Journal of Cell Science abig,C20Y UK 0XY, CB2 Cambridge, USA o:10.1242/jcs.126573 doi: 4647–4658 126, Science Cell of Journal 2013 July 9 Accepted uigivdpdafrainadivso fbreast of cells invasion cancer and MMP9 formation and invadopodia MMP2 during of trafficking regulates Rab40b Article Research yrlz opnnso h aeetmmrn,adhave and various mediating to membrane, in involved ability molecules basement key the as the possess emerged recently of MMP14), as components known hydrolyze also metalloproteinase- matrix MMP2 (MT1-MMP, membrane-type 2003). 1 as collagenases, categories well Nagase, as as and MMP9, multiple (Visse such and ECM gelatinases into specificity, multiple and substrate subdivided stromelysins cleaving their be on of can depending capable zinc-dependent MMPs of are family proteins. large Visse that a 2004; are a endopeptidases al., MMPs 2003). et of involves Nagase, (Polette secretion and targeted (MMPs) usually through metalloproteinases ECM al., disruption matrix the et of membrane degradation (Roskelley localized metastasis for Basement and required factors 1995). growth growth the tumor in various result of angiogenesis, also activation and cells and/or migrating lead release the can of degradation adhesion ECM increased Furthermore, to 1995; 1995). in Bissell, al., step and et (Roskelley important Roskelley metastasis an and is motility invasion function cell cell barrier cancer for this barrier of a Loss as invasion. extracellular and serves of which network proteins a of (ECM) up matrix made is membrane basement The Introduction invadopodia also during is vesicles VAMP4- Rab40b into cells. secretory words: that cancer sorting Key MMP2/9 demonstrate breast MMP2/9 we of of for metastasis Finally, transport required and degradation. is formation regulates matrix invadopodia Rab40b invasion extracellular Rab40b during siRNA cell that invadopodia-dependent cancer that Rab demonstrate breast for during human for show we functions required a required Rab40b Here, also that screened is cell shown transport. we and We have cancer MMP2/9 also transport, formation vesicles. during We of membrane MMP2/9. invadopodia of secretory of steps secretion the regulators and distinct containing for growth to key required two tumor protein are MMP2/9 least a of GTPases of as at aspects Rab GTPase targeting various Rab40b Because mediates identified regulate defined. and and that library be membrane machinery Basement to basement transport cells. MMP9 remains the and membrane cancer invasion of MMP2 breast components the Specifically, of (MMPs). hydrolyze However, metalloproteinases metastasis to matrix metastasis. during ability various role the of secretion major possess targeted a (MMP2/9) by plays mediated membrane is degradation basement membrane the of degradation Invadopodia-dependent Summary work this to equally ` contributed authors *These 5 4 3 2 1 Jacob Abitha aahR Junutula R. Jagath uhrfrcrepnec ( correspondence for Author eateto imdclSine nvriyo hfil,WsenBn hfil,S02N UK 2TN, S10 Road, Sheffield, Hills Bank Building, Western USA Trust/MRC Sheffield, 80045, Welcome of CO Research, University Aurora, Medical Science, Denver, for Biomedical Colorado Institute of of Cambridge Department University Cambridge, Campus, of Medical University Anschutz USA Medicine, Medicine, 94080, Clinical of CA 80045, of School CO Francisco, Department Oncology, Aurora, San Denver, Medical South Colorado of Way, of Division DNA University Campus, 1 Medical Inc., Anschutz Genentech Medicine, of School Biology, Developmental and Cell of Department 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. M2 M9 a4b naooi,Scein Invasion Secretion, Invadopodia, Rab40b, MMP9, MMP2, 1, ,Ja Jing Jian *, [email protected] 2 n yi Prekeris Rytis and 1, ,JmsLee James *, nvitro in ) ntebsso hs idns epooeta a4bmdae rfikn fMMP2/9 of trafficking mediates Rab40b that propose we findings, these of basis the On . 1, 2 ` eprSchedin Pepper , 95.Fnly M29adM1MPudroregulated undergo al., MT1-MMP et by and (Strongin pro-MMP2 MMPs MMPs MMP2/9 activate of Finally, and active MT1-MMP cleave 1995). Interestingly, inhibitors to the 2002). shown tissue Werb, was of and as (Egeblad inhibition such (TIMPs) the inhibitors, of by activation extracellular extracellular and and by research. MMP2 of controlled transcriptionally pro-enzyme of activities further the are area are this, to levels MMP9 active addition protein In an regulated. MT1-MMP tightly been the and have regulating mechanisms MMP2/9 MMPs the of tumors, production of potential metastatic increased and to Schmalfeldt 1998; al., cells linked et 2001). Itoh al., culture 1999; been et al., et tissue has (Itoh mice in MT1-MMP in metastasis and invasiveness partial MMP2/9 increased increased through observations, of part these expression with large Consistent epithelial-to- in proteolysis. as and are ECM (EMT), well MT1-MMP as transition (Chambers and angiogenesis mesenchymal tumor 2004; MMP2/9 stimulate metastasis al., Furthermore, to et 2009). known Polette and al., 2002; et Werb, growth Shah and Egeblad tumor 1997; Matrisian, of aspects 3 eas nrae eeso Mscreaewt increased with correlate MMPs of levels increased Because io .Gilbert M. Simon , 4 nrwA Peden A. Andrew , 5 , 4647 Journal of Cell Science naooi,tu fetn h naiecpct fbreast formed of newly Results capacity the invasive the to MMP2/9 cells. affecting targeting for cancer required thus and GTPase invadopodia, key transport the is we intracellular findings, Rab40b these of Rab40b basis that the On that propose motility. cell on show effect no having we invadopodia- Finally, trafficking for inhibits degradation. knockdown required MMP2/9 ECM is regulates and dependent We formation Rab40b degraded. invadopodia are that during Rab40b-containing they where demonstrate lysosomes, and also to of mistargeting MMP9 trans-Golgi VAMP4 in and results MMP2 the knockdown through from Rab40b endocytic vesicles. transport secretory on (TGN) on dependent relies not Network instead is secretion, MMP9 but MT1-MMP and unlike transport, MMP2 that, shown of have secretion We MMP2 MMP9. both of a and secretion the as for Rab40b required identified GTPase monomeric screen small This Rab targeting screen. a library and performed siRNA we transport GTPase secretion, membrane MMP2/9 regulates the that membranes. identifying machinery donor were the start with and to fusion have Thus, and transport such transport steps, GTPases transport sorting, membrane membrane cargo multiple as Rab of for are required regulators be families. organelles to shown key protein membrane-bound as multiple emerged of by targeting regulated Intracellular and MMP9. and MMP2 transport of secretion machinery contrast, targeted transport in By membrane involved 2003). the about al., known et is (Remacle nothing almost ECM regulating the in towards role activity a Furthermore, its is plays 2003). also al., MT1-MMP MT1-MMP of et endocytosis of Remacle selective invadopodia 2007; recycling and al., membrane et endocytic plasma (Bravo-Cordero the that to it targeting invadopodia shown in important been the 1993). 1997; al., et al., has at Monsky et 1998; al., It Nakahara enriched et 2008; Bourguignon 2006; al., be al., et et Artym Clark and to 2009; MMP2 al., shown MT1-MMP, et (Poincloux been emerge. to have beginning MMP9 of only transport are subcellular MMPs regulating 2008). mechanisms the Weaver, invadopodia, 2009; 2010). al., al., et et of (Quintavalle formation structures Clark the demonstrated invadopodia-like have 2008; studies recent al., Furthermore, potential metastatic et invadopodia- increased various an (Blouw of with correlate levels proteins expression forming high that shown been D-M9Mcclsepesadsceeenzymatically secrete and and express MDA-MMP2–Myc 1A,B, cells Fig. in MDA-MMP9–Myc shown As (MDA- MMP9–Myc). MMP9–Myc or expressing that (MDA-MMP2–Myc) lines MMP2–Myc To cell Rab either secretion. MDA-MB-231 for tet-inducible and screened created intracellular transport we we end, of MMP2/9 study regulate regulation this that the in GTPases transport, about MMP9 known and is MMP2 secretion little MMP9 and that MMP2 Given for required is GTPase Rab40b invadopodia of role The invasion cell 1999). cancer Gavrilovic, during to and kinase Murphy Src 2011; by invasion induced cell be ECM cancer to localized mediate shown of been sites have are al., and Invadopodia et degradation 2008). Poincloux al., ventral 2011; et Courtneidge, the Stylli and 2009; the (Murphy at cell invadopodia, located the forming of projections side of cellular sites finger-like the actin-rich at secretion and targeting 4648 ept h motneo h agtn fMP othe to MMPs of targeting the of importance the Despite ora fCl cec 2 (20) 126 Science Cell of Journal nvitro in naino D-B21cls while cells, MDA-MB-231 of invasion nvivo in nvivo in nvitro in sls eldfnd u thas it but defined, well less is Mrh n Courtneidge, and (Murphy sn nrvtlimaging intravital using nFg ,tesre dniidsvrlRbGPssta either that GTPases Rab several identified four screen shown of the As out 2, affected characterization. Fig. two further in least that for at considered candidates with were decreased siRNAs, treatment Only after siRNAs. or secretion re-screened MMP2/9 individual increased then four were and either MMP9–Myc Materials using or used that the MMP2–Myc was of see GTPases siRNAs secretion screen different Rab of four of description Methods). pool detailed a more cases, (for all In MMP9–Myc and 2). MMP2–Myc (Fig. of secretion regulate that GTPases MMP2/9. endogenous MMP2/9 to Myc-tagged that similar membrane suggest secrete manner data cytosol, plasma and a above in transport the basal the probably together, the Taken cells in d). these to and c found proximity 1E,F, (Fig. close also Organelles in (TGN) were S1). b), especially network Fig. and MMP2/9–Myc trans-Golgi material a containing the 1E,F, (supplementary (Fig. with VAMP4 region marker colocalized perinuclear they the MMP2/9–Myc where at and proteins, enriched MMP2–Myc secretory We of of were doxycycline- cells. expected localization these As subcellular in a MMP9–Myc. ECM 1D) the (Fig. analyzed in increased invasion next and doxycyline 1C) MMP9–Myc (Fig. Furthermore, degradation and manner. dependent MMP2–Myc active acrcl oiiyadivso Csele l,2007; this of remainder al., the for about GTPase et understanding known study. Rab40b on of is focused function (Caswell we little cellular Therefore, the contrast, regulates Rab40b. invasion of By that function 2012). and the GTPase al., MMPs. a motility et both Dozynkiewicz as of cell identified secretion cancer previously decreased was Rab40b Rab25 of and knockdown Rab25 affected but secretion, proteins only MMP9–Myc candidate or identified MMP2–Myc the either of MMP9–Myc. or most MMP2–Myc Interestingly, of secretion decreased or increased nue ees fI6(i.3)o F–G secretion GFP–hGH or 3G) LPS- (Fig. on effect IL6 little of of had MT1-MMP also secretion 3C,D). not release Rab40b (Fig. but decreased of induced 3E), Depletion (Fig. MMP9–Myc also the 3F). MMP9 MMP2/9 (Fig. and decreased and depletion MMP2 regulating also endogenous Rab40b MMP2–Myc in siRNAs Similarly, Rab40b of four all of Consistent secretion Rab40b. of transport, involvement encoding intracellular a mRNA effects the in of resulted in siRNAs MMP9– levels the with Rab40b the Rab40b and four all in MMP2–Myc 3B, tested of decrease Fig. of in role shown we secretion As Myc. the on secretion, siRNAs validate individual MMP2/9 To MMP2–Myc, GTPases. regulating Rab40b of between Rab40c overlap secretion functional and contrast, some is decreased By there that Rab40c secretion. suggesting MMP9–Myc of any Stein have or 2008; not knockdown MMP2–Myc did al., Rab40a on at et of depletion effect box (Piessevaux Interestingly, SOCS 2012). 3A) a and al., (Fig. of et Rab40a presence half GTPases members, the C-terminal of by Rab40 related their characterized of closely function is and other sub-family and Rab40c, two very a localization includes to is that belongs the There Rab40b about Rab40b. 2001). information their (Pfeffer, specific effector limited recruiting of targeting organelles These and and membranes. transport membrane-bound binding sorting, the regulate to by then proteins proteins function effector GTPases respective Rab MMP2 All of secretion and MMP9 sorting and for required is Rab40b et eue nEIAbsdsRAsre oietf Rab identify to screen siRNA ELISA-based an used we Next, Journal of Cell Science bec ftelssmlihbtrbflmcn ssonin shown As or bafilomycin. presence inhibitor the lysosomal in the hypothesis, of cells this absence test Rab40b-siRNA-treated lysosomes To incubated to cells. mis-sorted we Rab40b-depleted it are in MMP2/9, MMP2/9 degraded of that and levels possibility simultaneous intracellular the and in not raises data secretion resulted proteins, and the depletion 4C in post-TGN Rab40b (Fig. decrease other IL6 that or of Given 4A,B), CD63 levels shown). (Fig. receptor, on transferrin levels as effect such no MMP2/9 having and intracellular of while MMP2–Myc knockdown decreased of Surprisingly, levels analysis. Rab40b FACS intracellular using the determine MMP9–Myc measured To the cell. in we the result within this, would MMP9 intracellular and this MMP2 that of predict MMP2/9 accumulation would one regulate secretion, MMP9 to the is of one Rab40b that transport. of suggesting S1H) functions Fig. material (supplementary eas a4bdpeinlast eraei M2and MMP2 in decrease a to leads depletion Rab40b Because raelssatrdtruhtectsla ela in as well as small cytosol the arrows). in the (see organelles present lytic through enlarged 4E,F, were bafilomycin-induced Fig. scattered in MMP9–Myc shown organelles As and antibodies. MMP2–Myc marker) Rab40b- stained anti- (lysosomal and plasma we anti-Myc CD63 with cellular possibility, cells bafilomycin-treated this the and MMP9. test knockdown with further vesicles and to To and targeting secretory addition membrane. the MMP2/9 in affects MMP2 of block that directly fusion also suggesting secretory Rab40b of Rab40b, the sorting, of MMP2/9 rescue depletion levels not by caused did Rab40b intracellular of bafilomycin effects Interestingly, on the reversed treatment siRNA bafilomycin 4D, Fig. t ellrfnto.T hsed ecmae h oaiainof localization the compared we end, this about To clues function. provide cellular often can its protein a of localization Subcellular secretory VAMP4-containing vesicles to localized is Rab40b a4BrgltsMPscein4649 secretion MMP regulates Rab40B E o 4husi h bec or absence 1 the of in presence hours 24 Opti- for in MEM incubated were MMP9–Myc or MMP2–Myc dox-inducible expressing Myc. MMP9– or MMP2–Myc tet- inducible expressing lines cell MDA- MB-231 of Characterization 1. Fig. h en n ..o he independent three of s.e. and means are the shown Data Methods). the and see Materials details more (for zymography by analyzed was degradation ECM invadopodia-associated and were cells fixed hours, 20 for incubation After 1 of absence presence or the in incubated and coverslips and Fluor488-coated gelatin fibronectin-HiLyte on plated were Myc MMP9– or MMP2–Myc either inducible dox- expressing cells MDA-MB-231 (C) (B). zymography or (A) immunoblotting by analyzed or was MMP2–Myc MMP9–Myc secreted of and amount collected the then was medium MEM otd8- matrigel- coated on plated were MMP9–Myc or MMP2–Myc dox-inducible expressing cells MDA-MB-231 (D) experiments. 5 bar: Scale MMP9–Myc. or MMP2–Myc containing organelles Arrows cytosolic level. indicate coverslip optical the show at d sections and c level, panels TGN whereas the show at b sections and optical a Panels antibodies. Myc anti- mouse with and stained Rhodamine-phalloidin and fixed were (F) MMP9–Myc or (E) MMP2–Myc expressing cells MDA-MB-231 (E,F) s.d. experiments. and independent means three the from are shown data staining. The Violet Crystal using was analyzed matrix Matrigel to through cells invade of 1 ability of The absence doxycycline. or presence the in m m. AB D-B21cells MDA-MB-231 (A,B) m -oeflesadincubated and filters m-pore m /ldxccie Opti- doxycycline. g/ml m /ldoxycycline. g/ml nsitu in m g/ml Journal of Cell Science M agtn)ddnthv n feto M29secretion MMP2/9 on effect MT1- any shown). mediate have not to recycling not (data (known did regulate complex targeting) to Exocyst MMP (known the distinct or proteins by endosomes) of effector membrane depletion Rab11 this, MT1- plasma with various and Consistent to pathways. MMP2/9 targeted transport membrane that probably are present suggesting not MMP endosomes, al., was FLAG–Rab40b et recycling 5M–O, (Peden Fig. in marker in shown endosome As recycling 2004). Rab11- known and a FLAG–Rab40b of FIP1/RCP, 2007), localization al., the is et compared Similarly, next (Bravo-Cordero MT1-MMP we endosomes 1999). Because recycling MMP9–Myc the S1A–F). al., and in 2006; present Fig. MMP2–Myc et that al., material with Steegmaier (supplementary suggesting colocalized et 2011; also arrows), (Kakhlon al., VAMP4 the 5G–L, VAMP4 et (Fig. contain at Krzewski cell organelles vesicles the Rab40b VAMP4-containing VAMP4-secretory of are with periphery FLAG– these Consistently, colocalizes these TGN. that of the Rab40b from identity possible budding the vesicles is determine secretory unequivocally it does to images organelles, us the TGN of the allow resolution of the not edges Although, the arrows). by at present (marked was located FLAG–Rab40b vesicles 5A–F, VAMP4-containing (Steegmaier Fig. in at TGN shown As and 1999). well- vesicles al., a et secretory VAMP4, for of marker localization established the with Rab40b FLAG–tagged 4650 ora fCl cec 2 (20) 126 Science Cell of Journal h eta ieo h el(i.6–) FLAG–Rab40b- the site 6A–C). of (Fig. the mid-stage the at At cell arrow). enriched 6C, be (Fig. the invadopodia to forming observed of of were organelles side containing During at ventral invadopodia 6A). actin-rich (Fig. an formed the pores cells filter invasion, of and stages stages early Matrigel stained different and through at fixed invasion cells were of visualize cells to hours 36 Rhodamine-phalloidin or with 24 After Methods). and D-B21clswr eddo arglcae Transwell cell Matrigel-coated 8 on and during containing filters seeded forming were vesicles (Murphy cells the MDA-MB-231 Rab40b-containing invasion examined of to we invasion Thus, cell localization transported 2009). al., the et are cancer Poincloux 2011; MMP9 Courtneidge, during and invadopodia MMP2 (supplementary that secretion MMP2/9 on S2A–D). effect Fig. VAMP7, any contrast, material and have By VAMP3 medium. in namely not the vSNAREs, did shown in other of MMP9 As depletion and the MMP2 MMP9. reduced of of depletion on amounts and effect VAMP4 the S2B–D, S2A) the MMP2 Fig. Fig. material analyzed endogenous supplementary we material of VAMP4, (supplementary secretion of knockdown role VAMP4 the membrane. plasma examine the with To vesicles secretory MMP2/9 of fusion rvosfnig rmsvrllbrtre aedemonstrated have laboratories several from findings Previous for responsible R-SNARE the is VAMP4 that suggest data Our nvitro in ota n,FLAG–Rab40b-expressing end, that To . m oe frmr eal e h Materials the see details more (for pores m ifrn otecnrlat control the significantly to are different or line line grey grey bottom top below above values the experiments. All independent three and of means s.d. the are shown and Data Materials Methods. the see For details siRNAs. more Rab cells various in with (B) treated MMP9–Myc and (A) MMP2– Myc secreted the of quantification screen. siRNA from identified secretion- proteins MMP9 regulating and MMP2 2. Fig. ELISA-based P , 0.01. Journal of Cell Science oe ndgaaino h C uigbes acrcell cancer breast during ECM the of key degradation play MMP9 in and MMP2 roles that suggested has research and Previous invasion cell degradation cancer ECM breast Rab40b- invadopodia-dependent for required that is the Rab40b suggest to MMP2/9 data invasion. the cell of cancer these delivery during the invadopodia/pseudopodia all mediate vesicles together, containing Taken chamber S3). 8 during bottom the pseudopodia through the these invasion cell within to accumulated and MMP2–Myc also cells filter containing MMP9–Myc as organelles the these pore Similarly, 6A,F,G). filter through Finally, (Fig. entire to migrating 6E). the (Fig. 6A,D), filled close started (Fig. and membrane clustered thickened pore plasma pseudopodia Rab40b-organelles filter pseudopodia (probably contained the the into also pseudopodia invadopodia) which extended from derived MDA-MB-231 invasion, m oe(upeetr aeilFig. material (supplementary pore m nvitro in eai-otdgascvrlp vradwt fibronectin with on overlaid plated coverslips were cells glass the MDA-MB-231 secretion degradation. gelatin-coated to MMP whether ECM invadopodia-associated investigated MMP2/9 for we and required containing hypothesis, is this Rab40b vesicles test To targeting invadopodia. in involved of ability invasion VAMP4 the of inhibited material cells. VAMP4 effect supplementary MDA-MB-231 of in depletion the shown S2E, As tested Fig. invasion. cell also on we depletion on Rab40b- MMP2/9 on effect a present organelles, is no in in VAMP4 containing having Because resulted 7A,B). while Rab40b (Fig. Rab40a, invasion, motility not cell cell of in but decrease Rab40b, involvement significant of depletion the invade secretion, 8 with to (with cells Consistent filters on MDA-MB-231 knockdown of Rab40b Transwell of ability effect the the tested we Thus, metastasis. u ellrlclzto aasgetdta a4bis Rab40b that suggested data localization cellular Our a4BrgltsMPscein4651 secretion MMP regulates Rab40B m oe)cae ihMatrigel. with coated pores) m M9wsaaye yzymography. by analyzed was and MMP9 MMP2 on endogenous knockdown of Rab40b secretion of effect collected the then and was for Opti-MEM Opti-MEM hours. of 24 ml 1 and with dishes six-well incubated in plated were number cells equal of later, days Rab40b Two with siRNA#2. transfected were cells 231 three MDA-MB- of (E) s.d. experiments. and independent means the Data are blotting. shown western by analyzed MMP9– Myc and MMP2–Myc on of knockdown secretion Rab40b of and effect collected the then was for Medium medium hours. of 24 ml and 1 dishes with six-well incubated in plated of were number cells equal later, days Two Rab40b siRNAs. different four with transfected were (D) MMP9–Myc or (C) MMP2–Myc expressing cells MDA-MB-231 qPCR. (C,D) by determined Rab40b as of knockdown efficiency The (B) structure. domain Rab40b of representation MDA- cells. in MB-231 secretion MMP9 and decreases MMP2 knockdown Rab40b 3. Fig. aasonaetemasadsd of s.d. experiments. and independent means three the are shown The data ELISA. using of analyzed levels IL-6 the secreted and hours, collected 16 was for medium incubation mg/ml After 1 LPS. with of stimulated and six- dishes in well plated were cells MDA-MB-231 Rab40b-siRNA-treated or Mock- (G) experiments. independent s.d. and three means of the are shown Data MMP. MT1- membrane of plasma levels endogenous the measure to flow cytometry by analyzed and harvested were cells MDA-MB-231 treated VAMP4-siRNA- Mock-, or (F) Rab40b-siRNA- control. was negative cells as without used dish six-well a a from as collected used Opti-MEM was control. lane positive first the secreted in in MMP2/9) (rich serum bovine Fetal A Schematic (A) nvitro in using Journal of Cell Science rvosyrpre Cake l,20) M01almost GM6001 2007), al., et (Clark As reported in S1G). Fig. MMP2/9 MMP2/9- material previously the (supplementary of with SB3CT or inhibitor role GM6001 specific inhibitor the 2006; MMP validate al., broad-spectrum with are treated a To were et cells degradation, invadopodia 2009). (Hotary ECM invadopodia-associated al., activity whether et MT1-MMP or Poincloux on only reports degradation, Some dependent invadopodia- targeting. ECM for MMP2/9 required this are in associated MMP2/9 decrease whether whether a questioned unclear to have remains due it is effect invadopodia degradation, ECM on associated effect no S4). had Fig. material but (supplementary ECM formation 8A–G), invadopodia-associated (Fig. invadopodia in decrease for degradation a as required in resulted is Rab40b depletion identified degradation. Rab40b ECM invadopodia-associated 2011). whether be and al., formation tested et marker Murphy can we 2005; invadopodia al., Next, et the puncta (Courtneidge and 7I–K) contained (Fig. actin fibronectin Tks5 they which the because These structures, of invadopodia, matrix. degradation punctate localized gelatin actin-rich MDA- with 7C–H, formed associated Fig. in cells shown As MB-231 Fluor488. HiLyte to conjugated 4652 lhuhRb0 ncdw erae invadopodia- decreased knockdown Rab40b Although ora fCl cec 2 (20) 126 Science Cell of Journal epnil o agtn fMP/-otiigtransport cells. MMP2/9-containing cancer of invasion of during invadopodia are the targeting VAMP4 to vesicles and for Rab40b with that combined of data responsible suggest depletion These experiments 8I,J). imaging with (Fig. our alone compared VAMP4 pathway. not degradation or did same Rab40b also ECM the VAMP4 inhibit and in Rab40b further act alone of demonstrating probably knockdown MMP2/9 dual S4D,F), MMP2/9 Similarly, of and Fig. inhibition Rab40b material compared that or supplementary as Rab40b degradation 8H; (Fig. of ECM inhibit knockdown of further depletion with and not MMP2/9 did of inhibition shown Rab40b simultaneous As 8H, SB3CT. invadopodia- Fig. and siRNA in co-treated Rab40b for we MMP2/9 with invadopodia, is cells the responsible Rab40b MDA-MB-231 to cells, whether targeting test MMP2/9 partially MDA-MB-231 for further required To least in degradation. ECM at dependent that is together, indicate Taken secretion S4C,F). data 2006) Fig. Kleifeld, material these degradation 8H; and supplementary ECM (Fig. treatment Overall 8H; invadopodia-induced (Fig. 2005; number contrast, inhibited al., partially By invadopodia et only (Ikejiri S4B,F). on SB3CT Fig. with effect material no supplementary degradation, having ECM while invadopodia-associated blocked completely ihRb0 iN.Tody ae,clswere cells later, days Two siRNA. Rab40b transfected with were MMP9–Myc or expressing MMP2–Myc stably cells experiments. MDA-MB-231 independent (D) two are of three C means of in the s.d. shown and Data means the experiments. are independent B and Data A (C). in CD63 shown and (C) receptor (B), transferrin MMP9–Myc of (A), levels MMP2–Myc the intracellular measure by to analyzed cytometry and flow harvested were cells MDA- MB-231 Rab40b-siRNA-treated or Mock- MMP9. (A–C) and MMP2 of lysosomal degradation increases Rab40b 4. Fig. niois uliaesandbu ihDAPI. 5 with bar: blue Scale stained are (green) Nuclei anti-CD63 antibodies. or co- (red) and anti-Myc fixed with then stained with hours, treated 12 were for cells bafilomycin later, days Rab40b Two with siRNA. transfected were or (F) (E) MMP9–Myc MMP2–Myc expressing stably three cells of MDA-MB-231 s.d. (E,F) and experiments. means independent the are shown MMP9– Data and Myc. the MMP2–Myc measure intracellular to of cytometry levels flow by analyzed then were Cells (inset). secreted MMP9–Myc of and amounts MMP2–Myc the was measure medium to and collected hours of 12 absence for of bafilomycin presence the in incubated m m. Journal of Cell Science 02 oet ta. 04 hhe l,20) e h machinery the various yet 2009), Werb, al., of and et Shah Egeblad metastasis an 2004; 1997; al., and play et Matrisian, Polette MMP2/9 invasion 2002; and (Chambers that during cancers demonstrated role have important studies Multiple regulates Discussion also cancers. Rab40b non-breast that in secretion (supplementary suggesting MMP2/9 targeted S5C–F), degradation Fig. ECM S5A,B). area material the invadopodia-associated Fig. in decrease a material to of led (supplementary also knockdown cells Rab40b Importantly, cancer MDA-MB- to breast reminiscent cells. invadopodia 231 HMCB formed melanoma also invadopodia- human cells on HMCB in knockdown degradation Rab40b ECM of effect associated the tested next we ots hte a4bpasasmlrrl nohrcancers, other in role similar a plays Rab40b whether test To -NR,VM4 hc a ensont eit protein mediate to shown been with has colocalize which vesicles VAMP4, Rab40b-containing R-SNARE, that shown have we nesadtemcaim fMP/ rnpr,we during transport, Rab40b further MMP2/9 of invasion function to cell of and cancer breast Thus, localization MDA-MB-231 mechanisms the the was proteins. or investigated the GTPase, little Rab40b-interacting Rab40b acceptor study, of understand of function appropriate this cellular the identity vesicle Before the to about budding, 2001). known vesicle targeting (Pfeffer, Rab sorting, vesicle All compartment cargo and MMP9. transport regulating and transport membrane various by MMP2 of regulators both steps, is master siRNA that as of an GTPase act monomeric secretion GTPases used small plasma for we a the as study, required Rab40b with this identify to In fusion screen elusive. and remains transport membrane MMP2/9 regulates that a4BrgltsMPscein4653 secretion MMP regulates Rab40B –.Nce nCFIaesandblue stained DAPI. are with C,F,I in and Nuclei D–F J–L. in as images shown magnification area higher marks region boxed C I, In and Rab40b. and VAMP4 containing organelles J–L, In peripheral TGN. indicate the arrows of edges the at vesicles Rab40b-containing and VAMP4 arrows indicate D–F, In antibodies. (N,O) anti-FIP1 and (B,C,E,F,H,I,K,L) VAMP4 anti- anti- with (A,C,D,F,G,I,J,L,M,O), stained FLAG and fixed then were Cells coverlips. glass on collagen-coated plated and FLAG–Rab40b with transfected were cells MDA-MB-231 vesicles. secretory VAMP4-containing with colocalizes FLAG–Rab40b 5. Fig. nvitro in Interestingly, . Journal of Cell Science G,i slkl htRb0 srqie o prpit sorting appropriate for required is Rab40b the that at likely sites is budding it VAMP4-containing TGN, Because the MMP9. to by and localizes caused MMP2 Rab40b part, of in degradation least lysosomal at increased was, decrease transport This secretion. membrane MMP2/9 distinct by to secreted appear MT1-MMP and pathways. and MMP2/9 transported Thus, 2 be shown). (Fig. secretion siRNA- not and Exocyst data transport that the MMP2/9 and on of effect demonstrate components no of has We or complex Rab8 2008). of knockdown al., dependent factor work tethering et transport-vesicle protein a recent (Sakurai-Yageta a as function complex, MT1-MMP to Exocyst Furthermore, of known the complex targeting requires 2009). membrane Rab8, plasma to the al., al., to addition et in (Hotary et that demonstrated endosomes Poincloux recycling to by transported 2006; membrane be ECM to plasma mediates shown plasma was the that invasion, the MMP cell during to other degradation the transported VAMP4. MT1-MMP, and are Rab40b Interestingly, containing vesicles MMP2/9 described secretory by data MMP2/9 that membrane The in indicate decrease invasion. and a cell above Rab40b in MDA-MB-231 that results and demonstrated siRNA (Steegmaier secretion by membrane we depletion plasma Furthermore, VAMP4 the 1999). to al., TGN et the from transport 4654 n fteefcso a4bkokonwsadces in decrease a was knockdown Rab40b of effects the of One ora fCl cec 2 (20) 126 Science Cell of Journal M29sceinit h eim tsgiiatydecreased on degradation. significantly effect it ECM moderate medium, invadopodia-associated a the MMP2/9-dependent only into secretion had MMP2/9 knockdown Rab40b although degradation ECM mediate and they enriched where all invadopodia are the has MT1-MMP but at It and podosomes. MMP2/9 called that unclear, invadopodia, demonstrated are called been they remain are cells non-cancerous are cells in cancer invadopodia whereas and in structures cells, and actin differences generally culture functional podosomes tissue The podosomes. Invadopodia between in as to 2011). studied referred and Courtneidge, sometimes primarily the degradation and been ECM to (Murphy have mediating targeting in invasion implicated vesicle cell been MMP2/9 have that as well as TGN invadopodia- invadopodia. sorting the MMP2/9 regulates inhibited at Rab40b this that suggesting with and cells, knockdown MB-231 Consistent degradation defect, ECM membrane. Rab40b associated plasma targeting secretion MMP2/9 the MMP2/9 for observation, with required the fusion also is lysosomal and rescue Rab40b of that not inhibition suggesting did Interestingly, to transported membrane. degradation be to destined plasma vesicles the the to MMP9 and MMP2 of naooi r ci-ih igrlk ellrprojections cellular finger-like actin-rich, are Invadopodia nvivo in Mrh n ornig,21) Interestingly, 2011). Courtneidge, and (Murphy otiig8 containing filters Matrigel-coated on seeded were FLAG–Rab40b expressing stably invasion cell during organelles Rab40b- containing of Localization 6. Fig. nCEGadso el from cells show and C,E,G shown in images of rendering 3D are B,D,F invadopodia). from derived (probably pseudopodia or images invadopodia all indicate in Arrows in B,D,F. imaged panels stages invasion the A depict in Drawings antibodies. anti-FLAG and or fixed Rhodamine-phalloidin then with were stained Cells or (F). (B,D) hours hours 36 24 either for incubated pia etosdpce nE,G. the in of depicted level sections the optical indicate D,F in Lines and panels) nvitro in nvitro in X-Y m oe.Clswere Cells pores. m rgtpnl)planes. panels) (right . naino MDA- of invasion D-B21cells MDA-MB-231 Z-Y nvitro in (left Journal of Cell Science agtn fMP/ n T-M otesm location same the co- to Thus 1995). MT1-MMP al., and et MMP2/9 (Strongin targeting of to MMP2 shown targeting activate been overlapping In has and invadopodia. MT1-MMP the cleave required collagen, at that degrading are degradation outside to ECM pathways of addition membrane possible fidelity transport the plasma different ensure Exocyst to is two the the by at mechanisms it and present VAMP4 is also invadopodia, transporting Because remains are of pathways invadopodia? that complex distinct to endosomes question to two need The transported MMPs cells recycling cancer 2008). do is is al., why Exocyst-dependent et degradation MT1-MMP with at (Sakurai-Yageta the by consistent that of is degradation invadopodia This rest observations ECM MT1-MMP. The previous by 50%. decreased mediated only presumably for Rab40b by required However, invadopodia is invadopodia. of formed Rab40b newly that knockdown the suggest at secretion data MMP2/9 our together, Taken h ehnssta euaeMP/ agtn uigcancer during targeting MMP2/9 invasion. regulate cell that as identify well mechanisms to steps the as the future proteins, be invadopodia, will effector proteins to canonical Rab40b-SOCS-interacting targeting Rab40b secretion of MMP2/9 an cytokine identification as of Rab40b regulating of within regulator establishment motif the in important With box 2008). implicated proteins al., SOCS et of is (Piessevaux the sub-family Interestingly, proteins Rab40 2012). other the al., to et box unique (Stein SOCS proteins, and feature a Rab transport has a effector Rab40b All formation, domain, Furthermore, vesicle multiple 2001). Rab40b. (Pfeffer, still transport targeting recruiting to mediate is then bind it by which invadopodia, proteins function to effector GTPases targeting what and unknown sorting MMP2/9 for associated degradation ECM of invadopodia. amplification with an in result would lhuhti td eosrtsta a4bi required is Rab40b that demonstrates study this Although niois(,) cl a:1 bar: anti-Tks5 Scale and rabbit (J,K). fixed and antibodies were (I,K) cells Rhodamine-phalloidin hours, with 20 stained gelatin-coated for on incubation plated After were coverslips. cells MDA-MB-231 5 (I–K) bars: (F–H). Scale (C,E,F,H). invadopodia. cells Rhodamine-phalloidin indicate hours, with Arrows 20 stained for and incubation fixed After were H). Fluor488-coated and fibronectin-HiLyte (D,E,G and coverslips gelatin on plated were ehd) aasonaetemasadsd fthree of s.d. and * means experiments; and the independent was Materials are filter the shown the (see Data of staining Methods). side Violet the bottom and Crystal (A) the by hours to analyzed 16 migration or cell (B) hours of 8 extent 8- for incubated (B) then uncoated were or Cells (A) matrigel-coated on invasion plated cell MDA-MB-231 for required vitro is Rab40b 7. Fig. a4BrgltsMPscein4655 secretion MMP regulates Rab40B . AB ok rsRAtetdMAM-3 el were cells MDA-MB-231 siRNA-treated or Mock- (A,B) P . m .5 CH D-B21cells MDA-MB-231 (C–H) 0.05. m. m CE,1 (C–E), m m -oefilters. m-pore m m in Journal of Cell Science y rMP–y eecoe notepUHrtoia xrsinvector selected expression then were retroviral lines pHUSH cell 1 clonal the Stable, using CA). into Francisco, cloned San South MMP2– were (Genentech, lines, MMP9–Myc cell or stable MMP9–Myc Myc and MMP2–Myc (FBS), MDA-MB-231 serum 100 and bovine inducible penicillin fetal IU/ml heat-inactivated 100 with 10% supplemented g/l and and L-glutamine 4.5 with g/l DMEM 50% 5.84 lines and glucose, RPMI-1640 cell 50% in MDA-MB-231 cultured tet-inducible were cells of MDA-MB-231 generation and culture Cell Anti-VAMP4 PA). Grove, 2005). (West Laboratories al., ImmunoResearch Jackson et were as and from antibodies Alexa-Fluor-594- Seals purchased secondary 2010). anti-mouse domains al., 2008; and et anti-rabbit (Gordon al., Alexa-Fluor-488-conjugated Tks5-SH3-1/Tks5-SH3-4 described was et previously were antibody human (Blouw antibodies Anti-FLAG was described recombinant antibody Tex. anti-Tks5 previously polyclonal using Gene Rabbit MO). from Louis, generated with (St obtained conjugated Sigma from antibody was purchased Anti-Myc (GTX21261) CA). were (Temecula, anti-MMP9 HRP mouse Cruz Millipore and Santa from anti-MMP2 from Mouse purchased purchased CA). were Cruz, (Santa antibodies Biotechnology (9E10) anti-Myc monoclonal Mouse constructs and Antibodies Methods and Materials 4656 m /lo uoyi n rw sn h bv ecie medium described above the using grown and puromycin of g/ml ora fCl cec 2 (20) 126 Science Cell of Journal m tetmcn ocet tet- create To streptomycin. g onanVe,C).T nueete M2Mco M9Mcexpression, MMP9–Myc or MMP2–Myc 1 of presence either the in incubated were induce Laboratories, lines cells stable (Clontech To CA)]. FBS View, tet-free Mountain with supplemented medium [MDA-MB-231 o h iN cen 6wl irpae eecae ih100 with coated were microplates 96-well screen, siRNA the For assays ELISA 1.2 or at MMP9–Myc plates each 96-well either on against expressing plated siRNAs lines were different MMP2–Myc cell four stable of MDA-MB-231 interest. pool of a protein with performed was screen siRNA The screen library siRNA olce rmsRAtetdcls fe or ficbto,pae eewashed 100 were plates with incubation, of incubated hours and 2 After washed cells. siRNA-treated then from collected were 200 Plates with blocked were buffer. plates which blocking after wash, a by followed was This solution. (2 MMP2 n eeso ertdMP–y rMP–y nlzdb ELISA. by analyzed MMP9–Myc or collected MMP2–Myc then secreted was Medium of reader WI). levels Envision Madison, and Elmer (Promega, Perkin reagent by Glow monitored for Titer incubated was Cell viability using cells cell and The medium hours. serum-free 32 with another replaced then was Complete medium hours. growth 36 for incubated were cells in Transfected described protocol. manufacturer’s as the CA), Carlsbad, (Invitrogen, Max RNAi using oligonucleotides siRNA m /li B)o iue niMP (0.5 anti-MMP9 diluted or PBS) in g/ml 25 bars: Scale with (A–C). stained Rhodamine-phalloidin and fixed were of cells hours incubation, 20 After (D–F). coverslips Fluor488-coated HiLyte gelatin fibronectin on and plated were cells MDA-MB-231 Rab40b-siRNA-treated or Untreated (A–F) degradation. for ECM required invadopodia-associated is Rab40b 8. Fig. h en n ..o he independent three of experiments. s.e. and are means shown the Data cells. MDA-MB-231 treated Rab40b-siRNA- and untreated in degradation ubro el analyzed. cells of three number of s.e. and experiments. means independent the are shown Data cells. MDA-MB-231 or VAMP4-siRNA-treated Rab40b-siRNA- mock-, in ECM degradation of loading Quantification a (J) as control. used tubulin (top with blotting panels) VAMP4 western of by levels analyzed The were s.d. and shown means data the The are (graph). Rab40b qPCR encoding by mRNA analyzed of levels the harvested and were cells siRNA-treated or VAMP4 Rab40b-siRNA- Mock-, (I) analyzed. independent three of experiments. s.e. and are means shown Data the siRNA. Rab40b also with were treated cells indicated, Where inhibitor). (MMP2/MMP9 SB3CT or MMP inhibitor) (broad-spectrum GM6001 either treated with cells MDA-MB-231 in ECM degradation of Quantification (H) analyzed. m .()Qatfcto fECM of Quantification (G) m. 6 10 n n m /lo oyyln o 4hours. 24 for doxycycline of g/ml stettlnme fcells of number total the is stettlnme fcells of number total the is 4 est n otasetdwith co-transfected and density m /li B)antibody PBS) in g/ml n stetotal the is m fdltdanti- diluted of l m fmedium of l m lof Journal of Cell Science efre sn nelgn mgn noain Dne,C)three-dimensional software. CO) exploration (Denver, and Innovations rendering Imaging Intelligent using performed eeaanicbtdfr1hu tro eprtr (22 temperature room at hour 1 for incubated again were 90 with incubated and ok rRb0-iN-rae D-B21clswr nuae nthe in incubated were cells 37 MDA-MB-231 at medium Rab40b-siRNA-treated complete or Mock- assays Zymography 37 at incubated were cells MDA-MB-231 Rab40b-siRNA-treated or Mock analysis FACS 60 with coated were filters Briefly, Matrigel. with coated were Corning) pores, a were with Cells microscope deconvolution CA). 200M and Burlingame, Axiovert PBS, Zeiss in Laboratories, 63 inverted washed bovine an (Vector antibodies, fetal with specific 1% VectaShield imaged with and in BSA incubated 0.2% then mounted containing were PBS non- Cells and grown with saponin, serum. blocked 0.4% for coverslips, were containing permeabilized sites (PBS) minutes, glass saline specific 15 phosphate-buffered collagen-coated for in paraformaldehyde minutes on 4% 10 with plated fixed and were overnight cells nm. MDA-MB-231 570 at extracted density microscopy was optical Fluorescent filter measuring cotton the a by of with quantified side removed and bottom were acid the filter on acetic hours the remaining 2.5% 8 washes, cells of with or three from surface After dye assays) upper The Violet. the (invasion swab. Crystal on hours 0.1% remained 16 with that filter stained a cells assays, were After invasion cells matrix. In assays) chamber. Matrigel (motility bottom of with the hours coated to 48 added after was Transwell was medium six cells) (the with medium of conditioned incubation chamber MDA-MB-231 filter. top 8 in filter per with the resuspended cells filters Transwell to mm were (6.5 added cells using insets and siRNA-treated filter measured medium or was mock- serum-containing motility, fresh invasion measure or To assays. motility cell MDA-MB-231 vitro In mgduig10n -tp h DrneigwspromduigIntelligent exploration using and performed rendering were was three-dimensional rendering cells CO) software. 3D invading (Denver, The of Innovations z-step. Rhodamine- Z-stacks Imaging nm with 100 stained antibodies. and using anti-FLAG fixed (the chamber. imaged hours, or bottom medium 36 the or anti-Myc conditioned to 24 added MDA-MB-231 phalloidin, either was for cells) filter. incubated with per were incubation Cells the of cells hours rehydrate 50,000 to 48 after minutes seeded of medium 15 then density for were filter a FLAG–Rab40b the expressing at of stably sides cells both MDA-MB-231 to Matrigel. added then was Medium eupne n50m fPSadaaye yfo yoer sn Cytomics using cytometry flow CA). by Brea, analyzed Coulter, and (Beckman PBS cytometer flow of FC500 ml 500 in resuspended ytma ecie ntemnfcue’ rtcl h pia est t40nm Development 450 Spectrophotometer. ELISA at ND-1000 density NanoDrop DuoSet optical a The IL-6 using protocol. for determined manufacturer’s using collected the was performed in was samples. described normalize medium was as to hours, system assay analysis 16 Bradford ELISA for for harvested incubation IL-6 were cells After and LPS. analysis ELISA of mg/ml 1 with eddit i-eldse tadniyo 8 of density a at dishes six-well Spectrophotometer. into ND-1000 seeded NanoDrop a using nm 450 solution substrate at HRP density using optical measured and was MMP9–Myc or MMP2–Myc of amount piMM(nirgn albd A n el eeicbtda 37 at incubated were cells and CA) Carlsbad, (Invitrogen, Opti-MEM fMtie o ora 4 at hour 1 for Matrigel of upeetdmda el eete nuae ihanti-MMP14-APC serum- with and incubated HEPES-buffered then in were resuspended 37 Cells at and antibodies media. trypsinized supplemented then were iN-rae D-B21clswr nuae t37 at incubated were Cytomics cells using MDA-MB-231 cytometry siRNA-treated flow were CA). by cells Brea, for analyzed Coulter, washes, and FITC- (Beckman FBS) three PBS cytometer with of of flow minutes 2% FC500 set 30 ml 500 and for another in incubated After BSA resuspended and were antibodies. buffer Cells 1% hour. secondary FACS 1 conjugated with saponin, for paraformaldehyde, with antibodies times primary permeabilized 0.4% three 4% with then washed incubation with by containing were followed Cells fixed minutes, (PBS glycine. 30 and M buffer 0.1 trypsinized with FACS then quenching by were followed Cells hours. 64 sda eaiecnrl rel,Ot-E ape eedltdi h standard the in diluted were samples was Opti-MEM wells Briefly, and cell-free control. MMP2 from negative collected a in Opti-MEM as (rich control. used serum positive bovine as Fetal used Opti- zymography. was in MMP9) MMP9 by using and analyzed harvested MMP2 then secreted were of were lysates levels MEM The cell X-100. and Triton 1% collected containing was PBS medium Opti-MEM hours. 24 itr eeicbtda 37 at incubated were filters 6 ovsaieivdpda rnwl itrist 65m itr ih8 with filters mm (6.5 insets filter Transwell invadopodia, visualize To omauelvl fscee L6 ok rRb0-iN-rae el were cells Rab40b-siRNA-treated or mock- IL-6, secreted of levels measure To oaayepam ebaeMP4 ok,Rb0-iN-o VAMP4- or Rab40b-siRNA- mock-, MMP14, membrane plasma analyze To i-meso esadSnia ECDcmr.Iaepoesn was processing Image camera. CCD QE Sensicam and lens oil-immersion nainadmtlt assays motility and invasion ˚ o or el eewse he ie ihmedium, with times three washed were Cells hour. 1 for C ˚ m .Atr4 or ficbto,mdu a elcdwith replaced was medium incubation, of hours 48 After C. fmueat-y nioycnuae oHP Plates HRP. to conjugated antibody anti-Myc mouse of l ˚ ˚ ,atrwih55 which after C, o 0mntst lo arglt polymerize. to Matrigel allow to minutes 30 for C m oe,Crig Y tadniyo 100,000 of density a at NY) Corning, pores, m 6 10 5 m el.I- erto a induced was secretion IL-6 cells. fMtie a eoe and removed was Matrigel of l ˚ ) olwdb ah The wash. a by followed C), ˚ o 4hus Cells hours. 64 for C ˚ o another for C ˚ for C m m m l eswr ahdtiewt .%Tio -0 ormv D.Gl eethen CaCl were mM Gels 5 containing SDS. 8.0 remove pH to Tris-HCl, mM X-100 (50 Triton buffer 2.5% digestion in with electrophoresis, incubated Following twice concentrations. washed protein were lysate gels cell separated on then based were 3 normalized Samples containing gels agent. polyacrylamide reducing 7.5% a using without buffer sample SDS-PAGE M9(o ad a eetda ht ad nadr background. dark a on bands white as detected and band) was (bottom band) MMP2 endogenous (top by proteolysis MMP9 gelatin of extent The dye. Blue on oesisi 2wl ltswt .%gltn .%scoei B t37 at PBS in sucrose 2.5% gelatin, 2.5% with plates 12-well in coverslips round The situ In TeSa) h auso nrclua F eann fe erto were secretion FLOWJO after software remaining the AP21998. GFP of using addition intracellular before calculated fluorescence of sample. GFP secretion, was each using values for after normalized fluorescence analyzed The cells were GFP cells in (TreeStar). for live by mean gated remaining ice thousand cells Ten on live cargo The with scatter. trypsinized cytometer of side flow and and Fortessa amount BD PBS forward a the cold using analysed with measure were samples washed To ice, hours. on 2 placed AP21998 1 using were of initiated samples use GFP–FKBP-hGH at concentration before the plating overnight a of previously incubated before Secretion were at was Cells assay. counted 2010). plate. secretion it and 24-well al., 5. the a trypsinised et because in on Syntaxin were (Gordon well control, per cells or secretion cells positive hGH transfection, 50,000 Rab40b inhibits a after either depletion hours as 5 72 to used Synatxin six-well targeted was that on reported siRNA siRNA confluence with 5 25% Syntaxin transfected at plated and GFP 2010), were cell-associated plates al., GFP–FKBP-hGH the et expressing measuring (Gordon stably by GFP cytometry. cells with flow live by tagged in secreted) GFP– is (not quantified of hGH AP21998 fluorescence secretion be Because ligand synchronous can cells. FKBP and secretion the of aggregates these from Addition of aggregates, ER. hGH ligand-reversible solubilization the form in to in FKBP results S1H). GFP–FKBP-hGH of Fig. property trapping material the (supplementary on thus based 2010) is al., assay GFP–FKBP-hGH et This expressing (Gordon stably described cells HeLa previously used as we secretion hGH measure assay To secretion GFP–hGH cytometry-based Flow 2 from extracted was RNA Total quantitative (qPCR) and (RT-PCR) PCR reaction chain polymerase transcriptase Reverse yicbto t4 at incubation by pcfcfamnsaduiu rdcswr etdb etcreanalysis. melt-curve by tested the were by products calculated was unique quantification and Relative fragments specific naooi omto n oaie arxdgaain e admychosen quantify randomly ten To and 63 degradation, (using PBS CA). imaged matrix in Carlsbad, were localized X-100 fields and (Invitrogen, Triton formation fixed 0.4% Rhodamine-phalloidin then invadopodia were with cells with The permeabilized hours. stained 20 paraformaldehyde, for incubated 3% and well in each to added then were rpiaeb PRapiiainuigSb re PRMse i using amplification Mix qPCR Master The qPCR System. 50 Green PCR were: Sybr Real-Time conditions using ViiA7 in analyzed Biosystems amplification was Applied cells qPCR the siRNA-treated quantify hexamer by or To mock- random (Invitrogen). triplicate from cDNA polymerase using knockdown, Taq to (Invitrogen) of using percent III transcription performed was SuperScript Reverse PCR with protocol. primers. performed manufacturer’s was the cDNA to according (Invitrogen) Ctseeo,Dne,C)wste vrado o fcosikdgltnand gelatin 4 crosslinked at of hour top 1 for on dark overlaid the then in incubated was CO) Denver, (Cytoskeleton, upeetdwt 0 B AlnaBooias n 0 uSrm for Nu-Serum] [DMEM 10% medium and invadopodia Biologicals) (4 cells (Atlanta with MDA-MB-231 FBS equilibrated minutes. 20% 30 and with DMEM, supplemented with washed 1 eod) 60 seconds), (15 h eai a loe osta 4 at set to allowed was gelatin The el oprdwt oktetdcells. 5 mock-treated Rab40b-knockdown (sense, in with experiments) compared independent two cells from (averaged change fold noain Dne,C)tredmninlrneigadepoainsoftware. exploration Imaging and rendering Intelligent three-dimensional using CO) measured (Denver, areas were the the Innovations degradation, fluorescence on the of within FITC-fibronectin based area cells counted the invadopodia-associated lacking underneath measure were seen To cells spots To boundaries. degradation invadopodia, experiments. cell and independent with puncta three actin cells of least of presence at number in counted the were measure cells 260–330 of total nydgaainaesascae ihncl onaiswr analyzed. were boundaries cell within associated areas degradation Only 37 CTGCTTGCTGA-3 5 TCTACGGCCACTACTGTG-3 GTTATCG-3 9 -CCTGATGGCACCCATTTACACC-3 omaueteefc fRb0 ncdw nhHscein eacells HeLa secretion, hGH on knockdown Rab40b of effect the measure To ˚ o 44 or.Gl eesbeunl tie ihCoaseBrilliant Coomassie with stained subsequently were Gels hours. 24–48 for C nsitu in yorpyassays zymography 9 -AAAGACCTGTACGCCAACAC-3 yorpymti erdto sa a oeb otn 8mm 18 coating by done was assay degradation zymography/matrix 9 M9wsapiiduigfloigpies es,5 sense, primers: following using amplified was MMP9 . a4BrgltsMPscein4657 secretion MMP regulates Rab40B ˚ 1mnt) rmrpiswr eindt mlf mRNA- amplify to designed were pairs Primer minute). (1 C ˚ o 5mnts 50 A minutes. 15 for C 9 .MP a mlfe sn olwn rmr:sense, primers: following using amplified was MMP2 ). ˚ 2mnts,95 minutes), (2 C m npeamdcluemdu.T atsecretion, halt To medium. culture prewarmed in M 6 9 nisne 5 anti-sense, ; betv)prtetetfrec xeiet A experiment. each for treatment per objective) ˚ eoecosikn ih05 glutaraldehyde 0.5% with crosslinking before C 6 ˚ .Teds a trlzdwt 0 ethanol, 70% with sterilized was dish The C. 6 10 10 4 9 nisne 5 anti-sense, ; 7 ˚ el n2m fivdpdamedium) invadopodia of ml 2 in cells b 1 iue) 0cce t95 at cycles 40 minutes), (10 C atnsre snraiigcontrol normalizing as served -actin D-B21clsuigTRIzol using cells MDA-MB-231 m 9 m -GCACTGCAGGATGTCATAG-3 /lo eai.Sml odn was loading Sample gelatin. of g/ml DD /lslto fFITC-fibronectin of solution g/ml 9 nisne 5 anti-sense, ; C T ehd aasona the as shown Data method. 9 -CGACGGCATCCAG- 9 -GTCATACTC- 9 -CCCT- 2 )at ˚ ˚ C. C 9 . Journal of Cell Science odn .E,Bn,L . alne,D .adPdn .A. A. Peden, and A. D. Sahlender, M., L. Bond, E., D. Gordon, oay . i .Y,Aln . tvn,S .adWis .J. S. Weiss, and L. S. Stevens, E., Allen, Y., X. Li, K., Hotary, Z. Werb, and M. Egeblad, L. Tesfay, and F. D. Seals, I., Pass, F., E. Azucena, A., S. Courtneidge, oykeiz .A,Jmeo,N . apesn . rnly . a den van J., Grindlay, I., Macpherson, B., N. Jamieson, A., M. Dozynkiewicz, and G. W. Yarbrough, A., Kochaishvili, S., A. Whigham, B., Brown, S., E. Clark, M. L. Matrisian, and F. A. Chambers, lr,E . hga,A . abog,W .adWae,A M. M. A. A. Weaver, Weaver, and and S. G. E. W. Clark, Yarbrough, S., A. Whigham, S., E. Clark, W., K. Cheng, K., Clark, P., D. White, M., Parsons, J., H. Spence, T., P. Caswell, Megı R., Marrero-Diaz, J., J. Bravo-Cordero, orugo,L . uj-mt,Z n ia N. Iida, and Z. Gunja-Smith, Y., L. Bourguignon, A. S. Courtneidge, and B. Diaz, I., Pass, F., D. Seals, B., Blouw, ry,V . hn,Y,Sile-osiish . aaa .M n Mueller, and M. K. Yamada, F., Seillier-Moiseiwitsch, Y., Zhang, V., V. Artym, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.126573/-/DC1 online available material Supplementary months. 12 R.P.]. after to release DK064380 Komen for number by PMC [grant G. funded in Health partially Deposited Susan Cancer also of was Institutes the the project National This and the from R.P. R.P.]; to grants Colorado to of BCTR0706749 League by number supported [grant foundation was work This Funding manuscript. the writing in participated and project entire and oversaw the R.P. directed reagents. completed cell Vamp4 A.P. provided with and and assays S.G. us secretion assays. provided HGF secretion MMP P.S. with screen. helped and and siRNA lines J.L. the screen. siRNA with the helped performed J.Junutula and lines MMP2–Myc cell tet-inducible MMP9–Myc generated and J.Jing manuscript. participated as the well writing as experiments in the of the most for responsible for was A.J. AMC) Denver, contributions (UC Author Tentler John line. Dr cell to melanoma HMCB grateful three are of We s.d. and means Acknowledgements the are shown All Jolla, data assays. (La all software motility experiments. noted, 5.0d and independent Prism otherwise GraphPad invasion Unless using analysis, CA). done FACS were calculations secretion, statistical IL6 and MMP2/9 Student’s independent Two-tailed analyses Statistical 4658 ealpoes ra euae h aeetmmrn rnmgainprogram. transmigration membrane basement the Dev. Genes regulates triad metalloprotease mammalian in secretion constitutive for required SNAREs cells. identify to screen siRNA progression. cancer ege .V,vnTu,A,Mro,J . ore,C,Tmsn . Nixon, P., Timpson, C., Gourley, progression. P., cancer drive J. and Morton, endosomes/lysosomes A., Thun, al. et von C. V., P. Berghe, invasion. Biol. cell Quant. cancer Symp. and Harb. (invadopodia), podosomes Tks5, amplicon. substrate 11q13 SRC the in gene a cortactin, of levels M. A. Weaver, otci sa seta euao fmti ealpoens erto and secretion metalloproteinase secretion. matrix protease of of regulation regulator invadopodia. in essential degradation matrix an extracellular is Cortactin 3D al. in metastasis. et in migration I. metalloproteinases invasive K. promote Anderson, to J., integrin A. Messent, alpha5beta1 microenvironments. J., with M. associates Humphries, Rab25 B., G. pathway. Mills, exocytic Rab8-dependent novel a by 1499-1510. regulated is activity Garcı soito nmtsai ratcne cells. (MMP-9) cancer metalloproteinase breast matrix metastatic and in migration association cell tumor cytoskeleton-mediated vivo. in growth tumor in Tks5 Biol. protein scaffold podosome/invadopodia ealpoens tivdpda eiigtesae fivdpdafrainand formation invadopodia of stages the function. defining invadopodia: at metalloproteinase C. S. a .A,Ary,A .adMnoa .C. M. Montoya, and G. A. Arroyo, A., M. ´a, 87 Traffic 20) yai neatoso otci n ebaetp matrix 1 type membrane and cortactin of interactions Dynamic (2006). 555-567. , 21) a2 n LC olbrt opooeitgi eyln rmlate from recycling integrin promote to collaborate CLIC3 and Rab25 (2012). acrRes. Cancer ora fCl cec 2 (20) 126 Science Cell of Journal 20 11 2673-2686. , 1191-1204. , 20) grsieeso NC uosdpnso expression on depends tumors HNSCC of Aggressiveness (2009). a.Rv Cancer Rev. Nat. e.Cell Dev. 66 3034-3043. , 20) e ucin o h arxmtlortiae in metalloproteinases matrix the for functions New (2002). 70 167-171. , 13 u.J elBiol. Cell J. Eur. 496-510. , .Nt.Cne Inst. Cancer Natl. J. 20) e oefrcratni invadopodia: in cortactin for role new A (2008). t tsswr sdt nlz h eut of results the analyze to used were -tests 2 19) hnigveso h oeo matrix of role the of views Changing (1997). 161-174. , a,D,Genı D., ´as, .Cl Physiol. Cell J. acrRes. Cancer 19) D4381 sivle in involved is CD44v3,8–10 (1998). Oncogene 87 e.Cell Dev. 20) T-M proinvasive MT1-MMP (2007). 581-590. , 89 1260-1270. , s . Garcı L., ´s, 176 28 67 22 20) acrcell cancer A (2006). 20) oefrthe for role A (2008). 431-444. , 4227-4235. , 206-215. , 131-145. , 21) targeted A (2010). aGad,A., ´a-Grande, MOJ. EMBO odSpring Cold u.J Cell J. Eur. 20) The (2005). (2007). (2007). 26 , evr .M. A. Weaver, is,R n aae H. Nagase, and R. Visse, tli .S,Ky,A .adLc,P. Lock, and and A. H. G. A. Kaye, Grant, S., L., S. B. Stylli, Marmer, G., Bannikov, I., Collier, C. R. Y., Wade, A. and M. Strongin, Zerial, H., B. Habermann, S., Bernauer, M., Pilli, M., Stein, kjr,M,Brad,M . ofl .D,Tt,M,Cag . rda,R and R. Fridman, M., Chang, M., Toth, D., R. Bonfil, M., M. Bernardo, M., Ikejiri, temir . lmemn . oet,D . o,J .adShle,R H. R. Scheller, and S. J. Yoo, L., D. Foletti, J., Klumperman, M., S. Steegmaier, P. Patel, and K. H. Shukla, M., P. Shah, N., S. Shukla, D., F. Shah, oet,M,Nwok-ay . ils . lvl .adBrmat P. Birembaut, and C. Clavel, C., Gilles, B., Nawrocki-Raby, M., Polette, Liza R., Poincloux, el,D . zcn,E . r,Ps,I,Tsa,L,Gro,R,Worw M., Woodrow, R., Gordon, L., Tesfay, I., Pass, Jr., F., E. Azucena, F., D. Seals, resi . i-resa . at,J,Sen .N n toigr .L. J. Strominger, and N. J. Stern, J., Watts, A., Gil-Krzewska, K., Krzewski, A. S. Tooze, and R. Watson, B., Larijani, P., Sakya, O., Kakhlon, iseax . aes . ela,F n aene,J. Tavernier, and F. Peelman, D., Lavens, J., Piessevaux, cmled,B,Pehe,D,Ha D., Prechtel, B., Schmalfeldt, osy .L,Kly .adLn .Y. C. Lin, and T. Kelly, L., W. Monsky, and R. Suzuki, T., Yoshioka, H., Nishimoto, H., Matsuda, M., Tanioka, T., Itoh, ffe,S R. S. Pfeffer, aua-aea . eci . eDz . iaia .B,Dve,L,Cmns J., Camonis, L., Daviet, B., J. Sibarita, G., Dez, Le C., Recchi, M., Sakurai-Yageta, J. M. Bissell, and A. Srebrow, D., C. Roskelley, J. M. Bissell, and D. C. Roskelley, Murph A., Remacle, ee,A . cotih . hn . uuua .R,Shle,R .adPrekeris, and H. R. Scheller, R., J. Junutula, J., Chun, E., Schonteich, A., A. Peden, J. Gavrilovic, and G. Murphy, A. S. Courtneidge, and A. D. Murphy, Itohara, and H. Nishimoto, T., Yoshioka, H., Yoshida, M., Tanioka, T., Itoh, unaal,M,Ei,L,Cnoel,G n ornig,S A. S. Courtneidge, and G. Condorelli, L., Elia, M., Quintavalle, vrl,C .adKefl,O. Kleifeld, and and M. Y. C. Yeh, M., Overall, Seiki, H., Sato, W., E. Thompson, L., Howard, J., H., Maurer, Nakahara, Y., Kawakami, H., J. Tsai, A., P. Bromann, B., Diaz, A., D. Murphy, fmtlortiae:srcue ucin n biochemistry. and function, structure, metalloproteinases: of uorinvasion. tumour metalloprotease. membrane the of form Chem. activated the of Isolation collagenase. I. G. family—comparative Goldberg, GTPase selectivity. Rab binding human molecular of the determinants of reveals properties analysis interaction The (2012). 19) eil-soitdmmrn rti sipiae ntasGlinetwork trans-Golgi in implicated is trafficking. 4 vesicle protein membrane Vesicle-associated (1999). 194-202. uorivso n arxmetalloproteinases. matrix and invasion Tumour invadopodia. to trafficking MT1-MMP on focus a infcneo arxmtlortiae2ad9i ratcancer. breast in 9 and 2 metalloproteinase matrix of significance of invasion protease-driven the A. for cells. and S. function, cancer and Courtneidge, formation podosome and for required H. J. Resau, h OSbox. SOCS the ciao sascae ihporsinfo eint dacdoaincancer. ovarian advanced to Res. benign from plasminogen Cancer progression urokinase-type with the associated and is MMP-9, al. activator et (MMP)-2, W. metalloproteinases Kuhn, matrix M., of Schmitt, U., Berger, R., Fridman, AP-adVM7epesn eilsaebt eurdfrcttxcgranule cytotoxic for required both cells. NK are in vesicles exocytosis VAMP7-expressing and VAMP4- granules. secretory neuroendocrine of maturation for required is Metastasis Exp. Clin. M. Uehira, function. xcs ope srqie o uo elivso ontemo d4 n RhoA. and Cdc42 of downstream invasion Biol. cell Cell tumor J. for required is complex P. exocyst Chavrier, and C. D’Souza-Schorey, R. invadopodia. surface cell at metalloproteinase matrix mice. S. Cell inligrgltstsu-pcfcgn expression. gene tissue-specific regulates signalling regulates matrix extracellular function. the cell and is epithelial cells between mammary and information of pathways flow bidirectional different two surface. by cell the internalised to recycled is vitro. (MT1-MMP) in and metalloproteinase vivo in cells muscle smooth Biol. vascular Cell in J. formation podosome of control ealpoens niiosfrcne therapy. cancer for invasion. inhibitors metalloproteinase cell for USA required Sci. invadopodiais Acad. Natl. to Proc. docking metalloprotease T. type1-matrix W. Chen, Izpisu A., R. Stewart, Biol. Cell Opin. Curr. function.invadopodia: characteristics,formation and Chem. Biol. J. S. Mobashery, aha srqie o erlcetcl irto uigebyncdevelopment. embryonic during migration cell crest neural ONE for PLoS required is pathway 19) eue nignssadtmrporsini eaiaeA-deficient gelatinase in progression tumor and angiogenesis Reduced (1998). 20) h C-a1 ope euae noyi rti sorting. protein endocytic regulates complex RCP-Rab11 The (2004). 15 acrRes. Cancer 270 3530-3541. , rnsCl Biol. Cell Trends 5331-5338. , 6 20) a Tae:seiyn n eihrn rael dniyand identity organelle deciphering and specifying GTPases: Rab (2001). 19) xeietlmtsai ssprse nMP9dfcetmice. MMP-9-deficient in suppressed is metastasis Experimental (1999). 181 189 7 e22499. , acrCell Cancer 20) otci ntmrinvasiveness. tumor in Cortactin (2008). 2396-2404. , 280 20) oetmcaimbsdihbtr o arxmetalloproteinases. matrix for inhibitors mechanism-based Potent (2005). yoieGot atrRev. Factor Growth Cytokine raa .adCare,P. Chavrier, and F. ´rraga, 19) rnmmrn/yolsi oanmdae membrane domain-mediated Transmembrane/cytoplasmic (1997). 985-998. , 13-22. , .Ci.Neurosci. Clin. J. 19) ehns fcl ufc ciaino 2katp IV type 72-kDa of activation surface cell of Mechanism (1995). 33992-34002. , 58 o.Bo.Cell Biol. Mol. aBlot,J .adCutede .A. S. Courtneidge, and C. J. ´a-Belmonte, ,G n oh,C. Roghi, and G. y, 1048-1051. , 17 11 u.J Immunol. J. Eur. 177-181. , 614-621. , 7 20) arxmtlortiae n iseinhibitors tissue and metalloproteinases Matrix (2003). 155-165. , 11 94 .Cl Sci. Cell J. 19) rtoyi n elmgain raigapath? a creating migration: cell and Proteolysis (1999). 487-491. , 7959-7964. , 19) yai eirct eiie:acontinuous, a revisited: reciprocity Dynamic (1995). rig . Spa K., ¨rting, ice.Cl Biol. Cell Biochem. 10 15 1957-1972. , 19) idn n oaiaino ()72,000 M(r) of localization and Binding (1993). 21) h is n ot’o oooe and podosomes of ‘outs’ and ‘ins’ The (2011). 20) oad hr eeainmatrix generation third Towards (2006). 725-737. , 20) naooi:a h utn deof edge cutting the at Invadopodia: (2008). 116 20) h neato fIGP ihthe with IQGAP1 of interaction The (2008). 20) h dpo rti k5Fs is Tks5/Fish protein adaptor The (2005). 41 3905-3916. , 3323-3329. , 20) arxivso ytmu cells: tumour by invasion Matrix (2009). 19 rt e.Ocl Hematol. Oncol. Rev. Crit. 19) irrh fECM-mediated of hierarchy A (1995). 20) ebaetp I-matrix type Membrane (2003). r .Cancer J. Br. 371-381. , te . uk,S,Knk E., Konik, S., Rutke, K., ¨the, a.Rv Mol.CellBiol. Rev. Nat. .Cl Sci. Cell J. ur pn elBiol. Cell Opin. Curr. acrRes. Cancer acrLett. Cancer 73 20) nrae expression Increased (2001). 391-397. , 20) h ayfcsof faces many The (2008). ic Res. Circ. MOJ. EMBO LSONE PLoS 94 122 20) G function GGA (2006). ninJ Cancer J. Indian 941-946. , 21) Src-Tks5 A (2011). 21) MicroRNA (2010). 53 3015-3024. , 3159-3164. , 265 20) Clinical (2009). 25 92 157-166. , 49 1590-1602. , 12 7 827-839. , 7 o.Biol. Mol. 736-747. , ,179-186. ,413-426. e34870. , (2011). .Biol. J. (2004). Clin. 46 ,