smer ihntecl Hl n ulc,20;Ml and Mili 2009; Bullock, cellular and the (Holt specific for 2009). cell Macara, structural essential the and a within are polarity cellular asymmetry generating that to of maintenance hence and mRNA establishment of translation, distribution of localized asymmetric enables transport their compartment of species, localization the and to (Le proteins corresponds encoded mRNA of localization td eel oeua ehns ywihaparticular through mRNP a an . which of mRNA-binding an by transport with interaction the mechanism direct mediates molecular motor a reveals study delocalizes ZBP1 Teeatoscnrbtdeulyt hswork this to USA. equally 10461, contributed NY authors Bronx *These Medicine, of College Einstein undn rvne hno nvriyMdclClee hno,Guangdong China. Shantou, 515031, College, Province, Medical University Shantou Province, Guangdong pcfcitrcino I1 ihZP euae h rnpr of Song Tingting transport the regulates ZBP1 with b KIF11 of interaction Specific ARTICLE RESEARCH eevd1 uut21;Acpe 7Dcme 2014 December 27 Accepted 2014; August 15 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 in investigation that 2009). systematic Ephrussi, and and spatial (Martin A expression for mechanism of fundamental control temporal a is localization mRNA INTRODUCTION mRNA motility, Cell polarity, transport Cell ZBP1, KIF11, WORDS: KEY proteins two the of interaction the transport vitro mediate the of that to regions KIF11 with corresponding and KIF11 the ZBP1 characterize of colocalizes We ZBP1. ability KIF11 on dependent the that and is show mRNA, and We ZBP1 a with (mRNPs). of KIF11, interacts of component identification physically a and specific which isolation (also motor, the ZBP1 a report microtubule we the study, how elusive. of remains this cells transport In non-neuronal of in the the complex IGF2BP1) to as mechanism known Although contributes motor underlying asymmetry. molecular the structural established, of and mechanism polarity establish of localization ZBP1-modulated ABSTRACT e Gu Wei uhrfrcrepnec ([email protected]) correspondence for Author eateto ahpyilg,TeKyImnptooyLbrtr of Laboratory Immunopathology Key The Pathophysiology, of Department 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,10–00doi:10.1242/jcs.161679 1001–1010 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. atnmN n elmotility cell and mRNA - and . 0 fedgnu rncit r oaie n the and localized are transcripts endogenous of 70% 1, nvivo in ` irpino the of Disruption . 1, b b b atnmN oaiainhsbe well been has localization mRNA -actin ,Y Zheng Yi *, atnmsegrrbncepoenparticles ribonucleoprotein messenger -actin atnmN n fet elmgain Our migration. cell affects and mRNA -actin ´ 2 ue ta. 07.I ait fcl types cell of variety a In 2007). al., et cuyer eateto ntm n tutrlBooy Albert Biology, Structural and Anatomy of Department b 1, atnmN nbe elto cell a enables mRNA -actin ,Yrn Wang Yarong *, nvivo in Drosophila neato fKF1with KIF11 of interaction mro reveals embryos b atnmN is mRNA -actin 2 ahr Katz Zachary , b -actin in oo a epnil o h otn of sorting the for responsible MYH10) as was known signaling (also Rho-mediated IIB motor a and a through 2012), play operating al., pathway to et of Nalavadi shown transport 2011; been axonal al., and et have dendritic Va KIF5A the myosin and in Recently, roles MYO5A) 2003). as Singer, known (Fusco and filaments (also Oleynikov actin 2003; and/or al., and et both on Wilkie ZBP1– rely the 1998; to of seems al., transport 2008), et Singer, 2001; (Bassell Davis, microtubule the and mouse 2002). ZBP1 Yisraeli, human, of and Orthologs in 2012). (Yaniv al., impairs found et Katz be (si)RNA 2012; (Vikesaa al., can et formation interfering Gu invadopodia 2006; al., small and et motility by adhesion, to ZBP1 cellular unit single of a of motif as Knockdown zipcode functions recognition the region with KH34 ZBP1 interact ZBP1 the the that of 3 reveals characterization the Hu Biochemical 2003; within al., ‘zipcode’ et Farina of cis-acting (UTR) region and a untranslated domains, recognition (KH) recognizes RNA homology K two specifically hnRNP of four IGF2BP1), and combination as 2005; (RRMs) unique motifs trans- known a Singer, a (also neuronal on contains and ZBP1 relies which and process protein, Condeelis localization RNA-binding invasion The 1999; acting 2007). cell al., al., et polarity, et Lapidus cell (Zhang axons with extending plasticity of associated cones growth neuronal is to and cells migrating sflycmeetfrtasotadlclzto Draqe al., et (Darzacq 2005). that localization mRNP Johnston, and an St transport assemble 2003; for to competent order fully in proteins necessary is RNA-binding are motors between the interactions and unique model, the this which for evidence in valuable provided have mRNAs pair-rule ta. 02 tJhso,20) eea oe ugssta to that Januschke suggests 2002; model general Warrior, al., A et and 2005). Johnston, Schnorrer Duncan St 2000; 2002; 2002; al., al., al., et et RNA et Brendza Cha 1997; in of 2000; al., transport et implicated the (Long for been yeast required have is motor motors in Both localization 2011). of and Mowry, localization and and (Gagnon transport complex destination direct final to their revealed act to proteins RNAs action have motor coordinated the few or a studies process protein of motor the These one which in in localization. play mechanisms motors RNA molecular that of roles the illuminated lmnso hi agtmNswiedrcl rindirectly or directly Yeast while motors. mRNAs molecular localization target to recognize connecting their proteins of RNA-binding elements RNAs, localize 2 lhuhtemjrt flclzdRA r rnpre along transported are RNAs localized of majority the Although h oaiainof localization The eea eetsuisi es and yeast in studies recent Several i Liu Xin , 1 hoigChen Shaoying , Drosophila temire l,20;Ca ta. 2010). al., et Chao 2005; al., et ¨ttelmaier b atnmNst h edn deof edge leading the to mRNAs -actin oye,weesatp myosin V type a whereas oocytes, b atnmN Ca ta. 2010). al., et (Chao mRNA -actin b atnmN Rs ta. 1997; al., et (Ross mRNA -actin 1 oetH Singer H. Robert , Xenopus ASH1 ASH1 b RAi budding in mRNA b atnmN (Ma mRNA -actin Drosophila atnmN in mRNA -actin V1RBP/Vera) (Vg1 and b atnmRNP -actin Drosophila 2 and 1001 have 9

Journal of Cell Science ula oiin I1 a otyctpamcadclclzdwith colocalized and cytoplasmic mostly was KIF11 position. nuclear ein fZP n I1 hog hc h w rtisinteract. proteins two the which through KIF11 and ZBP1 of regions of transport ZBP1 motor, the kinesin with associates a physically of identification which and KIF11, cargoes. isolation mRNA the the report with associate we to Here, protein ZBP1 adaptor with an motor as transport actin acts or that microtubule properly a for to required is recognition order in therefore hypothesized be that could It 2001). al., et (Latham fibroblasts ARTICLE RESEARCH 1002 FLA sequenced was indicate band sequences migrating Red faster analysis. the sequence that peptide showed for (C) blots KIF11. sent human Western and of 5). excised sequences (lane was the extract band SDS-PAGE with actin-3 cell protein 4–12% match MDA231/GFP migrating on completely the upper resolved that in The were peptides not (B) Precipitates of but shown). cells. mass asterisks), (not 3) molecular 4, GFP–ZBP1 (lane with (lane MDA231/GFP bands extract protein and cell Two 2) GFP-ZBP1 Blue. (lane Coomassie the MDA231/GFP-FLAG-ZBP1 with of of stained component extracts and a in is ZBP1 and ZBP1 with to associated binds KIF11 1. Fig. transport the to contribute and ZBP1 proteins with motor associate potential directly identify to that order to In binds translation. localized ZBP1 cells, mobile In of component structural a b is and ZBP1 with associates KIF11 RESULTS of transport motility. cell the to regulates leads and ZBP1, mRNP with a through interaction KIF11, which direct by mechanism Our ability. novel migration a cell demonstrates the study alters hence and edge leading the cell the blocking or KIF11 of activity vivo motor the of inhibition Either a-l os mrofbolss(E)o nMSmuewr utrdo irnci-otdcvrlp,fxdadpoesdfrimmunofluorescence for processed for and oligonucleotides fixed Cy3-labeled coverslips, using fibronectin-coated experiments on FISH (E cultured extracts. by total with followed were Ext, and co-precipitated mouse [Prec.(c)]. (green) were MBS control KIF11 resin ZBP1 an against amylose–MBP and of antibodies using KIF11 (MEF) sample using precipitates. RT- control fibroblasts the the [Prec.(c)]. embryo in in control mouse precipitated antibodies not resin day-old were anti-ZBP1 amylose–MBP but mouse and (Prec.), using (Prec.) resin ZBP1 amylose–MBP-MCP amylose–MBP-MCP and of antibody precipitates of anti-KIF11 presence the rabbit the and detect [FT(c)], to performed resin was amylose–MBP PCR and (FT) resin MBP-MCP atnmRNP -actin neato fZP ihKF1delocalizes KIF11 with ZBP1 of interaction 9 UTR-MBS b 24 atnmN.W hrceie h corresponding the characterized We mRNA. -actin sn mls–B ei rayoeMPMPrsn oa Nswr sltdfo trigetat Et,fo-hog famylose– of flow-through (Ext), extracts starting from isolated were RNAs Total resin. amylose–MBP-MCP or resin amylose–MBP using , b atnmN n eitsits mediates and mRNA -actin b atnmN,aspecific a mRNA, -actin b atnmN nteRAsmls aeL N adr D etr lt oaayetepeec fKF1using KIF11 of presence the analyze to blots Western (D) ladder. DNA L, Lane samples. RNA the in mRNA -actin nvivo in b atnmN at mRNA -actin b atnmN complex. mRNA -actin oregulate to b -actin b atnmN.Saebr 10 bar: Scale mRNA. -actin b in atnmN a uiidfo yolsi xrcso E el xrsigthe expressing cells MEF of extracts cytoplasmic from purified was mRNP -actin , 0kaad10ka eeietfe ntepeiiae fteMDA231/FLAG- the of precipitates the in identified were kDa, 110 and kDa 90 n t soitdpoen.Teaiiyt uldown pull to ability The proteins. the associated of its (MCP) and the protein precipitate affinity coat to MS2-binding bacteriophage the RNA used then 3 We the 2011). to expressing targeted (cells an which gene was in actin cassette strain), site (MBS binding strain mouse MS2 embryos knock-in from 14-day-old fibroblasts a primary of kinesin-5 prepared specifically We the assay. was of pull-down KIF11 protein and whether ZBP1 motor migrating determine with a associated To slower KIF11, 1B). be the (Fig. to family not of it (data analysis revealed FLAG-GFP–ZBP1 of band spectrometric as bands Mass identified protein shown). was control distinct band the two migrating to 5), contrast lane by In , visualized 1A, 1A). (Fig. and (Fig. IgG-coated SDS-PAGE precipitation staining anti-FLAG 10% Blue Purified by used ZBP1. Coomassie with resolved and associated were proteins I proteins down pull the RNase treated human to 2012), with Sepharose FLAG-GFP-tagged al., et extracts a (Gu inserted cell which genetically was in gene ZBP1 cells carcinoma breast of process 0kaad10ka(i.1,ln )wr sltd h faster The isolated. were 4) lane 1A, (Fig. kDa 110 and kDa 90 A oimnpeiiainwspromdt dniytepoen directly proteins the identify to performed was Co-immunoprecipitation (A) ora fCl cec 21)18 0111 doi:10.1242/jcs.161679 1001–1010 128, (2015) Science Cell of Journal b atnmN,w rprdetat rmMDA231 from extracts prepared we mRNA, -actin m m. b b atnmN,w efre another performed we mRNA, -actin atnmN rd.Bu oo niae the indicates color Blue (red). mRNA -actin b atnM2mN)(ine tal., et (Lionnet mRNA) -actin-MS2 b b atnM2mRNA -actin-MS2 atnmN using mRNA -actin 9 T fthe of UTR b rmr 14- Primary ) -actin-MS2 G- b b - -

Journal of Cell Science eie rmtreidpneteprmns aaso h mean the show Data experiments. independent three from derived localized to mean normalized KIF11 the of show levels Data relative experiments. the independent reflect three from counted Student’s was using cells by 60 determine with 10 of to panel) bars: mean performed (lower A Scale was KIF11 cells. nuclei. FISH colchicine-treated or of min. panel) position of 30 ZBP1(middle the for presence co-precipitated indicates stimulated the detect color serum detect and to cultured utilized to were was performed cells blotting was MEF Western RT-PCR RT-PCR. amylose–MBP-MCP. for of control precipitates negative the and extracts starting S RA(i.1,Pe..Teseiiiyo h eut was Prec.(c)]. results 1D, the [Fig. was present protein of binding not MCP specificity which in experiments The control the Prec.). in tested 1D, (Fig. mRNA with co-precipitated MS2 were KIF11 and ZBP1 Both 1D). by (Fig. evaluated antibodies anti-ZBP1 RT-PCR then and anti-KIF11 by were with analysis precipitates immunoblotting assessed corresponding was The precipitation 1C). affinity (Fig. MCP by mRNA ARTICLE RESEARCH i.2 niiino I1 oo ucino ncdw fKF1epeso mar h oaiainof localization the impairs expression KIF11 of prepared. knockdown or with function KIF11 motor KIF11 of of Inhibition 2. Fig. MBS the from isolated lines MEF stable two and used ZBP1 We KIF11, between mRNA. relationship the tested then We ZBP1 with on KIF11 of association The to images panel). cell right 1E, and the (Fig. KIF11 superimposed colocalized of cell amounts We MEF substantial the that panel). of show 3 cytoplasm middle the the observed 1E, in in also distributed (Fig. was sites was KIF11 panel). and MS2-binding left granules 1E, 24 as (Fig. of mRNA the presence of the UTR to for owing edge, probes and oligonucleotide Distinct mRNA. MS2 KIF11 labeled for and fluorescently (MEFs) antibodies analysis immunofluorescence mouse using with MBS assay combined an a from performed fibroblasts primary with whether cultured and colocalized KIF11 was of localization KIF11 subcellular the investigate To with colocalizes KIF11 b b atnmN a infcnl eue nMA3-B1KF1sRA1tetdclsadMA3-B1KF1sRA2tetdcls h aawere data The cells. KIF11-shRNA-2-treated MDA231-ZBP1 and cells KIF11-shRNA-1-treated MDA231-ZBP1 in reduced significantly was mRNA -actin atnmN a uiidfo h xrcsuigayoersnatce B-C Pe.w n Prec.( and [Prec.(w) MBP-MCP amylose-resin-attached using extracts the from purified was mRNP -actin b atnmN sZP eedn.Ctpamcetat fMFMScls[x w]adZP-eee E/B el Et( [Ext cells MEF/MBS ZBP1-deleted and (w)] [Ext cells MEF/MBS of extracts Cytoplasmic dependent. ZBP1 is mRNA -actin t ts.()Wsenbossoigteepeso eeso I1 rti nMA3-B1KF1sRAtetdcls ubr eo h bands the below Numbers cells. KIF11-shRNA-treated MDA231-ZBP1 in protein KIF11 of levels expression the showing blots Western (D) -test. b atnmNswr eetda h elleading cell the at detected were mRNAs -actin b atnmN nfibroblasts in mRNA -actin b atnmN,w sltdand isolated we mRNA, -actin b atnmN depends mRNA -actin nsitu in b atn h rosidct h eetdpoen.W,wl ye E rp hwn httepretg fclswith cells of percentage the that showing Graph (E) type. wild WT, proteins. detected the indicate arrows The -actin. m b .()Telclzto of localization The (C) m. yrdzto with hybridization atnmN were mRNA -actin 6 b ...Saitclsgiiac a acltdb sn Student’s using by calculated was significance Statistical s.e.m. b b -actin- -actin -actin 9 b atnmN ttelaigeg ftecl a erae nmnsrl or monastrol- in decreased was cell the of edge leading the at mRNA -actin a opeiiae ntepeec fZP,btntco- not 2A, of but (Fig. association the expressed that ZBP1, not suggests with This KIF11 was of lanes). two gene right presence panel, ZBP1 lower the the when in precipitated 2012). co-precipitated al., et (Katz was in deleted was and, of gene expressed precipitation ZBP1 normally Affinity the was line, cell ZBP1 other line, the one In strain. mouse odtrieteefc fmnsrlteteto the on treatment monastrol of effect colchicine-treated the in 2C). (Fig. determine observed destroyed of was also structure To microtubule diminution the was which A in 17% cells, 2B,C). to (Fig. localization cells to decreased treated which was localization in edge, cells, leading untreated endogenous with , Compared fluorescent the (Mayer experiments. performing analyzed KIF11 (FISH) of and by activity 1999), distribution motor al., the et inhibits drug a this specifically monastrol, test the with that To in fibroblasts mRNA. primary role treated the a we of play assumption, localization could and and KIF11 ZBP1 transport that asymmetric with possibility the associated raised KIF11 mRNA that observation of The KIF11 localization of the knockdown impairs or expression activity KIF11 of Inhibition hc niae htbt h irtbl tutr n the and structure microtubule the both that anti- indicated an of with which levels cells examined and the antibody immunostained we cytoskeleton, 4 fteclssoe localized showed cells the of 34% ora fCl cec 21)18 0111 doi:10.1242/jcs.161679 1001–1010 128, (2015) Science Cell of Journal b atnmN oaiaini E el rae ihmnsrl Blue monastrol. with treated cells MEF in localization mRNA -actin b atnmN a B1dependent. ZBP1 was mRNP -actin b atnmN nMFcells. MEF in mRNA -actin b b atnM2mN hwdta KIF11 that showed mRNA -actin-MS2 6 atnmN nteRAsmls Cont, samples. RNA the in mRNA -actin ...Saitclsgiiac a calculated was significance Statistical s.e.m. D B1] oa Nswr sltdfrom isolated were RNAs Total ZBP1)]. b atnmN sn RT-PCR, using mRNA -actin b atnmN ttecell the at mRNA -actin b b atnmRNA -actin t -test. nsitu in atnmN.()Primary (B) mRNA. -actin , 9 nmonastrol- in 19% A h association The (A) b b D atnmRNA -actin atnmRNA -actin B1]were ZBP1)] hybridization b -actin 1003

Journal of Cell Science I1 ntecl extracts. cell the with in co-precipitated KIF11 ZBP1, fragments full-length ZBP1-RRM12 the that performed and indicate were ZBP1-RRM12+KH12 results blotting The western KIF11. and co-precipitated assays detect Pulldown to (C) study. this in ZBP1-KH12 used and ZBP1- ZBP1-RRM12 ZBP1, ZBP1-KH1234, full-length ZBP1-KH34, domains. recombinant RRM12+KH12, conserved of of drawing organization Representative the (B) showing ZBP1 of diagram rae el a oneuae o4%o t xrsinin expression 41% its to reduced of was it 44% cells shRNA-2-treated to in method. and downregulated cells (sh)RNA control was hairpin expression cells short of KIF11 treated control the internal which the using of to in Normalized down involvement lines knocked the cell was assess stable further MDA231-ZBP1 in To S1A,B). KIF11 Fig. material b ARTICLE RESEARCH 1004 KIF11 to binds ZBP1 of motif RRM12 The 3. Fig. ZBP1 of region putative the determine to the of order (termed zipcode In KH4 the 2010). and al., to K KH3 et binds hnRNP containing domain) ZBP1 four of KH34 indicate by RRM2, region reports followed Previous the and 3A). that RRM12) (RRM1 (Fig. canonical domains as motifs (KH1–4) six homology to recognition contains referred RNA that together two protein – RNA-binding domains an is ZBP1 vitro In KIF11 localization. of and importance transport the (supplementary suggest mRNA reduced results in significantly These S2). was Fig. edge material and leading ARPC5) cell as localization known the that (also showed Arp-16 on both results assays effect of FISH The same performed cells. the we KIF11-knockdown mRNAs, has in two KIF11 (Gu these and whether of dynamics address localization Arp-16 adhesion the To focal localizing 2012). for al., in important et KIF11 are roles which localizing either of plays to mRNAs, addition Thus, also downregulation in ZBP1, 2E). mRNA, or (Fig. that reported respectively activity previously impaired 16%, KIF11 expression or of 20% inhibition control to to delocalized 29% comparison KIF11 in of that, downregulation cells, indicated Experiments 2D). (Fig. atnmN xrsinwr nlee (supplementary unaltered were expression mRNA -actin dniiaino h B1dmi htbnst KIF11 to binds that domain ZBP1 the of identification b atnmN oaiain eetbihdtwo established we localization, mRNA -actin b atnmN oaiain ehave We localization. mRNA -actin b atn I1 nshRNA-1- in KIF11 -actin, b atnmN (Chao mRNA -actin nvitro in b a atnmN from mRNA -actin atnnmNsat mRNAs - . A schematic A (A) a -actinin b -actin eosrtn htteRM2mtf fZP specifically ZBP1 of 3C), (Fig. motifs KIF11 RRM12 KIF11. the recognized with that co-precipitated not demonstrating ZBP1- did were and ZBP1-RRM12 regions truncated RRM12+KH12 the KH1234 that However, or KIF11. proteins to KH34 ZBP1 bind KH12, Truncated ZBP1 individual KIF11. between truncated and interaction contained strong ZBP1 the a full-length showed of extract to the cellular experiments a ability proteins The from 3C). KIF11 fusion the (Fig. endogenous of down tested pull amounts to and protein(s) equal resin attached Fig. amylose then material (supplementary We resin S3A). amylose with proteins fusion ZBP1-KH34 in ZBP1- and domains ZBP1-RRM12, ZBP1-KH12 contained ZBP1-KH1234, as that RRM12+KH12, expressed, mutants and we ZBP1 truncation full-length KIF11, ZBP1 the with (MBP), proteins interaction fusion for maltose-binding required was that AROL(egre l,19) nodrt dniythe with identify interact and to recognize by order that In predicted KIF11 1995). were of al., KIF11 regions et potential within C- (Berger addition, a (motor, regions In PAIRCOIL and domain 4A). coiled-coil 361–761) (Fig. head 762–1056) (residues three (residues N-terminal domain domain tail stalk an terminal a of 1–360), composed residues ZBP1 to is binds KIF11 KIF11 of domain tail The rget eeefcieybudt B1 x,cl xrcs ,cnrlco- control C, beads. extracts; amylose cell MBP-coupled Ext, with ZBP1. experiment a (762–1056) to shows precipitation domain bound domain tail stalk effectively the the were and whereas fragments ZBP1 ZBP1, to with the bind interaction that not weak indicates does ZBP1 KIF11 of of for extracts domain blotting with head western incubated and fusion and Co-precipitation MBP–KIF11 beads cells. (C) amylose 293T ZBP1. to to to attached binding experiments were the for proteins in responsible used regions representative KIF11 the A of identify (B) fragments indicated. recombinant are of structures drawing coiled-coil predicted The form organization. KIF11. that domain of regions the showing domain KIF11 ZBP1-interacting of diagram the schematic of Identification 4. Fig. that suggested data These with 1–300). seen a was (residues binding domains. showed domain no alone and tail head ZBP1, (362–761) the to and domain binding weak stalk stalk relatively KIF11 both the truncation containing However, KIF11 the (272–1056) and KIF11 efficiently mutant full-length was the ZBP1 with endogenous down that with pulled found interact we cells, domain 293T stalk from and head the ZBP1 whether for tested we proteins first MBP–KIF11 S3B), fusion of We PCR Fig. amounts purified express material equal the the (supplementary to used identifying SDS-PAGE subcloned After pMalc by 4B). and proteins plasmid (Fig. PCR proteins the fusion by into various cDNAs generated fragments structure, KIF11 protein KIF11 truncated dissected we ZBP1, nvitro in ora fCl cec 21)18 0111 doi:10.1242/jcs.161679 1001–1010 128, (2015) Science Cell of Journal shrci coli Escherichia Fg C pe ae) sn xrcsprepared extracts Using panel). upper 4C, (Fig. Fg B,adafnt uiidthe purified affinity and 3B), (Fig. . nvitro in nvitro in ulonanalyses. pulldown A A (A)

Journal of Cell Science onZP,weeste7215 alfamn akn h 40 the lacking pull fragment effectively tail acid to 762–1056 amino the able whereas were with ZBP1, 557–1056 down KIF11 Two and extract. of 762–1056 cell a residues mutants in ZBP1 truncation down pull tail-containing to ability lower KIF11 strongest 4C, full-length the (Fig. experiments, has ZBP1 former the the with assess with interact to Consistent to panel). KIF11 domain of tail region the ZBP1. C-terminal of with potential the interacted on domains focused stalk then and We head than other regions ARTICLE RESEARCH I1.Ln ,cnrlpldw sa ihetat fuifce 9Tcells. 293T uninfected of extracts with against assay antibodies pulldown using control The 1, blotting Lane beads. western KIF11. Ni-Sepharose by performed using analyzed were cells were assays infected precipitates Pulldown of 3). extracts (lane the ZBP1- ZBP1-KH12 from His-tagged or expressing 2) lentivirus (lane with RRM12 infected were of Analysis cells 293T (D) KIF11. cells. blots 293T western of extract by cell analyzed total the and 1, 2) Lane 3), antibodies. (lane (lane ZBP1 (762–1056) (806–1032) using domain domain tail expressing tail KIF11 lentivirus KIF5A the with or of infected mutants cells truncation tail 293T fusion KIF5A of MBP The extracts (C) the ZBP1 cells. with 293T to performed of bound extract not cell was using total domain blotting 1, western Lane affinity by antibodies. amylose analyzed ZBP1 using were 4) precipitates (lane The or (762–1056) chromatography. 3) (lane domain (1–363) MBP tail KIF11 expressing 2), KIF11 lentivirus (lane the (1–300) with KIF11 infected of mutants were truncation that fusion cells performed 293T were of experiments domain extracts Pull-down tail with KIF11 ZBP1. The endogenous (B) with Blue. after interacted Coomassie Staining with respectively. performed with motif, was incubated ZBP1-RRM12 SDS-PAGE the were or fragment BSA tail KIF11 containing 3, buffer His-tagged and the 2 to Lanes Ni-beads ZBP1-RRM12. coupled or 1, with Ni-beads KIF11 incubated lane of were panel: proteins domain Right attached tail respectively. without the BSA, was with containing MBP-RRM12 incubated buffer 3, and with and beads were 2 maltose proteins Lanes to attached tail. attached without KIF11 beads His-tagged maltose tail with KIF11 1, incubated the lane panel: with Left motif 1056). RRM12 ZBP1 the of domain. interaction Direct 5. Fig. tail These KIF11 experiments. recombinant beads, co-precipitation purified amylose performed with and to beads fragment ZBP1-RRM12 this, the the test of MBP-tagged To incubated interaction domain. coupled direct tail the KIF11 we the through with be of domain recognition could ZBP1-RRM12 the ZBP1 that anticipated by we KIF11 results, above the tail on the Based with interacts KIF11 directly of ZBP1 domain of domain RRM12 sufficient The is (762–1056) ZBP1. with KIF11 interact of to domain results necessary tail a and These the ZBP1. displayed that for (802–1056) indicate affinity N-terminus binding the decreased at significantly residues acid amino nvivo in A B1RM2drcl neat ihteKF1ti oan(762– domain tail KIF11 the with interacts directly ZBP1-RRM12 (A) neato fteRM2dmi fZP ihendogenous with ZBP1 of domain RRM12 the of interaction nvivo in uloneprmnswere experiments Pulldown . I1 custruhtercrepnigdomains. corresponding their through occurs KIF11 n ietrcgiino h aldmi fKF1b h RRM12 the by ZBP1- KIF11 of ZBP1 mutual with domain of the tail domain indicate the complexes results of recognition These formed direct panel). and His- right fragment 5A, the (Fig. tail RRM12 next Consistently, KIF11 We the fragment. tagged with mixed panel). 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Crystal top the invasion 0.2% from scraped with PBS of were in cells stained Noninvasive formaldehyde min. and 3.7% 10 in invasive min The fixed 15 h. were 16 for chamber for the Matrigel the underneath through cells invade to allowed 500 were containing Cells dishes culture 24-well into xeiet.XL n ..cridotcl neto xeiet and experiments infection cell motility out cell carried and S.C. imaging and and live-cell Z.K. X.L. interpreted experiments. experiments. and biochemical out major carried out designed, carried Y.W. and designed Y.Z. and T.S. contributions Author interests. financial or competing no declare authors The interests motility Competing cell for School assistance. High technical Memorial their Pelham for in W.G. Xue analysis. of Alice laboratory thank Deling the also thank from We We Zheng manuscript. the Guiyu of and discussions Li valuable for Lin Stanley thank We Acknowledgements (2 were chambers cells transwell serum-starved the Briefly, into Matrigel. 400 Biosciences) in with (BD suspended pre-coated transwells using were performed that were assays invasion Cell assays invasion Cell odei,J n igr .H. R. Singer, H. and R. Singer, J. and Condeelis, C. S. Almo, M., Levy, V., Patel, Y., Patskovsky, H. A., R. J. Singer, Chao, and C. E. S. W. Almo, Theurkauf, Y., Patskovsky, and A., S. J. B. Chao, Koppetsch, R., L. Serbus, J., B. Cha, J. M. Justice, and A. Castillo, H. R. Singer, and B. Wu, R., A. Buxbaum, S. P. Kim, and M. Milla, T., Tonchev, E., Wolf, Taneja, B., D. H., Wilson, R. B., Berger, Singer, M., A. Femino, L., A. Byrd, H., Zhang, J., G. Bassell, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.161679/-/DC1 online available material Supplementary material release. Supplementary immediate for [grant PMC Health in of Deposited Institutes China R.H.S. National of to the Foundation GM84364] and Science number W.G.; Natural to National 31071152] the number by [grant supported was work This Funding manuscript. the prepared and study, experiments the coordinated biological plan, cell research out in the carried designed participated W.G. and manuscript. study the the revising coordinated R.H.S. assays. immunofluorescence rnz,R . ebs .R,Dfy .B n atn .M. W. Saxton, and B. J. Duffy, R., L. Serbus, P., R. Brendza, irtbl crosstalk. microtubule M. K. Yamada, oocyte. Drosophila the patterning in 1982-1991. roles interdependent have yeast. in Microbiol. differentiation daughter/mother and distribution asymmetric 21) B1rcgiino eaatnzpoeidcsRAlooping. target? RNA induces zipcode beta-actin of Dev. recognition ZBP1 (2010). complex. oocyte RNA-protein viral the a of to 15 coevolution the plasm for basis pole restricts exclusion cortical posterior. I-dependent Kinesin embryogenesis. pre-implantation of Commun. Res. maintenance Biophys. for essential translatability. its regulating for mechanism 419-422. a reveals neurons in S. culture. K. in Kosik, cones growth and and M. neurites 251-265. to I. protein Herman, and mRNA M., actin L. Lifshitz, L., K. o iei ntepseirtasoto sa RAadSafnprotein. Staufen and mRNA oskar of transport posterior the in Science I kinesin for correlations. residue pairwise USA of use Sci. by coils coiled Predicting 103-105. , 24 ora fCl cec 21)18 0111 doi:10.1242/jcs.161679 1001–1010 128, (2015) Science Cell of Journal il Cell Biol. 148-158. , 289 92 a.Cl Biol. Cell Nat. 6 614-620. , 8259-8263. , 2120-2122. , 97 20) ysnIArgltscl oiiyadactomyosin- and motility cell regulates IIA Myosin (2007). m fDE upeetdwt .%BAadplaced and BSA 0.5% with supplemented DMEM of l 97-110. , a.Cl Biol. Cell Nat. 4 357 592-598. , 20) h yolsi yenadknsnmotors kinesin and dynein cytoplasmic The (2002). 694-699. , 20) h iei eae oo rti,E5 is Eg5, protein, motor related kinesin The (2007). 6 20) o n h osbt-ci mRNA beta-actin does why and How (2005). 10 4 9 299-309. , el) h hmeswr inserted were chambers The cells). 21) Single (2014). 6 s.e.m. m fDE ih1%FBS. 10% with DMEM of l b 19) otn fbeta- of Sorting (1998). atnmN detection mRNA -actin a.Src.Ml Biol. Mol. Struct. Nat. 20) function A (2000). 20) Structural (2008). rc al Acad. Natl. Proc. .Neurosci. J. ur Biol. Curr. Science 20) RNA (2003). ur Opin. Curr. Biochem. (2002). (1995). 1009 343 12 18 , , ,

Journal of Cell Science ine,T,Calnk,K,Draq . hvTl . el,A . ho .A., J. Chao, L., A. Wells, Y., Shav-Tal, X., Darzacq, K., Czaplinski, T., Lionnet, H. R. Le Singer, and S. R. Adelstein, N., A. Tullio, H., E. Yu, M., V. Latham, u . az . u . ak .Y,L,D,Ln . el,A .adSne,R H. R. Singer, and L. A. Wells, S., Lin, D., Li, Y., H. Park, B., Wu, Z., Katz, W., Gu, L. K. Mowry, and A. J. Singer, Gagnon, M., J. Blanchard, M., H. S. Shenoy, R. B., Singer, Lavoie, N., and Accornero, R. D., Fusco, Darnell, K., Musunuru, S., Huttelmaier, L., K. Farina, W. P. Baas, and S. Tole, A., Falnikar, ARTICLE RESEARCH 1010 H. H. R. R. Singer, Singer, and and X. Zhu, M. H., S. E. Shenoy, Kislauskis, B., Wu, Y., H. Park, L., A. Wells, B., Z. M. Katz, J. Scholey, and C. G. Rogers, S., A. N. Kashina, Hirokawa, R., and Borup, N. T., Hansen, Dohmae, K., Y., L. Kanai, Nielsen, A., Krogh, J., Vikesaa, L., Jønson, St H., Lopez-Schier, A., J. Kaltschmidt, S., Dass, L., Gervais, J., Januschke, L. Hu S. Bullock, and E. C. Holt, aiu,K,Wcof . onin,G,Lrn,M,So,L,Condeelis, L., Soon, M., Lorenz, G., Mouneimne, J., Wyckoff, K., Lapidus, Lo J., Krauss, cyr . ohd,H,Prhsrty . l,C,Bbk . eoia T., Cerovina, T., Babak, C., Alm, N., Parthasarathy, H., Yoshida, E., ´cuyer, tlae,S,Znlsn . eee,M,Dcebr,J,Lrn,M,Meng, M., Lorenz, J., Dictenberg, M., Lederer, D., Zenklusen, S., ttelmaier, ¨ ak .Y,d urs . oe-oe,M n igr .H. R. Singer, and M. Lopez-Jones, V., Turris, de Y., H. architecture Park, cellular organizing in role M. prominent H. function. a and Krause, reveals and P. localization Tomancak, mRNA R., T. Hughes, beta- localizes myosin fibroblasts. through in operating mRNA pathway actin signaling Rho-dependent A chemotaxis. n iet elmigration. cell separation. directs and centrosome driving (2012). motors mitotic Acta bipolar Biophys. Biochim. essential : granule. C. RNA-transporting an 525. F. of characterization Nielsen, and isolation and J. granules. ribonucleoprotein Christiansen, IMP1 of H., composition A. Johnsen, cooperation. I Kinesin and 1971-1981. A. Dynein Guichet, requires and oocyte S. Drosophila Roth, the H., A. Brand, D., Johnston, ZBP1. of phosphorylation H. 512-515. Src-dependent R. Singer, by and translation J. beta-actin Condeelis, J., G. Bassell, X., motility-related matters. it and why adhesion and cells cell IMP1/ZBP1. by of cells expression cancer breast local in mRNAs of Regulation (2012). cells. E. mammalian living Bertrand, in movement and probabilistic H. R. granule localization, mRNA attachment. beta-actin cytoskeletal facilitate and domains formation, KH ZBP1 Two (2003). migration. neuronal modulates 1561-1574. protein, motor associated .S n igr .H. R. Singer, and S. J. motility. cell augment 1270. synthesis protein and localization localization. RNA ysnVrgltsokrmN oaiaini h rspiaoocyte. 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Curr. 229-239. , 19) h iCfml of family bimC The (1997). of regulation Spatial (2005). 20) lblaayi of analysis Global (2007). 160 20) oa rnpr in transport Polar (2002). 6 .Cl Biol. Cell J. 77-87. , 798-811. , 125 o.Bo.Cell Biol. Mol. 20) Molecular (2007). 13 81-91. , Neuron 161-167. , ur Biol. Curr. Nature 136 21) A (2011). 43 (2001). 1263- , (2009). 513- , Curr. 438 12 22 , , , cnre,F,Bhan .adNu and K. Bohmann, F., Schnorrer, aen,V . alai . rvne .W,J,Stl-ivia .R,Sotelo, R., J. Sotelo-Silveira, Jr, W., D. H. Provance, R. A., Singer, Calliari, P., and V. L. Salerno, K. Taneja, H., E. Kislauskis, Y., Oleynikov, F., A. Ross, a,F,Hu F., Pan, H. R. Singer, and Y. Oleynikov, and T. Takumi, M., A. Swanson, P., Picard-Fraser, E., L. Griffin, C., V. Nalavadi, G. I. Macara, and S. Mili, ai,K n irei .K. J. Yisraeli, and K. Yaniv, I. Davis, and S. G. Wilkie, ae,T . aor .M,Hgat,S . ig .W,Shebr .L and L. S. Schreiber, W., R. King, J., S. Haggarty, M., T. Kapoor, U., T. Mayer, A. Ephrussi, and C. K. Martin, hsaoa .A,Sne,R .adCnels J. Condeelis, and H. R. Singer, A., E. Shestakova, hn,H . igr .H n asl,G J. G. Bassell, and H. R. Singer, L., H. Zhang, Christiansen, M., U. Wewer, R., Borup, L., Jønson, V., T. Hansen, J., P. Vikesaa, S. Gilbert, and T. M. Valentine, D. Johnston, St R. D. Soil, H. R. Singer, a . aa,J . u .S,Cle,B . ho .V n aee N. Tanese, and V. M. Chao, P., B. Culver, S., M. Jansen, Yu, N., and J. K. Savas, Nasmyth, B., Ma, I., Gonzalez, X., Meng, H., R. Singer, M., R. Long, irbat rmmlil organs. multiple from A. fibroblasts J. Mercer, and R. J. protein. zipcode-binding mRNA beta-actin Biol. a Cell. of Characterization (1997). 8340-8351. idn fZP obt-ci RAdrn transcription. during mRNA beta-actin to ZBP1 of binding localization. and transcription during mRNA Biol. beta-actin with association Va. myosin by axons in J. dynamics G. Bassell, oaieaial ydni-eitdtasoto N particles. 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Cell Trends 19) aigtp wthn nyatcnrle yasymmetric by controlled yeast in switching type Mating (1997). 199-207. , 19) h s fcmuesi nesadn o nmlclscrawl. cells animal how understanding in computers of use The (1995). tlae,S,Sne,R .adG,W. Gu, and H. R. Singer, S., ttelmaier, ¨ a.Rv o.Cl Biol. Cell Mol. Rev. Nat. 8 17 165-170. , 20) ihasfrmN transport. mRNA for Highways (2008). 2158-2165. , 163 19 21) euaino icd idn rti transport 1 protein binding zipcode of Regulation (2012). 20) oigmsae:teitaellrlclzto of localization intracellular the messages: Moving (2005). 19) ml oeueihbtro ioi pnl bipolarity spindle mitotic of inhibitor molecule Small (1999). 75-81. , Cell rc al cd c.USA Sci. Acad. Natl. Proc. 43-104. , a.Cl Biol. Cell Nat. 20) N-idn Mspooecl deinand adhesion cell promote IMPs RNA-binding (2006). 136 MOJ. EMBO 20) ysnV eitsRAdsrbto nprimary in distribution RNA mediates Myosin-Va (2008). 19 20) N oaiainadplrt:fo (C to A(PC) from polarity: and localization RNA (2009). 20) h novmn facnevdfml fRNA of family conserved a of involvement The (2002). 20) rspiawnls n arrl transcripts pair-rule and wingless Drosophila (2001). 719-730. , 156-164. , Science 20) RAlclzto:gn xrsini the in expression gene localization: mRNA (2009). elMtl Cytoskeleton Motil. Cell 25 6 .Neurosci. J. senVlad C. sslein-Volhard, ¨ 363-375. , 1456-1468. , 20) eltm iulzto fZBP1 of visualization Real-time (2003). 2 277 20) ose rntt tp How step? to not or step To (2007). 185-190. , Science 383-387. , b atnmN nrtneurons. rat in mRNA -actin 19) ertohnrglto of regulation Neurotrophin (1999). 98 32 286 7045-7050. , 15133-15141. , Cell 971-974. , 20) h physiological The (2001). 20) B2facilitates ZBP2 (2007). 134 20) h molecular The (2000). 65 722-723. , o.Cl.Biol. Cell. Mol. 422-433. , Cell Gene .Cl Biol. Cell J. 105 287 209-219. , 49-54. , (2011). Curr. Curr. 147 Mol. Nat. Sci. 27 , ,

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