arnDeebr,Mnk eram w ik ee cmee n ihe Krauss* Michael and Schmieder Peter Fink, Uwe Beerbaum, Monika Diesenberg, Katrin EGFR downregulation of ubiquitin-dependent regulates negatively SEPT9 ARTICLE RESEARCH ß eevd2 uut21;Acpe 4Nvme 2014 November 24 Accepted 2014; August 21 Received ([email protected]) correspondence for *Author terminal Germany. Berlin, elongation 13125 the filament fu for Leibniz-Institut occupies allow thereby which might cells SEPT9, SEPT9 can non-neuronal positions. dividing contain subgroup from Octamers complex same additionally 2011). SEPT2–SEPT6–SEPT7 SEPT6 al., the the and et in (Sellin other SEPT2 of each the for members of substitute members Individual of and subgroups. usually SEPT7 are complexes tissues, of recruitment these state, composed and hexameric the the cells In hexamers octamers. apolar of and from consisting orchestrate assemblies purified rod-shaped form septins When that as effectors. described binding downstream been scaffolds further have with filaments molecular association septin for Accordingly, partners. interfaces extensions, al., C-terminal provide and et N- to by which (Sirajuddin flanked them is allows G-domain complexes All The that 2007). sequence hetero-oligomeric 2003). domain into GTPase on (Kinoshita, central assemble subgroups SEPT2, Based a the SEPT7 contain into manner. isoforms and classified tissue-specific SEPT6 been have SEPT3, and isoforms these cell- similarity, expressed differentially a are that in isoforms 13 guanine-nucleotide-binding comprising of family proteins a constitute septins Human INTRODUCTION Ubiquitylation Sorting, CIN85, EGFR, SEPT9, WORDS: pathway. KEY degradative how the into of Taken EGFRs of impairs explanation membrane, sorting ubiquitylation. plasma mechanistic the the at EGFR a exclusively acting provide though reduced SEPT9, data in these resulting together, Cbl EGFR CIN85, ligase ubiquitin regulates the with of negatively association the SEPT9 preventing by that degradation be is demonstrate SEPT9 to we manner. CIN85-dependent where Finally, CIN85–SEPT9 a (also membrane, in receptors show plasma CIN85 EGF-engaged to that the recruited further protein to domain We adaptor exclusively N-terminal localized SH3KBP1). the SEPT9 as with the known association within its factor motif supports growth identify consensus epidermal We degradation. of a receptor levels enhancing by surface (EGFRs) receptors depletion decreases that underlying show SEPT9 we mechanisms of been Here, molecular understood. have poorly the isoforms are but pathogenesis Several disease, processes. in biological implicated of variety involved are a that proteins in GTP-binding of family a constitute Septins ABSTRACT 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,3747doi:10.1242/jcs.162206 397–407 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. ¨ oeuaePamklge(M) Robert-Ro (FMP), Pharmakologie Molekulare r ¨ seSrß 10, ssle-Straße nldn uoieei n erdgnrto Dlte al., et (Dolat diseases, neurodegeneration certain of these of and development the misregulation of tumorigenesis with the importance including correlated that been surprising fundamental and has not isoforms the (Caudron is it transduction Given processes, signal 2009). and Barral, membranes division, cell trafficking including of processes, cellular diffusion membrane compartmentalization of range form the broad a thereby during support phosphoinositides They that 2009). as barriers al., such et phospholipids, (Tanaka-Takiguchi (Kim charged incorporated negatively variant splice individual 2011). the al., et on of depending length superstructures, septin the of formation the abrogate or Kwnt ta. 03,apoierc idedmi n a motifs and 2002; domain proline-arginine middle al., association proline-rich membrane atypical in et a implicated region 2003), to coiled-coil Soubeyran C-terminal al., binding et 2002; SH3-domains, three (Kowanetz confer al., encodes CIN85 et 2002). which al., interaction its (Petrelli et on Szymkiewicz depends Cbl function This downregulation EGFR. with the as in such involved RTKs protein of adaptor an is 2003). ESCRT-dependent SH3KBP1) al., et an Lu and in 2003; al., pathway et of degradative multi- (Haglund of level manner sorting the the support at into to receptor However, believed EGFR debate. are of moieties drive ubiquitin matter plasma induces endosomes, a to the still essential at is is thereby endocytosis receptors ubiquitylation Whether dimerized and polyubiquitylation. to Cbl (Schmidt triggers Cbl membrane of binding been ligase ligand recruitment receptor ubiquitin concentrations, have high the E3 factor At the sorting 2005). by Dikic, growth and regulated degradative be epidermal to and on shown the well-studied In internalization effects 2008). the (EGFR), al., profound and et of dynamic have (Sigismund endocytosis under case duration RTKs and Hence, is strength of turn 2009). signaling in Goh, trafficking which and intracellular surface, receptors (Sorkin (Witsch of cell number regulation progression the the been on cancer at depends has signaling exposed of RTK (RTKs), 2010). stages al., kinases et several tyrosine with cell receptor correlated of the that particular underlying obscure. cancers. largely mechanisms of remain SEPT9 development of molecular functions the of biological levels support the elevated or al., al., that However, et induce suggesting et Scott abscission might 2010), 2011a; Kim al., al., SEPT9 the et et 2010; Stanbery (Connolly of been al., tumors 2005; have several misregulation SEPT9 et of in levels (Estey a detected protein midbody increased to (Surka Conversely, the 2011). defects owing cytokinetic at for 2002), cause SEPT9, machinery to al., of shown Depletion et been 2012). has Cossart, example, and Mostowy 2014; etncmlxsbn ommrn ufcserce in enriched surfaces membrane to bind complexes Septin b-neatn rti f8 D CN5 lokonas known also (CIN85, kDa 85 of protein Cbl-interacting in receptors, their and factors growth of misregulation The 397

Journal of Cell Science significant. 4h el eeaaye si ( B in as analyzed were cells h, 24 o ifrne ntelvlo ufc GR ausfrdgae n recycled and degraded for values EGFR, surface of level the in differences for ie h udmna oeo ET uigdvlpetadits and development during SEPT9 of role fundamental the Given degradation EGFR controls SEPT9 molecular ubiquitin- RESULTS control. the growth regulates cellular linking to negatively thereby SEPT9 of degradation, domains, associating activity by EGFR SEPT9, SH3 that dependent show CIN85 we Here, with 2009). al., et (Zhang ARTICLE RESEARCH 398 9 at cells HeLa siRNA-treated of surface the to bound was AF647–EGF EGFR. (B,C) of trafficking antibodies. endosomal indicated controls SEPT9 1. Fig. RNA upon receptors factor al., growth of et levels (Estey surface in differentiation changes and division Fu 2010; cell in involvement h elsrae Recycled surface. cell the Degraded 0ng/ml 20 h muto internalized of amount The eaclswr nuae ih15ng/ml 1.5 with incubated were cells HeLa bound o h niae ie.EF erdto a eetdb muoltig t immunoblotting. by detected was degradation ( EGFR microscopy times. epifluorescence indicated by the quantified for was cell per ligand 125 ctae ta. 01,w e u oivsiaepotential investigate to out set we 2011), al., et ¨chtbauer 125 -G ( I-EGF 125 -G o 5mnadcae o 0mn pnrccig natEFi eesdit h elmdu,btamnrfato ean tahdto attached remains fraction minor a but medium, cell the into released is EGF intact recycling, Upon min. 40 for chased and min 15 for I-EGF -G elcsteaon frdociiyfudi h uentnso C rcpttsdrvdfo elmdu n ellsts oaccount To lysates. cell and medium cell from derived precipitates TCA of supernatants the in found radioactivity of amount the reflects I-EGF n 5 ) G iN-rae eaclswr ernfce fe 4hwt ullnt F)SP9o nHA-tagged an or SEPT9 (FL) full-length with h 24 after retransfected were cells HeLa siRNA-treated (G) 3). 125 125 -G a hscluae steaon frdociiydtce nTApeiiae rmtecl eimadfo nai wash. acid an from and medium cell the from precipitates TCA in detected radioactivity of amount the as calculated thus was I-EGF -G a eemnduo usqetlsso h el.Frec iepit internalized time-point, each For cells. the of lysis subsequent upon determined was I-EGF n 5 ) l uniaiedt r eitda h mean the as depicted are data quantitative All 8). 125 -G t37 at I-EGF ˚ o h niae ie.Surface-bound times. indicated the for C A etnsRAvrfcto.sRAtetdHL el eeaaye yimnbotn sn the using immunoblotting by analyzed were cells HeLa siRNA-treated verification. siRNA Septin (A) n 5 0.()sRAtetdHL el eesiuae ihEFi rsneo cycloheximide of presence in EGF with stimulated were cells HeLa siRNA-treated (D) 10). 1/2 125 aflvso GR( EGFR of half-lives , -G eenraie ottlinternalized total to normalized were I-EGF GR,w etivsiae h fet fSP9dpeinon depletion SEPT9 of effects the investigated next we EGFRs, S1B). Fig. S1C). material Fig. (supplementary material as (supplementary such EGFR proteins, of surface Other levels supplementary levels decreased slightly 1A–C; total surface also (Fig. SEPT9 the EGFR of cells Knockdown in S1A). control Fig. reduction material HeLa with 50% SEPT7-depleted compared near not when a but SEPT9-, displayed Strikingly, cells cold. the (AF647–EGF) Alexa-Fluor-647–EGF in applying by EGFR quantified We surface SEPT9. of depletion (RNAi)-mediated interference steedt ugs osberl o ET nstabilizing in SEPT9 for role possible a suggest data these As 6 ...( s.e.m. ora fCl cec 21)18 9–0 doi:10.1242/jcs.162206 397–407 128, (2015) Science Cell of Journal n 125 xeiet) * experiments); ˚ .Saebr:10 bars: Scale C. n -G a eoe ya cdws n quantified. and wash acid an by removed was I-EGF 5 ) E iN-rae eaclswr nuae with incubated were cells HeLa siRNA-treated (E) 3). P , .5 ** 0.05; m b .()Teaon fsurface-bound of amount The (C) m. -nern eandunaffected remained 1-integrin, 125 125 -G a omlzdt surface- to normalized was I-EGF P -G ( I-EGF , D -uat fe nadditional an After N-mutant. .1 *** 0.01; n 5 ) F siRNA-treated (F) 3). P , .0;ns,not n.s., 0.001;

Journal of Cell Science nw odpn nlgn ocnrto Sgsude al., et (Sigismund concentration ligand is on endocytosis EGFR depend of mode to the known As EGFR. of uptake endocytic fewer Fig. and material cells, supplementary control 1E; (Fig. of S1F). recycled that were with receptors compared as incubation splmnaymtra i.SE.Nx,w efre pulse- inhibitors performed lysosomal we and Next, proteasomal S1E). recovered blocked Fig. both h were EGFR 1.2 material of processes (supplementary and addition of degradative the material h levels when 1.1 by cells supplementary surface to knockdown 1D; Accordingly, in h Fig. 2 S1D). substantially siRNAs; from Fig. SEPT9 half-lives independent of in approximately (two an decrease Loss by reflected twofold EGFR. degradation, receptor of accelerated sorting endosomal ARTICLE RESEARCH C,ttlcl yae t idtp;F,fl egh C ev chain. heavy coi c HC, CC, HeLa length; antibodies. from indicated full purification the FL, Affinity with type; immunoblotting (C–E) wild by antibodies. analyzed wt, indicated was lysate; the material cell with Affinity-purified immunoblotting total baits. as by TCL, proteins analyzed GST-fused were indicated Precipitates the extracts. using cell A431 from SEPT9 (SEPT9 CIN85. only with G-domain complex the a forms SEPT9 2. Fig. a cells, knockdown In EGFR of pathways. of fraction recycling larger sorting or ligand-induced degradative monitor into to experiments chase atrdgaainmgtb h osqec fenhanced of consequence the be might degradation Faster 125 -aee G a erddatr4 i of min 40 after degraded was EGF I-labeled D )i niae eo h oansrcue B w ifrn I8-pcfcatbde oimnpeiiaeendogenous co-immunoprecipitate antibodies CIN85-specific different Two (B) structure. domain the below indicated is N) A oansrcueo ET rncitvrat3(sfr )adCN5 eeinvrato ET comprising SEPT9 of variant deletion A CIN85. and c) (isoform 3 variant transcript SEPT9 of structure Domain (A) GR ae oehr hs eut hsdmntaethat of level demonstrate the internalization. at thus receptor EGFRs affecting results of without of sorting endosomes these neither degradative endocytosis modulates affected together, SEPT9 depletion clathrin-independent Taken SEPT9 EGFR. ligand. nor material of when (supplementary clathrin-dependent doses high EGFR and S1G) of 1F) Fig. rates (Fig. low internalization with the stimulated determined we 2005), GRlvl lotcmltl eoee,weesSEPT9 whereas recovered, of surface completely re-expression background, almost upon SEPT9-knockdown levels a EGFR Importantly, in 2A). SEPT9 Fig. full-length 1G; (Fig. alone EGFR G-domain the might full-length comprising mutant of (SEPT9 that truncation a Expression of mechanisms or experiments. SEPT9, molecular knockdown-rescue carried we the EGFRs, out of into stabilization SEPT9-dependent the CIN85 insight underlie protein adaptor gain the with To complex a forms SEPT9 D ) i o infcnl le h eeso surface of levels the alter significantly not did N), ora fCl cec 21)18 9–0 doi:10.1242/jcs.162206 397–407 128, (2015) Science Cell of Journal e-oldomain; led-coil l extracts ell D 399 N,

Journal of Cell Science oan fCN5 utemr,ordt ugs htthis that results receptors. suggest rather activated to filaments, data our monomers, SEPT9-containing SH3 SEPT9 our recruit the than Collectively, to to Furthermore, SEPT9 serves CIN85. links interaction 2E). motif (Fig. of PR atypical domains 2011) present an when al., that only et demonstrate but was SH3A-C, (Kim CIN85 SEPT9 SEPT2/6/7-containing SEPT9 GST-fused with on Interestingly, co-purified assemble were to SEPT9. known unable with filaments was aa334-end) interact (CIN85 domains to of SH3 variant the deletion A lacking 2010) S2A). CIN85 al., Fig. to material et similar supplementary (Schroeder 2D; extracts, dynamin-2 (Fig. cell including from ligands, SH3 SEPT9 other with associate to sufficient CIN85– the that direct. indicating is interaction S2C), SEPT9 Fig. material (supplementary 2) mn h aiu H oan etd nytetreSH3 three the only tested, domains SH3 various to Fig. the material Among SEPT9 S2A). (supplementary link rearrangements might cytoskeletal sorting and/or region endosomal in this implicated proteins that SH3-domain-containing speculated therefore N- We EGFR. SEPT9 of the sorting of endolysosomal role during crucial restore domain a to terminal suggests unable was This domain, expression. N-terminal EGFR the lacking mutant the ARTICLE RESEARCH oiindi ls rxmt otences n othinner to and nucleus, the 400 to S3A,B; proximity structures 2002), close fiber-like Fig. al., prominent in et to material (Surka positioned localized reports SEPT9 previous (supplementary endogenous with Consistent CIN85 1A). Fig. and against raised at indirect SEPT9 antibodies applied investigate using we To complex, microscopy 2004). this sorting immunofluorescence enter al., might plasma et endolysosomal SEPT9 Kowanetz stage remains the which 2002; and that it al., of et complex – internalization (Haglund Cbl signaling level during ligase EGF-induced ubiquitin the an assembled the of at with part together forms EGFR – where with membrane, associates cell the CIN85 at receptors EGF activated surface to SEPT9 recruits CIN85 (GST–SEPT9 domain native terminal of with fraction 2B). (Fig. substantial co-immunoprecipitated extracts a cell be from found to CIN85 and SEPT9 probed interaction We endogenous sorting. potential EGFR a to CIN85 vesicular SEPT9 considered for linking thus the factor we candidate and and a of 2010), degradation as al., steps internalization, et (Havrylov including various recycling EGFRs, in of implicated transport extracts an cell is protein CIN85 from matrix. SEPT9 adaptor affinity retain (GST) to glutathione-S-transferase a able on were CIN85 of domains iia idn atr a bevdwe h sltdSH3 (His isolated the CIN85 when observed of was domains pattern A binding binding. for similar dispensable largely were motifs PR219/220) remaining and two (PR26/27 the between whereas CIN85, abolished with conserved completely formation complex almost PR129/130AA) highly (SEPT9 motif this is of inactivation Mutational particular PR129/130) S2B). Fig. the One material (supplementary (SEPT9 2003). species fit al., motif et CIN85 would (Kowanetz with PR which 2A) interactions (Fig. N-terminus, mediate domains. to SH3 its known in sequence consensus motifs proline- atypical SEPT9 (PR) three the of arginine of presence the inspection revealed Closer sequence CIN85. primary with (GST–SEPT9 associate G-domain to isolated unable Fig. the material contrast, supplementary By 2C; S2C). (Fig. CIN85 with interaction the sepce,teS3dmisdrvdfo I8 were CIN85 from derived domains SH3 the expected, As iceia apn xeiet eeldta h ET N- SEPT9 the that revealed experiments mapping Biochemical residues. proline in enriched is domain N-terminal SEPT9 The 6 CN5S3-)wr probed were SH3A-C) –CIN85 aa1–220 a ufcett promote to sufficient was ) D )was N) otepam ebaei I8/ET-eedn manner, CIN85/SEPT9-dependent a in membrane recruited surface- are plasma filaments with the septin that to SEPT2 idea the of SEPT9 supporting Moreover, EGF, colocalization bound 3D). the Fig. 2E; decreased (Fig. SEPT2, depletion depleted instance was for CIN85 other SEPT9, when for with observed be co-assembling could isoforms effect similar septin A 3B,C). and (Fig. knockdown CIN85 SEPT9 upon of between decreased colocalization significantly of Dikic, structures degree EGF-positive and the (Schmidt note, S3D) Of 2005). Fig. of material downregulation (supplementary and EGFRs ubiquitylation ligand-induced and ligase EGF ubiquitin SEPT9 mediating a with AF647-labeled c-Cbl, colocalized (HA)-tagged of also with epitope haemagglutinin CIN85 surface colocalization with sites, the these its At at 3A). by EGFRs (Fig. shown activated as to CIN85 cells, of facilitated EGF recruitment of application the expected, As EGF. with stimulation filamentous from organelles. SEPT9 CIN85-positive endogenous onto of structures relocation the sufficient EEA1-containing was trigger early CIN85 to of from overexpression distinct Importantly, clearly endosomes. accumulated were and which material S3C) (supplementary 2009), Fig. LAMP1 as al., such et markers late-endosomal (Zhang as endosomes, previously aberrant mRFP-tagged enlarged, described of of formation Overexpression the periphery. induced CIN85 cell the in filaments hscmlxdrn rso fe endocytosis. from after plasma excluded soon is is the or but during – at manner, complex endosomes this CIN85-dependent EGFRs a of ligand-engaged in level and membrane to the SEPT2 recruited at partners though transiently – sorting binding SEPT9 degradative that cognate indicate regulating results its these conclusion, and In SEPT7. a SEPT9 held in same for the membranes Interestingly, to true 4B,C). cytosol (Fig. the of manner from translocation dose-dependent Grb-2 the adaptor triggered signaling As EGF the starvation. pool during with small membranes when stimulation a with only anticipated, associated temperatures, found be low we to filaments), SEPT9 at to of localized (e.g. not with conditions is SEPT9 stimulation these upon SEPT9 Under of EGF. distribution insight subcellular gain the to into experiments therefore fractionation We biochemical 2009). performed al., a et (Tanaka-Takiguchi that phosphoinositides endosomes. possibility on assembled the is CIN85 SEPT9. exclude and SEPT9 not of largely Vps4A(E228Q) complex but native data expected, negative these as Collectively, CIN85, dominant endogenous accumulated of (Baumga overexpression stalled of endosomes formation degradative by the However, during of bodies. potential complex site a multivesicular SEPT9–CIN85 considered the the also to of we components 2014), function al., ESCRT of et of role (Renshaw recruitment the abscission and the 2006) complex in (Sadoul, sorting septins machinery (endosomal transport) ESCRT for the CIN85 required of of assembly implication the functional the during (supplementary Given S4A,B). respectively Fig. eGFP–Rab11, material wild-type to of or eGFP– or early GTP-locked Rab5 to of stimulus overexpression translocate the upon not internalized of endosomes did recycling duration SEPT9 with or Furthermore, min) 4A). presence colocalization (15 (Fig. the late no of irrespective and the displayed EGFRs, cell min) during and the (5 in localization filaments periphery, early with associated its to remained SEPT9 monitored endosomes. AF647–EGF we of EGFRs, transport of sorting multimers. or mono- SEPT9 than rather ete nlzdtelclzto fedgnu ET upon SEPT9 endogenous of localization the analyzed then We etn a soit ihmmrn ufcserce in enriched surfaces membrane with associate can Septins degradative during SEPT9 of role potential a investigate To re ta. 01 splmnaymtra i.S4C) Fig. material (supplementary 2011) al., et ¨rtel ora fCl cec 21)18 9–0 doi:10.1242/jcs.162206 397–407 128, (2015) Science Cell of Journal

Journal of Cell Science EERHARTICLE RESEARCH i.3 ET n I8 atal ooaiewt ciae GRa h lsamembrane. plasma the at EGFR activated 9 with at colocalize AF647–EGF partially ng/ml CIN85 500 and SEPT9 3. Fig. K eesi el pnsiuainwt G Fg 4D; were (Fig. signaling ERK1/2 S4D,E). EGF Fig in remained material with changes (supplementary levels no detectable stimulation AKT contrast, Total By upon S4D). unaffected. phospho- cells Fig. reduced material in significantly supplementary also levels SEPT9 AKT of depletion cells, loecneitniypoie ln ie eitdi h egdiae.Bxdaesaesona ihrmgiiaini h nes B I8 siR CIN85 (B) insets. the in treat 10 magnification were bars: cells higher Scale HeLa at siRNA-treated (D). shown (C,D) SEPT2 antibodies. are or indicated areas the (C) Boxed using SEPT9 immunoblotting images. for by merged analyzed stained the were cells in HeLa depicted siRNA-treated lines verification. along profiles intensity fluorescence uniidb acltn h muto etnfudi G-oiiesrcue dt eecretdfrttlEFbudt h elsrae ( surface) cell the to bound EGF total for corrected were (data structures mean EGF-positive the in as found depicted septin are of data amount quantitative the calculating by quantified ocmtn ihtedces nsraeEF nknockdown in EGFR surface in decrease the with Concomitant ˚ n tie o ET n I8.Nt htSP9flmnsaedsutduo ramn tlwtmeaue.Rgtpanels, Right temperatures. low at treatment upon disrupted are filaments SEPT9 that Note CIN85. and SEPT9 for stained and C 6 ...( s.e.m. n m xeiet) * experiments); mi mgs,1 images), (main m P , .5 ** 0.05; m ist) ooaiaino ufc-on F4–G ihspi was septin with AF647–EGF surface-bound of Colocalisation (insets). m P ple ula antcrsnne(M)setocp ogain to spectroscopy (NMR) and resonance To approach magnetic endosomes? structure-based nuclear a on applied followed not we acts and question, it this membrane if answer plasma EGFR the of at sorting degradative exclusively modulate SEPT9 can How EGF of receptors ubiquitylation Cbl-dependent regulates SEPT9 , 0.01. ora fCl cec 21)18 9–0 doi:10.1242/jcs.162206 397–407 128, (2015) Science Cell of Journal A etpnl,cnoa mgso eaclstetdwith treated cells HeLa of images confocal panels, Left (A) n da nAand A in as ed 5 ) All 3). NA 401

Journal of Cell Science Fg D.Teesrcua n iceia aasgeta suggest data biochemical exclusive and mutually structural is domains These Affinity CIN85-SH3 5D). to (Fig. 5C). Cbl (Fig. a and of binding the SEPT9 the of CIN85 lacking that peptide confirmed further presence control motif mutant PR-signature of conserved the a of or in domain binding peptide SEPT9-derived performed same SH3A experiments Accordingly, the site. chromatography occupied on binding peptides same surface Cbl-derived the and that for indicating SEPT9- compete 2013), al., ligands PR- in et other of direct (Ceregido both changes to 5B) domain binding induced (Fig. in the motifs SH3A implicated peptide signature acids the amino the confirmed of with of shifts chemical peptide titration experiments Moreover, SEPT9 CIN85. the Our of 5A). association (Fig. CIN85 EERHARTICLE RESEARCH 402 the with with peptide it association incubated SEPT9-derived its and a for needed CIN85, designed motif We PR-signature the CIN85. containing with SEPT9 association the of underlying mechanisms molecular the into insights stimulation. EGF upon 37 septins at of AF647–EGF recruitment Membrane 4. Fig. mgs,1 images), ihEFfr5mnadsbetdt LS omauepopoATSr7 ee ( level phospho-AKT-Ser473 measure ( to C ELISA in shown to is subjected and and * quantified min experiments). was 5 SEPT9 for cytosolic EGF to with membrane-associated of ratio The immunoblotting. m ist) BC ebaeadctslfatoaino G-tmltdHL el.Eulaonso ohfatoswr nlzdby analyzed were fractions both of amounts Equal cells. HeLa EGF-stimulated of fractionation cytosol and Membrane (B,C) (insets). m P , ˚ .5 ** 0.05; o h niae ie,sandfrSP9 h oe ra r hw thge anfcto nteist.Saebr:10 bars: Scale insets. the in magnification higher at shown are areas boxed The SEPT9. for stained times, indicated the for C P , 0.01. 15 -aee HAdmi of domain SH3A N-labeled A ofcliae fHL el pnsavto,o pntetetwt 0 ng/ml 100 with treatment upon or starvation, upon cells HeLa of images Confocal (A) infcnl oeuiutnta GRfo oto cells for explanation control cells. molecular SEPT9-depleted incorporated from a in observed provide degradation had EGFR accelerated results the than cells These 5E,F). ubiquitin SEPT9-knockdown (Fig. more of significantly lysates affinity-purified EGFR from that with this revealed cells HeLa analyses the of Quantitative test stimulation in ligand. upon EGFRs SEPT9 Cbl- of To of ubiquitylation absence or of regulate degradation. presence extent the might and assessed we SEPT9 ubiquitylation directly, which EGFR by mediated mechanism molecular hmfo erdto n nrae h rcino receptors of fraction the increases protecting and by EGFRs of degradation stabilizes the subset from SEPT9 with a them CIN85. association in by of the SEPT9 domains encoded promotes SH3 which of motif variants, PR role splice conserved function SEPT9 regulatory This highly EGFR. key a of levels involves novel expression surface a the modulating identify we Here, DISCUSSION n 5 ) l uniaiedt r eitda h mean the as depicted are data quantitative All 3). ora fCl cec 21)18 9–0 doi:10.1242/jcs.162206 397–407 128, (2015) Science Cell of Journal n 5 ) D iN-rae eaclswr stimulated were cells HeLa siRNA-treated (D) 3). 6 ...( s.e.m. m n (main m

Journal of Cell Science oto etd.(,)EF bqiyaini iN-rae eacls eletat eesbetdt muorcptto sn nEGFR-specif an using immunoprecipitation to from subjected quantified were were extracts ubiquitin Cell of cells. amounts HeLa relative siRNA-treated The in ubiquitylation (F) EGFR (E,F) peptide. control EERHARTICLE RESEARCH uigedsmlsrig(aln ta. 02.Our phosphatidylinositol 2002). that our hypothesize on al., Based we internalization. after assays, et transiently, soon or fractionation complex during EGFRs this (Haglund released joins is activated and SEPT9 with sorting that suggest associated observations remain endosomal to during known is cell the CIN85 at receptors EGF inhibits activated of surface to SEPT9 effects and recruits observed CIN85 CIN85 the sorting. for thus to EGFR SEPT9 data on explanation our depletion that binding molecular SEPT9 together, Taken a demonstrate for cells. provide in of we EGFRs Cbl of level Finally, ubiquitylation the with the on CIN85. dependent competes at membrane, of plasma act of the presence recruitment to not the exclusively triggers affecting in SEPT9 does stimulation EGF involvement without SEPT9 Instead, its endosomes. membrane, sorting, despite plasma However, degradative the internalization. to receptor back recycling ubiquitylation. EGFR regulates and CIN85 to binding for c-Cbl with competes SEPT9 5. Fig. eiusfrwihppieidcdceia hf hne eeosre r ihihe nrdadoag.()HL el eesiuae ih50ng 500 with B2Z8. stimulated structure were X-ray cells the HeLa on (D) based orange. SH3A and CIN85 red of in 0.5 model highlighted peptide, ribbon are (no and observed PxxxPR Sequence were peptide (B,C) changes 9 SEPT9-derived peaks. shift at the the chemical EGF of to peptide-induced amounts next which increasing shown for containing is Residues samples assignment from resonance spectra The with equivalents). alone A SH3 CIN85 ˚ n ujce oafnt uiiainuigteS3Admi fCN5a at xrcshdbe upeetdwt ET-eie ra or SEPT9-derived a with supplemented been had Extracts bait. as CIN85 of domain A SH3 the using purification affinity to subjected and C n 5 xeiet.TL oa ellst.Dt hwtemean the show Data lysate. cell total TCL, experiments. 4 ,-ipopae[PI(4,5) 4,5-bisphosphate lse,SP9sostefsethdoyi ae(etand (Zent rate hydrolysis fastest septin the other al., of shows et members (Kim SEPT9 to oligomers comparison classes, SEPT9 higher-order in Interestingly, of of 2011). formation isoforms the long stabilize whereby with manner, interaction non-stoichiometric be their progression. to extent cancer remains to what 2014; It contribute al., 2010). to might et al., CIN85 and Connolly et SEPT9 whether 2010; Stanbery tumors long al., 2011b; determined various et al., note, in (Amir et upregulated lines Connolly protect Of be cell to to cancer 1G). SEPT9 shown and (Fig. been of have degradation isoforms isoforms long 2009). from septin contain al., CIN85-associated EGFRs to et experiments, need (Tanaka-Takiguchi rescue filaments surface our their to cell support SEPT9- According and the of membrane at plasma recruitment retention the initial to the filaments aid containing might EGFRs stimulated rshsht [PI(3,4,5) trisphosphate ET a idt ET–ET–ET opee na in complexes SEPT2–SEPT6–SEPT7 to bind can SEPT9 ora fCl cec 21)18 9–0 doi:10.1242/jcs.162206 397–407 128, (2015) Science Cell of Journal A vra fthe of Overlay (A) P P 3 cuuaigi h iiiyof vicinity the in accumulating ] 2 n hshtdlnstl(3,4,5)- phosphatidylinositol and ] 1 H, 6 15 ... * s.e.m.; SCsetaof spectra HSQC N P , 0.05. 6 ,1 6 ,2 6 15 ,10 N-labeled cantibody. ic 6 molar 403 /ml

Journal of Cell Science ol ntaetefraino ifso are,wihwould which barrier, diffusion a of formation septin the local initiate of could remodeling of a induce state thereby SEPT9 of structures. and activity nucleotide-binding with stability GTPase filaments the interactions affect in that and could the possible region is N-terminal formation the it on monomers, septin filament individual depend As additionally 2014). Wittinghofer, ARTICLE RESEARCH ET,aercutdt tmltdMtdrn nainof invasion 404 during Met stimulated including septins, to several recruited Listeria Interestingly, c-Met are as 2002). such SEPT9, al., kinases, of tyrosine et levels receptor surface other (Petrelli the of have of also CD2AP regulation but paralog the EGFR, in its implicated and been CIN85 only studies. will not future interactome of EGFR how subject the and of be Whether composition space. serves the and thereby modulates time and SEPT9 in membrane, as activities far their plasma coordinate as the to least at at assembled complexes, EGFR are those they of in SEPT9 formation that involved the unlikely not regulates proteins is also It including trafficking. subcellular 2011; 2004), and al., signaling et al., (Buchse CIN85 et of domains Kowanetz SH3 the with associate to Fig. material (supplementary of is localization endosomes process the S4B). recycling this for in evidence on SEPT9 no found of SEPT9 we role (Schroeder that direct EGFRs given a unlikely, of However, recycling 2010). in al., implicated et CIN85 negatively that been note thereby We recently the endosomes. and has of with level we membrane, EGFR the at of plasma machinery ESCRT association account, the ubiquitin-dependent the of into ubiquitylationregulates level EGFR or of the observations extent 2003), the at limits al., these SEPT9 that et hypothesize EGFR Taking (Lu attenuates 2004), al., Tsg101 between et degradation. (Hislop subunit interaction RNAi by ESCRT-I the Tsg101 (Sigismund depleting the perturbing complex and Furthermore, ESCRT-0 HRS 2005). the associates of al., EGFR component ubiquitylated et a that shown HRS, For been with 2006). al., has an et it has (Huang instance, degradation however, EGFR EGFR Ubiquitylation, during during 2010). role manner essential al., cooperative et a (Goh in that endocytosis observation act the with mechanisms line several in internalization, EGF of pathways clathrin affect significantly not does thereby CIN85, SEPT9 with ubiquitylation SEPT9-sensitive ubiquitylation. that formation receptor attenuating complex suggest for further c-Cbl data with competes biochemical and structural the Our to EGF of receptors ubiquitylation contribute Cbl-dependent different regulates question. SEPT9 might open between an cytoskeleton remains crosstalk trafficking 2012). EGFR the of local Cossart, modulation of and such (Mostowy components other whether cytoskeleton and the septins However, between of interplay further elements functional have studies the Several cytoskeleton. septin highlighted actin between the crosstalk a and for and to filaments allowing (Nagata binds potentially factor 2005), domain septin exchange Inagaki, N-terminal Russell, nucleotide other SEPT9 and guanine of the (Hall RhoA-specific example, complex or same For binding domain the 2012). further N-terminal in recruit SEPT9 co-assembled that isoforms the scaffolds of the as these of partners Moreover, serve subdomains 2009). might in Barral, filaments and complexes (Caudron signaling membrane of plasma retention the aid etnflmnsasmldi ls rxmt oatv EGFRs active to proximity close in assembled filaments Septin eie -b/b-,nmru te atr aebe found been have factors other numerous c-Cbl/Cbl-b, Besides and Shigella noHL el Msoye l,2009). al., et (Mostowy cells HeLa into dpneto clathrin-independent or -dependent sfr )adhmnCN5v B050)wr lndit pGEX (BC021102; into cloned SEPT9_v3 were human (BC015806) CIN85_v1 of human and variants c) deletion isoform and Full-length Plasmids METHODS AND MATERIALS al., et (Connolly explanation cancer molecular with SEPT9 novel 2011a). of a association provides genetic the study for our regard, (Fu migrate this from cells to in wild-type shown with derived than might been line slowly fibroblasts have more In SEPT9 mice embryonic cells. SEPT9-knockout that cancer cultured conditional of hypothesis, suggests potential this this migratory the migration, modulate cellular during ojgtd(F4)EFwsprhsdfo ieTechnologies. Alexa-Fluor-647- Life from Mouse purchased Peprotech. was from (AF647)–EGF purchased conjugated was EGF Human antibodies and Chemicals N-terminal study: this in 1–220), used acids were (amino SEPT9 SH3 The region 1– 256–333), of following 334–end). acids acids acids truncations (amino (amino (amino The region C following A C-terminal SH3 1–333), SH3 96–163), BioTez). acids Site-directed study: (amino acids A-C this (amino (MWG, cells. in B used SH3 were sequencing mammalian 69), were constructs CIN85 dsDNA in of All truncations PCR. by expression overlapping by for verified performed was tags EGFP mutagenesis HA, mRFP with fusion in-frame an or encoding expression plasmid pcDNA3-based bacterial a for or (Novagen) pET28a+ or Biosciences) (Amersham CONH o h ET nioy h etdsNH peptides the antibody, SEPT9 the For ptp a eindsc httersligatbd eet l long all detects antibody resulting the that such designed was epitope nfn-uigEF inln.Gvnterl fPI(3,4,5)P of role the Given levels signaling. SEPT9 of phospho-ERK1/2 EGFR functions fine-tuning contrast, additional in indicating By the cells upon unchanged, that EGF. detectable SEPT9-knockdown remained note phospho-AKT with in to of levels EGFR interesting stimulation reduced various is surface with at It of correlate progression 2010). levels cancer al., reduced et promote (Witsch to stages known is determined. receptors be to remains well during as function process CIN85 and this regulates (Veiga SEPT9 Met Whether on of 2005). ubiquitylation depend Cossart, Cbl-mediated to as appears well bacteria as of CIN85, entry subsequent the Moreover, muie gis h etd NH were rabbits peptide antibody, the SEPT7 affinity- the were against For antisera specificity. immunized All for peptides. tested different and two purified injecting by rabbits in ImageJ. from plugin performed blot was western blots the western using from Quantification #70796-3). (Novagen, G3 a bandfo abohmaddsovdi MO NH DMSO. in dissolved and Calbiochem and (NEX42800). from Elmer (H-7112) obtained Perkin Bachem was from MG132 from obtained was obtained experiments was recycling for experiments uptake for EGF Acm a192,popoEK/ Sga M19 n His ERK1/2 and #4060), #M8159) (Sigma, D9E, phospho-ERK1/2 clone #9272), #ab17942), Signaling, Signaling, (Abcam, (Cell (Cell AKT1/2/3 #610112), phospho-AKT-Ser473 #HPA018481), Transduction, (Sigma, (BD SEPT2 signaling, Grb2 #HPA005665), Signaling, Cell (Cell (Sigma, P4D1, SEPT6 EGFR (clone #3936), ubiquitin #610441), #T5168), Bioscience, (Sigma, (BD transduction, tubulin 2 #4267), (BD dynamin TD.1), c-Cbl (clone #610245), chain western heavy For clathrin against #555801). Sigma), used Pharmingen, were antibodies (BD experiments, LAMP-1 blotting and #sc-101) Cruz, #610456), transduction, (BD b EEA1 Babco/Convance), HA.11, (clone HA water. in dissolved and Roth from obtained was xeiet,a iN-eitn,utge eso fSP9was SEPT9 of version untagged siRNA-resistant, generated. an experiments, -nern(M ilpr,#A18) GR(ln -,Santa R-1, (clone EGFR #MAB1981), Millipore, (EMD 1-integrin oylnlatbde gis ET,SP9adCN5wr raised were CIN85 and SEPT9 SEPT7, against antibodies Polyclonal o muoloecneeprmns nioiswr sdagainst used were antibodies experiments, immunofluorescence For ngnrl h eeuaino rwhfcosadtheir and factors growth of deregulation the general, In 2 n NH and ora fCl cec 21)18 9–0 doi:10.1242/jcs.162206 397–407 128, (2015) Science Cell of Journal 2 -CTELSIDISSKQVEN-CONH D aioais28ed.Frrescue For 238–end). acids (amino N 2 -CYEFPETDDEEENKLV-COOH. ctae ta. 01.Also 2011). al., et ¨chtbauer 2 -CSTQKFQDLGVKNSEP- 2 eec-netd The co-injected. were b atn(ln AC-15, (clone -actin 6 125 -tag 4 Cl I- 3

Journal of Cell Science xxR NH PxxxPR, AAGAGA-3 CUUGCAGCUGUGACUUAUA-3 GUC-3 fedgnu I8 n ET,orSP9seii nioywas antibody SEPT9-specific our Link Lightning the SEPT9, using Atto565 and to coupled directly CIN85 detection parallel endogenous for allow of To antibody used. SEPT9 was (H00010801-M01) the Abnova Here, from the the 2A. throughout for Fig. not used in but experiment was immunoblotting, coimmunoprecipitation and antibody immunofluorescence at This for located motif study v3. PR whole isoform the contain of that v8) 129-130 and position v3 v2, (v1, SEPT9 of isoforms ARTICLE RESEARCH ltdHis Eluted vitro In with incubated and 4 above at h described 2 as for proteins prepared GST-tagged purified were extracts matrices affinity Cell GST on purification Affinity His purification and expression Protein KCl, mM 100 7.4, pH HEPES mM MgCl assay, (20 mM buffer ubiquitylation 2 extract EGFR in the lysed were except cells experiments biochemical all For Immunoprecipitation h. 48 after analyzed and were (Invitrogen) Oligofectamine samples using the cells into transfected were transfection). (Polyplus siRNAs JetPrime using cells Dulbecco’s into transfected in 0.1 were penicillin, Plasmids calf grown U/ml fetal (0.1 were antibiotics 10% and cells with (FCS) serum supplemented A431 (Invitrogen) and medium clone) Eagle’s modified M (HeLa cells HeLa transfection and culture Cell 3 a 5 with #1, SEPT9 synthesized follows: GUCAACAUCAAC-3 and as were MWG sequences from siRNA overhang. purchased were siRNAs All sequences peptide and siRNA nioisue nimnfursec,prfe His purified immunofluorescence, in used antibodies EVLGHKTPEPAAARTE-COOH. oa f15po fGTtge rtiswsicbtdwith incubated was proteins GST-tagged of His pmol of 115 amounts of 180,000 equimolar at ultracentrifugation total by experiment A the to prior immediately i-eetNce fiiyGl(im)o S idRsn(Novagen) Resin Bind instructions. GST manufacturer’s or the (Sigma) to Gel according from Affinity purified Nickel and (DE3) His-Select BL21 in expressed were ntutos o h eeaino I8-pcfcatbde sdduring used antibodies NH peptides CIN85-specific the immunoprecipitation, of generation manufacturer’s the the For to instructions. according #351-0010) Biosciences, (Innova System eewse ihetatbfe n on rtiswr ltdwith eluted were proteins bound and buffer. buffer sample extract SDS-PAGE with washed were he.Baswr ahdwt xrc ufradbudpoen were proteins bound buffer. and sample buffer SDS-PAGE extract with with eluted washed were Beads wheel. to (conjugated Probes Molecular conjugated). peroxidase from (horseradish Dianova antibody or CIN85 purchased dyes) Secondary fluorescent immunoblotting. A were for antigen. used an was antibodies 179.1.E1) as (Clone injected Upstate was from 429–end) acids (amino LSaaoe(at rzBoehooy o t4 at h Protein-A/G- 2 on for at Biotechnology) immobilized Cruz centrifugation antibodies (Santa with by PLUS–agarose incubated EGF- precleared were (Sigma, were Extracts using added Lysates were experiments #P5726). 13,000 cocktails For and inhibitor #P0044 (Sigma). phosphatase cells, cocktail stimulated inhibitor protease n NH and oaigwel ed eewse ihetatbfe n bound and buffer added. was and extract binding imidazole During with mM buffer. 10 sample washed washing, SDS-PAGE with were eluted were Beads proteins wheel. rotating 6 tge vco E2a rGTtge rtis(etrpGEX) (vector proteins GST-tagged or pET28a) (vector -tagged 9 g 2 I8,5 CIN85, ; idn assay binding CQLEDEKR-OH eec-netd o CIN85 For co-injected. were -CKQLLSELDEEKKIRL-CONH2 o i n lrcnrfgdfr1 i t180,000 at min 15 for ultracentrifuged and min 1 for 6 tge rtiswr lae rmpsil precipitates possible from cleared were proteins -tagged 9 2 2 h eune fteppie sdaea olw:SEPT9 follows: as are used peptides the of sequences The . %Tio -0)splmne ih1m MFand PMSF mM 1 with supplemented X-100) Triton 1% , ELHTEART-OH ET xxA NH PxxxAA, SEPT9 -EVLGHKTPEPAPRRTE-COOH; 9 -GGCACAGAAUGAUGAUGAA-3 9 ET 2 5 #2, SEPT9 ; 6 tge rtisfr3 i t4 at min 30 for proteins -tagged 9 oto,5 Control, ; 2 -CLQMEVNDIKKALQSK-COOH ˚ 9 narttn he.Beads wheel. rotating a on C -AGACCAUCGAGAUCAA- 9 -AUCGUUGACUUAC- shrci coli Escherichia TM m /lstreptomycin). g/ml ai Conjugation Rapid 6 ˚ narotating a on C CIN85 -tagged 9 9 ET,5 SEPT7, ; -GGAGGAG- ˚ Cona using 9 TT g g 2 9 - - . . o M studies, NMR For spectroscopy NMR o nsrmfe eim el eesiuae o h indicated the for stimulated were Cells medium. starved serum-free and coverslips in glass h Matrigel-coated 3 on for seeded were cells HeLa uptake EGF with or min 10 for at 4% PBS with min in 5 fixed sucrose and for coverslips 4% methanol Matrigel-coated (PFA), on paraformaldehyde seeded were Cells microscopy Immunofluorescence 10 of presence in EGF ng/ml 500 for incubated with and medium times serum-free in indicated h 4 the for starved were cells HeLa degradation EGFR Mo 0m o h ihrcnetain oaoddlto fthe of dilution avoid to concentrations higher z- the of self-shielding for solution stock mM a a 50 from with added or was mM equipped peptide 5 the cryoprobe titration, the TCI For gradient. a using frequency) ape Five sample. nteidvda pcr sn h oml shift protein. formula the the saturate using positions peak completely spectra the from to individual calculated the made were shift in was chemical in attempt Differences No 1:10. f521)181N ope onsadat a and points complex 512(1H)*128(15N) of ucoe nopT8)wsprfe n2 MTi-C H8 0mM 50 4 8, at overnight pH cleavage Tris-HCl thrombin mM to 20 subjected in and purified NaCl was pET28a) into subcloned sn h -a tutr 28b elcn h mn cdsd chains side acid Sybyl. CCPN amino created of using the was energy replacing to model the University by molecular minimizing B2Z8 transferred and A 3. structure assignment. X-ray subsequently for the SPARKY using 2005) and al., Kneller, et Francisco) (Vranken and San (Goddard California, to converted format first were data UCSF processed The Germany). Karlsruhe, Biospin, dimensions. respective 120 t ouetdpeiul,w efre esrmnst ofr the 600 confirm of one sample to A 2013). the resonances. measurements al., from protein the et differed performed of slightly (Ceregido assignment we here others previously, used by construct documented provided protein kindly the was Because protein the of nlsso pfursec aawspromdwt ImageJ. with performed by was Volocity. or with data performed Inc.) was images Data epifluorescence (Improvision confocal MicroManager. of software Quantification of of control Volocity Inc.) the analysis of under Elmer Axiovert microscopy Zeiss control (Perkin epifluorescence a the microscope on acquired confocal under were Images spinning-disc washing. and FCS 200M-based for 10% used containing was and medium in PBS blocking performed proteins, was surface decoration antibody of immunostaining For immunostaining. 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AV600 frequency) shielding a 1H on performed MHz (600 were Experiments K. 298 H,max H,max N,max Dd l pcr eercre n rcse sn osi ..(Bruker 3.1. topspin using processed and recorded were spectra All M xeiet osuyteitrcinwt SEPT9-derived a with interaction the study to experiments NMR 15 15 15 ( 15 m -OS-SCuigegtsas aasz f512( of size data a scans, eight using N-TOCSY-HSQC N) N) (i) (i) 5 H- a sdi -mNRtb tatmeaueof temperature a at tube NMR 5-mm a in used was SH3-A M N)/10) 6 6 25m.Tepoenppiertows10 :.,11 : and 1:2 1:1, 1:0.5, 1:0, was ratio protein:peptide The ms. 42.5 5 5 46( 80( . s n NAuigegtsas aasz f512( of ms. size 10.6 data a scans, eight using HNHA a and ms; 9.6 ora fCl cec 21)18 9–0 doi:10.1242/jcs.162206 397–407 128, (2015) Science Cell of Journal 1 1 2 1 )cmlxpit,t points, complex H) t points, complex H) ,where ], H, 15 H,max -SC eercre sn orsas aasize data a scans, four using recorded were N-HSQCs 15 g -aee His N-labeled 5 n uentnswr ujce owsenblot western to subjected were supernatants and Dd 12m,t ms, 51.2 2 stedfeec nceia hf nthe in shift chemical in difference the is 20 ˚ .Sadr rtcl eeue for used were protocols Standard C. 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Journal of Cell Science ooeie.Eulaonso ohfatoswr nlzdby analyzed were fractions thoroughly both and of buffer sample amounts following immunoblotting. SDS-PAGE 440,000 Equal hot discarded at in homogenized. h dissolved were 1 was for debris pellet ultracentrifugation cell by in 1000 separated and cycles at freeze-thaw Nuclei centrifugation three nitrogen. following liquid Germany) Heidelberg, (HGM, o nsrmfe eim el eeicbtdwt 0 ng/ml 500 with incubated were Cells 9 at medium. min serum-free starved 30 and for in AF647–EGF coverslips h glass 3 Matrigel-coated on for seeded were cells HeLa EGF of binding Surface 37 at AF647–EGF containing medium serum-free with times ARTICLE RESEARCH 406 125 125 13 at min 60 for and EGF ice of on cooled concentrations medium, indicated serum-free in the h with 3 treated for starved were cells HeLa fractionation cytosol and Membrane 13 and at ice min on 30 cooled for medium, serum-free EGF ng/ml in 100 h with 3 treated for starved were cells HeLa ubiquitylation EGFR ed eewse ihRP ufradbudpoen eeeue with eluted were proteins bound and buffer RIPA with washed were Beads eu-remdu otiig01 S n 0m EE H7.4. in pH h HEPES 4 mM for 20 with starved and stimulated and were BSA plates Cells 0.1% 24-well containing in medium seeded serum-free were cells HeLa 1996). gels. parallel the from analysis, was determined For immunoprecipitated was sample SEPT9-knockdown condition. of which amount control EGFR, the immunoprecipitation for To in the corrected were the to ubiquitin level compared of immunoblotting. amounts of gel EGFR the volume by onto in loaded 1.6-fold analyzed decrease a the and samples, for buffer compensate sample SDS-PAGE ahdtiewt B otiig2m MgCl mM 2 containing PBS with twice washed nitreto ak u loecneitniyo hsms was mask this of EGF. intensity of intensity fluorescence fluorescence total Sum to with normalized mask. calculated was intersection EGF of surface amount an surface- with The colocalizing with microscopy. protein confocal studies respective by colocalization the analyzed For were normalized condition. cells and EGF, the control fluorescence. calculated bound the from then background of was staining decrease that cell DAPI to to per the intensity needed of fluorescence was Sum area signal epifluorescence the EGF quantitative of of by surface subtraction quantification analyzed A were were For cells microscopy. cells temperature, level, microscopy. room EGF at immunofluorescence surface min 10 by for PBS analyzed in sucrose PFA/4% 4% bt at rzBoehooy o t4 at h 2 for of Biotechnology) Protein-A/G-PLUS–agarose mg Cruz on 1 Santa phosphatase of immobilized (both 180,000 and total at (sc-101) min A (Sigma) 15 EGFR-antibody #P5726)]. for cocktail ultracentrifuged and was deoxycholate, #P0044 inhibitor lysates sodium (Sigma, 1% protease cocktails X-100, inhibitor Triton PMSF, NaCl, 1% mM mM SDS, 150 1 7.4, 0.1% pH EDTA, Tris-HCl mM mM [50 1 buffer RIPA in lysed were Cells c-odPScnann MMgCl mM 2 containing PBS ice-cold rvn nenlzto.Clswr cae n2 MHPSp 7.4, pH HEPES MgCl mM mM 20 2 in KCl, scraped mM were 100 Cells internalization. prevent aHfr6 i.Rdociiyo h yaewsmeasured of was was to excess time. internalized to 300-fold of relative ratio binding lysate a The plotted values. of was all EGF presence Non-specific the from surface-bound M subtracted the 1 was in and of EGF). with EGF time-point cold lysed each Radioactivity internalized and for min measured min. 5 to 60 for temperature (corresponding for and room The collected at ice. NaOH on dried was 300SL). min (HIDEX were EGF) 5 counter scintillation for Cells surface-bound a NaCl using M measured to was 0.5 radioactivity (corresponding acid, acetic wash M 0.2 acid with wash acid an pnfxto ih4 F/%scoei B o 0mn el were cells min, 10 for PBS microscopy. in confocal sucrose by PFA/4% analyzed 4% with fixation Upon ahdtieo c ihPS Surface-bound PBS. with ice on twice washed -G paewspromda ecie rvosy(onlv tal., et (Kornilova previously described as performed was uptake I-EGF -G uptake I-EGF g o i.Ctsladmmrnswere membranes and Cytosol min. 5 for 125 ˚ 2 ,taserdt c n ahdtiewith twice washed and ice to transferred C, n rce sn elcakrdevice cracker cell a using cracked and -G nsavto eima 37 at medium starvation in I-EGF 2 pnfxto ihpre-cooled with fixation Upon . ˚ opeetinternalization. prevent to C 125 ˚ narttn wheel. rotating a on C -G a eoe by removed was I-EGF 2 tro temperature. room at g n nuae with incubated and g h membrane The . ˚ ˚ and C and C ˚ Cto rsneo 0-odecs fulbldEFadwr utatdfrom fractions, subtracted were the both and in EGF values. and unlabeled time-point of all a each excess NaOH, 300-fold for in a M measured of 1 presence analyzed were counts in were Non-specific dissolved Wizard). pellet, was and pellet supernatant The min. 5 eetrtafciga eitdi i.1 a lte stertoof ratio the as leftover plotted was as 1E EGF. Fig. degraded EGF. in defined to internalized depicted recycled total and as was to degraded trafficking normalized Recycled, all cell. Receptor lysate were the EGF in TCA- internalized the remained leftover the lysate. had in that and in EGF radioactivity supernatant internalized the radioactivity medium of both sum precipitable EGF. of the recycled as by fractions defined determined TCA-soluble and 100%. wash was acid to was EGF the supernatant set Degraded in medium and radioactivity the the well of to one precipitates added TCA from in derived found fractions Radioactivity all in radioactivities hraooi,Bri,Gray o h raino h tutrlmdladDr and model structural the of creation the for Germany) Berlin, Pharmakologie, odtos diinly yae eesbetdt etr ltanalysis levels. blot AKT1/2/3 western starved total to under control subjected were for cells to were parallel control lysates in to pSer473-Akt1/2 Additionally, processed normalized conditions. were was of Values which experiment. were curve, 40 levels each standard of a Lysates Total using concentration determined final #P5726). instructions. a phosphatase and manufacturer’s at 13,000 and assay at #P0044 (Sigma) min ELISA 5 cocktail (Sigma, for mM 20 centrifuged inhibitor cocktails NaF, mM protease 10 mM inhibitor NaCl, PMSF, 20 mM 500 X-100, mM 7.4, 1 pH Triton Tris-HCl mM 1% 20 in EDTA, ice on lysed and PBS 125 125 itcnlge rses egu)frteasgmn ftersnne fthe of voor resonances Instituut the (Vlaams of Nuland assignment van the N.A.J. for 1 Dr Belgium) and Brussels, Ceregido Biotechnologie, M.A. Dr thank We Acknowledgements (EGFR one-sample a 5F Here, used. Fig. SEPT9). material was of knockdown in test supplementary after and level shown (EGFR SEPT9) S1B data Fig. of unpaired the knockdown the after for ubiquitylation using except experiments performed quantified were analyses Statistical Statistics for stimulated and FCS 37 0.1% at pSer473- containing min were medium the 5 cells in using HeLa #ADI-900-162). overnight measured Sciences, starved Life was (Enzo Ser473 kit ELISA at Akt1/2 AKT of Phosphorylation signaling downstream EGFR . al H45 niefr3mn oalwfrendosomal for allow To medium min. starvation 3 in pre- incubated for with further 4 twice containing acetate, and ice washed medium sodium were starvation on M cells warmed (0.2 receptors, 4.5) wash EGF-bound pH acid of progression mild NaCl, a by M removed 0.5 was surface cell C-rcpttd(etoe nenl natEF n TCA-soluble and min. 30 above. 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EGF the intact recycled, separate to serves nsrmfe eimcnann .%BAad2 MHPSp 7.4. ng/ml pH 20 h HEPES with mM 4 stimulated for 20 starved and were and BSA Cells plates 0.1% 24-well containing in medium seeded serum-free were in cells HeLa 1996). al., et 5mnadwse wc niewt PBS. with ice on twice washed and min 15 TA rcptto 5 C,05 S;oengta 4 at acid overnight trichloroacetic BSA; a 0.5% to TCA, subjected (5% and collected precipitation was (TCA) medium the ice, on H, oa nenlzdEFwscluae stesmo measured of sum the as calculated was EGF internalized Total -G eyln a efre sdsrbdpeiul (Kornilova previously described as performed was recycling I-EGF -G eyln n degradation and recycling I-EGF 15 -SCo HAo I8,D .Kas LinzIsiu fu (Leibniz-Institut Krause G. 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Journal of Cell Science aln,K,Sgsud . oo . zmiwc,I,D ir,P .adDikic, and P. P. Fiore, Di I., Szymkiewicz, S., Polo, S., Sigismund, K., Haglund, A. I. Dikic, Sorkin, and and I. Szymkiewicz, S. N., Shimokawa, Gygi, K., Haglund, W., Kim, F., Huang, K., L. Goh, Fu se,M . iCaoOier,C,Fos,C . eie .T n rml,W S. W. Trimble, and T. M. Bejide, D., C. Froese, C., Ciano-Oliveira, T. Di P., E. M. Estey, Spiliotis, and Q. Hu, L., Dolat, V., V. Bernard, N., Simmons, C., Tazearslan, E., Adler, G., H. Hoang, D., Connolly, S., Coniglio, H., M. Oktay, N., Simmons, M., Castaldi, C. Z., Montagna, Yang, D., and Connolly, P. Verdier-Pinard, I., Abdesselam, D., Connolly, Lo S., Casares, L., J. Ortega-Roldan, A., Garcia-Pino, A., M. Ceregido, ado,F n arl Y. Barral, and F. Caudron, uhe . ors . efr,E,Kytl . obl . cuan M., Schumann, S., Korbel, G., Krystal, E., Lenfert, N., Horras, T., Buchse, o M pcrsoy ..adPS efre n nepee M analyses. NMR interpreted and performed P.S. and protein M.B. spectroscopy. recombinant purified NMR U.F. biochemical, for performed experiments. K.D. microscopy experiments. and of biology design molecular the to contributed authors All contributions Author interests. financial or competing no declare authors The interests Competing E228Q. EGFP–Vps4 encoding plasmid (Universita Krausslich Hans-Georg ARTICLE RESEARCH Baumga J. N. Mabjeesh, and M. Golan, S., Amir, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.162206/-/DC1 online available material Supplementary material agency Supplementary funding research M.K. German to the SFB958/A11] from number grant [grant a Forschungsgemeinschaft by Deutsche supported was work This Funding wrote M.K. and P.S. K.D., manuscript. experiments. the the oversaw and planned conceived, M.K. i,M . ree .D,Ety .P n rml,W S. W. Trimble, and P. M. Estey, D., C. Froese, S., M. Kim, A. Sorkin, and S. Gygi, X., Jiang, D., M. Kirkpatrick, F., Zastrow, Huang, Von and A. Marley, N., J. Hislop, L. V. Buchman, and J. M. Redowicz, S., Havrylov, E. S. Russell, and A. P. Hall, hbur . asn .B,Jne,A . oad . urg,A S., A. Quiroga, J., Howard, B., A. Jensen, B., L. Lassen, A., chtbauer, ¨ oobqiiaino I8 sivle nrglto fligand-induced of regulation I. in involved receptors. is EGF of CIN85 degradation the of of monoubiquitination endocytosis receptor. clathrin-mediated factor growth regulate epidermal collectively mechanisms stability. cellular and formation filament Fu 392 septin and in S. involved F. is Pedersen, Septin9 B., A. Sørensen, S., Warming, 21) itntrlso etn nctknss ET eitsmidbody mediates SEPT9 cytokinesis: in septins of roles abscission. Distinct (2010). pathology. and physiology tissue. breast tumor and 395 peritumoral C. in Montagna, expression and isoform-specific mouse H. M. and Oktay, M., Castaldi, human during changes progression. epigenetic cancer breast and C. localization Montagna, and P. expression, Verdier-Pinard, J., M. I. Fazzari, A. Azuaga, tumorigenesis. in and roles Septin A. N. Cbl-b*. proline-rich and Nuland, atypical CD2 two from with van CIN85/CD2AP sequences of J., binding differential and Bravo, Multimeric O., Mayorga, uaytcmembranes. eukaryotic iulzto fdnmc fHVbdigst neatoswt nESCRT SHIP-1. M. for Tiedge, an role and cells-specific with S. Mikkat, interactions E., Krause, site budding HIV of component. dynamics of visualization Kra pathway. factor-1 cancer hypoxia-inducible prostate of human Res. disruption of Cancer with angiogenesis concomitant and growth cells tumor inhibits SEPT9_v1 cuistetria oiin nspi caesadmdae polymerization- abscission. mediates in and octamers functions septin dependent in positions terminal the occupies domain. multiubiquitination kinase by degradation the and within internalization receptor EGF of regulation G lysosomes. non-ubiquitinated to a of receptor trafficking protein-coupled endocytic in proteins protein-sorting vacuolar malignancy. and migration adhesion, Traffic transport, vesicle-mediated in Ruk/CIN85 compartmentalization. and morphogenesis 287-299. cellular in roles with degradation. and 20) utpemnuiutnto fRK ssfiin o hi endocytosis their for sufficient is RTKs of monoubiquitination Multiple (2003). usih .G,Bra G., H. ¨usslich, 157-167. , 769-777. , re,V,Iacek,S,Dpn,A,Srev . iea,P W., P. 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