lsbtaArgenzio Elisabetta and adhesion cell regulates CLIC4 ARTICLE RESEARCH eevd2 aur 04 cetd1 coe 2014 October 13 Accepted 2014; January 24 Received ` Netherlands. Research, The Sanquin Amsterdam, Biology, 1066CX Cell 125, Molecular Plesmanlaan of 121, Department Plesmanlaan address: Institute, *Present Netherlands. Cancer The Netherlands Amsterdam, The 1066CX Biology, Cell 2006), of Division al., et 2012; al., (Gagnon et hearing In al., (Padmakumar 2009). 2010), al., proteins, et healing et (Jiang wound al., Ulmasov CLIC function and et the macrophage angiogenesis Qiu and of platelet 2012; roles in including physiological 2010). non-redundant al., review, et a Littler 2014; (for membrane al., processes et actin other in Jiang and formation, see membrane- implicated transport vacuole and ion been trafficking, reorganization, soluble have intracellular conserved both They highly remodeling, al., in et are forms. exist Littler that 2005; associated and al., proteins vertebrates et globular Littler among are 2014; al., Harrop CLICs et 2001; 2010). Jiang al., et 2001; (Dulhunty al., appear functions et but cellular to (GSTs), distinct related S-transferases have glutathione structurally to of are class that omega (CLIC1–CLIC6) the consists members family six protein of (CLIC) channel intracellular chloride The INTRODUCTION Rab35 trafficking, acid, Integrin Lysophosphatidic adhesion, Cell CLIC4, WORDS: KEY antagonizes and activity Rab35 of suppresses regulation Rab35-dependent CLIC4 that of show recycling LPA-induced or serum- b the LPA and for motility. internalization required the is both cell CLIC4 endosomes. to Rab35-positive increases in CLIC4 and of membrane it recruitment whereas the stimulates signaling, spreading integrin cell adhesion, and cell–matrix cells, MDA-MB-231 decreases and and knockdown HeLa in CLIC4 that (LPA) show CLIC4 we of Here, target(s) elusive. acid exocytic–endocytic subcellular remain and in function CLIC4 lysophosphatidic the for However, role trafficking. by possible to a suggesting membrane recruited serum, rapidly plasma to is in formation CLIC4 the implicated channel remodeling. both ion is membrane in from intracellular ranging and exists processes, forms, (CLIC4) cellular diverse membrane-associated 4 and protein soluble channel intracellular Chloride ABSTRACT n otrH Moolenaar H. Wouter and eieCI4a euao fRb5atvt n eu-adLPA- and serum- and activity Rab35 of regulator a as CLIC4 define eedn nerntrafficking. integrin dependent h LChmlgEC4daaial mar omto and formation impairs dramatically EXC-4 homolog CLIC the uhr o orsodne(.rezonin;[email protected]) ([email protected]; correspondence for Authors nern u o o G eetrtafcig utemr,we Furthermore, trafficking. receptor EGF for not but integrin, 1 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,58–23doi:10.1242/jcs.150623 5189–5203 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. eetreigsuisi iehv eu orva essential, reveal to begun have mice in studies targeting Gene ` or agdn* ail etnPi,Hn ase,Ke aik rodSonnenberg Arnoud Jalink, Kees Janssen, Hans Leyton-Puig, Daniela Margadant*, Coert , anradtselegans Caenorhabditis ` b nerntafcig u results Our trafficking. integrin 1 b nerna h plasma the at integrin 1 irpinof disruption , euaino nerntafcig ecnld htCI4i a of is trafficking CLIC4 agonist-regulated that the conclude in Rab35-dependent We player antagonizes new trafficking. integrin and of activity, regulation Rab35 of suppresses serum/ recycling and internalization LPA-induced the both stimulates CLIC4 CLIC4 of endosomes. integrin recruitment integrin-mediated the and stimulates for LPA to adhesion required that is and cell CLIC4 adhesion, cell that on find MDA-MB- focusing We and behavior. cells, HeLa carcinoma in CLIC4 231 of redistribution of LPA-induced recruitment agonist-induced membrane. a rapid plasma the in the very to about functions CLIC4 however, known CLIC4 route; is that little trafficking on This possibility exocytic–endocytic dependent 2009). the regulated al., strictly et raises manner (Ponsioen integrity a observation F-actin in and activity stimulation, upon we RhoA membrane serum plasma model, translocation the or of transient a regions LPA distinct but to as rapid cytosol the undergoes cells from constituent CLIC4 neuroblastoma that serum al., N1E-115 showed et major (Moolenaar Using are stimulation serum a (LPA) receptor 2004). to LPA responses to is cellular acid attributable many fact LPA in and lysophosphatidic 1993) 2009). al., et in mediator al., (Eichholtz agonists, et lipid receptor (Ponsioen the G-protein-coupled by particular initiated 2006). pathway Ivaska, adhesion, and Pellinen cell 2011; al., of et al., both Caswell et regulation 2012; Integrin Margadant al., the 2009; et manner. (Bridgewater to trafficking, migration key stimulus-dependent and spreading is a recycling endocytic–exocytic in endocytic cytoskeleton. the and to undergo constitutively matrix extracellular the Integrins Cell link transmembrane unknown. heterodimeric that al., integrins, is receptors by et mechanism mediated (Padmakumar is underlying adhesion adhesion the cell However, in defects 2012). healing, to wound impaired due and 2009) erosions apparently skin al., spontaneous et as (Ulmasov well as vacuolization and angiogenesis defective irto n on eln.Thus, for healing. role a wound to differentiation, and angiogenesis, points as evidence migration processes al., al., diverse Growing et such 2007). et (Shukla in nucleus al., CLIC4 the Chuang et to actin-based even Suh also 2003; and 2009; can 2010) and al., Goldenring, it et organelles Chuang but and 1999; protein, vesicles, (Berryman diffusible intracellular structures a and to as expressed cytosol localize ubiquitously the is in CLIC4 regulation detected unknown. and largely function(s) still precise its are yet member, family studied 2003; al., et (Berry 2006). Hobert, functions and distinct Berry vertebrate have and but vertebrate CLICs that tube, invertebrate indicating phenotype, excretory the rescue intracellular cannot CLICs the of maintenance ntepeetsuy estott xmn h ucinand function the examine to out set we study, present the In signaling a in player new a as CLIC4 identified previously We mn h i amla LCpoen,CI4i h best- the is CLIC4 proteins, CLIC mammalian six the Among b b nerna h lsammrn n nRab35-positive in and membrane plasma the at integrin 1 nerntrafficking integrin 1 b nern utemr,CLIC4 Furthermore, integrin. 1 Clic4 2 / 2 b iedisplay mice integrin. 1 5189

Journal of Cell Science hLC) erie LC-pcfcplcoa antibody polyclonal CLIC4-specific a raised (denoted CLIC4 We containing against (shRNAs) vectors shCLIC4). RNAs CLIC4- lentiviral hairpin studies. using small adhesion obtained different cell were are epithelial which cells in cells, depleted models MDA-MB-231 used and down knocked HeLa widely we in CLIC4, expression of function(s) CLIC4 cellular the cells examine CLIC4-knockdown To of analysis and Generation RESULTS ARTICLE RESEARCH hncnrlcls(i.2, n i.2,,respectively). 2E,F, Fig. cells 5190 shCLIC4 and that revealed 2A,B cells (Fig. spreading spread of cells staining less Vinculin serum- appeared control in cells fibronectin HeLa or than collagen-I CLIC4-depleted on medium, plating after free Yamada, h and 1 (Geiger At spreading 2011). cell to and phosphorylation unresponsive protein cells the LPA. rendering panel). by motility, stimulation leads right in further depletion cell CLIC4 directionality 1F, maximal that (Fig. cell suggest to cells results affect cells these CLIC4-depleted not random together, CLIC4-depleted Taken did or of the LPA control that panel). enhanced not either left but factor, 1F, cells, (Fig. motility control of established motility adhesion. cell an reduced than with LPA, consistent faster collagen-I, significantly on CLIC4- cells time- migrated conditions, control using cells serum-free by MDA-MB-231 Under depleted motility microscopy. cell video affected lapse cells CLIC4-depleted was the fibronectin to panel). right adhesion 1E, Cell (Fig. panel). rescued green middle and similarly red 1E, of to (Fig. number collagen-coated adhesion the cells counting on cell by seeded quantified rescued as and GFP–mCLIC4 collagen-I, 1E, ratio, of (Fig. 1:1 Expression alone were a mouse surfaces. in mCherry mixed shCLIC4(#3) or shRNA-resistant panel), (GFP–mCLIC4) an left containing CLIC4 either of rescue cells with version To or transfected HeLa substrates. these transiently shCLIC4(#3) phenotype, to either well this less containing adhered cells, in shCLIC4(#5), HeLa plating CLIC4-depleted MDA-MB-231 1D, after Fig. min and in integrin-binding 30 shown As the at medium. fibronectin, to serum-free and vector) collagen-I empty and substrates CLIC4-depleted (containing of adhesion cells of control level the determined first We assembly adhesion motility focal adhesion, and cell integrin-dependent regulates CLIC4 cells, integrin and control adhesion cell–matrix did in CLIC4 than function. our of focused role plates therefore the We on function. the studies integrin and in to plating) defect a after efficiently suggesting h 24 less cells (at spread CLIC4-depleted adhered well that less morphology observed appeared cell we consistently assessing shown; however, When argues not 2009). adhesion, (results which al., and remodeling et cells, actin Ponsioen in also control CLIC4 see fibers for to role stress similar a against developed stimulation, RhoA- LPA and mitogenic showed in upon contraction role cells cytoskeletal direct CLIC4-depleted no mediated has Furthermore, CLIC4 material that signaling. (supplementary MAPK3 indicating AKT1 as S1), and known Fig. EGF- respectively) (also modulate it ERK1/2 MAPK1, did of and nor activation proliferation, LPA-induced cell of or did rate distinct most knockdown CLIC4 the the shown). affect five not not were data Among and shCLIC4(#5) 1B,C; (Fig. and (qPCR). effective shCLIC4(#3) western PCR by tested, quantitative knockdown shRNAs CLIC4 and stable blotting confirmed and 1A), (Fig. nernlgn idn ed ofclahso formation, adhesion focal to leads binding Integrin-ligand of adhesion cell–matrix reduced the how examined next We eitdeet,ntbycl–arxahso,cl spreading, integrin- motility. cell cell of adhesion, and regulation assembly cell–matrix adhesion the focal notably for required events, is mediated CLIC4 we results, that these From conclude spreading. cell and FAK- reduced assembly with adhesion less consistent focal 2I,J), significantly (Fig. conditions similar showed under pY397 cells CLIC4-depleted seeding. fitgi inln Pros 03.Uo ltn neither at on reached FAK- maximum in a plating with increase time, an over Upon levels showed pY397 cells 2003). HeLa fibronectin, (Parsons, or collagen-I signaling integrin readout the well-established a of measured (FAK-pY397), Y397 residue We as on known PTK2) respectively). also (FAK, kinase fibronectin, adhesion focal 2E,G,H of autophosphorylation or Fig. and collagen-I 2A,C,D (Fig. on to assembly adhesion compared of focal indicative when shControl), impaired number (denoted shRNA and control size containing cells adhesion focal reduced had ebaeuo P tmlto.W oue nthe on focused We stimulation. we plasma LPA the processes, at integrin-mediated upon integrins to in recruited membrane is CLIC4 CLIC4 whether the of examined Given including 2009). types, role al., et cell (Ponsioen apparent various cells HeLa in and stimulation but membrane A431 N1E-115, plasma serum rapid the or undergoes to LPA cytosol CLIC4 upon the that from translocation reported transient previously have to We CLIC4 recruits stimulation LPA al 1,nrddi fettelvl ftepeusradmature and precursor the of levels the of affect forms it CLIC4 did flow nor that S1), of Table expression established Through surface other cell we the or conditions. affect blotting, not did western depletion steady-state and cytometry under expression to integrin- recruitment CLIC4 how investigate To serum- recycling and LPA- induced and internalization, integrin regulates CLIC4 CLIC4–integrin- some although cytosolic, mainly was distribution whereas CLIC4 cells, serum-starved In the and GFP–CLIC4 integrin of images super-resolution to dual-color down obtain resolution depletion optical exceptional ground-state (Fo the method by imaging using cells HeLa microscopy, LPA-stimulated super-resolution and serum-starved both in subunit fibronectin. and collagens expressed ubiquitously LC iecn ncl deinaentsml u to due simply not are adhesion cell expression. integrin in on differences silencing CLIC4 compartments. intracellular in and membrane These to plasma CLIC4 the 3D). recruits at (Fig. stimulation both LPA cells that MDA-MB-231 show ultra-thin results LPA-treated gold-labeled of using microscopy sections electron by confirmed an using by active shown recognizes as that conformation, antibody active the the in note, predominantly Of 3A,B). (Fig. internal compartments with intracellular colocalized in detected CLIC4–integrin- prominently enhanced it integrin; where 1-oleoyl-LPA membrane, with plasma stimulation Following (1 3A). (Fig. observed eeaie h oaiaino LC n h integrin the and CLIC4 of localization the examined We P-nue ooaiaino LC and CLIC4 of colocalization LPA-induced m ;2mn,sgiiatymr LC a eetda the at detected was CLIC4 more significantly min), 2 M; ora fCl cec 21)17 1950 doi:10.1242/jcs.150623 5189–5203 127, (2014) Science Cell of Journal b b b b uui nttlitra elcinfursec mode. fluorescence reflection internal total in subunit 1 -eitdcl dein edtrie integrin determined we adhesion, cell 1-mediated b b nernwsmil on ttepam membrane, plasma the at found mainly was integrin 1 nernpo htclclzdwt LC was CLIC4 with colocalized that pool integrin 1 and - nern(i.4) hs h bevdefcsof effects observed the Thus, 4B). (Fig. integrin 1 a sbnt Fg A upeetr material supplementary 4A; (Fig. -subunits lige l,20) hstcnqeoffers technique This 2008). al., et ¨lling b nern hc eit deinto adhesion mediate which integrin, 1 b nernol Fg 3C). (Fig. only integrin 1 b ooaiainwsalso was colocalization 1 b b integrin 1 ooaiainwsalso was colocalization 1 , b 0n,alwn sto us allowing nm, 10 nernmgtaffect might integrin 1 b nernwas integrin 1 , 0mnafter min 60 b b integrin, 1 integrin 1 b b 1 1

Journal of Cell Science EERHARTICLE RESEARCH n P tmlto erisCI4t nern oha the at both We integrins 3). (Fig. 2007), to compartments Integrin al., intracellular CLIC4 in et 2001). and recruits (White membrane al., plasma LPA stimulation et and LPA serum Roberts and by cell 2011; controlled in al., is role recycling et Norman, and key Caswell Margadant 2012; a al., 2006; et plays (Arjonen motility recycling and adhesion and internalization integrin sd rmitgi xrsin h yai euainof regulation dynamic the expression, integrin from Aside D-B21wr nuae ihanti- 4 with at incubated (TS2/16) an and employed were HeLa shCLIC4 first and MDA-MB-231 We shControl which cytometry. in assay, flow antibody-based and assays the biochemical of affect might CLIC4 of trafficking depletion whether examined therefore nernwsiiitdb rnfrt eu-remdu t37 at medium serum-free to transfer by initiated was integrin ora fCl cec 21)17 1950 doi:10.1242/jcs.150623 5189–5203 127, (2014) Science Cell of Journal ˚ .Teefe,itraiaino elsurface cell of internalization Thereafter, C. b nern sn ofclmicroscopy, confocal using integrin, 1 *** 5th– * the percentiles. represent 95th whiskers by the indicated line, is the 75th median to the 25th and the percentiles, represents box The cells). ( experiments in independent panel) three (right directionality (left and distance panel) the represent Data ImageJ software. using the analyzed and were the cells directionality and the h, by 8 covered during distance min 2 were every Images captured imaging. time-lapse to prior 1 and with overnight stimulated h. starved 48 serum for were adhere Cells to allowed on and plated collagen were cells cell MDA-MB-231 random motility. and CLIC4 (F) experiments. plots mean The represent software. ImageJ the counted using was cells green-stained and number red- The of A. in as treated and and collagen fibronectin, 1:1 on in seeded mixed and were ratio cells Middle panels: control. right loading and as used was actin CLIC4; and RFP GFP, against using antibodies analysis immunoblot by detected GFP–mCLIC4 was and mCherry of (mCLIC4). Expression GFP–CLIC4 the of or version mCherry mouse with transfected were shCLIC4 cells panel: Left CLIC4- phenotype. the deficient of Rescue (E) experiments. rsa ilt h lt ersn the represent plots The Violet. with Crystal stained were cells For quantification, fixed. were and cells away adherent washed non- were plating, cells after adherent min 30 or At collagen-I fibronectin. on plated were shCLIC4 cells and shControl assays. Cell–matrix adhesion (D) qPCR. by were determined GAPDH) to mRNA (normalized CLIC4 levels (C) reference. cell as HeLa lane the control using indicated, is software) (ImageJ densitometry by of determined amount CLIC4 relative The control. loading using antibody; lysate anti-CLIC4 cell total of by immunoblotting determined B). efficiency panel Knockdown in (B) C (labeled vector using empty obtained was The population shown. control are CLIC4 targeting distinct shRNAs Two Methods. the and in Materials described as particles with lentiviral infected were stable cells For knockdown, cells. MDA-MB-231 in and knockdown HeLa CLIC4 (B,C) loading. equal protein shows fusion staining GST Ponceau-S as proteins. purified were CLIC4. CLICs for Distinct specificity antibody the showing cell motility. affects and adhesion knockdown CLIC4 1. Fig. mean P , 6 .0;n,ntsignificant. not ns, 0.001; ..o he independent three of s.d. 6 m independent two of s.d. P P rlf untreated left or LPA M b , -nernantibody 1-integrin b .5 ** 0.05, atnwsue as used was -actin A muolt(IB) Immunoblot (A) P , 0.01, n 5 62 5191 b b ˚ C - 1

Journal of Cell Science EERHARTICLE RESEARCH 5192 ora fCl cec 21)17 1950 doi:10.1242/jcs.150623 5189–5203 127, (2014) Science Cell of Journal ftreidpneteprmnsare experiments independent three out of one of blots Representative loading control. as used were immunoblots and Anti-FAK antibody. pY397 anti-FAK- using analyzed immunoblotting was by FAK indicated of the Activation for time. (J) fibronectin collagen or on (I) seeded were cells HeLa shCLIC4 and shControl by activity. measured FAK as signaling, Integrin (I,J) **** percentiles. 5th– 95th the represent whiskers the by line, indicated the is 75th median to the 25th percentiles, the represents box The experiments; (two independent software ImageJ using number (D,H) adhesion focal and number (C,G) adhesion focal (B,F), cell area of quantification the 10 represent bars: Plots Scale shown. (blue) are DAPI cells and (green) phalloidin vinculin with (red), stained cells Confocal of PFA. sections with fixed Adherent were min. cells 60 for (E–H) coverslips fibronectin-coated or on (A–D) seeded collagen-I- were cells HeLa shCLIC4 signaling. integrin and assembly adhesion cell focal reduces spreading, depletion CLIC4 2. Fig. hw ne h lt.**** blots. the under shown (mean analysis Densitometric shown. 6 ..)o he xeiet is experiments three of s.e.m) P , 0.0001. hoto and shControl n P 5 , 0cells). 50 0.0001. b -actin m m.

Journal of Cell Science EERHARTICLE RESEARCH i.3. Fig. e etpg o legend. for page next See ora fCl cec 21)17 1950 doi:10.1242/jcs.150623 5189–5203 127, (2014) Science Cell of Journal 5193

Journal of Cell Science el eesiuae ih1 with and stimulated (green) were CLIC4 Cells show Dual- images GFP–CLIC4. with super-resolution transfected color with were CLIC4 coverslips collagen-coated of on colocalization cells induces LPA 3. Fig. 1.5 5 ag n ml odprils epciey rospitto membrane. point Arrows plasma respectively. PM, particles, cytoplasm; gold small and and CLIC4 large sectioning. ultrathin for 1 collected with stimulated collagen-I-coated were on Cells dishes cells. (green), MDA-MB-231 LPA-stimulated GFP–CLIC4 of show microscopy images Confocal min. 2 active and for overnight LPA starved collagen-coated with on serum stimulated seeded GFP–CLIC4, were with cells transfected HeLa coverslips, stimulation. LPA upon integrin mean ( represents experiments plot The membrane. the plasma inside area CLIC4-positive the EERHARTICLE RESEARCH 5194 al., et Powelka 2013; al., et internalized Margadant of Recycling 2012; 2004). al., et (Arjonen EIA rtcl ouigo integrin on focusing assay immunosorbent protocol, enzyme-linked (ELISA) and biotin-labeling a of using recycling synchronized important LPA-regulated, is CLIC4 and plasma that LPA internalized serum- appears using the thus the as obtained It were for to shown). cells, results not CLIC4-depleted Similar pool (data alone 5C. in Fig. integrin reduced in quantified internal strongly was contrast, the marked membrane In of internalized cells. control of redistribution in min) occurred recycling 15 membrane rapid plasma to 5A,B), Fig. 5 in shown (within As serum. with stimulation LC-eltdcls(i.6) eyln nteasneof reduced absence addition, In the points. time in in later after at recycled Recycling reduced cells had also 19% 6A). was control CLIC4 (Fig. only in whereas cells stimulation, membrane CLIC4-depleted serum integrin plasma of the min internalized above 5 to the the back with of recycled Consistent 55% Methods). and results, Materials (see 2001) m oqatf hspoes emaue nerntrafficking integrin measured we process, this quantify To .Bxdaesaesona ihrmgiiaino h ih.Saebar: Scale right. the on magnification higher at shown are areas Boxed m. m .()Clclzto ewe LC and CLIC4 between Colocalization (B) m. b nern(nioy9G,rd n AI(le.()Immunoelectron (D) (blue). DAPI and red) 9EG7, (antibody integrin 1 n b 5 integrin. 1 el) * cells). 7 P , m .5 C LC ooaie ihactive with colocalizes CLIC4 (C) 0.05. P o i rlf nrae.Saebar: Scale untreated. left or min 2 for LPA M m P o i,adfxdclswere cells fixed and min, 2 for LPA M b -nernpstv ra(0% tthe at (100%) area 1-integrin-positive b nernwste nue by induced then was integrin 1 b 6 nernwr tie with stained were integrin 1 b b ...o he independent three of s.e.m. nernbc othe to back integrin 1 nernwsmaue as measured was integrin 1 a 5 b b a integrin. 1 Rbrse al., et (Roberts 1 5 b b nern(red). integrin 1 a already had 1 b nern Cyt, integrin. 1 A HeLa (A) b 1 ebae thsrcnl eoecerthat clear plasma the become to integrin recently returned Integrin particular is has that S2B). It system integrin endosomal membrane. of Fig. the amount in the material sorting determine subsequent supplementary and internalization 6B; (Fig. cells sseii.W oue nteEFrcpo EF) hc has which (EGFR), integrin receptor with co-traffic EGF to the shown on trafficking been integrin focused on We CLIC4 specific. of effect is the whether trafficking asked EGFR next for We required not is CLIC4 o6h.Cmaal otetroe bevdi te cell other in observed turnover the in to incubated (up Comparable recycling were and h). internalization 6 cells for were to allow integrins the to surface medium subsequently Cell complete therefore degradation. and 2010; We increased al., CLIC4-knockdown biotinylated, 2012). to in et al., led defect Lobert et recycling cells 2012; Steinberg the whether 2012; al., investigated al., et et Dozynkiewicz the Margadant 2012; that al., of and tail et cytoplasmic sorting receptors, the on in depends factor signals internalization and growth recycling between like balance much lysosomes, ytm,mr hn5%of 50% than more systems, le h taysaelvl fEF ttepam ebaein membrane plasma the at EGFR not of did depletion levels CLIC4 steady-state 6D, the Fig. in alter shown As 2009). al., et Muller h osiuieedctssadtesrm n LPA-regulated and serum- the of and recycling endocytosis the constitutive in the observed were 6C). degradation (Fig. integrin cells CLIC4-depleted of kinetics Similar eyln of recycling noyoi ymauigteitraiaino integrin of internalization the measuring by endocytosis S2A). Fig. material (supplementary CLIC4 anti- against an using assay, based atr LS.W on httepo fitraie integrin internalized of pool the by that followed a found protocol We ELISA. biotin-labeling capture aforementioned the using hf rm4 from shift 5 i.4 LC ncdw osntaffect not does knockdown CLIC4 4. Fig. I)aayi fttlcl lysates. cell total of analysis (IB) b affect of not levels does surface knockdown three the CLIC4 of that out indicate one of experiments negative histograms independent a Representative as line). used black was (dashed only control antibody secondary with Staining were cytometry. cells anti- MDA-MB-231 with and incubated HeLa trypsinized, shCLIC4 and shControl (A) ae oehr hs eut hwta LC euae both regulates CLIC4 that show results these together, Taken eas xmndwehrCI4wsrqie o integrin for required was CLIC4 whether examined also We b nerni hoto n hLC el a nlzdb immunoblot by analyzed was cells shCLIC4 and shControl in integrin 1 udrsrmfe odtos,a nue yatemperature a by induced as conditions), serum-free (under 1 ora fCl cec 21)17 1950 doi:10.1242/jcs.150623 5189–5203 127, (2014) Science Cell of Journal ˚ Cto37 b b nern ihu fetn nerndegradation. integrin affecting without integrin, 1 nerni eea a ofre naFACS- a in confirmed was general in integrin 1 ˚ b ,wsrdcdby reduced was C, a nern B xrsino rcro n mature and precursor of Expression (B) integrin. 1 5 b ,i otdt aeedsmsand endosomes late to sorted is 1, b nioyadtodsic shRNAs distinct two and antibody 1 b -nernatbd n nlzdb flow by analyzed and antibody 1-integrin b a atnwsue slaigcontrol. loading as used was -actin 5 b b n bqiyain(Bo ubiquitylation and 1 a erddwti h. 6 within degraded was 1 a , 5 b b 0 nCLIC4-depleted in 50% nernexpression. integrin 1 Csele l,2008; al., et (Caswell 1 b nern in integrin, 1 ¨ttcher a 5 b 1,

Journal of Cell Science 2hat37 nern(re)adDP-tie bu)cls cl as 10 ( bars: indicated Scale is cells. (blue) experiments DAPI-stained and (green) integrin EERHARTICLE RESEARCH B21()clso olgncae oesiswr eu tre n aee ihanti- with labeled and starved serum were coverslips collagen-coated on recycling. cells integrin (B) serum-stimulated MB-231 regulates CLIC4 5. Fig. ˚ nsrmfe eim nernrccigwssiuae y1%FSfrteidctdtm eid.Iae ersn ofclscin of sections confocal represent Images periods. time indicated the for FBS 10% by stimulated was recycling Integrin medium. serum-free in C n 5 17–26). m .()Tepretg fclswt recycled with cells of percentage The (C) m. AB nioybsditgi eyln sa.sCnrladsCI43HL A n MDA- and (A) HeLa shCLIC4#3 and shControl assay. recycling integrin Antibody-based (A,B) b -nernatbd TS2/16. antibody 1-integrin ora fCl cec 21)17 1950 doi:10.1242/jcs.150623 5189–5203 127, (2014) Science Cell of Journal b nerna n 5mnfo w independent two from min 15 and 5 at integrin 1 b nernitraiainwsalwdfor allowed was internalization integrin 1 5195 b 1

Journal of Cell Science EERHARTICLE RESEARCH 5196 37 at min 30 for proceed to allowed was Internalization NHS-S-S-Biotin. with integrin surface-labeled of were recycling cells and HeLa internalization shCLIC4 the regulates CLIC4 6. Fig. needn xeiet.()CI4kokonde o fetitgi erdto.Boi-aee eaclswr nuae t37 at incubated were cells HeLa Biotin-labeled degradation. integrin affect not does knockdown CLIC4 (C) experiments. independent on.()CI4kokonde o fetEF erdto.Clswr etutetdo tmltdwt 0 gm G t37 at EGF ng/ml 100 with stimulated or untreated left were Cells degradation. EGFR affect not does knockdown CLIC4 (G) point. eue nernitraiain el eeboi-aelda nA olwn nenlzto o h niae ie t37 at times indicated the for internalization the Following against A. antibody in monoclonal as a biotin-labelled using were ELISA Cells capture internalization. by integrin determined reduces was integrins biotin-labeled internalized, nlzdb lwctmtya nE h ltrpeet means represents plot The E. in as cytometry flow by analyzed noyoi.Clswr aee ihat-GRatbd si n usqetysitdt 37 to shifted subsequently and E in as antibody anti-EGFR with labeled were Cells endocytosis. eN t4 at MesNa xeiet r hw.()CI4kokonde o fetEF eyln.Clswr nuae ih2 gm G o nie n hnsitda 37 at shifted then and ice, on indepe h two 1 of for out EGF one ng/ml from 20 with results incubated b Representative were analyzed line). Cells and black recycling. antibody EGFR (dashed anti-EGFR affect control with not negative incubated does trypsinized, as knockdown CLIC4 were used (E) Cells was shown. levels. only are surface antibody experiments cell secondary EGFR with affect remaining Staining not of cytometry. does amount knockdown The CLIC4 FBS. (D) 10% experiments. plus DMEM in periods soitdboi a eue t4 at reduced was biotin associated GRlvl eedtrie yimnbo I)aayi fttlcl yae sn niEF antibody. anti-EGFR shown. using are lysates experiments cell two total of of out analysis one (IB) from immunoblot by blots determined were levels EGFR o 5mn(us)t lo GRitraiain el eeaaye yfo yoer.FrdtisseMtrasadMtos h ltrepresents plot The Methods. and Materials see details For cytometry. flow by analyzed were Cells internalization. EGFR allow to mean (pulse) min 15 for 6 ...o w needn xeiet.I ahasy 000clswr nlzdfrec iepit F LC ncdw osntafc EGFR affect not does knockdown CLIC4 (F) point. time each for analyzed were cells 50,000 assay, each In experiments. independent two of s.e.m. ˚ ,adteclswr nuae ih1%FSa 37 at FBS 10% with incubated were cells the and C, ˚ .Teaon finternalized of amount The C. a 5 6 b ...o w needn xeiet.I ahasy 000clswr nlzdfrec time each for analyzed were cells 20,000 assay, each In experiments. independent two of s.e.m. nernwsdtrie si ,) aarpeetmean represent Data A,B). in as determined was integrin 1 ˚ o h niae ieprost rmt eyln fteitra ol h mutof amount The pool. internal the of recycling promote to periods time indicated the for C a 5 b nernwsdtrie si .Dt nABrpeetmean represent A,B in Data A. in as determined was integrin 1 a 5 b ,btnto EGFR. of not but 1, ora fCl cec 21)17 1950 doi:10.1242/jcs.150623 5189–5203 127, (2014) Science Cell of Journal A LC ncdw eue nernrccig hoto and shControl recycling. integrin reduces knockdown CLIC4 (A) ˚ o h niae iepito nenlzto.Clswere Cells internalization. of point time indicated the for C b atnwsue slaigcnrl Representative control. loading as used was -actin a nernsbnt B LC knockdown CLIC4 (B) subunit. integrin 5 ˚ ˚ with reduced was biotin surface Cell C. srmfe odtos,membrane- conditions), (serum-free C 6 ...o w independent two of s.e.m. ˚ o h niae iepoints. time indicated the for C ˚ 6 o h niae time indicated the for C ...o three of s.e.m. ndent flow y ˚ C

Journal of Cell Science lsammrn ylo o)adi nenlcmatet oag o) oe ra nACaesona ihrmgiiain cl as 10 bars: Scale magnification. higher at shown are A–C in areas Boxed box). (orange compartments internal in and box) (yellow membrane plasma 2 (green), niae,LAwsaddfr2mn e rosidct oprmnspstv o LC n a3.TeMne’ ofiin (mean coefficient Mander’s The Rab35. and CLIC4 for positive compartments ( indicate software arrows ImageJ Red min. the 2 using for 2 added was (green), LPA GFP–CLIC4 indicated, show images Confocal (B) EERHARTICLE RESEARCH lsisadsrmsavdoengt el eete tmltdwt P o i rlf nrae.()Cnoa mgsso F–LC gen,EEA (green), GFP–CLIC4 show images Confocal (A) untreated. left or min 2 for LPA with stimulated then were Cells and endosomes. overnight. (red) Rab35-positive starved to serum CLIC4 and directs plasmids stimulation LPA 7. Fig. 6 b nern(le.Rdarw,cmatet oiiefrCI4 E1and EEA1 CLIC4, for positive compartments arrows, Red (blue). integrin 1 y–a3 rd and (red) Myc–Rab35 n 5 5cls rmtreidpnetexperiments, independent three from cells, 25 b nern(le nLAsiuae el.Arwed on otiatt ooaiaino LC,Rb5and Rab35 CLIC4, of colocalization tripartite to point Arrowheads cells. LPA-stimulated in (blue) integrin 1 6 y–a3 rd n AI(le.Arw niaecmatet oiiefrCI4adRb5 Where Rab35. and CLIC4 for positive compartments indicate Arrows (blue). DAPI and (red) Myc–Rab35 eaclso olgnIcae oesiswr rnfce ihteindicated the with transfected were coverslips collagen-I-coated on cells HeLa P , .05btentetocniin) C ofcliae hwGFP–CLIC4 show images Confocal (C) conditions). two the between 0.0005 b nern elwarw,cmatet oiiefrCI4adEEA1. and CLIC4 for positive compartments arrows, Yellow integrin. 1 ora fCl cec 21)17 1950 doi:10.1242/jcs.150623 5189–5203 127, (2014) Science Cell of Journal 6 ... a calculated was s.e.m.) b nerna the at integrin 1 m m. 5197 1

Journal of Cell Science EERHARTICLE RESEARCH 5198 and late meet compartments early, might trafficking the of CLIC4 identify where populations To distinct endosomes. membrane recycling define of regulators and key are family trafficking Rab the of GTPases Small endosomes positive with colocalizes CLIC4 of trafficking the manner. specific regulates a CLIC4 ligand- in that integrin for required conclude these of not CLIC4 time, we basis the is results, On of CLIC4 EGFR. over of independent that degradation and appears trafficking manner decreased induced thus a It levels 6G). in (Fig. EGFR degradation, of stimulation, 6E,F). indicative (Fig. EGF 2008) as al., endocytosis, et After EGFR (Sigismund cytometry affect flow ligand- it by affect did measured nor not recycling, did EGFR silencing induced CLIC4 Furthermore, cells. HeLa xrsigHL el,CI4ddntclclz ihArf6, with colocalize GFP–CLIC4- not In did lysosomes. a CLIC4 and (LAMP1), cells, endosomes HeLa 1 late expressing protein for effector membrane Rab-5 endosomes, marker early the lysosomal-associated of marker as a and well (EEA-1), 1 as antigen endosome markers, early endosomal as GTPases b nerni al n Rab35- and early in integrin 1 b nern eue eea Rab several used we integrin, 1 b 1 oivsiaeterltosi ewe LC n Rab35 and CLIC4 between relationship in the activity investigate To activity Rab35 affects CLIC4 oiiecmatet ls otepam membrane. plasma to the to CLIC4 close of compartments positive also recruitment that the compartments promotes was intracellular for in Rab35 the stimulation contained at and Rab35 LPA and with membrane colocalization Upon plasma increased CLIC4 showed cells. CLIC4 plasma min, of non-stimulated 2 the colocalization compartments in at intracellular observed found Some in extent, serum- mostly 7B). lesser In (Fig. was 2006). a al., to Allaire Rab35 et and, 2010; Kouranti membrane cells, 2010; al., HeLa al., et et starved Chua (Allaire 2013; recycling al., et endocytic of 7A). regulator (Fig. endocytosis integrin observed small on the CLIC4 with a of consistent effect endosomes, However, with early colocalize EEA1-positive shown). to in found not was CLIC4 of (data subpopulation in nor cells serum-starved in neither serum-stimulated LAMP1, or Rab11A Rab7, Rab4a, enx xmndRb5 hc a eetyeegda a as emerged recently has which Rab35, examined next We ora fCl cec 21)17 1950 doi:10.1242/jcs.150623 5189–5203 127, (2014) Science Cell of Journal b nern(i.7) ecnld htLAsignaling LPA that conclude We 7C). (Fig. integrin 1 b nerntafcigadLAsgaig we signaling, LPA and trafficking integrin 1 rsiuae ih1 with untreated stimulated left or and overnight starved serum 2 with cells transfected shCLIC4 were and shControl assay. down pull- GTP-Rab35 LPA- manner. an independent in activation Rab35 knockdown stimulates CLIC4 (C,D) qPCR. determined by were GAPDH) levels to mRNA (normalized Rab35 B, In control. antibody; anti-Rab35 using (IB) by immunoblotting determined was A, efficiency In knockdown vector. empty were using cells obtained Control shown. are targeting Rab35 shRNAs distinct and Two Materials Methods. the in described as were obtained cells knockdown cells. Rab35 HeLa Stable in knockdown Rab35 (A,B) trafficking. Rab35- integrin on regulated effects opposing has activity and Rab35 suppresses CLIC4 8. Fig. ersnaieboso n u fthree of out one of blots Representative loading. GST–RBD35 equal shows staining Ponceau-S (5%). lysate total cell of analysis immunoblot by determined was Rab35 by Total analyzed analysis. was immunoblot beads the to GTP-Rab35 bound cells. transfected cell from total lysates with incubated was GST–RBD35 needn experiments. independent Data 7B. means Fig. represent in described as treated and NHS-S-S-Biotin were with cells surface-labeled HeLa shRab35 and shControl endocytosis. integrin increases knockdown Rab35 (F) experiments. independent Data 7A. Fig. mean in represent described as treated and biotin-labeled were Cells affect recycling. not integrin does knockdown Rab35 (E) (mean experiments independent three of analysis shows densitometric D shown. are experiments b atnwsue sloading as used was -actin 6 b 6 ...o two of s.e.m. nerni Rab35- in integrin 1 ...o two of s.e.m. m P o min. 2 for LPA M 6 Myc–Rab35, b integrin 1 6 s.d.).

Journal of Cell Science a3 dt o hw) et emaue h eeso active of levels the measured of we localization Next, subcellular shown). the not CLIC4 alter (data whereas of Rab35 membrane, detectably ability plasma not the the did to depletion a affect CLIC4 versa of not recruit vice to expression did LPA and (or mutant) depletion Rab35 expression, Rab35 dominant-negative Rab35 CLIC4 Furthermore, that distinct affect 8A,B). showed (Fig. not two analyses did using qPCR knockdown lines and cell Immunoblot shRNAs. HeLa Rab35-depleted generated ARTICLE RESEARCH ta. 09,w o hwta P tmlto rmtsthe promotes stimulation LPA that show with (Ponsioen CLIC4 now of LPA we colocalization to 2009), response and rapidly in al., is adhesion membrane CLIC4 et plasma that cell–matrix the finding to previous epithelial recruited our on in Building serum- CLIC4 motility. the in of CLIC4 for importance role of new recycling LPA-stimulated a and uncovers study present The DISCUSSION integrin of not recycling was LPA-induced Rab35- Rab35 or in CLIC4, serum- a cytometry Unlike the for flow cells. required and control versus protocol depleted ELISA CLIC4 and between interplay affected labeling observed Rab35 the how and examined next We on Rab35 trafficking and integrin CLIC4 of effects signaling. Opposing Rab35 LPA suppresses of of CLIC4 cells independent presence that manner and appears a CLIC4-depleted absence in thus the activity, in It in 8C,D). both Rab35-GTP (Fig. cells, LPA in control to increase compared observed we twofold Interestingly, 2011). a al., et (Fukuda (1 assays stimulation pulldown before cells LPA HeLa after CLIC4-depleted and and shControl in Rab35-GTP otn eisadsalGPsso h r n a families Rab Ivaska, and and Pellinen Arf 2011; al., the et Margadant of 2009; al., GTPases et small (Caswell kinases, and including nexins families protein requires sorting diverse and of function, action integrin concerted regulate the to mechanism pivotal a as o ohtecntttv nenlzto fintegrin of internalization required constitutive be to the appears both CLIC4 for endosomes. Rab35-positive in and integrin of internalization the contrast, affect not did hsahso eetms ieyudristeipie wound impaired less the underlies and adhere in likely keratinocytes, observed most wild-type to healing skin study, defect by secreted adhesion in latter found matrix this adhesion the a were cell In to efficiently 2012). reduced keratinocytes al., to CLIC4-deficient et and cell (Padmakumar 2010), reduced keratinocytes al., to cells epithelial et leads pigment retinal (Chuang in depletion motility increased CLIC4 previous and with adhesion that line in it showing are results adhesion studies, Our below), cell and Rab35. CLIC4-regulated requires (discussed activation whether motility and examined Rab35 endocytosis be depletion. opposes to integrin remains CLIC4 CLIC4 promotes upon that thereby motility find focal cell we adhesion, Although and cell–matrix assembly, in might differences findings adhesion These observed recycling. the LPA-dependent explain or serum- its as of suppressor a is Rab35 that , n P-tmltditgi eyln,weesCI4and CLIC4 internalization. integrin whereas on effects recycling, opposing serum- have regulates integrin Rab35 Rab35, not LPA-stimulated but CLIC4, and that indicate results our 5 0 pnRb5kokon(i.8) togysuggesting strongly 8F), (Fig. knockdown Rab35 upon 50% nerntafcigtruhteedsmlsse a emerged has system endosomal the through trafficking Integrin b Fg E aantson.Frhroe a3 depletion Rab35 Furthermore, shown). not data 8E; (Fig. 1 b nerndgaain(aantson.I marked In shown). not (data degradation integrin 1 Clic4 b nerntafciguigtebiotin- the using trafficking integrin 1 2 / 2 b a ie(amkmre l,2012). al., et (Padmakumar mice m nerna h lsamembrane plasma the at integrin 1 5 ;2mn yuigRab35-GTP using by min) 2 M; b b nenlzto.I conclusion, In internalization. 1 nern n nesoe the underscores and integrin, 1 a 5 b b a nrae by increased was 1 1 a 5 b swell as 1 nbt nenlzto n tmltdrccigof recycling stimulated impacts and intriguingly which internalization trafficking, both CLIC4 integrin on identify in now player data new Our a as 2013). Norman, and Rainero 2006; a esrd ssc,ti e-pde o xld the exclude not does set-up this such, cell As complexes the measured. integrin–antibody on internalized appearance was the previously and of stripped surface were antibodies bound eotdt ooaiewt integrin with colocalize general. to in reported trafficking. trafficking on vesicular acts on EGFR CLIC4 than that rather affects suggesting cargo depletion, specific CLIC4 were CLIC4 by degradation affected and whether not recycling endocytosis, tested EGFR, EGFR notably Importantly, integrins most also active that receptors, Given factor we 2012). their growth 2012; al., with al., et in co-traffic Margadant et can 2010; mainly Dozynkiewicz the al., 2012; et consist with al., Lobert agreement et integrins (Arjonen in internal conformation conformation, that internalized active with notion their colocalizes in predominantly integrins CLIC4 that seems yooe,adt rtc integrin protect to and lysosomes, h bec fCI4 hs LC n LC c ndistinct in act CLIC3 and CLIC4 Thus, CLIC4. in reduced of not integrins explains are absence internalized levels also the integrin of and steady-state that sorting pathways, observation degradation the the and for that recycling suggests required between strongly not This is degradation. in CLIC4 integrin detected affect depletion not CLIC4 not is and does compartments, CLIC4 lysosomal however, or CLIC3, endosomal (Dozynkiewicz late Unlike membrane 2012). plasma al., the et to recycling its promoting LC ooaie with colocalized CLIC4 effects. compensatory exert other the not that do suggesting members shown), not family (data proteins of CLIC other fate of the regulate differentially internalized and compartments subcellular nooe ugssta h CLIC4–integrin- the that suggests 2001). endosomes al., localizes the CLIC4 with that et observation with through The endosomes. (Roberts colocalized Rab35-positive recycling CLIC4 compartment Rab11, integrin Instead, with recycling or long-loop a recycling, perinuclear Rab4, stimulates integrin with short-loop colocalize which fast not of did mediator CLIC4 However, endosomes. 03.Ipraty ntelte td,clswr nuae for incubated were 37 cells at study, latter h 2 the in that Importantly, reported 2013). authors the increased however, knockdown study, Rab35 subsequent a also on In study Rab35 2010). integrin previous of a effect fine-tune results, no to our found with mechanism agreement Rab35-positive In regulatory to endocytosis. a CLIC4 of is recruitment suggests compartments inhibition the cells through Possibly, endocytosis Rab35. CLIC4-depleted integrin of in stimulate can higher Rab35 CLIC4 is that not activity Instead, thus Rab35 is Rab35 that stimulation. and on activity CLIC4 effects LPA opposing and Rab35 exert internalization, serum, upon that integrin or suppresses observation LPA altered by the detectably triggered Rab35 with recycling that integrin find consistent We affect 2006). al., not 2013; et either al., does Kouranti et be 2012; Allaire can al., 2010; effects et al., Chesneau its et system, (Allaire inhibitory cell or and stimulatory cargo the on depending steps pathway. distinct trafficking at integrin occurring the phenomenon, short-lived along a be to likely nte ebro h LCfml,CI3 a been has CLIC3, family, CLIC the of member Another ossetwt LC euaigitgi internalization, integrin regulating CLIC4 with Consistent a3 euae noyi rfikn fdvrepoen,and proteins, diverse of trafficking endocytic regulates Rab35 b nerni utasbe falEA-o Rab35-positive or EEA1- all of subset a just in integrin 1 ora fCl cec 21)17 1950 doi:10.1242/jcs.150623 5189–5203 127, (2014) Science Cell of Journal ˚ ihat-nernatbde,atrwihsurface- which after antibodies, anti-integrin with C b nern LC elto i o fettelevels the affect not did depletion CLIC4 integrin. 1 b b noyoi.Teuepce finding unexpected The endocytosis. 1 nerni E--oiieearly EEA-1-positive in integrin 1 b b nernrccig(lar tal., et (Allaire recycling integrin 1 nernrccig(lar tal., et (Allaire recycling integrin 1 b a a nerni ustof subset a in integrin 1 5 5 b b nlt nooe and endosomes late in 1 rmdgaainby degradation from 1 b neato is interaction 1 b nern It integrin. 1 5199

Journal of Cell Science h LC)(tue n etc,21) ie htCl that Given 2013). Jentsch, and (Stauber CLICs) the noyoi n o LC upessRb5activity. Rab35 cell CLIC4-mediated studies suppresses in such with difficulty Rab35 One established. of CLIC4 be to remains role adhesion possible how a internalization, on Furthermore, and Rab35 and increased CLIC4 endocytosis of of effects opposing apparently result of here. absence find the we the what actually with in consistent ‘re-surfacing’ is enhanced Rab35 the that possibility ARTICLE RESEARCH uiiduigN-hoaorpyadglflrto.Hstgwas His-tag filtration. gel 5200 and Ni-chromatography using BL21 purified in expressed was CLIC4 His-tagged generation antibody CLIC4 (acti-stain were phalloidin-red iodoacetamide and EGF and Sigma. MesNa from fibronectin, puromycin, 1-oleyl-LPA, Reagents METHODS AND MATERIALS Cl with conjunction in Cl pump and regulated proton channels is 2009). V-ATPase-driven normally in acidification the unknown al., Vesicular by is acidification 2012). mechanism al., et phagosome underlying et (Jiang the (Ulmasov to again, but, contributes mechanism macrophages CLIC1 cells, unknown fusion Similarly, endothelial vacuolar an In and acidification through regulation. intravesicular pH supports CLIC4 intravesicular in proteins 2014), al., thus et to pathway, (Jiang given CLIC4 integration a signal allow along facilitating partners might distinct interactions with dynamic. Therefore, interact highly transient briefly strong and/or proteins. and/or a weak weak be other for to Such evidence and likely compelling are CLIC4 interactions al., no CLIC4 between yet cytoskeletal et binding as shown). Suginta include is direct not 2003; there CLIC4 (data but Goldenring, 2001), interaction and for (Berryman direct partners components a binding we detect screen, Candidate to proteomic unable a were In signaling. integrin found receptor specific in acts of and CLIC4 how Identification clarify trafficking to challenge. help should major partners binding a remains GTPase- members 2010). al., et and Chua 2013; al., et factors (Chaineau proteins activating guanine-nucleotide-exchange by regulated is activity Rab35 multiple its with (2) that consistent effectors, and and distinct processes, elusive, numerous six remains mediating least CLIC4 at of on function acts precise Rab35 the (1) that is ridrcl,VAPs ciiyo l hne ucinin function channel ClC explore or to compartments. activity endocytic interesting distinct V-ATPase endocytic– be directly indirectly, either to modulate, will might or CLICs) other key it (and CLIC4 events, whether is trafficking acidification exocytic intravesicular dependent rmRce H-SBoi a rmPec.Atbde sdwere: used Antibodies Pierce. from from anti- was was monoclonal NHS-SS-Biotin collagen were tablets Roche. Type-I cocktail from inhibitor Invitrogen. protease EDTA-free from BioMaterials. Inamed were Inc.) Cytoskeleton, ln b1 abohm n oylnl ln 05 at Cruz Santa 1005, (Invitrogen). Chicago, antibodies clone secondary Group, Alexa-Fluor-conjugated (ProteinTech polyclonal, and IL) anti-Rab35 and (monoclonal, polyclonal anti-EGFR Calbiochem, Biotechnology), Glukhova), Ab-1, Marina from clone gift (a monoclonal Signaling), anti-vinculin (Cell anti-FAK polyclonal 9E10, monoclonal below), a (clone and see anti-FAK-pY397 9GE7, anti-Myc in-house, (made monoclonal clone anti-CLIC4 Vestweber), polyclonal and Covance), Dietmar Bank from gift Hybridoma kind Studies anti-human Developmental monoclonal TS2/16; Pharmingen), (BD integrin fitrs,eegn vdnesget oefrCLIC for role a suggests evidence emerging interest, Of family its and CLIC4 of action of mode the Elucidating the of basis mechanistic the address should studies Future b nerni F–LC muorcptts u we but immunoprecipitates, GFP–CLIC4 in integrin 1 b 2 atn(ln C1,Sga,mncoa anti- monoclonal Sigma), AC-15, (clone -actin /H + xhneso h l aiy(neae to (unrelated family ClC the of exchangers .coli E. TM n LC rti was protein CLIC4 and 5 hlodnfrom phalloidin 555 b nern(clone integrin 1 b 1integrin 2 a 2 5 - ouncnann uiidCI5 h rs-dobdsrmwsthen a was through Scientific). AminoLink serum passed the a cross-adsorbed using first purified The obtain was affinity CLIC5. To serum purified immunization. the containing CLIC4, column rabbit against before antibody protease specific TEV with removed LC n a3 tbekokonadcnrlclswr eetdand 2 selected transfection. with were after cells medium h control calcium in and 48 the maintained knockdown collected stable using was Rab35 hairpins virus and RNA CLIC4 the single and HEK293T as the protocol, infection, and with used phosphate stable transfected were for pLKO.1 and were particles vectors cells vectors lentiviral Empty TRCN0000380003 generate used. lentiviral To were Sigma controls. the respectively, library; v2.0; TRC005, in shRNA pLKO library shRNAs human Rab35 shRNA TRCN0000380335) (TRC human human two CLIC4 (TRC and human TRCN0000044358-TRCN0000044362) distinct Sigma five studies, itnecvrdb h el n eldrcinlt eeaaye using analyzed were directionality cell and software. cells ImageJ the by covered distance ua eaadMAM-3 el eegoni Dulbecco’s in grown bovine 37 were fetal at 10% (FBS) cells with serum supplemented (DMEM) MDA-MB-231 medium Eagle’s and modified HeLa transfections Human and infections culture, Cell ahdtiewt B n nuae o i ihDP ro to prior DAPI with min were 5 Immu-Mount for Cells incubated with and antibodies. PBS mounting secondary with min twice Alexa-Fluor-conjugated 30 at washed for indicated min incubated the the 30 then with and for PBS, h with with PBS, 1 twice in for washed antibody, BSA incubated primary 2% were for Coverslips with PBS temperature. blocked in room fibronectin-coated BSA subsequently 0.2% Fixed and or X-100, temperature. min room Triton at 10 0.1% collagen-I- min with 10 permeabilized on for were PFA cells grown 4% with were fixed and coverslips cells imaging, analysis For image and microscopy Fluorescence times 10 three BSA. washed 0.1% and with trypsinized supplemented were DMEM cells with assays, adhesion cell assays For motility and adhesion Cell %PAfr1 i tro eprtr n emaiie ih0.1% with permeabilized and with temperature fixed room to PBS, at prior with min overnight briefly 10 starved for washed serum PFA were were 4% Cells cells LPA. h, with 24 stimulation After coverslips. 1.5 (Fo method (63 imaging microscope confocal TCS-SP5 Leica a on examined GA 0m aprpopae 0m Na mM 10 Na-pyrophosphate, mM 20 EGTA, rtaeihbtrcocktail. inhibitor protease ls05 S) el eeicbtdfr3 i t37 at min 30 (DMEM for buffer incubated blocking were with Cells blocked BSA). 1.5 and 0.5% coating, with plus no or coated fibronectin plates 96-well ntutos el eelsdi Sbfe 5 MHPSp 7.5, pH HEPES MgCl mM mM (50 1.5 X-100, buffer Triton 0.5% JS glycerol, 1% in or NaCl, lysed (Roche) mM 150 9 were X-tremeGene Cells with instructions. performed were Lipofectamine studies imaging for xoet20Mmcocp qipdwt etd(37 heated Zeiss a a with with equipped acquired microscope were M images 200 1 Phase-contrast Axiovert with untreated. stimulated left either or and LPA overnight starved serum 2012). plates, al., six-well et with (Margadant analyzed F-actin described and for as with antibody software stained ImageJ anti-vinculin were using and Cells Inc.) coverslips above. (Cytoskeleton, fibronectin-coated described phalloidin or as collagen- min 60 on for plated were were cells 4% cells number, Fixed Plates with min. min. nm. 30 10 for 490 with fixed SDS for at 2% stained with read ethanol) were lysed were and and 2% water buffer twice in with cells washed washing washed mg/ml extensively (5 Attached min, using Violet 10 washes BSA). Crystal for five (PFA) 0.1% by paraformaldehyde plus removed were (DMEM cells adherent CO o ue-eouinmcocp sn h rudsaedepletion ground-state the using microscopy super-resolution For omauerno elmtlt,clswr eddo collagen-coated on seeded were cells motility, cell random measure To adhesion focal and size adhesion focal spreading, cell quantify To 2 5)cnrle.Iae eetkneey2mndrn n the and h 8 during min 2 every taken were Images controller. (5%) ora fCl cec 21)17 1950 doi:10.1242/jcs.150623 5189–5203 127, (2014) Science Cell of Journal H Ivtoe)raet codn otemanufacturer’s the to according reagents (Invitrogen) ˚ lige l,20) el eeclue n2 m no. mm, 24 on cultured were cells 2008), al., et ¨lling ne %CO 5% under C TM m /lprmcn lsi transfections Plasmid puromycin. g/ml 2 m Tem cetfc.Sie were Slides Scientific). (Thermo o LC n a3 knockdown Rab35 and CLIC4 For . /lclae yeI 1.5 type-I, collagen g/ml H lsImblzto i (Thermo Kit Immobilization Plus 3 VO 5 el eesee into seeded were cells 4 upeetdwith supplemented ) ˚ 6 )cabrand chamber C) objective). ˚ n non- and C 2 ,5mM m g/ml m M

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Scientific) integrin Thermo (Nunc plates 96-well ecie above. described m o nernrccigasy,clswr aee ihboi sabove, as biotin with labeled were cells assays, recycling integrin For odtrieitgi erdto,clswr aee ihboi and biotin with labeled were cells degradation, integrin determine To P o i rlf nrae.GuahoeSpaoebeads Glutathione–Sepharose untreated. left or min 2 for LPA M b nernrccigasywspromda ecie previously described as performed was assay recycling integrin 1 a ora fCl cec 21)17 1950 doi:10.1242/jcs.150623 5189–5203 127, (2014) Science Cell of Journal b 5 m -nernatbd ttepam ebaewssrpe with stripped was membrane plasma the at antibody 1-integrin b ˚ oa ellst o 0mna 4 at min 30 for lysate cell total g o .Clswr ahdtiewt c-odrecycling ice-cold with twice washed were Cells h. 1 for C nernwsdtce ycpueEIA sn MaxiSorp using ELISA, capture by detected was integrin 1 ˚ o .Ecs fEFwswse u ytwo by out washed was EGF of Excess h. 1 for C ˚ o 0mn(us) el eecoe nice on cooled were Cells (pulse). min 30 for C b -nernatbd S/6(1 TS2/16 antibody 1-integrin m uiidGTRD5wr incubated were GST–RBD35 purified g ˚ o .Clswr ahdtwice washed were Cells h. 1 for C a 5 b ˚ o h niae recycling indicated the for C a eetdb LS as ELISA by detected was 1 6 ˚ o 0mnt allow to min 30 for C ˚ .Baswr washed were Beads C. y–a3 n serum and Myc–Rab35 ˚ o 5mn(pulse). min 15 for C m /lo anti- of g/ml ˚ m o the for C ˚ o the for C e 10 per g 5201 a ˚ C 5 6

Journal of Cell Science awl,P . arv,S n omn .C. J. Norman, and S. Vadrevu, T., P. and Caswell, D. Boettiger, W., M. McCaffrey, J., A. Lindsay, M., Chan, T., P. Caswell, C. J. Norman, and T. P. Caswell, Bo etakMtuoiFkd Thk nvriy edi aa)frpoiigthe providing for Japan) Sendai, University, (Tohoku Fukuda Mitsunori thank We Acknowledgements Student’s unpaired significance, statistical of determination For analysis Statistical ARTICLE RESEARCH 5202 L. B. Tang, and S. Y. Lim, M., E., Fukuda, C. I., Chua, Kouranti, M., Machicoane, D., Dambournet, L., Chesneau, and F. E. Knol, E., A. Zuurbier, M., Stahle-Backdahl, H., Janssen, J., Calafat, T. P. Caswell, and C. J. Norman, E., R. Bridgewater, R. J. Goldenring, and A. M. Berryman, Bu L., O. K. Berry, Hobert, and J. L. Ivaska, K. and Berry, S. Veltel, J., S., Alanko, Konefal, A., E., Arjonen, Sadr, Seyed M., Chaineau, M., Sadr, and Seyed S. P. D., McPherson, P. G., Allaire, Paolo, Di C., Dall’Armi, L., A. Marat, D., P. Allaire, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.150623/-/DC1 online available material Supplementary material Supplementary for Organisation Dutch Netherlands the the (NWO). and of (K.W.F.) Research part Society Scientific is Cancer which Dutch STW, the Foundation by Technology supported was work This manuscript. the Funding wrote W.H.M. and and supervised A.S. W.H.M. C.M., study. E.A., the study. supervised the and and coordinated data C.M. the experiments. microscopy analyzed microscopy super-resolution electron A.S. performed performed H.J. and studies. designed analyzed experiments K.J. and and experiments D.L.-P. the data. performed the and designed study, the conceived E.A. contributions Author interests. competing no declare authors The interests Competing support. NKI technical the for and facilities reagents; Cytometry for Flow Francis) and Paris, Microscopy Curie, Mu (Institut (NCI, Institute, Gluckhova Janssen Planck Marina Lennert (Max discussions; Vestweber Norman helpful Dietmar Jim for Amsterdam), and UK) Amsterdam) Glasgow, (NCI, Institute, Secades (Beatson Pablo Dene Innocenti, USA), Metello OH, Athens, Littler, University, (Ohio Berryman Mark construct; GST-RDB35 hag .Z,Mle,T . h,M n ug .H. C. Sung, and M. Zhu, A., T. Milner, Z., J. Chuang, hieu . ono,M .adMPesn .S. P. McPherson, and S. M. Ioannou, M., Chaineau, eepromduigGaha rs6sfwr.Teindicated The show plots software. percentiles. Box 75th * conditions. and Prism6 25th control and to median GraphPad compared are using values significance performed were P thr .T,Srme,C,Mvs . ee,H,Wdae,M,Teg .Y. H. Tseng, M., Widmaier, H., Meyer, A., Meves, C., Stremmel, T., R. ttcher, ¨ lh5ea nernadEF1t rmt elmgaini 3D in migration cell promote to EGFR1 and microenvironments. integrin alpha5beta1 Norman,J.C.migration. the to binding by integrins Fa and noyi eyln n ucsflcytokinesis. A. successful and Echard, recycling and endocytic B. Goud, transport. endocytic of slaves granules. cytoplasmic of subpopulation a in factor-alpha A. Egesten, glance. a of midbody and centrosome the at AKAP350 cells. mammalian with cultured colocalizes and junctions maintenance. and formation tube intracellular 302 for required protein like vivo. in proteins (CLIC) channel intracellular inactive and S. active P. McPherson, coordinate and to F. migration. recycling R. and cargo controls adhesion Maestro, Arf6 cell Del and Rab35 B., between Ritter, Interplay (2013). D., Maret, M., Fotouhi, endosomes. early from exit cargo-specific B. Ritter, nrclua hoiecanlp41i soitdwt ag dense-core large with associated is neurons. hippocampal p64H1 rat in channel vesicles chloride intracellular roles. modulating actin with GTPase small regulating Asadeffectors. and GAPs , .5 ** 0.05, 2134-2137. , slr R. ¨ssler, 21) h oncenDN oan E o a3 mediating Rab35 for GEF a domain: DENN Connecdenn The (2010). .Cl Sci. Cell J. Traffic P 19) ua ooye n etohl tr rnfriggrowth transforming store neutrophils and monocytes Human (1997). o,H . al .H n oet O. Hobert, and H. D. Hall, E., H. low, ¨ , .1 *** 0.01, 21) otn ei 7peet yooa erdto of degradation lysosomal prevents 17 nexin Sorting (2012). 7 20) a-opigpoencodntsrccigof recycling coordinates protein Rab-coupling (2008). 14-21. , b .Cl Biol. Cell J. integrins. 1 Traffic 125 3695-3701. , P b elMtl Cytoskeleton Motil. Cell -nerntail. 1-integrin 14 , .Cl Sci. Cell J. 20) apn ucinldmiso chloride of domains functional Mapping (2006). 21) nAF/a3 Taecsaefor cascade GTPase ARF6/Rab35 An (2012). a.Rv o.Cl Biol. Cell Mol. Rev. Nat. 1109-1117. , .0,**** 0.001, 20) nerntafcigadtecnrlo cell of control the and trafficking Integrin (2006). 183 Traffic 143-155. , .Neurosci. J. 13 126 20) LC serce tcell-cell at enriched is CLIC4 (2003). P 610-625. , 21) a3 eiua traffic- vesicular a – Rab35 (2010). a.Cl Biol. Cell Nat. o.Cell Mol. vle r niae sfollows: as indicated are -values P 722-731. , .Ml Biol. Mol. J. , 0.0001. 20) nern:msesand masters Integrins: (2009). ur Biol. Curr. 19 21) itntrccigof recycling Distinct (2012). 21) nerntafcigat trafficking Integrin (2012). 56 20) .eeasCLIC- elegans C. A (2003). 37 2919-2928. , ESLett. FEBS 159-172. , 370-382. , Blood 10 ntr emn)and Germany) ¨nster, 21) a3:GEFs, Rab35: (2013). 14 359 843-853. , 22 584-592. , 19) 9kDa 29 A (1999). 1316-1333. , 147-153. , 90 ,1255-1266. 584 1-6. , Science t -tests b 1 oek,A . u,J,L,J,Go . hw .M,Snebr,A n Hsu, and A. Sonnenberg, M., L. Shaw, M., Gao, J., Li, J., Sun, M., A. Powelka, i,M . in,L,Mthe,K . conale,S . ufe,T,Mangin, T., Kuffner, M., S. Schoenwaelder, I., K. Matthaei, L., Jiang, R., M. Qiu, K. Jalink, D., Littler, M., Berryman, M., Langeslag, L., Zeijl, van B., J. Ponsioen, Ivaska, and T. Pellinen, T. J. Parsons, A., Ryscavage, E., K. Masiuk, S., Pal-Ghosh, K., Speer, S., C., Karim, V. Padmakumar, H., E. Tan, P., M. Iwanicki, B., Doyle, T., P. Caswell, A., P. N. Muller, B. Giepmans, and A. L. Meeteren, van A. H., W. Sonnenberg, Moolenaar, and G. Zambruno, A. M., Sonnenberg, Kreft, and C., C. Margadant, J. Norman, J., A. D. Sonnenberg, Groot, de and M., C. Kreft, J. C., Margadant, Norman, N., H. Monsuur, C., Margadant, orni . ahe . ruh,N,Gu,B n cad A. Echard, and B. Goud, N., Arouche, M., Sachse, I., Wilk, Kouranti, P., A. Duff, V., A. Sokolova, J., S. Harrop, J., Yu, M., J. Phang, L., Jiang, oet .H,Beh . eesn .M,Wsh,J,Opl,A,Mlrd L. Malerød, A., Oppelt, J., Wesche, M., N. Pedersen, A., Brech, H., V. Lobert, Jiang, V., A. Mynott, M., J. Phang, C., S. Goodchild, J., S. Harrop, R., D. Littler, in,L,Slo . i . yik,J . ae,R . u,X . h,X X., X. Shi, W., X. Luo, M., R. Yates, M., J. Rybicka, H., Li, K., Salao, L., M., Jiang, S. Valenzuela, T., Reztsova, D., W. Fairlie, Z., M. DeMaere, J., S. Harrop, ite,D . sad .N,Hro,S . rw,L . akus,G J., G. Pankhurst, J., L. Brown, J., S. Harrop, N., N. Assaad, R., D. Littler, egr .adYmd,K M. K. Yamada, and B. Geiger, N. Ohbayashi, and K. Ishibashi, H., Kobayashi, M., Fukuda, Fo H. C. Sung, and Y. S. Chou, Z., J. Chuang, ann .H,LnoGes .M,Brya,M,Si,J . alr .W., K. Saylor, B., J. Shin, M., Berryman, M., C. Longo-Guess, H., L. Gagnon, H. W. Moolenaar, and I. Fahrenfort, K., Jalink, T., Eichholtz, P. Board, and G. Chelvanayagam, S., Curtis, P., Gage, A., Dulhunty, den van J., Grindlay, I., Macpherson, B., N. Jamieson, A., M. Dozynkiewicz, ln,J,Bsi . ok . ed,R,Wr,C . en . aos S., Jakobs, B., Hein, A., C. Wurm, R., Medda, H., Bock, M., Bossi, J., lling, ¨ R6adRab11. and ARF6 W. V. eeainadcaatrzto fmc ihnl uaino h chloride al. the et of M. mutation S. null Valenzuela, with D., mice gene. Connor, 1 of channel J., intracellular characterization Low, and E., Generation J. Joseph, P., RhoA. by CLIC4 protein channel H. W. Moolenaar, and wound corneal and skin 1409-1416. reduced al. et and L. mice. Tessarollo, erosions CLIC4(NULL) V., characterize Coppola, skin healing C., J. Spontaneous Edwards, S., (2012). Hwang, L., S. Dengler, recycling. al. integrin et promoting P. Gosselin, by L., invasion R. 1341. drives Ludwig, p53 A., D. Mutant Gillespie, N., Lukashchuk, signaling. acid lysophosphatidic of outs turnover. adhesion and dynamics integrin regulates integrin regulating in kindlin and talin trafficking. of roles Distinct (2012). trafficking. and activation integrin of Mechanisms euae nedctcrccigptwyesnilfrtetria tp of steps terminal the for essential pathway actin recycling al. the cytokinesis. endocytic to et an membranes M. regulates of S. coupling Valenzuela, the gun? N., and smoking S. A moesin cytoskeleton: Breit, radixin, H., ezrin, proteins, Alkhamici, E., K. irbatmgaintruhlssmldgaaino fibronectin-integrin of degradation lysosomal through complexes. migration fibroblast H. Stenmark, and scaffolding enzymes, al. proteins, et redox N. channels, S. Ion proteins? Breit, proteins: J., CLIC L. the Brown, of M., enigma Mazzanti, M., S. Valenzuela, L., ufe,T,Ta,V . uan,Y tal. et Y. Husaini, W., V. Tsai, T., al. Kuffner, et CLIC1 channel K. ion Warton, chloride resolution. intracellular L., the 1.4-A Jankova, of at form R., (NCC27) soluble M. a of Qiu, structure R., Crystal Tonini, M., Mazzanti, rti LC euae arpaefnto hog ouainof modulation through function macrophage acidification. regulates phagosomal CLIC1 protein uin,P,Aulr .I,Mzat,M,Brya,M . ri,S .e al. ion et chloride N. redox-regulated S. the Breit, of A., form CLIC4. soluble M. protein the Berryman, channel of M., structure Mazzanti, Crystal I., (2005). M. Aguilar, P., Luciani, arxadhesions. matrix return. single-molecule W. and S. depletion Hell, and C. Eggeling, hne rti LC sepesda ihlvl nhi elseeclaadis and stereocilia cell hair function. in ear levels inner high normal at R. for expressed K. essential is Johnson, CLIC5 and protein G. channel P. 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Journal of Cell Science tue,T n etc,T J. T. Jentsch, and T. Stauber, hka . ai,M,Ctisn . o . ree,T,Sh .S n Yuspa, and S. K. Suh, T., Friesen, Y., Ho, C., Cataisson, M., Malik, A., Shukla, J. Norman, and P. Sluijs, der van A., Woods, S., Barry, M., Roberts, C. J. Norman, and E. Rainero, ARTICLE RESEARCH iimn,S,Agni,E,Tsn,D,Cvlao . oo .adD Fiore, Di and S. Polo, E., Cavallaro, D., Tosoni, E., Argenzio, S., Sigismund, function. inln u ipnal o degradation. for dispensable but signaling P. P. 3. and phospho-Smad2 of Biol. stabilization Cell consequent Nat. and CLIC4 of translocation H. S. spreading. early and adhesion from cell 1402. integrin for alphavbeta3 necessary is of endosomes recycling cells rab4-dependent how PDGF-regulated dictates that way are a receptors in matrix pathway endosomal cell migrate. late invasion: the and through migration trafficked cell integrin-mediated during 20) ltrnmdae nenlzto sesnilfrssandEGFR sustained for essential is internalization Clathrin-mediated (2008). 20) G-easgaln srgltdb cnri2dpnetnuclear Schnurri-2-dependent by regulated is signalling TGF-beta (2009). BioEssays nu e.Physiol. Rev. Annu. 11 777-784. , 35 523-532. , 21) aeedsmladlssmltrafficking lysosomal and endosomal Late (2013). 75 453-477. , 21) hoiei eiua rfikn and trafficking vesicular in Chloride (2013). e.Cell Dev. 15 209-219. , ur Biol. Curr. 11 (2001). 1392- , ht,D . awl,P .adNra,J C. J. Norman, and T. P. Caswell, P., D. White, lao,B,Buo . odn . atet .E n dad,J C. J. Edwards, and E. M., M. Hartnett, J. N., Gordon, Crutchley, J., Bruno, A., B., Ulmasov, Ryscavage, L., Li, T., Mutoh, M., H. Mutoh, S., R. K. Ashley, Suh, and A. Aitken, N., J. P. Karoulias, Cullen, W., and Suginta, D. M. Bass, J., K. Heesom, F., Steinberg, lh5ea nernrccigptwy itt ontemRokinase Rho downstream dictate migration. cell pathways persistent regulate to recycling signaling integrin alpha5beta1 cdfcto fvcoe ln h nrclua uuoei pathway. tubulogenic intracellular the along supporting Pathol. by vacuoles angiogenesis of in functions acidification protein-4 channel intracellular Chloride H. differentiation. S. keratinocyte Yuspa, 2640. Ca2+-induced and for C. Cheng, required A., R. Dumont, a isoforms. in 14-3-3 I and dynamin tubulin brain to actin, directly containing binds complex (p64H1) CLIC4 protein channel intracellular recycling and lysosomal between sorting by pathways. degradation from integrins protects ora fCl cec 21)17 1950 doi:10.1242/jcs.150623 5189–5203 127, (2014) Science Cell of Journal 174 .Cl Biol. Cell J. 1084-1096. , 197 219-230. , .Cl Biol. Cell J. 20) LC eitsadis and mediates CLIC4 (2007). 20) lh ea and beta3 v alpha (2007). ice.J. Biochem. .Cl Sci. Cell J. 177 20) Chloride (2001). 515-525. , 21) SNX17 (2012). 359 120 55-64. , 2631- , (2009). m J. Am. 5203

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