nterboytei rfikn,itraiain eyln and 2007; 2011). recycling al., internalization, al., et availability (Margadant trafficking, et depends degradation The biosynthetic strongly 2009). surface (Takada their cell al., the on on ligands et integrins Caswell of diverse functionality 2006; and al., for et specific heterocomplexes Humphries different are 24 forming that cells, human in expressed nvriyo uk,250Tru Finland. Turku, 20520 Turku, of University aut,IsiuefrMdclIfraisadBoer IB,Technische (IMB), Biometry and Informatics Universita Medical for Institute Faculty, Ato o orsodne([email protected]) correspondence for *Author of University Biotechnology, Germany. Molecular Heidelberg, and 69120 Pharmacy Heidelberg, of Institute and BioQuant edleg Germany. Heidelberg, nRA cenietfe I1 sanvlrgltro the of Eskova Anastasia regulator novel integrin a of as trafficking KIF15 endocytic identifies screen RNAi An ARTICLE RESEARCH ß eevd2 ue21;Acpe 0Fbur 2014 February 20 Accepted 2013; June 21 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This 1 differentiation. and migration, survival 18 cell Currently, proliferation, as such transduction, functions, numerous signal and in receptors, involved adhesion are cell-surface major they the of one are Integrins INTRODUCTION Dab2 KIF15, trafficking, Endocytic Integrin, WORDS: KEY a ABSTRACT n thsbe mlctdi ueostrmoi n immune and thrombotic numerous in implicated been diseases. has it and rgoi nbes acr urnl,vr itei nw bu the trafficking and about of expression known cell-surface is the regulates little that poor very mechanism Currently, and cancer. metastasis breast in increased prognosis with associated is downregulation atya Lisauskas Tautvydas niio fteedctctafcigof trafficking significant endocytic transferrin. a the as of the motor, inhibitor a or molecular revealed microtubule-based on a screen (EGF) KIF15, siRNA for the effect role factor of validation observed growth and interrogation of no Further epidermal regulators with of putative be expression, internalization to and/or found were trafficking 83 these, of Of accumulation intracellular the affected these of 122 that cytoskeleton- 386 on of genes -regulatory impact or associated the investigated we assay, RNAi based Erfle Holger h lentv ltrnaatrDb,tu mign npathways on impinging thus regulate of Dab2, that localization adaptor membrane clathrin plasma alternative mediating in the KIF15 for role novel iQat nvriyo edleg 92 edleg Germany. Heidelberg, 69120 Heidelberg, of University BioQuant, 2 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,23–47doi:10.1242/jcs.137281 2433–2447 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. a b 2 nerni n ftems motn olgnbnigreceptors, collagen-binding important most the of one is integrin 1 b nern ee sn uniaiefluorescence-microscopy- quantitative a using Here, integrin. 1 ¨ rse,037Dedn Germany. Dresden, 01307 Dresden, t a 2 a b nerninternalization. integrin 2 nerni oettmu upesr n its and suppressor, tumour potent a is integrin 1 a 1 nernad8 and integrin n yat Starkuviene Vytaute and 5 nertv iifraisadSsesBooy DKFZ, Biology, Systems and Bioinformatics Integrative 1 etn Knapp Bettina , 1 anrPepperkok Rainer , b nerncan r nw obe to known are chains integrin a nernedctssadfound and endocytosis integrin 2 4 ML eehftae1 69117 1, Meyerhofstraße EMBL, a nern u aasgeta suggest data Our integrin. 2 3 etefrBiotechnology, for Centre 2 ooaMatelska Dorota , 1, * 4 oetRussell Robert , 2 a a Medical integrin 2 integrin. 2 1 uan Reusing Susanne , nern Csele l,20) niemn te endocytic other many Unlike 2009). al., different et clathrin-independent of internalization (Caswell and the Arjonen integrins for clathrin-dependent 2011; elucidated been al., and have pathways et 2012), (Valdembri al., Both internalized 2000). et be surface Howell, can (non-clustered) (also and non-bound integrins efficiently homolog (Wang and to (clustered) TGN38 membrane trans-Golgi ligand-bound the plasma to at the binding TGOLN2) (Tiwari reach or require state TGN46 and inactive as al., the known et 2011a) in Huang al., complex 1989; Golgi et al., the et traverse (Heino 1997), reticulum endoplasmic the in Zage l,20) n ysn6pooe h nenlzto of internalization the in promotes 6 as filopodia myosin role ligand-bound and in 2004), such a adhesion al., et motors, cell play (Zhang al., integrin-mediated actin-based et regulate motors Day instance, 10, 2011a; myosin molecular For al., several trafficking. et Tiwari integrin addition, 2010, al., In et SNAP23– 2011). Veale the 2010; (Skalski (e.g. al., 6) syntaxin et recycling and 3 integrin syntaxin complex, in been 2007), syntaxin-4–VAMP3 have function al., fusion) et to membrane Caswell 2004; of described 2006; al., (mediators al., SNAREs et et endocytic diverse Pellinen Powelka and of 2012; 2001; al., regulators al., et et crucial Arjonen (Roberts are Rab7, trafficking Rab25) Rab5, integrin (Rab4, and family Rab21 Rab and are the Rab11, of degradation GTPases or integrins Small recycling emerging. internalization, integrin other in involved of 2006). Norman, and trafficking (Caswell the stimuli of extracellular composition heterodimer, type, cell on or the integrin also on only but recycling not state, depends activation Bo trafficking promote integrin integrin 2011; of of al., pathway might binding exact et al., The integrins (Mai Competitive et retention Tiwari to 2012). intracellular 2010; al., regulators al., al., et et diverse Lobert et Dozynkiewicz non-lysosomal 2012; Ligand-bound Valdembri al., or 2012; 2011b; 2012). et lysosomal al., Rintanen al., et to 2009; 2001; (Arjonen et directed pathways be al., Arjonen degradation also 2004; et can or al., integrins (Roberts (long-loop) et pathways Rab11-specific Powelka internalized (short-loop) through the either Rab4-specific and of (Caswell 2006) membrane majority plasma the Norman, The to back as recycled 2009). known are integrins al., (also 2009, proteins al., et ARH et Ezratty or Teckchandani 2007; sorting Kaibuchi, Numb and (Nishimura Dab2, clathrin-associated LDLRAP1)] instance, requires for [CLASPs; clathrin-dependent often the through pathway internalization integrin cargoes, irtbl-ae oo I1 rmtstedlvr of delivery the promotes KIF1C motor microtubule-based nernfo h eiula eyln oprmn othe to a However, 2012). compartment al., et recycling (Theisen cells migrating perinuclear of edge the trailing from integrin 1 oadEils Roland , el yteie nern r sebe noheterodimers into assembled are integrins synthesized Newly ept h opeiyo rfikn ahas regulators pathways, trafficking of complexity the Despite a 5 b 1,5 nern(admr ta. 09.The 2009). al., et (Valdembri integrin 1 1 nt Arjonen Antti , oan Ivaska Johanna , 3 , 3 asKaderali Lars , tce ta. 2012). al., et ¨ttcher a 2433 2 5 , b 1

Journal of Cell Science npatcdse.I re oqatf h muto intracellular of amount the quantify to order a In dishes. plastic on o n hnitraie t37 at internalized then and h 1 for rfikn ieis(awl ta. 09 n ih have might 2009), selected and al., the 2009) et of analysis al., (Teckchandani the requirements et molecular (Caswell different different kinetics exhibit different heterodimers 12 different trafficking with As 2002). heterodimers (Hynes, forms chains it however, machinery trafficking; molecular missing. still the is trafficking integrin of regulating characterization comprehensive ARTICLE RESEARCH 2434 high and transfection of of ease trafficking characterized their b previously the for with (chosen efficiency) RNAi cells HeLa of endocytosis in the compared we trafficking, endocytic whether define To endogenous of trafficking Endocytic RESULTS the with mechanisms. only molecular specific delineate motn eetr fclae Kae n ak,1989; 2000), Marks, al., et and Emsley (Kramer 1990; most al., collagen the et of of one Hemler As receptors cells. endothelial important and epithelial leukocytes, and keratinocytes, platelets fibroblasts, in expressed is heterodimer atr,19;d ogrle ta. 00.Ipraty the Importantly, 2000). al., in et involved 2000; also wound Fougerolles al., of is et expression de and It (Eckes 1999; diseases 2005). epidermis immune Santoro, Eble, and 2002; the thrombotic angiogenesis, al., numerous of et organs, (Chen renewal healing tubular aggregation, of platelet morphogenesis hemostasis, n eatssadtu ihpo rgoi Zte ta. 1998; al., et (Zutter 2011). prognosis al., poor et with Ramirez thus and metastasis and and 08 pae l,20) idn of Binding al., et 2004). Karjalainen al., 2012; et al., Upla et 2008; (Rintanen EV1 bodies of multivesicular Binding inactive 2011). al., of a et and Fleming clustering rotavirus 2004; induces al., al., (EV1), et et Londrigan 1 Feire 1992; 2003; al., echovirus et (Bergelson as cytomegalovirus human such pathogens, several Rnae ta. 02.B otat itei nw bu the about known is little pathway contrast, non-clustered degradation By of same route 2012). trafficking the al., to et heterodimer (Rintanen the direct might o a2 idn othe to binding Rab21 for h oeua oo I1.Ordt upr h novel the the support of Dab2. adaptor localization data clathrin membrane alternative of plasma Our motor-dependent internalization KIF15. through the that motor suggestion of molecular role the of inhibitors detail the validated more of strongest context in the of the of characterize one in to accumulation were regulators chose novel 122 We intracellular were endocytosis. these these, of the half Of and affect integrin, cytoskeleton. to cellular membrane to demonstrated the related the functionally and are of that trafficking proteins regulators chosen 386 identify of of to trafficking screen endocytic RNAi microscopy-based the facilitates 2011). which (GTPase RASA1), of RasGAP as p120 recycling known the also of protein; binding for activating role a and endocytosis nern h eann elsraeintegrin–antibody cell-surface remaining the integrin, 2 nern Cell-surface integrin. 1 nernt api-eedn erdto aha in pathway degradation calpain-dependent a to integrin 2 b ee eue nubae uniaiefluorescence- quantitative unbiased an used we Here, nerni rqetyue oivsiaeintegrin investigate to used frequently is integrin 1 a 2 b oneuaincreae ihicesdcl invasion cell increased with correlates downregulation 1 a a 2 2 b b b nernt h lsammrn Mie al., et (Mai membrane plasma the to integrin 1 nernsbnt(ye,19) and 1992), (Hynes, subunit integrin 1 nerni togyrdcdi ratcancer, breast in reduced strongly is integrin 1 a nernudrosheterodimer-specific undergoes integrin 2 a 2 a b a a nern etree h expression the targeted We integrin. 2 nernas csa orcpo for co-receptor a as acts also integrin 1 nernwsatbd-aee t4 at antibody-labeled was integrin 2 nernctpamcti ofacilitate to tail cytoplasmic integrin 2 a hi ih emr sflto useful more be might chain a a 2 ˚ 2 o ayn egh ftime of lengths varying for C b b n iet virus-bound directs and 1 nern pr rmarole a from apart integrin, 1 a a a 2 integrin 2 b nerni regulated is integrin 2 a nernt collagen to integrin 1 nerntrafficking, integrin 2 a a nerninteracts integrin 2 2 b scuilfor crucial is 1 a integrin 2 a 2 b a ˚ C a 1 2 yokltldnmc eeicue ae nltrtr data. literature and/or on based trafficking included membrane were major regulating dynamics three myosins. proteins cytoskeletal (the 37 and addition, subunits chains) light dynein In cargo-binding ten 13 and including chains, chains proteins, heavy heavy was motor R.P.) kinesin mammalian by most 44 (supplementary (provided include library genes to targeted 386 designed The targeting S1). Table siRNAs material of RNAi 1084 accumulation fluorescence-microscopy-based with intracellular a screen performed the we of integrin, regulators identify To assays RNAi by identified of regulators Potential yooa erdto aha nfiinl.Approximately peripheral the inefficiently. of pathway 70% degradation the of lysosomal 10% that suggesting Only 2D), (Fig. 2A). (Fig. GTPase positive peripheral Rab5a were that overexpressed structures for peripheral with internalization, colocalized of integrin h 1 were following GFP–Rab-GTPases for for stained results were several cells and overexpressed, internalization, following h. 1 Internalized cytoplasmic after and 2011)]. 1A,D) area (Fig. juxtanuclear al., structures the buffer et at acidic efficiently an (Erfle accumulated with cells Methods, treating [see by removed were complexes rvosy(itnne l,21;Kraanne l,20;Upla 2008; reported al., et as 2004). Karjalainen al., 2012; 1C), et (Fig. al., et localization (Rintanen any previously membrane hardly with plasma structures residual punctate cytoplasmic in accumulated antibody-clustered the contrast, in By reduction 2009). progressive a for was measured There cell-surface 1A). of (Fig. amount incubation h prolonged 6 a of after time structures cytoplasmic scattered the pdra rwhfco eetri ciae Nn ta. 2007) al., of et (Ning clustering in activated upon is pathway or receptor factor caveolin-dependent growth also Chao epidermal is a pathway 2007; by latter by The Kaibuchi, utilized 2008). Sottile, and and and (Shi 2009) (Nishimura myofibroblasts Kunz, types cell and various in of 2B,C). (Fig. overexpressed were Rab4a of and Rab11b localization perinuclear unlike that indication Interestingly, strong a – 2B,C) a (Fig. positive Rab4a and Rab11b A1o N2wssfiin oihbtteintracellular the inhibit to sufficient CLTC, of was expression of DNM2 target the accumulation Fig. in the or material reduction of (supplementary incomplete CAV1 levels an transfection Even of the S1). h in 48 reduction after proteins 50–80% was which a staining, siRNAs immunofluorescence demonstrated respective or and blotting the western Doherty of by tested efficiency 2009; The Nichols, 2009). and McMahon, and (Hansen (CAV1) (DNM2)] caveolin-1 (CLTC), caveolae-dependent the dynamin-2 chain and in heavy [clathrin clathrin-dependent role pathways a of play components pathways key these To of of trafficking mechanisms. endocytic both cell-type-specific whether possible investigate suggesting 2004), nernetr hr-adln-oprccigpathways. recycling long-loop and short- enters integrin 2 nodrt eemn h noyi ot of route endocytic the determine to order In rvosRA xeiet eosrtdta h endocytosis the that demonstrated experiments RNAi Previous b nernocr hog h ltrndpnetpathway clathrin-dependent the through occurs integrin 1 ora fCl cec 21)17 4324 doi:10.1242/jcs.137281 2433–2447 127, (2014) Science Cell of Journal b a nern(oek ta. 04 roe ta. 2012), al., et Arjonen 2004; al., et (Powelka integrin 1 -nerncnann tutrswr a7 positive Rab7a were structures 2-integrin-containing a nerni vra acnm el hnthe when cells carcinoma ovarian in integrin 2 b nerni eacls(ekhnaie al., et (Teckchandani cells HeLa in integrin 1 a nernb 08%(i.1F,G). (Fig. 70–80% by integrin 2 a b a 2 a a nern(oek ta. 2004), al., et (Powelka integrin 1 nerni eacls edpee the depleted we cells, HeLa in integrin 2 a b nernitaellraccumulation intracellular integrin 2 nern(i.1,) iia othat to similar 1B,E), (Fig. integrin 2 a -nerncnann tutrswere structures 2-integrin-containing nernb niois(pae al., et (Upla antibodies by integrin 1 nernetr h Rab7a-dependent the enters integrin 2 a nernwsrdcdwhen reduced was integrin 2 a . nernlclzdlreyto largely localized integrin 2 0 fteintracellular the of 70% a nern(e Methods) (see integrin 2 a nern iia to Similar integrin. 2 a a integrin 2 integrin 2 a a 2 2

Journal of Cell Science EERHARTICLE RESEARCH hl cen(e ehd) eqatfe the spotting quantified one We in Methods). prepared the (see of were reproducibility screen and These efficiency whole high 2007)]. arrays a cell ensuring al., experiment, on primary et transfection the [reverse the (Erfle For with Lipofectamine pre-spotted proteins. and were that adaptor siRNAs chambers Lab-Tek and different used 5 we scaffold screen, microtubule-associated phosphatases, 23 these 9 and 14 of and chains, and factors) kinases 34 GAPs exchange actin-associated 53 proteins, nucleotide GTPases, 41 (guanine Rho and GTPases, GEFs Ras 62 small 28 and included group This surlal,w oue nclswt efficient with of cells on amount focused the we in unreliable, cell is variation large over large intracellular samples a control of internalized the revealed level of during the populations Inspection ratio signal-to-noise which screen. best the the at achieving thereby time-point 1D), (Fig. the internalization, intracellular of after cells h individual for 1 signal fluorescence intracellular specific a a a nerni el ihlwacmlto efficiency accumulation low with cells in integrin 2 nernwstehgeti h oto sample control the in highest the was integrin 2 nerni eacls sqatfcto of quantification As cells. HeLa in integrin 2 a a 2-integrin- integrin 2 b integrin ahsRAnraie ota ftengtv otos(see with siRNAs controls considered We negative integrin. the of negative that Positive to Methods). normalized high- siRNA yield each to negative analysis the further with for transfected (see used data. were cells quality subpopulations these HeLa and of control, cell 40–50% intracellular Mixture in of separate Gaussian found levels high experiment the automatically Usually, every and Methods). to for S2), population Fig. Model cell material respective (supplementary the of function define distribution levels cumulative to high empirical with the used cells subsequently for threshold a intracellular set We accumulation. hc 1 niie n ceeae h intracellular the accelerated 7 of screen, our and in hits inhibited of as assigned accumulation 115 were genes 122 which total, In S2). than less cenht eedfndacrigt individual to according defined were hits Screen ora fCl cec 21)17 4324 doi:10.1242/jcs.137281 2433–2447 127, (2014) Science Cell of Journal 2 z soe niaerdcdaonso intracellular of amounts reduced indicate -scores 1( a . nern(eie iulydrn mgn)and imaging) during visually (defined integrin 2 1)a rmr is(upeetr aeilTable material (supplementary hits primary as |1|) a nern(i.3) et esprtdthe separated we Next, 3A). (Fig. integrin 2 z soe niaeicesdaonsand amounts increased indicate -scores * experiments); independent (three of E, accumulation and D in Intracellular 100% respectively. to set was point and time-point h surface 1 the at integrin internalized the of amount The (E). cell-surface and internalized (D) the of Quantification (C). and antibody-clustered (B) non-clustered surface (A), cells. of endogenous trafficking Endocytic 1. Fig. 20 bars: Scale control. Ctrl, (F,G). cells HeLa in caveolin-dependent and P # m .5 ** 0.05, .Dt hwtemean the show Data m. z iulzto finternalized of Visualization soe fgetrta or 1 than greater of -scores a nerna h time- h 0 the at integrin 2 P # a .1 *** 0.01, nerni HeLa in integrin 2 a nerni clathrin- is integrin 2 a a nernwere integrin 2 integrin 2 a integrin 2 P # z soe of -scores 0.001. 6 s.e.m. 2435 a a 2 2

Journal of Cell Science EERHARTICLE RESEARCH aeilTbeS) o ntne h oneuainof caveolae-dependent downregulation the the in 2436 instance, implicated For protein a S2). were syndecan-4, genes Table that (supplementary other effect downregulation DNM2 larger 26 material and a of CLTC showing by total caused some A that inhibitors, 3B). strong (Fig. as arrays classified cell on traffic of scores ( strong score into hits primary , 15) oneuaino LCadDM produced DNM2 and CLTC of Downregulation |1.5|). 2 .,crepnigt togihbto of inhibition strong a to corresponding 1.5, z -score . 15)adwa fetr ( effectors weak and |1.5|) a integrin 2 z z - - nenlzto fitgis(ase l,21) e oastrong a to led 2011), al., et ( (Bass inhibition integrins of internalization mgs,5 (mean images), Rab of respective lower percentage the the the show in with panel figures each The of Rabs. image overexpressed right arrows, the thin of structures; images; localization the colocalizing overlay perinuclear by arrows, in indicated Thick structures region images. non-colocalizing the upper arrowheads, of the row view in lower magnification box The higher white internalization. a the after shows h images 1 of and at (C) (green) Rab4a (D) GFP-tagged Rab7a (B), GFP-tagged Rab11b mCherry-tagged (A), Rab5a tagged intracellular Endogenous of Colocalization 2. Fig. 8hsoe erdcbebtwa niioyefc ( effect inhibitory for CAV1 weak of but depletion reproducible The a 115). showed of out h 48 (90 effectors weak a Rnaz ta. 07 hthsbe rvosysonto shown previously ARF6 been as of scored ( was has GAP accelerator It 2012). strong a that al., a et ASAP1, (supplementary (Onodera 2007) recycling is integrin ‘accelerators’ al., regulate these called of et One were (Randazzo S2). primary screen Table Seven the material S1). Fig. of material hits (supplementary period siRNAs incubation short with relatively the during achieved knockdown score nldd4 u f47ietfe o-eudn opnnsof components non-redundant screen identified Our 467 a of complexes. out integrin-primed 45 of included components migration the cell and published positive previously were a 2008). (5.5%) al., in genes et and 161 (Simpson the assay al., of assay our out et including in 9 that both (Mise-Omata formation, found both We PTEN adhesion 2005). for phosphatase focal (15%) hits phosphatidylinositol genes and 161 primary of out accumulation 24 were identified 2009; We al., that genes 2008). et al., (Winograd-Katz 161 et migration addition, of Simpson cell regulators In identify and to adhesions 3C). screened (Fig. focal and also were al., 2008) experiments Caswell et our al., (Winograd-Katz from 2009; et screens migration Simpson those RNAi Kunz, with published 2009; cell screen and relevant our of two and Chao results of the compared 2013; dynamics we 2008), al., Norman, adhesion et focal (Morgan of regulation of accumulation intracellular be to hits the 3C). EGF appeared our (Fig. screen to or of our transferrin only 122 from either hits of specific primary of out the 83 regulators 39 and as of that (EGF) endocytosis, reported found factor regulators were we growth for 2010), (32%) our epidermal al., screen et comparing and significant genome-wide (Collinet transferrin While (11%) a on of no observed. to endocytosis based library was hits S2), (assigned terms) primary class screened S1, Ontology functional the Gene protein Tables of in representation material comparison representation in (supplementary their effectors strong with among (19%) over-represented recycling. inhibited intracellular of accumulation enhanced n yolsi yen1haycan1(YCH).Arecent A intracellular (DYNC1H1)]. 1 the chain for heavy of search 1 regulators dynein cytoplasmic potential and as of assigned accumulation were 19 nern h aoiyo h niioswr hrceie as characterized were inhibitors the of majority The integrin. 2 5 eosre arylreoelpbtenorpiayhits primary our between overlap large fairly a observed We the for fundamental is integrins of trafficking endocytic As were which phosphatases, and kinases of exception the With b and 1 52 ora fCl cec 21)17 4324 doi:10.1242/jcs.137281 2433–2447 127, (2014) Science Cell of Journal .4.I at hsmgtrfettelvlo protein of level the reflect might this part, In 1.14). m a b lwrrwo images). of row (lower m z a 4 -nernitrcigpoen (Bo proteins 1-integrin-interacting -score nern(e)wsclclzdwt h vrxrse GFP- overexpressed the with colocalized was (red) integrin 2 b nern Hmhise l,20)ad fthese, of and, 2009) al., et (Humphries integrins 1 a 52 nern[..temlclrmtr KIF2C motors molecular the [e.g. integrin 2 z -score .8 fteitaellracmlto of accumulation intracellular the of 1.78) 6 5 ...Saebr:10 bars: Scale s.d.). .6;ti fetwscue ythe by caused was effect this 2.26); a nernwt a GTPases. Rab with integrin 2 a a nernintracellular integrin 2 nern ieydeto due likely integrin, 2 a nernta colocalizes that integrin 2 tce ta. 2012) al., et ¨ttcher m uprrwof row (upper m a integrin 2 z -

Journal of Cell Science irto (right). migration of accumulation which within boundaries ( (the screen primary the in genes tested 386 EERHARTICLE RESEARCH aeilTbeS)(ioaae l,20) Validation intracellular 2009). was of pool under accumulation the al., when plates Intracellular inhibited multi-well as et conditions. considered non-coated transfection in (Hirokawa (supplementary direct screen performed S1) primary were the assays Table selected in essentially the tested and material of were trafficking, integrin One kinesins role in the all proteins). motors about known molecular (23 is these little hits very of as primary kinesins, the the included selected groups of we above, fifth described screen a siRNA the in of identified regulators were potential the validate To accumulation of intracellular regulators potential of Validation chromosome inhibited of strongly 2007), component 3B). al., (Fig. key et of accumulation (Mayr intracellular a the mitosis in KIF18A, congression example, of regulators revealed novel for screen potential Our GAP of 1997)]. [a al., number RALBP1 et a and (Matsubara and Cdc42 1999)] GTPases and al., Rac1 small et of integrins, (Tu pathways connects LIMS1 signaling 2009), (supplementary that EGF al., protein hits et adaptor (Moser primary FERMT1) [an and as protein as known study, integrin-activating (also the our assigned kindlin-1 included in These were S2). tested Table were these material 11 of which of four proteins, 96 yielded ( hits Strong S2). of Table trafficking material endocytic supplementary of regulators potential identifies screen fluorescence-microscopy-based A 3. Fig. z -score , 1)aesoni le B xmlso nrclua cuuainof accumulation intracellular of Examples (B) blue. in shown are |1|) a nernwt h euaoso rnfri n G noyoi lf) n euaoso oa deinfrainadeihla cell epithelial and formation adhesion focal of regulators and (left), endocytosis EGF and transferrin of regulators the with integrin 2 a -nernseii loecnewsmaue.Saebr 100 bar: Scale measured. was fluorescence 2-integrin-specific a nernwe downregulated when integrin 2 a z a soeo h otefciesRAi hw o vr gene, every for shown is siRNA effective most the of -score nerntafcigthat trafficking integrin 2 integrin 2 z -scores a nerntrafficking; integrin 2 . 15)aelse n hw nrd ekht ( hits weak red; in shown and listed are |1.5|) a integrin 2 a -nernseii loecneo elary.Wiecrlsidct spot indicate circles White arrays. cell on fluorescence 2-integrin-specific eue rti xrsin od hs emaue h total the or measured trafficking we inhibited this, of do of To consequence amounts expression. direct protein a reduced are integrin KIF13A include hits. not validated did of we list screen, the with primary not in the but in for siRNAs, used additional ones the regulator the intracellular with only the obtained recycling was affected result and a 2009), al., of S4) KIF13A, accumulation et S3, (Delevoye only Tables re- melanocytes the that in We material non-hits screen. (supplementary were demonstrated our that screen in kinesins hits primary traffic-related more primary cells 13 as the tested identified beyond HeLa important investigation kinesins KIF15, our are nine extended kinesins in of we namely As effectors regulators, 1). trafficking expressed as three, (Table validated accumulation are were S3), intracellular KIF23 nine Table and that primary KIF18A the the material kinesins From in as (supplementary S4). hit effect Table same two primary material the least showed (supplementary at when siRNAs screening validated four as of hits out potential scored We by control. reduced was splmnaymtra i.S) steeknsn eeshown were kinesins downregulated these As were S3). KIF23 Fig. and material KIF18A of (supplementary KIF15, levels when Expression affected 1). (Table kinesins et etse hte erae eeso intracellular of levels decreased whether tested we Next, m ora fCl cec 21)17 4324 doi:10.1242/jcs.137281 2433–2447 127, (2014) Science Cell of Journal .()Cmaio fptnilrgltr fteintracellular the of regulators potential of Comparison (C) m. a nernfloigdwrglto ftevalidated the of downregulation following integrin 2 a nerni h aiaineprmns sthis As experiments. validation the in integrin 2 . z -score 0 oprdwt hto h negative the of that with compared 40% z soe fohrsRA r hw nthe in shown are siRNAs other of -scores , a integrin. 2 15)aesoni re n non-effectors and green in shown are |1.5|) A itiuino the of Distribution (A) a nernwr not were integrin 2 a integrin 2 z soe of -scores 2437 a 2

Journal of Cell Science aiae euaosta nii h nrclua cuuainof accumulation intracellular of the levels inhibit that regulators Validated TN139.11 PTPN11 y1 36.43 NF2 Myo1A KIF23 KIF18A KIF15 TB 7.38 ITGB1 RGP 42.03 ARHGAP6 B252.78 ABL2 ARF1 eaiecnrl1010100 100 100 Intracellular DNM2 control Negative of accumulation intracellular name the Gene of regulators Validated 1. Table ARTICLE RESEARCH 4,)adldt togihbto fteintracellular 2438 decrease a the with Consistent of S3A,B). 70% Fig. inhibition a material supplementary strong in resulted a a h to 48 Fig. of led material for accumulation (supplementary and KIF15 levels protein of S4A,C) KIF15 RNAi for in described far. reduction be been on has so to focused function we appeared KIF15 trafficking-related screen, no what targeted as our regarding KIF15, by insight revealed regulators detailed novel more gain To the of in half of Internalization changes nearly to For of level attributed (43%). expression the be hits affected primarily could 23 intracellular effect the of accumulation this of targets out the 10 for screen 35%). nearly (by numbers in cell reduction reduced the significantly to due largely total was downregulation ARHGAP6 intracellular on effect observed on seen effects role of a accumulation play might in proteins proteins in these however, these reduced downregulated; Myo1A for when and role ITGB1 of a trafficking. levels expression ARHGAP6, the suggesting alter ABL2, not integrin, these did of NF2) (ARF1, two and strong of (ARF1 screen expression proteins as the classified Suppressing primary PTPN11). also and the Myo1A were these in of screen Five inhibitors S4). primary Table intracellular of material the levels reduced strongly inhibition in scored proteins a RNAi-mediated these were siRNAs of S2). that seven of multiple molecules Table the material with chose (supplementary we effectors functional this, diverse as do to belonging To hits groups. primary of additional number and 14 the tested KIF15 in change of significant 1). a downregulation (Table induce cells whereas not cells did twofold, of KIF18A cells number nearly of siRNAs. number respective the al., the by the reduced et calculated with expression Tanenbaum KIF23 transfection we of of 2007; Downregulation h 2005b), al., 48 et al., after (Mayr remaining et mitosis Zhu in role 2009; a play to nernepeso ee n elnmes(al 1; (Table numbers cell and level expression integrin 2 supplementary 1; (Table assays validation the in integrin 2 ogtabte vriwo h pcfct forsre ere- we screen our of specificity the of overview better a get To nsmay ecudrcptlt h eut fteprimary the of results the recapitulate could we summary, In a nerntafcig sihbto fteintracellular the of inhibition as trafficking, integrin 2 a nernepeso.Dwrglto fol PTPN1 only of Downregulation expression. integrin 2 a nernrpeettemean the represent integrin 2 a nernepeso Tbe1.B otat the contrast, By 1). (Table expression integrin 2 a a a nernwtotasgiiatcag in change significant a without integrin 2 nernwsmr rnucdta the than pronounced more was integrin 2 nerni I1 dependent KIF15 is integrin 2 a a nern hra h te hits other the whereas integrin, 2 35.59 25.40 51.12 27.80 49.17 27.20 nernlvl pnAL and ABL2 upon levels integrin 2 6 ...of s.e.m. 6 6 6 6 6 6 6 6 6 6 6 .976.83 5.19 .054.12 3.40 .095.89 3.10 77 64.72 92.69 17.79 6.36 .497.64 9.54 .596.50 4.45 .543.14 6.35 .456.84 9.94 .399.11 7.43 .382.45 6.63 a a a nernexpression integrin 2 a nern()Total (%) integrin 2 integrin. 2 -nernseii loecnedrvdfo el hwn fiin noyoi n l el,respectively. cells, all and endocytosis efficient showing cells from derived fluorescence 2-integrin-specific a integrin 2 a nernwtotchanging without integrin 2 a 2 a integrin 2 noeospoen splmnaymtra i.S4B,D) the Fig. of that material 3.3-fold (supplementary was al., which et protein) of (Tanenbaum level endogenous KIF15 expression cell-surface GFP-tagged (the in 2009) ectopic moderate increase of Moreover, concomitant S3C). expression Fig. a material (supplementary to integrin led expression in I1 srssatt ua iNs epromdrescue of accumulation performed intracellular we the inhibitory on siRNAs, siRNA-mediated KIF15 the of human effect that showed to which resistant experiments, murine punctate is GFP-tagged membrane, As KIF15 microtubules. plasma and influence. the 4A), structures (Fig. to apparent cytoplasmic cytoplasm localization no the occasional to had with localized conditions mostly these KIF15 of GFP-tagged Overexpression under internalization. alone of h GFP 1 intracellular after of region perinuclear amount the in by increase an induced ntetafcigo G hti erddi lysosomes in with agreement degraded In to 1983). is recycled al., is et that that (Dickson transferrin membrane EGF and plasma 1976) the of Cohen, downregulation and KIF15 trafficking of (Carpenter effect the whereas the intracellular tested on then cells, the We 5A,D). inhibited the (Fig. control, strongly of negative within KIF15 accumulation the of accumulated with downregulation gradually transfected cells integrin In depleted. of accumulation Fig. material (supplementary cells of types S3D). together, of different accumulation in Taken intracellular observed integrin the 4E,F). inhibited were KIF15 (Fig. of cytoskeleton depletion downregulation significant microtubule No KIF15 cell and Methods). following actin whole (see in the assay cells changes quantifying microscopy-based of subpopulation the in defined a for difference versus assays these the as in population to might downregulated This attributed 1). was (Table be assay KIF15 addition, microscopy-based the when integrin with In of compared 25%) rate inhibition 4C). (around lower (Fig. a trafficking revealed assays 2001) Both 2012) 4D). al., al., et (Fig. (Arjonen assay et antibody-based biotin-capture quenching ratiometric Roberts (Margadant using of 2012; endocytosis (ELISAs) by al., assays in confirmed et immunosorbent KIF15 further of enzyme-linked was role The trafficking GFP- of overexpression KIF15. following 4B) tagged (Fig. 30% by lessened was 6 a 6 6 6 6 6 6 6 6 6 6 nern()Cl on (%) count Cell (%) integrin 2 76 99.16 17.67 .266.07 3.72 36 85.77 13.64 .4126.42 49.11 4.04 33.23 67 98.64 16.74 .0106.44 2.30 .099.93 7.10 .375.59 4.33 .972.05 4.49 37 98.56 13.77 et eaaye twihsaeteintracellular the stage which at analyzed we Next, , a nernitaellrlvl,sprsino KIF15 of suppression levels, intracellular integrin 2 0 Fg AB,with 4A,B), (Fig. 40% ora fCl cec 21)17 4324 doi:10.1242/jcs.137281 2433–2447 127, (2014) Science Cell of Journal a nernepeso r hw nbl.Itaellradtotal and Intracellular bold. in shown are expression integrin 2 b nernwsas niie,a esrdb a by measured as inhibited, also was integrin 1 a a nerntruhu h sa 56 min) (5–60 assay the throughout integrin 2 nernwsafce hnKF5was KIF15 when affected was integrin 2 a nernacmltn tthe at accumulating integrin 2 6 6 6 6 6 6 6 6 6 6 6 28.10 3.19 31.44 17.32 7.24 18.60 37.85 6.88 22.74 46.71 7.75 a nernendocytic integrin 2 a a integrin 2 integrin 2 a a a 2 2 2

Journal of Cell Science EERHARTICLE RESEARCH atraiecahi dpos r eurdfrintegrin for required CLASPs Teckchandani several 2007; are Kaibuchi, that and shown adaptors) (Nishimura been internalization has clathrin It trafficking integrin-related (alternative KIF15. the of investigate further function Dab2 to of localization sought cell-surface time-point We for required any is KIF15 at downregulation KIF15 was 5C,F). trafficking by (Fig. the endocytic influenced to EGF transferrin contrast, not of cargo By recycling inhibited membrane. the to plasma 5B,C,E,F). due with the (Fig. is Presumably, or 5B). 5E), effect (Fig. (Fig. observed EGF region time-points perinuclear downregulation the either in significantly KIF15 accumulating were early of trafficking by transferrin internalization affected of at steps the later However, cargoes on influence KIF15 transferrin of downregulation no 2010), al., had et (Collinet results previous ge elwt rvossuis(hoadKn,2009; integrins Kunz, of and levels of inhibits strongly surface (Chao Dab2 Furthermore, internalization whereas that studies showing effect, 2012) the previous weaker al., et data with a Our Teckchandani inhibition. well had significant these no agree ARH showed of Numb of of role suppression same RNAi potential the 6A–C). (Fig. the and assess 6D), a (Fig. to in transcripts used CLASPs Dab2 respective were the as conditions caused h of RNAi 48 such experiments, for level our Maurer Numb In and CLASPs 2012; transferrin 2006). ARH al., Dab2, al., of et the upon et Keyel 2006; Teckchandani of Cooper, dependent 2009; and internalization al., not et contrast, (Teckchandani is By 2009). receptor al., et a2dfcetHL el Tccadn ta. 2009). al., et (Teckchandani cells HeLa Dab2-deficient nernedctsswsotie ydpeino Dab2 of depletion by obtained was endocytosis integrin 2 ora fCl cec 21)17 4324 doi:10.1242/jcs.137281 2433–2447 127, (2014) Science Cell of Journal a nerntafcig h togs niiinof inhibition strongest The trafficking. integrin 2 b nernwe downregulated. when integrin 1 ci F.Saebr:20 bars: Scale or (F). (E) actin microtubules affect not does *** and integrin of accumulation intracellular that the show (D) assay quenching- antibody-based and (C) assay based ELISA- Biotin-capture (C,D) inhibition. of the rescue of Quantification Ctrl, (B) KIF15. control. GFP-tagged and GFP of levels high expressing cells GFP.Asterisks, not but of KIF15, overexpression GFP-tagged the by lessened is of appearance intracellular the prevents RNAi by trafficking of endocytic of the role in the KIF15 of Validation 4. Fig. mean the show Data is downregulated. KIF15 when inhibited is P a integrin. 2 # 6 .0.Dwrglto fKIF15 of Downregulation 0.001. ... * s.e.m.; a b nerntrafficking integrin 2 . a b and 1 nern n hseffect this and integrin, 2 0 euto nthe in reduction 80% nern respectively, integrin, 1 A elto fKIF15 of Depletion (A) P # .5 ** 0.05, a nraein increase 2 m m. P # 0.01, 2439 a 2

Journal of Cell Science EERHARTICLE RESEARCH 2440 inhibite * of strongly internalization experiments). is of independent membrane min plasma (three 60 the s.e.m. to after indicate transferrin controls bars of negative recycling to the normalized contrast, cargo, By each affected. not of is (C) internalization EGF 20 the and for (B) required transferrin of specifically internalization is KIF15 5. Fig. nrclua a2i h eiula einfloigthe play following might KIF15 region that indicating perinuclear 7D), (Fig. the KIF15 of in was depletion changed distribution of Dab2 Dab2 accumulation not frequent in supplementary more intracellular was KIF15 2.7-fold a of 7B; cells by (Fig. role demonstrated in further The conditions S4F). Dab2 Fig. these of material level under structures cytoplasm total the Dab2-specific significantly to The of membrane 7B). plasma fraction (Fig. the large from redistributed a being with fluorescence 7C,D), reflection (Fig. of internal Downregulation a 7A). induced total (Fig. KIF15 modes in epifluorescence imaged and intracellularly (TIRF) and cells membrane structures HeLa plasma punctate the in to on both localized found Dab2 al., were 2011), that et al., (Teckchandani et studies Chetrit previous 2009; with agreement In KIF15. enx etdwehrDb sfntoal eedn upon dependent functionally is Dab2 whether tested next We m .Qatfcto fteedctctafcigof trafficking endocytic the of Quantification m. . 0 oso h elsraeascae Dab2 cell-surface-associated the of loss 40% a nern() rnfri T)()adEF() ie ersn h vrgditaellritniisof intensities intracellular averaged the represent Lines (F). EGF and (E) (Tf) transferrin (D), integrin 2 P # 0.05. a integrin. 2 a nernad1 i fitraiaino rnfri n G.Cr,cnrl Error control. Ctrl, EGF. and transferrin of internalization of min 15 and integrin 2 oneuaino I1 niisteitraiainof internalization the inhibits KIF15 of Downregulation oei eyln ahas upiigy osignificant no Surprisingly, pathways. and recycling 37 Dab2 between in colocalization role a ta. 02 egoele n hrs,21) Integrin 2010). Cheresh, and cancer Desgrosellier of Shin 2012; 2008; al., processes al., et et crucial cell–extracellular-matrix (Mosesson metastasis and cytoskeleton, progression, progression initiation, the cycle and alterations cell interactions numerous signaling, to leads in integrins of endocytosis Defective DISCUSSION rpigo nerna o eprtrsi eurd as required, is temperatures low at for integrin demonstrated of trapping ufc oaiaino a2ta,i un ih euaethe regulate might turn, in cell- the that, mediates Dab2 of KIF15 internalization of that localization suggest data surface our together, Taken ˚ ,rgrls fKF5epeso ees ti osbethat possible is It levels. expression KIF15 of regardless C, ora fCl cec 21)17 4324 doi:10.1242/jcs.137281 2433–2447 127, (2014) Science Cell of Journal a integrin. 2 b nern(ekhnaie l,2009). al., et (Teckchandani integrin 1 a nernwsotie at obtained was integrin 2 a nern() u the but (A), integrin 2 B.Saebar: Scale (B). d a 2 b 1is

Journal of Cell Science EERHARTICLE RESEARCH Rmrze l,21) oee,ltl skonaotthe that about here metastasis known show is cancer We heterodimer. little breast this however, of endogenous of 2011); trafficking suppressor al., endocytic potent et a (Ramirez as known 20 bar: Scale control. Ctrl, RT-PCR. Numb quantitative Dab2, by encoding shown by mRNAs as shown of ARH as downregulation and Dab2, The of (D) efficiency blotting. downregulation western The (C) (B). effect of accumulation of intracellular accumulation the inhibits CLASPs a of Depletion 6. Fig. aaso h mean the show Data oeta euaos,adol ee oeue nrae the increased molecules of seven of only regulators accumulation intracellular and reduction potential regulators), a potential Predominantly, of as S2). in Table accumulation scored material supplementary intracellular molecules S1), Table the 122 material (supplementary mammalian tested with most were proteins, encompassing motor and organization, cytoskeletal 3). (Fig. cell-surface screen the RNAi microscopy-based affecting of proteins the expression of proportion substantial antibody-clustered the by endocytic a taken the pathway Nevertheless, degradation manner. dependent unbound mechanisms sequential of a intriguing caveolin-dependent route in an and act opens clathrin- might and data (Doherty that our responses possibility Alternatively, cellular 2009). individual broad intracellular McMahon, of invoke perturbation the could RNAi-based pathways however, of parallel; inhibition in act caveolin-1 strong 2). also (Fig. of comparably but accumulation structures clathrin a specific only not induced Rab11- plasma of the and depletion to Interestingly, Rab4- recycled by potentially is membrane and compartment specific eesont eueteoealepeso of expression inhibitors overall the the of reduce some to shown experiments, were validation subsequent the integrin. 2 nern(itnne l,21) ihtega fdfnn a defining of goal the With 2012). al., et (Rintanen integrin 2 nttl 8 ee soitdwt ebaetafcand traffic membrane with associated genes 386 total, In a nernitaellracmlto a eodd(o 115 (for recorded was accumulation intracellular integrin 2 oneuaino a2adAHpeet h intracellular the prevents ARH and Dab2 of Downregulation a a nerni eaclspse hog Rab5- a through passes cells HeLa in integrin 2 nern(,) hra oneuaino ubhsno has Numb of downregulation whereas (A,B), integrin 2 a a 6 nern epromdafluorescent a performed we integrin, 2 a nern(i.1,) ohptwy might pathways Both 1F,G). (Fig. integrin 2 .. * s.d.; nerndfessrnl rmtecalpain- the from strongly differs integrin 2 P # .5 ** 0.05, a nernwe oneuae.In downregulated. when integrin 2 P # 0.01. a nern(i.3A; (Fig. integrin 2 a nernin integrin 2 m m. nern(al ) sepce,tevldto aeo kinesins of rate validation of the trafficking expected, endocytic As the 1). (Table inhibited KIF18A integrin (KIF15, strongly three KIF23) hits, Of primary assays. and as our recorded of (supplementary kinesins fidelity screen nine high the indicating primary S4), the S2, in Tables effect material no had that kinesins ta. 00] neetnl,nal afo h neatn partners interacting the of half (Collinet of nearly the 3C; Interestingly, with [Fig. 2010)]. 122) endocytosis of al., out EGF et (39 and hits unsurprisingly, transferrin our and, of of contrast, overlap regulators cells large By HeLa fairly a in was libraries. there performed siRNA were normalization screens different both testing read-out data and microscopy), the multiparametric), procedures confocal Possibly, versus data versus between 2010)]. (monoparametric field differences EGF al., reflect (wide or screens et acquisition the transferrin of of (Collinet outputs regulators different 3C; as [Fig. identified not endocytosis screen were of our accumulation they of intracellular as hits the primary to 83 specific KIF15, that be as might found such We trafficking, identified. also of were inhibitors specific however, cells; loefcosof effectors also niaigafntoa rstl ewe h rfikn of trafficking the 2009)], between al., integrins. crosstalk ligand-bound et focal and functional unbound of (Winograd-Katz a regulators 3C; potential [Fig. indicating as formation of known regulators adhesion are potential levels 122 trafficking expression of and/or out integrin context 24 cellular Surprisingly, different integrin. a of to tagged due is and this proteomics overexpressed the the with of found a partners be interacting could of overlap analysis little but S2), Table ta. 01 ipo ta. 02,btifrainaottheir observed about We on information missing. Paul al., effect but is trafficking 2010; 2012), et no 2009; integrin al., al., Zhu in et al., specifically et 2010; Simpson roles (Collinet al., 2011; et al., trafficking et (Tanenbaum et membrane Neumann regulation and 2003; 2005a) Vale, cycle and cell Goshima the in motors. of performed been molecular already context has microtubule-based kinesins of analysis kinesins, Systematic were for role analysis direct more traffic. a integrin suggesting in 1998), it proteins al., these and to et 1996), binds (Obremski Fehon, NF2/Merlin endocytic cells the and that the (McCartney contrast, reported to cells was By localizes S2 in NF2/Merlin 2005). compartment of Giovannini, al., ortholog a et and actin Drosophila (Li providing be McClatchey the organization cannot by membrane and 2012; regulating ARF1 proteins indirectly thus of membrane-associated cytoskeleton, act functions between might other linkage NF2/Merlin however, 2012); excluded. al., for demonstrated et was as membrane plasma ARF1 trafficking Potentially, 2005). important al., inhibits et most Volpicelli-Daley and the 2006; D’Souza-Schorey Chavrier, 1994; a2 of Klausner, and one primarily COPI (Donaldson the regulators – and of influence component complex ARF1 key to a coatomer GTPase, is ARF1 appeared levels. small expression of others integrin a accumulation – whereas and them intracellular integrin, Merlin) of the two as influenced only predominantly known Curiously, of (also 1). heterogeneous seven (Table the NF2 and in validated functionally S2) siRNAs were Table multiple them material 14 by (supplementary hits screen as contained primary recorded were subset that molecules One hits. nerni T00cls(eas ta. 02.Potentially, 2012). al., et (Uematsu cells HT1080 in integrin 2 nte usto oeta euaoscoe o validation for chosen regulators potential of subset Another primary the of subsets several chose we validation, data For b nern(Bo integrin 1 ora fCl cec 21)17 4324 doi:10.1242/jcs.137281 2433–2447 127, (2014) Science Cell of Journal a nerntafcigb euaigisrccigt the to recycling its regulating by trafficking integrin 2 a nerntafcigwe oneuaig13 downregulating when trafficking integrin 2 a tce ta. 02 rsn norlbaywere library our in present 2012) al., et ¨ttcher nerntafcig(upeetr material (supplementary trafficking integrin 2 b nerni Schwann in integrin 1 a nern(Krndija integrin 5 a integrin, 2 2441 a a a 2 2 2

Journal of Cell Science EERHARTICLE RESEARCH raiaino ioa ioi pnls(uih ta. 2000; KIF15 2442 addition, al., In et 2013). Ohi, (Sueishi and Sturgill spindles 2009; al., mitotic the et in Vanneste bipolar 2011) Mayer, agreement as of and known Florian in (also organization 2009; Eg5 is al., by et replaced observation be (Tanenbaum can KIF11) latter KIF15 any that The having notion the numbers. without with 5A,D) cell Fig. on 4C,D; effect Fig. 1; (Table approaches of accumulation intracellular a inhibited of KIF15 accumulation of intracellular Downregulation of effectors validated 2012). al., stomatitis et vesicular (Simpson of virus) mutant of trafficking temperature-sensitive biosynthetic (a expression the ts-O45-G of actual the regulators potential changed likely as them identified most of of are none multiple level ones the as with validated regulators targeted trafficking the regulators the However, of siRNAs. that than lower was and 2 nti td,w aefcsdo I1,oeo h strongest the of one KIF15, on focused have we study, this In b a nern,a hw ysvrlcomplementing several by shown as integrins, 1 nern ned I1 n I2 aebeen have KIF23 and KIF15 Indeed, integrin. 2 a integrin. 2 ci rmcouuectseeo n etooe,rln u the out ruling in centrosomes, alterations Altered and apparent cells. cytoskeleton induce microtubule normal not or and did actin cancer KIF15 of in plays levels trafficking KIF15 expression integrin that in A549 indicating role a S3D), Fig. lung inhibition material induced (supplementary in KIF15 BJ- of fibroblasts observed of downregulation human A normal Furthermore, was the 5ta. structures. and cells pattern punctate carcinoma at epithelial and localization localized microtubules the similar to localization is membrane, occasional with cells, plasma largely HeLa it localized in KIF15 cytoplasm murine the bundles – to GFP-tagged actin 2003). neurons al., 2000), different post-mitotic et types al., terminally (Buster in remarkably et microtubules and (Sueishi cell fibroblasts a cells in HeLa various shows is in KIF15 centrosomes a and in 2010). in interphase progression al., localization cycle et in cell (Neumann of expressed screen regulator RNAi a as genome-wide identified not was a nernitaellracmlto nteecells these in accumulation intracellular integrin 2 ihsRA agtn I1 eeaaye.Saebr:20 bars: Scale analyzed. were transfected KIF15 cells 200 targeting and siRNAs (Ctrl) cells with control 160 negative microscopy. the confocal with by transfected visualized of Dab2 as perinuclear loss arrows), of to (white appearance leads and downregulation (arrowheads) KIF15 Dab2 (D) peripheral A. in line dashed membrane. plasma the ( Dab2-specific from Position arrows, redistributed Black are modes. that Epi structures and Dab2-specific TIRF showing by plot, structures profile intensity Fluorescence (C) p n IFmds aaso h mean the show Data in modes. imaged TIRF intensities and plotting. Epi fluorescent profile Dab2-specific intensity of fluorescence Quantification the for (B) line, used dashed was overlaid; that are position images TIRF Epi). and the mode, to Epifluorescence imaging mode) (epifluorescence imaging space (TIRF membrane intracellular plasma the with the associated from Dab2 membrane. redistributes plasma downregulation KIF15 7. Fig. ora fCl cec 21)17 4324 doi:10.1242/jcs.137281 2433–2447 127, (2014) Science Cell of Journal x ai)idctstedsac rmtelf n fthe of end left the from distance the indicates -axis) A oneuaino I1 ipae Dab2 displaces KIF15 of Downregulation (A) 6 s.e.m; n 5 5 * 15; P # m m. 0.05.

Journal of Cell Science splmnaymtra i.SC.Teedt somewhat data These downregulation S3C). KIF15 Fig. following cell-surface material measured increased (supplementary was addition, depletion KIF15 of expression In by inhibition 4A,B). caused rescued (Fig. accumulation partially intracellular siRNAs, integrin human to 4E,F). resistant (Fig. changes cytoskeletal of general to accumulation owing intracellular altered the that possibility ARTICLE RESEARCH ih eivle ntesraedlvr fa nr factor entry an of delivery surface the for in required involved be might not (Collinet was 5B,C,E,F; [Fig. transferrin KIF15 2010)]. and of al., depletion EGF et On the of compartment. by internalization influenced recycling perinuclear contrary, the function the might from it that trafficking suggesting in 5B), respectively. at (Fig. transferrin 2005), region of perinuclear accumulation al., the induced et KIF15 of (Hoepfner the Downregulation plus-end- recycling in transferrin reduction microtubule a and of induced the localization KIF16B 1996). and cell-surface of al., KIF1C et motors downregulation (Boleti directed motor instance, this of For directionality the contradict nw oseiial regulate specifically to known ta. 05.I gemn ihti,w hwdta endocytic that showed we this, Ezratty with non-clustered 2009; agreement of trafficking Kunz, In 2005). and al., Chao et 2007; Kaibuchi, and (Nishimura ta. 09,wihmgtrfetteueo different a of with use adaptor the the reflect Numb, might of for which preference Depletion 2009), methodologies. al., the on depletion ARH et of of effect expression no contrast, surface By 6B). of (Fig. accumulation 40% intracellular LDLR by of the of downregulation inhibited 2006), endocytosis also Cooper, and the ARH adhesions Maurer the to 2006, al., being Similar focal et Dab2 (Keyel 6). (Fig. with of Our observations, effector 2009). these strongest disassembly microscopy-based confirms Kunz, largely a the and assay using Chao for data 2007; RNAi as Kaibuchi, and well (Nishimura are as FXNPXY-signal- Numb and the cells, ARH, that Dab2, 2006) known for al., required is et in (Keyel It CLASPs pathways 1F,G). sorting (Fig. clathrin-dependent cells also HeLa but clathrin-independent nern sDb skont nii h nenlzto of internalization the inhibit to known of is accumulation unbound Dab2 intracellular the As in integrin. change significant no in oneuaino I1 Fg D,btde o colocalize not ce-Dab-1, does agreement, following In but conditions. compartment the these 7D), under (Fig. perinuclear transferrin KIF15 with the of at downregulation non- displacement accumulates of internalization their and the that inhibits therefore, membrane plausible, clustered plasma is the It from 2009). al., unbound et and to shown Morris were colocalize 2009; structures to al., Dab-2-specific addition, et In Teckchandani 2001). 2011; Cooper, al., et by are (Chetrit AP-2 These cells colocalize and to 7A,C). shown clathrin (Fig. were they with as membrane the structures, endocytic plasma that likely most show the could the Dab2 from punctate We of of structures analysis. redistribution effector induced further KIF15 strongest of for downregulation the non-clustered Dab2 be on of to focused appeared accumulation and intracellular 2012) al., et ae oehr u aaidct h osblt htKIF15 that possibility the indicate data our together, Taken is which KIF15, murine GFP-tagged of Overexpression anradtselegans Caenorhabditis b a nern(ekhnaie l,20;Teckchandani 2009; al., et (Teckchandani integrin 1 nern a2i eitiue rmteperiphery the from redistributed is Dab2 integrin. 2 b a nernitraiaini ubro migrating of number a in internalization integrin 1 a nernitraiain ned LSsare CLASPs Indeed, internalization. integrin 2 nern(ekhnaie l,20) resulted 2009), al., et (Teckchandani integrin 5 b nernwsrpre (Teckchandani reported was integrin 1 b a rhlgo a2 osntcolocalize not does Dab2, of ortholog a nernby integrin 1 . nernmgtrqientol the only not require might integrin 2 5 0 nHL,NHT n ES-2 and NIH3T3 HeLa, in 70% b nern(hie ta. 2012) al., et (Theisen integrin 1 b nerninternalization integrin 1 a nernendocytosis integrin 2 . 5 (Teckchandani 25% a nern we integrin, 2 a nernis integrin 2 a a integrin 2 integrin 2 a a 2 2 motn olgnrcpos seegn sa exciting an as emerging is receptors, collagen of important trafficking in overexpressed endocytic is KIF15 2013). as al., et context, (Bidkhori tumours disease various a in investigate ytmtcietfcto fnvlrgltr fteintracellular the the in of of is expression helpful regulators and/or that is novel accumulation assay of and integrin-mediated identification microscopy-based applications systematic with a high-throughput prevention for present associated the suitable we diseases for Here, essential of adhesion. is treatment knowledge and This area. research o h ees rnfcino elary,5 arrays, cell on transfection reverse the cDNAs For and siRNAs of Transfection Rene from gift a anti- monoclonal were Mouse Institute). antibody Cancer Netherlands anti-KIF15 (The Medema and GFP- University). plasmid Heidelberg (DKFZ/BioQuant, MmKIF15 Brady a Nathan were from mCherry–Rab4a gift and mCherry–Rab11b GFP–Rab7a, GFP–Rab5, Materials (BSA). albumin serum bovine (w/v) 0.01% with respective supplemented the medium in kept were 100 cells starvation, and serum and penicillin requiring serum experiments U/ml For DMEM calf 100 fetal of glutamine, mM 10% mixture contained 2 4:1 (FCS), media a All (Sigma-Aldrich). in 199 cultured BJ-5ta Medium (Sigma-Aldrich), were F-12K CRL-4001) in (ATCC cultured cells were A549 CCL-185) (Sigma-Aldrich), MEM (ATCC in cells cultured were CCL-2) (ATCC cells HeLa culture Cell METHODS AND MATERIALS n abtplcoa nidnmn2wr rmAcm Mouse Abcam. from were anti-clathrin monoclonal anti-dynamin-2 mouse anti- polyclonal Biosciences, monoclonal rabbit BD rabbit from and anti-caveolin-1 Merck-Millipore, was polyclonal (clone from anti-Dab2 rabbit 52/p96) monoclonal Mouse Biotechnology. and was Cruz Santa from were (H-110) P1E6) anti-Dab2 (clone monoclonal antibody integrin a ie ih3.5 with mixed was S1. Table material supplementary the in listed are siRNAs and Qiagen. other (SI03042634 from All were SI03199581) ARH and (SI04256994 SI02780386), Numb and siRNAs and SI00111657) (SI02780316 Invitrogen. Dab2 from and targeting were EGF–Alexa-Fluor-555 conjugates Transferrin–Alexa-Fluor-568, phalloidin–Alexa-Fluor-647 were HRP R&D. to coupled Invitrogen, antibodies from from anti-mouse-IgG and were were Healthcare (HRP) 647 GE peroxidase Fluor from horseradish Alexa to and coupled antibodies 488 antibodies anti-rabbit-IgG Fluor anti-rabbit-IgG Alexa to and coupled anti-mouse-IgG secondary Technology, Knie l,21) h mrigrl fKF5i h trafficking the caveolae in KIF15 to of role recruitment emerging of The cargo 2014). al., enhance et that (Kanai demonstrated KIF13B, recently trafficking was like clathrin-independent it as kinesins, presently, in out ruled KIF15 cycle be in cannot of cell 2011). function al., involvement et of and (Chetrit The cycle context localization cell the throughout cell-surface the change remainsendocytosis Dab2 within indirectly 6 as particularly or 2002), myosin progression, answered, al., motor et be Morris molecular to 2007; as al., actin-based binding, et direct the contribute(Spudich through might of for distribution fate KIF15 demonstrated Dab2 endocytic 2009), the influences Kunz, of KIF15 and regulation the Chao Ezratty to 2009; 2012; the al., As al., through 2012). et et occurs (Teckchandani al., integrins pathway et clathrin-dependent of (Fu the internalization transferrin by of Dab2-mediated recycling including affected the regulate proteins, be to numerous shown might was which distribution Dab2, mislocalized transferrin By the 2006). Cooper, contrast, and (Kamikura structures Rab11-positive with lcdtn h oeua ehnssifunigthe influencing mechanisms molecular the Elucidating a nern n osbyohrcros ilb neetn to interesting be will cargoes, other possibly and integrin, 2 ora fCl cec 21)17 4324 doi:10.1242/jcs.137281 2433–2447 127, (2014) Science Cell of Journal a tbln(ln MA)wsfo elSignaling Cell from was DM1A9) (clone -tubulin m fLpfcaie20 Ivtoe)ad3 and (Invitrogen) 2000 Lipofectamine of l a 2 b nern n ftemost the of one integrin, 1 a integrin. 2 a nern Whether integrin. 2 m lof30 m /lstreptomycin. g/ml m siRNA M 2443 m lof a 2-

Journal of Cell Science a2atbde a efre o t4 at h anti- 1 and for anti-KIF15 the performed with was incubation antibodies and PBS-Tween, Dab2 in with milk blocked were 5% proteins buffer, membrane unspecific The (PVDF) Laemmli Merck-Millipore), SDS-PAGE. difluoride 8% (Immobilion-P, hot polyvinylidene by to of separated transferred ml and were DTT proteins 80 mM in 100 lysed with cDNAs, supplemented and siRNAs respective iNswssotdot orLbTkcabr,wt –2negative 6–12 1084 with of chambers, library Lab-Tek whole four The rows. onto 32 spotted and was columns siRNAs 12 in Star’ organized of 1125 spots ‘Microlab to size set spot robot was pins a contact distance solid handling giving for eight using 600), used liquid (Nunc) (PTS slides and Lab-Tek automated one-chamber 7.25 (v/v), on the with fibronectin (Hamilton) mixed for 0.01% with was in incubated it printing (w/v) Thereafter, and gelatine temperature. sucrose 0.2% room M at 0.4 min 20 containing (Invitrogen) Opti-MEM ARTICLE RESEARCH 2444 10 with overlaid h, 14 for starved serum were Cells Fluorescence-microscopy-based system 2 PCR the after expression by Real-Time Numb calculated and StepOnePlus was ARH RNAi reaction, a Dab2, the oligo-dT Relative using for Biosystems). was and used (Applied performed was (Ambion) cDNA Mastermix was PCR transcriptase which protocol. Green SYBR reverse manufacturer’s Power MMLV primer. reagent the TRIzol with using to prepared performed was according RNA (Invitrogen) cellular total of Preparation RT-PCR and RT-PCR Quantitative 1.5 respectively. blotting assay, Western the was before cDNAs h and 24 siRNA with and cells h of 48 Transfection performed well. per seeded cells 5 layout. each across randomly distributed siRNAs control ado h ltwsepesda h rao h ekatrsbrcinof blot. subtraction relevant whole after the peak the the the of of of level using area intensity system background the as average using The by expressed ECL the was option. by blot Gel’ the the recorded quantified of ‘Analyze band using and was (NIH) (Intas) ImageJ by luminescence the system detected detection The were chemiluminescence Healthcare). interest of (GE proteins The rmrBATo sda ulse rvosy(alne l,2007). al., et (Jaulin using previously of designed published sets as either three used were or to the Primers BLAST up against transcriptome. Primer analysis with and BLAST RT-PCR genome nucleotide by of by human genes determined tested of were was that Expression cells primers quantification. HeLa the for in reference interest a as GAPDH of nern uliwr tie ih0.1 with stained were Nuclei integrin. ioetmn 00acrigt h auatrrspooo ih2 with protocol manufacturer’s the to eight-well according 2000 in Lipofectamine with cDNAs transfection direct or Liquid-phase siRNAs chamber. Lab-Tek per seeded were ihiecl E-S ormv xesatbd n nuae with incubated and twice 37 antibody washed at briefly excess MEM-BSA then remove pre-warmed ice, to on MEM-BSA h ice-cold 1 with for MEM-BSA in antibody integrin rel ahdtiewt c-odMMBAadicbtdwt the with were incubated 37 cells and Then at MEM-BSA each. 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Alexa-Fluor-568 cells pH NaCl, assay, mM trafficking 150 glycine, mM (50 pcfcfursec a 95,tgte aigu o8.%o the of 88.7% to up making together 69.5%, total was and image 19.2% fluorescence was for and specific stripping obtained acid imaging after was obtained intensity integrin for fluorescence parameters average total highest same The the analysis. average using the internalization compared non-clustered intracellular we and stripping, integrin total acid of of efficiency vales the estimate to a eoe yGusa lrfle n niiulsrcue of structures with analysis, the individual for used and were cells/Rab five filter than more of Blur noise regions algorithm, Gaussian ball by rolling . The a ImageJ. removed using in subtracted was performed was was background plane image confocal single multi-channelled respectively. 647, Fluor was Alexa TIRF and 488 respectively. Fluor Alexa 647, of TIFR Apo excitation Fluor Nikon with Alexa microscope TIRF Ti 60 and Eclipse mCherry Nikon 488, 568, a Fluor on Alexa performed Fluor or GFP of Alexa excitation the or for used were nm 633 0pxl nsz eebnrzdadoelpe.I oa,peripheral total, In overlapped. and binarized were size in pixels 10 6 A14 betv.Lsrlnso 8 n 4 mwr sdfrthe for used were nm 640 and 488 of lines Laser objective. 1.49 NA a a nernfluorescence. integrin 2 nern(0%.Telvlo o-lsee intracellular non-clustered of level The (100%). integrin 2 ora fCl cec 21)17 4324 doi:10.1242/jcs.137281 2433–2447 127, (2014) Science Cell of Journal Ratmtdivre irsoe(lmu Biosystems) (Olympus microscope inverted automated ˆR a nernseii loecne surface-localized fluorescence, specific integrin 2 Raqiiinsfwr.A10 A software. acquisition ˆR a nern nectto aeegho 2–4 nm 426–446 of wavelength excitation an integrin, 2 m /l el eefxdwt %PAfr2 min 20 for PFA 3% with fixed were Cells g/ml. 6 m imtrpnoe ae ie f48 6 and 561 488, of lines Laser pinhole. diameter m 6 .5N i betv UlnAo Olympus (UPlanSApo; objective air NA 0.75 i-meso betv HXP P 63 APO PL (HCX objective oil-immersion a b nernwt vrxrse asi the in Rabs overexpressed with integrin 2 a nernitraiainassay internalization integrin 1 nernitraiainassay internalization integrin 2 a nernwsmaue sdescribed as measured was integrin 2 b a nernlbln.Atrcell After labeling. integrin 1 ˚ nernfloig6 i of min 60 following integrin 2 o ifrn egh ftime. of lengths different for C 6 . 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Journal of Cell Science eino neet h ehdalwdubae aaquantification. data unbiased allowed method of majority the touching the interest, without As of cell 70–90% separation. region reach the cell-to-cell to of clear optimized intracellular area were secure densities to maximum Cell were confluence a ones. expanded cells were encompass neighboring individual nuclei the to of of regions as nuclei the and The so module, control. intensity by visual identified to a subjected applying were by [Scan subtracted algorithm was ball signal rolling background intensity, fluorescence ARTICLE RESEARCH edlegUiest.Dpstdi M o meit release. immediate for PMC in Landesgraduiertenfo Deposited the University. by Heidelberg number supported [grant is CellNetworks A.E. Cluster EXC81]. Excellence and University HEALTH-F5-2011-278758]; Heidelberg number the [grant MEHTRICS 0313923]; FP7 number Union [grant European ViroQuant fu Systembiologie Bundesministeriums der by Forschungseinheiten supported was work This Funding R.P. and J.I. T.L., interpretation. R.E., data analysis; the data evaluation; to bioinformatics data contributed performed statistical and R.R. performed A.A. and L.K. S.R., and A.E., D.M. manuscript; B.K. the experiments; wrote performed and H.E. project the designed V.S. and A.E. contributions Author interests. competing no declare authors The interests Competing the of reading thorough comments. for and Finland) Turku, manuscript of We University KIF15. Biotechnology, encoding of cDNA and Fa The KIF15 Reinhard Amsterdam, labeling thank Institute, antibodies Cancer the (Netherlands for Medema Netherlands) H. Rene thank We two-tailed Student’s Acknowledgements by experiments the tested between variance. for was uneven taken difference with were study samples of population this transferrin in significance For throughout cells manually. Statistical all set assays was analysis. separating trafficking endocytosis, threshold, a EGF low that and and except high screening, with primary were analysis the cells the in of as steps same other All the analyzed. and taken were images/well intracellular the screen. primary on the effect in tested siRNA siRNAs acceleratory every eight or than of more of inhibitory of out accumulation consistent out siRNAs siRNAs two a least three at least had siRNAs, at tested or primary two tested a of six as out considered one was was either Welch’s gene when A one-sample ratio hit values. one-sided, significance This compute a to spot. and used calculated every was array. in cell siRNA each median number on controls the cell negative the Eventually, cells of total such median the of the against number cells normalized by the of by intensity divided signal multiplied and median was the accumulation Then, efficient spot. with every intracellular for median calculated was The distributed. of intensities normally signal are efficiencies low cells and subpopulation Gaussian high (with A subpopulations automatically experiment. two to of the into used cells was within the 1997) samples Azzalini, cluster all and (Bowman for and Model same controls, Mixture negative the the kept was according that separately, was threshold experiment a every using for trafficking set endocytic weak with cells effects a removed spatial calculate for B- to account and the would applied artifacts. which averaged was between-plate with spot, 2003) and were each cells for al., for intensities factor et of correction excluded cell (Brideau 2% remaining were normalization score spot, the intensities imaged first, fluorescence package each At R integrin-specific data, used. highest screening were (http://www. primary Bioconductor the bioconductor.org/) from of cellHTS and analysis (http://cran.r-project.org/) statistical the For o h aiaineprmnsi eight-chamber in experiments validation the For of accumulation intracellular the quantify reliably To a nernitaellracmlto) suigta neach in that assuming accumulation), intracellular integrin 2 a -nernseii loecnewscutrdi the in clustered was fluorescence 2-integrin-specific se MI atnre,Gray n ele aii(Centre Hamidi Hellyeh and Germany) Martinsried, (MPI, ssler ¨ a nern oeta 00clswr nlzdfor analyzed were cells 3000 than More integrin. 2 a nernadcl ubr fec subpopulation each of numbers cell and integrin 2 z soeo – niiulrpiae fevery of replicates individual 5–8 of -score RAayi oue(lmu).Alimages All (Olympus)]. module Analysis ˆR rBlugudForschung; und Bildung ¨r reugflosi of fellowship ¨rderung m a sie,30–42 -slides, nern we integrin, 2 t t ts was -test ts for -test oykeiz .A,Jmeo,N . apesn . rnly . a den van J., Grindlay, I., Macpherson, B., N. Jamieson, A., M. Dozynkiewicz, oado,J .adKase,R D. R. Klausner, and G. J. I. Donaldson, T. Pastan, H. and McMahon, and C. J. M. G. Doherty, Willingham, A., J. Hanover, B., R. Dickson, P. Chavrier, and C. D’Souza-Schorey, oei . asni .adVro,I. Vernos, Bo and E. Karsenti, H., Boleti, Nowzari-Dalini, A., Moeini, S., Ashtiani, Hosseini Z., Narimani, G., W. Bidkhori, R. Finberg, and E. M. Hemler, M., B. Chan, P., M. Shepley, M., J. Bergelson, ho .T n uz J. Kunz, C. and J. W.-T. Chao, Norman, and S. Vadrevu, T., P. Caswell, egoele,J .adCeeh .A. D. Cheresh, and S. J. Desgrosellier, H., Ohno, S., Uzan-Gafsou, J.-B., Sibarita, D., Tenza, I., Hurbain, C., Delevoye, G., Chi-Rosso, L., C. Nickerson-Nutter, G., A. Sprague, R., C. A. Hu, Fougerolles, and de D. Humphrey, D., Corbin, N., Hasan, A., K. Riggs, P., Day, Sto C., M. Collinet, Ehrlich, and I. N. Smorodinsky, T., Bielik, G., Horn, M. L., M. Barzilay, D., Zutter, Chetrit, and A. S. Santoro, G., D. Grenache, G., T. Diacovo, J., Chen, utr .W,Bid .H,Y,W,Slwk,J . Chauvie M., J. Solowska, W., Yu, H., D. Baird, W., D. Buster, A. Liaw, and B. Pikounis, B., Gunter, C., Brideau, A., Byron, D., J. Humphries, D., R. Nunan, C., R. Williamson, D., M. Bass, awl,P . pne .J,Pros . ht,D . lr,K,Ceg .W., K. Cheng, K., Clark, P., D. White, M., Parsons, J., H. Spence, T., P. Caswell, J. Norman, and P. Caswell, C. J. Norman, and T. P. Caswell, S. Cohen, and G. Carpenter, omn .W n zaii A. Azzalini, and W. A. Bowman, J. Ivaska, and S. Veltel, J., Alanko, A., Arjonen, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.137281/-/DC1 online available material Supplementary material Supplementary thr .T,Srme,C,Mvs . ee,H,Wdae,M,Teg H.-Y. Tseng, M., Widmaier, H., Meyer, A., Meves, C., Stremmel, T., R. ttcher, ¨ ege .V,vnTu,A,Mro,J . ore,C,Tmsn . io,C. Nixon, P., Timpson, C., Gourley, P., J. Morton, A., Thun, von V., P. 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Journal of Cell Science asn .G n ihl,B J. B. Nichols, and G. C. Hansen, D. R. Vale, and G. Goshima, en,J,Int,R . elr .E,Cos,C n Massague and C. Crouse, E., M. Hemler, A., R. Ignotz, J., Heino, F. J. Collawn, and Z. Bebok, M., S. Rowe, P., L. Tang, A., Rab, L., Fu, U. T. Mayer, and S. Florian, lmn,F . rhm .L,Tkd,Y n olo,B S. B. Coulson, and Y. Takada, L., K. Graham, E., F. Fleming, T. Compton, and H. Koss, G. L., G. A. Feire, Gundersen, and E. E. Marcantonio, C., G. Bertaux, J., G. E. Gundersen, Ezratty, and A. M. Partridge, J., E. Ezratty, rl,H,Ekv,A,Ryan .adSakvee V. Starkuviene, and J. Reymann, A., Eskova, H., Erfle, rl,H,Nuan . ibl . oes . ed . atr . lebr,J. Ellenberg, T., Walter, M., Held, P., Rogers, U., Liebel, B., Neumann, C. H., R. Erfle, Liddington, and J. M. Barnes, W., R. Farndale, G., C. Knight, J., Emsley, Holtko D., Kessler, P., Zigrino, B., Eckes, A. J. Eble, rdj,D,Mu D., Krndija, N. Traub, Marks, and and C. H. S. R. Watkins, Kramer, E., J. Heuser, R., Roth, K., S. 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Hemler, EERHARTICLE RESEARCH 2446 rtisi ioi:cmrhnieRA nlssi h rspiaS elline. cell S2 Drosophila the Biol. in Cell analysis RNAi J. comprehensive mitosis: in proteins 643. euaino elahso eetr ytasomn rwhfactor-beta. growth transforming subunit. 1 beta by common a receptors share Chem. that integrins adhesion of regulation cell Concomitant of Regulation endocytosis. independent the of trafficking post-endocytic and regulator. endocytosis conductance of transmembrane regulator fibrosis key cystic a is Dab2 spindles. bipolar assemble to Hklp2/Kif15 eemnnso h pcfct frtvrsitrcin ihtealpha2beta1 the with interactions rotavirus of specificity integrin. the disintegrin-like of conserved Determinants highly a via in cytomegalovirus disassembly domain. human adhesion for receptors focal for endocytosis cells. integrin migrating mediates Clathrin adhesion focal and dynamin by mediated kinase. is disassembly adhesion focal induced ihcnetscreening. high-content cenn microscopy. screening R. Pepperkok, and integrin by recognition collagen of 47-56. basis Structural (2000). Biol. Matrix T. Krieg, Des. Pharm. 145. u o ffakn ein nteitgi ea uui eursascainwith association requires subunit beta2 integrin subunit. the alpha in the regions flanking of not but KIF16B. kinesin endosomal M. the Zerial, by degradation and H. Stenmark, transport. intracellular and proteins family. motor integrin the in 1) beta 3 (alpha Y. eeeaiglvr3(R-)pooe elmgaintruhArf-activity- through migration cell promotes (PRL-3) 3 G. Wichert, liver von and regenerating F. Oswald, T., Seufferlein, cells. melanoma human on adaptors. cargo-selective by governed integrin- cargo alpha2beta1 M. L. in participates caveosomes. entry to sorting Pak1-regulated dependent Raft-derived, Hyypia A H., G. Renkema, P., Liberali, caveolae. to LRP1 recruiting Caenorhabditis by in LRP1 of secretion protein for adaptor an elegans. Ce-DAB-1, of localization cells. MDCK in KIF5B by membrane Cell apical the Dev. to transport selective dependent 110 Arf6. adhesion. and Rac1 of regulator dual a as 2 J. RCC2 identifies M. complexes Humphries, and glance. aeedsmslssmsaddiecne progression. cancer drive and endosomes/lysosomes late al. et eedn tmlto fintegrin of stimulation dependent ra51. , 19) utpelgn idn ucin o L- apa2bt )adVLA-3 and 1) beta 2 (alpha VLA-2 for functions binding ligand Multiple (1990). 673-687. , 20) igecmo otlfrcahi-eitdedctsso distinct of endocytosis clathrin-mediated for portal common single A (2006). 21) a2 n LC olbrt opooeitgi eyln from recycling integrin promote to collaborate CLIC3 and Rab25 (2012). 264 a.Cl Biol. Cell Nat. .Cl Sci. Cell J. .Bo.Chem. Biol. J. rc al cd c.USA Sci. Acad. Natl. Proc. Traffic 20) irbatmti neatosi on eln n fibrosis. and healing wound in interactions Fibroblast-matrix (2000). 13 20) olgnbnigitgisa hraetcltargets. pharmaceutical as integrins Collagen-binding (2005). 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