill Centre, Medicine, Research of Cancer Department Goodman Morris Montre and University, Rosalind McGill Biochemistry, of Department 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. naooi,MtRK a1 otci,Mti eoeig elinvasion Cell remodeling, Matrix Cortactin, Gab1, RTK, Met Invadopodia, 2 [email protected] oge Zuo Dongmei , 1,2 ehyHavrylov Serhiy , ) 2 ihlL Tremblay L. Michel , a,Que ´al, a,Que ´al, 2,3, a,Que ´al, ,Ksa Zaoui Kossay *, bcHA11 Canada 1A1, H3A ´bec bcHA11 Canada 1A1, H3A ´bec bcHA16 Canada 1Y6, H3A ´bec 1,2 eetrtrsn iae,we eeuae ncne,converge multiple cancer, that in possibility deregulated Mader the when raise 2011; kinases, also findings tyrosine al., (RTK) These receptor et 2011). kinases al., (Eckert tyrosine et biogenesis derived receptor invadopodia platelet as (PDGF) promotes well as factor (EGF) Activation factor growth 2010). growth al., carcinoma epidermal et the breast Smith-Pearson of 2011; MDA-MB-231 al., in et biogenesis (Mader structures in cells involved cellular are these and invadopodia, of to localize Arg, al., and 2011). 2010; al., et al., et often et Bowden Mader Balzer 2010; 2009; 2008; are al., al., al., et et Oikawa which et Kelley 2007; al., (Ayala et fibroblasts, Webb podosomes 2006; transformed as to of invadopodia- referred in and many structures cells then, cancer in like in Since activity invadopodia kinase 2002). Src of increased formation al., for the role et a (Hauck established activation have oncogene constitutive studies sarcoma v-Src with linked Rous the were of by and in 1989), identified transformed (Chen, originally virus fibroblasts were ECM embryonic structures degrade chicken cellular to capacity Invadopodia-like the emerge. with to beginning only n oa Park Morag and 2,3, nadto oSc te o-eetrtrsn iae,Abl kinases, tyrosine non-receptor other Src, to addition In are biogenesis invadopodia to leading mechanisms Molecular ,RcadVaillancourt Richard *, a,Que ´al, bcHA13 Canada 1A3, H3A ´bec 1,2,3,4, ` 1,2 ate Stuible Matthew , eerhArticle Research 1,2 , Journal of Cell Science epnet G,atnplmrzto spooe following in promoted formation is polymerization invadopodia actin During EGF, to 2011). response (Murphy Courtneidge, metabolism , lipid regulatory of and actin regulators as by well regulated as kinases, is protein to which of formation contributing the biogenesis invadopodia progression. promote metastatic to signals opopaiyioio ,-ipopae[PtdIns(3,4) 3,4-bisphosphate recruited phosphatidylinositol is complex al., protein Tks5 to et a (Yamaguchi of Src, nucleation from Downstream recruitment actin 2005). branched allowing Wiscott– promote activates (N-WASP) to which Arp2/3 protein 2009), syndrome al., 2008; al., Aldrich et a form et (Stylli to Nck, (Blouw complex protein, adaptor Tks5 trimeric the recruits protein Tks5 scaffold al., 2005). al., the et et Seals as (Bowden well also cortactin kinase as Src of 2006), 2009). the phosphorylation al., tyrosine of et (Oser promotes release cofilin localized protein, severing and actin phosphorylation tyrosine cortactin ciiyaedpneto erimn fGb htlclzsto localizes that Gab1 of Met recruitment by on induced dependent invadopodia that are show activity We capable ECM. structures remodeling invadopodia-like of form Tpr-Met, the receptor, of Met variant Met oncogenic activated active constitutively an fibroblasts the that that with and transformed cells demonstrate carcinoma we 1998; in invadopodia al., Here al., promotes et RTK 2010). et Wu 2008; al., Okuda al., 2005; et et Tuynman al., Ponzo 2007; et al., al., invadopodia Lengyel et et Sawada coordinate 2007; Cruz 2008; 1999; al., to al., et et Kammula Met (Camp 2003; addressed of been with not tumors ability has aggressive formation of the hallmark prognosis, a is poor and invasion cell cancer 2007). Park, and Peschard 2009; 2003; al., al., et et survival,Lai Birchmeier 2007; and cell Comoglio, migration and increased promote (Benvenuti as and invasion that well 2000) 2000; as 2010) al., remodeling, al., et al., actin (Lamorte et et Rap1 (Maroun and (Abella Rac Ras–MAPK Nck/N-WASP 2000), 1999), al., et al., proteins Schaeper et these PI3K– (Maroun complex, including cascades, Akt Met–Gab1 signaling multiple a of al., activation to et trigger (Lock recruited domain SH3 which Once C-terminal 2002). its of through receptor-bound (Grb2) two factor 2 growth protein motifs, protein adaptor the rich al., constitutive with the et in association proline implicated Cunnick are al., five) six 2000; and four et al., motifs (proline-rich contains Lamorte et Maroun 2000; Gab1 1999; al., 2001). al., et et Gual Maroun 1999; 2000; al., subunit et SH2-domain-containing P85 Garcia-Guzman Nck, numerous PLC Crk, and Shp2 including PI3K, for of proteins, signaling sites and docking adaptor as residues these al., multiple phosphorylation, serve et Upon on 2007). (Birchmeier phosphorylated al., receptor et Met and Peschard 2003; activated to to an recruited by Gab1 is residues tyrosine tethers and PtdIns(3,4,5) 1999) which al., with et domain, interactions (PH) through homology membranes an scaffold pleckstrin exerts the containsa Gab1 through Met (Gab1). 1 dispersal, primarily protein 2003). GRB2-associated-binding protein program al., cell morphogenic et important epithelial invasive are (Birchmeier which induce development growth, ligand, during invasive that its and and remodeling signals Met epithelial tissues, activate normal cancer In in HGF, 2003). implicated al., often proto-oncogene et (Birchmeier a is receptor] (HGF) 2008). and al., domain et (PX) (Oikawa biogenesis homology invadopodia phox initiates its through regions membrane h ne tutr fivdpdacnit fa ci-ihcore, actin-rich an of consists invadopodia of structure inner The lhuhaern ciaino e slne oincreased to linked is Met of activation aberrant Although factor growth hepatocyte as known [also RTK Met The c Ael ta. 00 capre l,2000; al., et Schaeper 2010; al., et (Abella P 3 (Maroun P 2 ]-rich e T nue naooi hog a12941 Gab1 through invadopodia induces RTK Met Results to response structure–function in invadopodia, signals. functional Met By oncogenic of is interaction assembly kinase. Gab1–cortactin for a of required that Src established phosphorylation have of we tyrosine analysis, independent increased Met key that to cortactin and a invadopodia, contributes to cortactin, cortactin activity with demonstrate with colocalizes We Met invadopodia. directly within that dynamics interacts actin of and regulator structures these eue ice a T F33 irbat rnfre ihthe with possibility, transformed this fibroblasts (FR3T3) investigate or 3T3 To rat invadopodia Fischer protrusions ECM. used produce we of membrane remodeling to active enable to cells that proteolytically due these part actin-rich in of least similar ability at are by acquired produce receptor transformed Met the to the cells phenotypes, of of malignant cells variants potential that oncogenic these possible metastatic is and of it tumorigenicity Therefore ability 2010). Schoumacher 2009; al., the al., et metastatic et Buccione with 2008; and (Gimona, linked invadopodia fibroblasts invasive transformed tightly malignantly that and is cells suggested cancer of have potential studies Recent in rosettes invadopodia fibroblasts of formation induces Tpr-Met RT irbat cur h blt opoueinvadopodia produce to ability the findings acquire our fibroblasts fibroblasts Hence, oncogene, Tpr-Met FR3T3 FR3T3 1A–C). the with (Fig. transformation upon Tpr-Met-transformed results that demonstrate similar of obtained and clones to independently using due experiments derived To these was repeated 1B). we fibroblasts (Fig. effects, FR3T3 clonal state Tpr-Met-transformed steady invadopodia by at of formation rosettes cells observed the of through that possibility penetrated 35% the and exclude in cell), matrix per gelatin rosette one the (usually 50% cells in found the indeed were of rosettes, cellular invadopodia These were call S1). we which Fig. actin structures, structures material the markers supplementary with 1E; invadopodia colocalization observed (Fig. their with rosettes showed and the cells cortactin and these Tks5 that stained we confirm matrix, invadopodia, To gelatin Movie fluorescent material supplementary 1). 1A,D; degraded (Fig. invadopodia of of FR3T3 typical areas associated time, filaments underlying actin same with of rosettes the ventral but fibers, prominent At stress produced few 1A). formed Tpr-Met (Fig. with transformed fibroblasts matrix to unable gelatin were actin and staining), remodel extensive phalloidin by formed defined we fibroblasts (as assay, fibers FR3T3 stress this control In gelatin. that fluorescent observed the on actin-rich plated remodel ventral Tpr- when produce and protrusions and control to fibroblasts of invadopodia ability FR3T3 Met-transformed the of examined we formation matrix, extracellular the induce could 2002). al., et 1) Saucier (Fig. distinct 1995; capacity al., invasive inhibition et a and FR3T3 contact (Fixman migratory formed lost developed increased whereas foci, and acquired formed and Tpr-Met culture, spread morphology, cell with study in elongated this transformed findings, monolayer in previous fibroblasts our contact-inhibited used with line cells a al., In et FR3T3 2002). Fixman al., 1996; control tumors al., et develop et to Saucier (Fixman as mice 1997; well nude many as the in ECM, metastases including the and acquire cells, through invade cancer to fibroblasts transformed ability malignantly FR3T3 of Tpr-Met- features Upon transformation, Tpr-Met. receptor, Met mediated the of variant oncogenic oetbihwehritouto fteTrMtoncogene Tpr-Met the of introduction whether establish To Journal of Cell Science ihulbldclae o 4husadimnsandfrivdpdamresTs n otci oietf naooi oets h oe regio boxed The 10 rosettes. bars: invadopodia Scale identify panel. to fourth cortactin the and in Tks5 magnification markers higher invadopodia at for shown immunostained is and ( image hours matrix. gelatin third 24 the for (arrowheads) collagen below unlabeled and (arrows) with above from invadopodia of View acrcl ie:a naiebslbes acnm elline cell carcinoma breast basal invasive different an two promote lines: used could we cell to cells purpose, sought cancer cancer this we in For 2009), Met biogenesis. al., invadopodia of et activation (Lai whether previous metastasis verify of and of promote poorly number can invasion Met formation a from cancer downstream are induce and signaling aberrant could fibroblasts, that reports cells, active in Tpr-Met, constitutively rosettes Met, cancer a invadopodia of that form protrusive invasive observation oncogenic active our of Given proteolytically understood. structures, similar al., biogenesis in et and RTKs cellular Corso different of 2003; invadopodia roles al., The of 2009). et invasive al., (Birchmeier et with Lai cells associated 2005; cancer is and of is malignancies capacity Met, in including kinases, observed tyrosine often receptor of activity Increased in formation invadopodia cells for cancer required is activity Met Courtneidge, and (Murphy oncogene fibroblasts Src 2011). and the cells cancer with by transformed produced those to similar rosettes, rose actin-rich as defined are invadopodia Active experiments. independent three of ( means phalloidin shown. the with are Met- stained are images ( were Values Representative remodeling. Cells Tpr-Met. cells. matrix matrix). to the overlaying (gelatin response of gelatin Oregon-Green-conjugated in plane with ventral invadopodia coated the active coverslips at glass taken on were hours images 24 for invadopodia. cultured form were fibroblasts 4) FR3T3 Tpr-Met-transformed 1. Fig. 2942 P pe) e n ci.( actin. and Met (pMet), ora fCl cec 2 (12) 125 Science Cell of Journal D C ofclZscin eecletd eovle sn MRSsfwr n ouernee orcntutte3 image. 3D the reconstruct to rendered volume and software IMARIS using deconvolved collected, were Z-sections Confocal ) D-AEwspromdo ellstsfo RT el rF33clssal vrxrsigTrMtadpoe for probed and Tpr-Met overexpressing stably cells FR3T3 or cells FR3T3 from lysates cell on performed was SDS-PAGE ) ( A RT el rF33clssal vrxrsigTrMt(p-e n Tpr-Met and 3 (Tpr-Met Tpr-Met overexpressing stably cells FR3T3 or cells FR3T3 ) m m. eepoe ncdw fMtepeso sn specific a matrix using expression gelatin activity, Met Met the on of dependent remodel knockdown is this employed and that we confirm invadopodia To MKN45 2E–H). form profound of (Fig. inhibitor, ability a to the molecule in abrogated small and cells resulted morphology, a 2003), cell in using al., specific change et RTK Strikingly, (Christensen Met 2E–H). the PHA665752 (Fig. the in of stimulation invadopodia 2A,C,D). inhibition (Fig. HGF form cells of cells MKN45 absence non-stimulated of 30% approximately with by Approximately 2B), invadopodia compared (Fig. of receptor number enhanced twofold, the active to increased the also led recognizes but phosphorylation- that HGF, a using antibody of visualized specific as presence receptor Met Met the the of on express activation in plated also cells gelatin but MDA-MB-231 2010) kinase of fluorescent Stimulation al., tyrosine 2B). et (Fig. (EGFR) Pichot receptor 2007; receptor al., factor et of (Nam growth activation to epidermal response amplified in the MDA-MB-231 formation is 1993). invadopodia al., Met increase et which cells (Fushida active in constitutively MKN45, is a line and and dependent cell HGF carcinoma is gastric activation Met which in MDA-MB-231, E B RT el xrsigTrMtwr ltdo ls oesiscoated coverslips glass on plated were Tpr-Met expressing cells FR3T3 ) uniiaino h blt fF33clst omatnrste or rosettes actin form to cells FR3T3 of ability the of Quantification ) n confocal and ttes nthe in n Journal of Cell Science rcnrlsRAadpoe o Met- for probed and siRNA control or ( rae ih5 MsRAtreigMto oto iN.Clswr rpiie 8husatrtetetadpae ngltnmti n utrdfra for cultured ventral and the matrix at (63 gelatin acquired magnification were on lower images a plated at confocal and taken and treatment (blue) cortactin DAPI after or and hours (phalloidin) (red) 48 actin actin with trypsinized invadopodia, ( stained of were cells markers Cells of for images siRNA. stained DIC mar control cells. were invadopodia or Cells the Met hours. for targeting 24 stained siRNA were additional nM Cells hours. 50 3 with additional treated an for HGF ( nM experiments. 0.5 signaling. with RTK stimulated Met and to response ( hours ( in cortactin. 3 invadopodia for or form matrix MKN45, (phalloidin) cells, gelatin actin cancer on gastric cultured and were MDA-MB-231, cells cells, 231 cancer breast Invasive 2. Fig. H F C , , D-AEwspromdo ellstso K4 el rae ih0.1 with treated cells MKN45 of lysates cell on performed was SDS-PAGE ) G D h oso naooi omto nMN5clsi epnet ramn ihMtihbtrPA672o iN-eitdkokono Met. of knockdown siRNA-mediated or PHA665752 inhibitor Met with treatment to response in cells MKN45 in formation invadopodia of loss The ) h blt fMAM-3 el ofr naooi nrsos oHFsiuainwsqatfe.Vle r h en ftreindependent three of means the are Values quantified. was stimulation HGF to response in invadopodia form to cells MDA-MB-231 of ability The ) E K4 el eeclue ngltnmti ntepeec f0.1 of presence the in matrix gelatin on cultured were cells MKN45 ) B D-AEwspromdo ellstso D-B21clssiuae ih05n G n o-tmltdcells. non-stimulated and HGF nM 0.5 with stimulated cells MDA-MB-231 of lysates cell on performed was SDS-PAGE ) P pe) e n uui.Saebr:10 bars: Scale tubulin. and Met (pMet), e T nue naooi hog a12943 Gab1 through invadopodia induces RTK Met m m e niio r5 MsRAt e rtersetv eils(DMSO) vehicles respective the or Met to siRNA nM 50 or inhibitor Met M m. m e niio H655 rDS o 4hus K4 el were cells MKN45 hours. 24 for DMSO or PHA665752 inhibitor Met M 6 r hw ntergt ersnaieiae r shown. are images Representative right. the on shown are ) ( A MDA-MB- ) ln fthe of plane kers n Journal of Cell Science omto fatnrste rmti eoeig(i.4A–C; (Fig. the remodeling initiate matrix to the or failed In rosettes when Tpr-Met actin examined. rosettes of of was invadopodia expression formation matrix form Gab1, gelatin of to absence fluorescent cells on these cultured of ability the naooi omto.TrMtwssal xrse in of expressed knockout in with stably Gab1 fibroblasts for ( was Gab1 embryonic requirement the mouse Tpr-Met tested immortalized formation. we 1997), are invadopodia Tpr-Met al., 1997; al., of et et (Fixman 1356, scaffold Nguyen Gab1 and the of 1349 recruitment for resides, required tyrosine both cells cancer Because by and induced fibroblasts formation in invadopodia Met for required is Gab1 phenotype. transformed of indicative the 3A,B), al., of (Fig. reversal et fibers the stress Nguyen actin enhanced 1997; cells formed these fibroblasts, al., also FR3T3 parental to et manner similar (Fixman a In active 1997). catalytically Tpr- mutant is this though Met even ( 3A,B), matrix Fig. gelatin Tpr-Met; by the WT expressing remodel replaced to unable were Y1356 and considerably ( and produced rosettes actin Y1356F], Y1349 fewer Y1349, both [Tpr-Met actin with phenylalanine of with mutants number Tpr-Met Tpr-Met the ( of in rosettes residues decreases ( slight Y1356 rosettes to fibroblasts, or led FR3T3 Y1349 phenylalanine Tpr-Met-transformed of with substitution comparison gelatin. fluorescent In on rosettes invadopodia Y1349F active produce carrying proteolytically to ability oncogene, their quantified Tpr-Met and lines substitutions, Y1356F the and/or cell of stable form established mutants to expressing fibroblasts we FR3T3 rosettes, of invadopodia ability functional the 1997). determine al., also et Tpr-Met Nguyen 2000; al., phosphorylated et Lock a 2003; al., Grb2 to through et indirectly, (MBD) (Lock and 1996), al., domain et (Weidner Met-binding residue Y1349 Gab1 the interaction through directly, of 1997), both Tpr-Met al., to recruited et a be (Nguyen to can Gab1 Tpr-Met domain SH2 of Grb2 residue Grb2 the al., Y1356 between of phosphorylated the et binding interaction Lock direct Whereas for 1997; requires 1996). Tpr-Met al., essential and al., et are Fixman et 1995; Ponzetto and al., 2003; et 1994) and (Fixman oncogene RTK al., Tpr-Met the Met of et activity Nguyen transforming Ponzetto 1996; and biological al., 1997; et Fournier al., 1997; et al., scaffold et the (Fixman and Gab1 Shc, and protein residues Grb2 proteins, phosphorylation-dependent adaptor These the for receptor). of site recruitment and docking Met multiprotein Y1349 wild-type a to the C- create corresponding of the Y489, residues of and Y1356 residues (Y482 tyrosine region key terminal two accompanied is at oncogene auto-phosphorylation Tpr-Met by the of activation Constitutive docking multiprotein Tpr-Met intact of an site by invadopodia determined functional is form rosettes to fibroblasts extracellular of Ability and that biogenesis signals invadopodia elicits remodeling. increased HGF, matrix dependent with both in Met, stimulation result of is of activation signal independent cells, demonstrate Met and cancer data invasive of these Together, in inactivation 2E–H). that (Fig. upon half MKN45 invadopodia by of decreased ability form the to phenotype: same cells the observed and siRNA 2944 oass hte inl rmtemlirti okn ieof site docking multiprotein the from signals whether assess To Gab1 , , ora fCl cec 2 (12) 125 Science Cell of Journal 0;Fg AB.Hwvr RT el expressing cells FR3T3 However, 3A,B). Fig. 30%; 0)a ela rtoyial cieinvadopodia active proteolytically as well as 20%) 2 / 2 Es HlaoMduaadWn,20)and 2003) Wong, and (Holgado-Madruga MEFs) , 0 hto el xrsigW Tpr-Met) WT expressing cells of that 20% , %ta fcells of that 5% a1wsrsudb nrdcino F-ue ain fthis of variant GFP-fused a of protein, introduction scaffold of by expression rescued when was However, Gab1 2). Movie material supplementary ontemsgaigcsae Brhee ta. 03 Lai diverse 2003; al., trigger et that Tpr-Met (Birchmeier proteins or cascades receptor, many signaling Met recruits downstream phosphorylated Gab1 a oncogene, to binding Upon Tpr-Met- of fibroblasts of invasion transformed formation and for rosettes required invadopodia are functional Gab1 of motifs Proline-rich 10 bars: and Scale mutants tubulin. 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Fig. eedn ati acrcells. of Met- cancer in formation gastric and the fibroblasts dependent in transformed role Tpr-Met essential in these both an invadopodia, Together plays Gab1 4D–F). that (Fig. support formation data fourfold MKN45 a invadopodia to in in led expression, decrease expression Gab1 Decreased Gab1 cells. cancer performed gastric of we knockdown cells, cancer siRNA-mediated in formation invadopodia dependent stress actin reduced and 4A). ruffles (Fig. fibers actin peripheral increased in to upon expression lead addition, Gab1 In S2C,D). of 4A–C; Fig. rescue (Fig. manner 3, fibroblasts Movie a material FR3T3 in supplementary rosettes, Tpr-Met-transformed actin to active similar proteolytically form to ability oetbihwehrGb xrsini locuilfrMet- for crucial also is expression Gab1 whether establish To Gab1 2 / 2 Esepesn p-e curdthe acquired Tpr-Met expressing MEFs Gab1 ( A RT el tbyoverexpressing stably cells FR3T3 ) 2 m / m. 2 Es p-e signals Tpr-Met MEFs, B h blt fTpr- of ability The ) Journal of Cell Science aaiyo hs el yfufl Fg CE.Furthermore, 5C,E). (Fig. fourfold of capacity by reduced the cells with consistent invasive these a the increased of at expression Gab1 (Matrigel) capacity WT of matrix rescue level, extracellular basal reconstituted invade could omatnrste,teewsamr hn5%rdcini their in reduction 50% than more a was there rosettes, actin form Gab1 using rescued xrsigTrMt(i.5,,) nta,clsexpressing cells Instead, 5A,B,D). (Fig. Gab1 Tpr-Met expressing o hs ersudGb xrsinin expression approach. 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Fig. ecetefraino ci oetsin rosettes actin of formation appreciably to the failed Gab1, Gab1 of rescue motifs and proline-rich domain, fifth and binding wild- fourth Gab1 to Met mutants, contrast deletion the Gab1 In the lacking protein. of or two scaffold Gab1, Gab1 (WT) deletion type the various of using mutants point (2B-Tpr-Met-1) Tpr-Met expressing omatnrste eedcesdb 5,cmae ihcells 5B,D,F). with to compared (Fig. ability 75%, Gab1 their by WT decreased and expressing were fibers, rosettes stress actin actin form prominent formed Gab1, Although D B n Gab1 and MBD Gab1 C a1nl p-e el rclsrsudwt F–a1wr rninl rnfce ihRPatnadsbetdt ielpevideo time-lapse to subjected and RFP–actin with transfected transiently were GFP–Gab1 with rescued cells or cells Tpr-Met null Gab1 ) 2 / 2 D Esepesn p-e (2B-Tpr-Met-1) Tpr-Met expressing MEFs B rGab1 or MBD D 45mtnst iia eesa WT as levels similar to mutants P4/5 D 45dlto uat to mutants deletion P4/5 Gab1 Gab1 D D 2 K4 el eetetdwt 0 McnrlsRAo mrpo iN gis a1fr4 hours. 48 for Gab1 against siRNA smartpool or siRNA control nM 100 with treated were cells MKN45 ) 45 isn the missing P4/5, / 2 2 Gab1 / 2 p-e MEFs Tpr-Met Esstably MEFs 2 / 2 D MBD, MEFs ( A a1nl el tbyoeepesn p-e Gb ulTrMt n ece with rescued and Tpr-Met) null (Gab1 Tpr-Met overexpressing stably cells null Gab1 ) e T nue naooi hog a12945 Gab1 through invadopodia induces RTK Met upr omto fatnrste n eceivsvns of invasiveness rescue and rosettes actin Gab1 of formation support h alr fteGab1 the whether determine of To failure 2003). through al., the mediated et Met, (Lock protein to adaptor recruitment Grb2 the indirect and in 4 implicated motifs are proline-rich 5 Gab1 and receptor, WT Met phosphorylated expressing cells with 5C,E). compared (Fig. Gab1 when capacity, invasive ie el n ielpevdomcocp nlssof analysis microscopy the examined video and rosettes actin time-lapse form on that and analysis cells confocal FR3T3-Tpr-Met both cells performed Gab1 we fixed this, the For of invadopodia. ability the examined 6H–J). 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E D , 45mtn olclz to localize to mutant P4/5 F D h blt fMN5clsto cells MKN45 of ability The ) B eeinmtnsto mutants deletion MBD D 45wsrecruited, was P4/5 P B Proteins ) (pMet), Journal of Cell Science ievra ots hshptei,w efre reciprocal performed we hypothesis, or this rosette test actin emerging To of sites versa. to vice cortactin a 2010; as recruit serves al., sequences Gab1 to et that possibility scaffold (Rubini the consensus raises cortactin This 1996). of al., xPPxPxKPx et domain transformed Sparks SH3 Tpr-Met to and the in by similar recognized rosettes Murphy are actin of fibroblasts, formation 2009; actin for initiate crucial al., 2005). which al., et et Arp2/3, Yamaguchi regulatory 2011; and Courtneidge, (DesMarais actin promote N-WASP with to polymerization thought association Nck, is Cortactin by proteins 2009). formation al., et invadopodia Oser DesMarais 2006; 2009; and 2007; Kapus, al., al., and 1999; cells et et Cosen-Binker Clark 2008; al., 2008; cancer al., Weaver, et and et in Cortesio Clark 2006; (Bowden both al., et fibroblasts invadopodia, Bowden of transformed as cortactin component oncogenically protein adaptor key the a identified have studies Previous interaction in involved cortactin are with Gab1 of motifs Proline-rich other proline-rich possibly fifth and/or and fourth Gab1. 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(Fig. (Murphy nomenclature rosettes proposed actin recently a active with form accordance to proteolytically ability upon the acquire of that ventral fibroblasts variant Tpr-Met, invasiveness demonstrate oncogenic receptor, active Met We the constitutively cancer invadopodia cells. a increase with cancer in transformation induce and human can fibroblasts, in 2007), implicated biogenesis Park, in and rosettes is Peschard invadopodia 2003; al., which et (Birchmeier RTK, Met eerhpromds a a trbtdidcinof induction attributed has far so performed Research eew eotfrtefrttm htsgasfo nactivated an from signals that time first the for report we Here ora fCl cec 2 (12) 125 Science Cell of Journal a Ekr ta. 2011). al., et (Eckert yTrMt(i.6,btfist eceasml fatnrosettes actin of assembly the rescue to whereas is fails but Met 6), 6), (Fig. to (Fig. Tpr-Met Grb2 formation by of rosette (Lock recruitment actin Gab1 Met direct to that for Given Gab1 dispensable Grb2 2002). recruits the al., bind indirectly direct also et motifs which P4/5 provide involved protein, The protein 7). these a adaptor 5, cortactin, (Figs that of dynamics domain actin shown SH3 in actin the have for for sites (P4/5) and binding Gab1 of formation motifs rosette proline-rich two of a actin-rich requirement of provides assembly 2010). periphery. 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Gab1 invadopodia Movies dependent of material knockdown supplementary 4; mediated expression (Fig. by rescued Gab1 is of phenotype this and rosettes invadopodia n ornig,21) erfrt hssrcuea an the as of a as amplification structure or HGF, genomic ligand, this the of with to activation engagement result that through either show refer Met, also of we we Importantly, rosette’. 2011), ‘invadopodia Courtneidge, and rsbtnilydcesdfloigMtkokon(i.2) (Fig. knockdown Met following decreased substantially gastric of or amplification in genomic contrast, carry By MET which signals. cells, upstream MKN45 multiple carcinoma by driven a is that formation be suggesting invadopodia 2011), promoting al., machinery et molecular Mader 2009; similar al., et of activation (Oser those (EGF) EGFR factor resemble the growth epidermal morphologically to response 2) in 2007; (Fig. formed stimulation al., HGF et upon 1998). 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Met types Courtneidge, cell and many Murphy invadopodia 2006; In al., as et known secretion (Artym remodeling maturation targeted ECM regulate for membrane that metalloproteases of actin-rich signals precursor by signals of followed initial protrusions, establishment involve the These trigger 2008). that al., et (Oikawa invadopodia the is biogenesis. signal invadopodia Met of the driver upstream cells, major cancer Met-addicted in that indicating sn tutr–ucinapoc ehv dniidthe identified have we approach structure–function a Using naooi bevdi aa ratcne D-B21cells MDA-MB-231 cancer breast basal in observed Invadopodia eunilsgasaeivle nasml ffunctional of assembly in involved are signals Sequential Gab1 omto fivdpdai blse yMtinhibition Met by abolished is invadopodia of formation , D 45mtn tefi tl erie oadphosphorylated and to recruited still is itself mutant P4/5 2 / 2 irbat,TrMtfist nuefrainof formation induce to fails Tpr-Met fibroblasts, MET ou,increases locus, Journal of Cell Science rnfre irbat,i ntae yteacmlto of accumulation Src in the , by the initiated that is PtdIns(3,4) al., proposes et fibroblasts, model (Oser transformed formation second in end A resulting barbed cofilin, 2009). increased of and tyrosine activation severing on and actin cortactin release of promotes is phosphorylation al., residues et precursors subsequent (Uruno early complex and Arp2/3 the the 2001) to through mediated cortactin be of to Artym thought Recruitment 2009; that al., 2006). precursors et al., early (Oser et at Arp2/3 and initiated invadopodia N-WASP is model, cortactin, EGF contain one to response In in signals. assembly multiple is from Gab1 downstream 5). of (Fig. formation motifs invadopodia proline-rich Met-dependent for through required mediated interaction in K4 el eemitie nRM otiig1%FS h eeainof generation The Dulbecco’s (FBS). 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Technologies NY), and Institute, Placid, pMet1234/35 Research Gab1 (Lake Medical gift Biotechnology kind antibodies: (Sanford-Burnham a was (1736) Courtneidge Commercial antibody Tks5 Sara 1991). al., et from Rodrigues 1999; al., et (Maroun ofclmcocp,uls etoe otherwise. mentioned unless microscope, confocal eppi.Frimnpeiiain yae 1m)wr nuae ihthe with incubated 4 were at mg) hours (1 2 for lysates antibodies immunoprecipitation, indicated For leupeptin. MM n 000clswr ltdadicbtda 37 at incubated and minutes. plated 20 with were for washed ethanol cells were 70% 50,000 with coverslips and quenched gelatin-coated DMEM, and Oregan-Green-conjugated PBS with Finally times three washed nlsso etr lt a efre sn dse software. Odyssey Densitometric using NE). performed was Lincoln, blots (Li-Cor, (Amersham western with washes system of four detection incubated detection analysis After ECL respectively. Odessey an hour, hour, with or 1 1 visualized Biosciences) and were for hours proteins Inc., bound 2 20) TBST, for nitrocellulose Laboratories, with TBST Tween 8.0, ImmunoResearch in a pH PA) 0.1% (Jackson Grove, Tris to antibodies West EDTA, mM secondary (10 transferred mM and TBST 2.5 and in primary BSA NaCl, SDS-PAGE 3% mM in the by 150 in blocked times were resolved three Membranes washed and membrane. was complex buffer immune The lysis hour. additional an for added 25 complexes, cetfc(itbrh A n mgswr curduiga100 Thermo a from using acquired Immunomount were using images mounted and were PA) Samples (Pittsburgh, previously Scientific 2010). paraformaldehyde described al., 4% as with et protocol, fixed immunofluorescence (Abella regular were with Cells continued otherwise. and specified unless hours 24 m m /li .%ucnuae eai Se elTechnologies, Cell (Stem gelatin unconjugated 0.1% in g/ml f5%sur fete rti-–o rti-–ehrs was protein-G–Sepharose or protein-A– either of slurry 50% of l m /lo uoyi.pDAcratnW n the and WT pCDNA-cortactin puromysin. of g/ml ˚ ihgnl oain ocletimmune collect To rotation. gentle with C m /lartnnad10 and aprotinin g/ml ˚ Cin5%CO 6 ˚ betv na on objective o hour, 1 for C m 2 g/ml for Journal of Cell Science ucoe . adei .adAaa I. Ayala, and G. Caldieri, R., Buccione, odn .T,Oioi . lc,R,Moi . oea . aaa .M and M. K. Yamada, T., Yoneda, A., Myoui, C. R., S. Slack, Mueller, E., Onikoyi, and T., I. E. R. Bowden, Glazer, D., Thomas, M., Barth, T., E. Bowden, A. S. Courtneidge, and B. Diaz, I., Pass, F., D. Seals, F. B., G. Blouw, Woude, Vande and E. Gherardi, W., Birchmeier, C., Birchmeier, azr .M,Wipe .A,Topo,K,Bgs .E,Soi,J,Co .H., E. Cho, J., Slovic, E., A. Boggs, K., Thompson, A., R. Whipple, M., Mariggio E. Balzer, F., Attanasio, G., Caldieri, G., Giacchetti, I., Ayala, evnt,S n oolo .M. P. Comoglio, and S. Benvenuti, ry,V . hn,Y,Sile-osiish . aaa .M n Mueller, and M. K. Yamada, F., Seillier-Moiseiwitsch, Y., Zhang, V., V. V., Artym, Sangwan, D., Zuo, G., M. Ponzo, M., M. Frigault, R., Vaillancourt, V., J. Abella, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.100834/-/DC1 online available material for Health Supplementary PMC in of Deposited Institutes Genetics. Cancer Canadian in release. Chair immediate Diane the the Guerrera holds Sal from M.P. M.P.]. and Research to and grant MOP-106635 K.Z.]; Cancer number [grant and operating Research Integrated S.H. an to McGill FRN53888 Health by the number [grant of from Program Training research Institutes Sante cancer [grant Canadian en in Initiative Recherche US R.V.]; la Research the de to Research/Fonds Cancer C.V.R.]; XWH-09-1-00 Breast to number Defense CGD-96470 of number of Institutes Department [grant Canadian from Research fellowship a Health by supported was work This Holgado- Funding We Marina FACS. and HGF with for help Inc. his Genentech for for IMARIS Madruga thank to using McDonald like Z-stacks Ken would confocal thank for of We Spurmanis deconvolution software. Aleksandrs the with and on help comments Brown helpful their Claire their for thank lab We Park manuscript. the of members thank We 10 PMSF, Acknowledgements mM 1 to glycerol, 10% transferred EDTA, mM Hepes 2 and mM 10 constructs, (20 NaCl, and A mM aprotinin indicated cortactin buffer SDS-PAGE 120 lysis either in the 7.5, with proteins pH by incubated fusion with GST–SH3–W525K were or membranes transfected GST–SH3 separated The transiently membranes. nitrocellulose and were cells immunoprecipitated 293 HEK blotting Far-western chamber, environmental transparent CO (v/v) small 5% microscope, a with Inc.) inverted with Zeiss, Inc.) (Carl equipped Climabox Zeiss, the and (Carl on camera, M positioned digital 200 were 100 Axiovert mm) with (35 the equipped coverslips on glass stage gelatin-coated motorized on grown Cells imaging cell Live 2952 sn niGTantibodies. anti-GST using rvnb xoiinL otae(alZis n..Temtrzdstage motorized 30 The for collected Inc.). were Zeiss, photographs (Carl and locations software minutes. pre-programmed LE to AxioVision advanced by driven tutrsfrtefcldgaaino h xrclua matrix. extracellular the of degradation focal Rev. the for structures cieivdpdai ua ratcne cells. cancer breast human with in invadopodia associates active C. PKCmu S. and Mueller, degradation. matrix extracellular cortactin, of sites of at complex invadopodia invasion-related An vivo. in growth tumor in Tks5 Biol. protein scaffold podosome/invadopodia more. and motility metastasis, arn,M . oea . ule,S .adMri,S S. metastasis. 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