Teeatoscnrbtdeulyt hswork this to equally Sciences, contributed of authors Academy *These Chinese China. Physics, 100049, Energy Beijing High of Institute Nanosafety, China. 116044, China. 100049, ulagFang Guoliang to Cep68 and cohesion C-Nap1 between maintain interaction an mediates Centlein REPORT SHORT eevd3 uy21;Acpe 1Jnay2014 January 31 Accepted 2013; July 31 Received ` the phase, and (Nigg G2 MTOC duplicated single the a the as into 1 function to and G1 order in from duplicated, linked remain ‘G1–G2 progress is the proximal cells centrosome their or from As 2010) emanating al., 2011), ends. Stearns, et and to (Barrera (Nigg referred tether’ often cohesion is end centrosome which distal linker, the as cycle proteinous as a tip cell through the connected the and with during end axis, proximal long cilia the (Go its as along to of polarized referred ultrastructure is and base formation The the 2011). the distal that become Dynlacht, shows to for and harboring cortex (Kobayashi body functionally cell quiescence centriole, the basal and to mother migrate a structurally can the are appendages, subdistal Only Raff, microtubule- and and two distinct. (Nigg centrioles primary The (PCM) two material the 2009). comprises pericentriolar and surrounding is the (MTOC) centrosome center organizing mammalian The INTRODUCTION C-Nap1, Cep68, Centlein, cohesion, Centrosome WORDS: KEY the by C-Nap1–centlein–Cep68. maintained of is complex Both our identified cohesion Collectively, splitting. newly centrosome substrates. that centrosome Nek2A demonstrate novel in data are the results Cep68 to and turn, Cep68 during centlein of in centrioles recruitment and, and impairs of C-Nap1 centrosomes centlein ends between of link Depletion proximal molecular a Cep68. the as functions at and interphase Cep68 with complexes and centlein (also Moreover, C-Nap1 C-Nap1 Cep68. centriolar both and proximal with the Cep250) interacts the as directly that to known and localizes show centrioles CNTLN) the we of as ends study, known (also this centlein In unknown. are growing, cohesion largely is centrosome microtubule-organizing of still regulation cohesion splitting and of composition the single precise list to the the leads Although a cohesion centrosomes. interphase of centrosome proteinaceous of the as Impairment a that center. function ensures as centrioles, centrosome(s) regarded parental between mostly linker cohesion, Centrosome ABSTRACT uhr o orsodne(ildmd.d.n [email protected]) ([email protected]; correspondence for Authors olg fLf cecs nvriyo hns cdm fSine,Beijing Sciences, of Academy Chinese of University Sciences, Life of College 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,13–69doi:10.1242/jcs.139451 1631–1639 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. nz,21) h w etilswti 1cnrsm are centrosome G1 a within centrioles two The 2012). ¨nczy, 3 2 e aoaoyfrBooia fet fNnmtrasand Nanomaterials of Dalian Effects University, Biological Medical for Dalian Laboratory Cell, Key Stem Cancer of Institute 1, ,DcunZhang Dachuan *, 1, ,HiogYin Huilong *, 1 uZheng Lu , etooechso ean ob lcdtd ee we Here, elucidated. be of tethering to composition of precise remains number the growing are identified, cohesion a been LRRC45 centrosome have Despite and that mitosis. Barrera rootletin proteins of at 2007a; centrosomes C-Nap1, onset the al., from which the displaced et and of Nek2A (Graser (He by 2010), phosphorylated LRRC45 CDK5RAP2 rootletin 2007a), al., and 2000), al., et et 2013) al., (Graser et al., Cep68 (Mayor et 2005), C- Cep250) al., include Proteins et centrioles as 2003). (Bahe parental known for the Fry, (also of preparation and Nap1 tethering the in (Faragher in phosphorylation, assembly implicated separation spindle centrosome Nek2A-kinase-mediated is mitotic cohesion with centrosome by concomitant mitosis, of onset disassembled the At 2011). Stearns, NL)addmntaeta eteni opee ihC- cohesion. as centrosome with for known complexed required is is (also and centlein Cep68, centlein and that Nap1 protein demonstrate and centriolar CNTLN) the characterize ii n etils etenwsdtce stodt that dots and two the pericentrin for within as staining localized by against marked also detected primary was but which antibody of was PCM, tubulin marker acetylated an Centlein a with centrioles. – colocalized using 2007b) and by al., cilia et visualized (Graser tubulin readily centriole, becomeacetylated daughter cell are a cells RPE-1 and an which centriole RPE-1 stage, body/mother this basal withdrawal, At a cilia. possesses primary serum develop and Upon quiescent cells. antibodies. the the RPE-1 of of patterns 20% staining confirming the below S1C), of Fig. specificity to the material the (supplementary immunofluorescence reduced in control from centlein negative scattered Silencing signals S1C). signals the Fig. punctate material marker centrosomal (supplementary the as with colocalized detected and antibodies, cytoplasm the was with staining centlein immunofluorescence material (supplementary By of lysates S1B). cell band Fig. RPE-1 of and a depletion U2OS in siRNA-mediated recognized centlein after 7H2 diminished greatly (‘antigen’, clone was which that protein antibody showed rat blotting rat centlein Western the S1A). and recombinant Fig. material mouse the supplementary both 901–1191, of and generated 89–437 respectively, residues we spanning antibodies centlein, monoclonal centrioles of characterize ends proximal To the to localizes Centlein DISCUSSION AND RESULTS i.1;splmnaymtra i.SD.I ocuin the conclusion, 2012; In al., S1D). et Fig. (Arquint proximal material by Cep135 supplementary the S1D); 1B; and Fig. with Fig. C-Nap1 material substantially markers supplementary colocalized centriolar 1B; as centlein (Fig. (Kim known centrioles contrast, 2011) of (also ends al., distal CP110 the et of and markers centlein are two centrin2 which that the CCP110), from revealed with distinctly examination associated localized is Further centlein 1A). (Fig. that indicating centrioles (Lopes 2011), PCM-1 by al., marked et were which satellites, centriolar the and olclz etena h etooepeiey ecoet use to chose we precisely, centrosome the at centlein localize To 1 ioi Bi Xiaolin , 2,3, ` n iYuan Li and 1, ` , c 7 kDa, 170 c -tubulin, -tubulin 1631

Journal of Cell Science ADG;1 (A,D–G); mgso h etooe.()Qatfcto ftenraie loecneitniyo eten -a1 otei n e6 ttesltcentro split the at Cep68 and rootletin against C-Nap1, antibody ce centlein, an for of and specific intensity duplexes (green) fluorescence ** siRNA antibodies normalized or cells. indicated the siRNA U2OS of the nonspecific siRNA-treated Quantification a with indicated (H) with co-stained h centrosomes. then the 72 were of for and images transfected Cep68 were cells or U2OS rootletin (D–G) C-Nap1, nonspecific. NS, experiments. independent three HR REPORT SHORT eten(6 D) oee,teseiiiyo h nioywas antibody 1632 the of human specificity of the mass however, molecular kDa); predicted (162 the detect centlein to to supposed corresponding is band fragment it 803–953), GST-tagged a Because residues 2008). a acid al., against (amino centlein et generated of (Makino been previously kDa had has 90 antibody centlein, of the band against single antibody a polyclonal detected analysis, a blotting western using 2008; lysates, al., HeLa by In et 2008). (Makino al., unknown et was Sakamoto function precise its however, centrioles. of ends proximal the the label recognize and centlein protein specifically antibodies monoclonal siRNAs using by cells U2OS and centrosome RPE-1 of the from was views of Cep68 centrioles enlarged Images or two show (red). rootletin between insets centlein C-Nap1, distance The against centlein, the (blue). antibody of when nuclei an depletion split the and upon as stain (green) splitting counted to antibodies centrosome were used indicated of Centrosomes was the percentage DAPI with The (red). co-stained (C) centlein cohesion. were enlarged. centrosome against cells for antibody RPE-1 required an and (B) centrioles and centrosomes. of (green) ends proximal antibodies the indicated at the localized is Centlein 1. Fig. etenwsfrtietfe yNknsiadcolleagues; and Nakanishi by identified first was Centlein m Aist;B;2 B); insets; (A m m DGinsets). (D–G m P , .1cmae ihN,mean NS, with compared 0.01 , 7 -kDa 170 6 s.d., n . etooesltighsbe bevdpeiul ncells in previously observed been has splitting Centrosome nteosre oiiyo h protein. discrepancies the of explains mobility which observed 2008), the al., in et (Makino verified not el.We h itnebtenteprna etilsis centrioles parental the between distance the . When interphase increased by centlein-depleted an cells. the protein noticed in and the centrosomes cells depleted between U2OS distance we and RPE-1 centlein, in of siRNAs using function the explore centrosomes To to and Cep68 cohesion of centrosome recruitment for required is Centlein 0prgopi he needn xeiet.Saebr:10 bars: Scale experiments. independent three in group per 50 . 2 2 m m ,i stre cnrsm pitn’(rsre l,2007a). al., et (Graser splitting’ ‘centrosome termed is it m, .** m. ora fCl cec 21)17 6113 doi:10.1242/jcs.139451 1631–1639 127, (2014) Science Cell of Journal P , .1cmae ihN,mean NS, with compared 0.01 A h uecn P- el eec-tie with co-stained were cells RPE-1 quiescent The (A) c tbln(e) nesso enlarged show Insets (red). -tubulin 6 s.d., n . 0 e ru in group per 300 oe nthe in somes m m were s ntlein, .

Journal of Cell Science 07) eqatfe etooesltigi el lacking al., cells et negative in Graser with comparison 2005; splitting in Cep68 al., or centrosome whereas rootletin et C-Nap1, quantified centlein, 2000), (Bahe We al., centrioles 2007a). proximal parental et the from of (Mayor emanating ends fibers decorate protein C- Cep68 is and of docking it rootletin that localization suggests centriolar The centrioles parental 2007a). of a al., variation ends proximal et large the Graser to with Nap1 2005; albeit al., Cep68, et or (Bahe rootletin C-Nap1, lacking REPORT SHORT rcnli ihCp8i 2Scls oeta high-level that Note cells. U2OS in Cep68 C-Nap1 with with centlein centlein either overexpressed or we Cep68, and Nap1 S3E). Fig. rootletin material between interaction (supplementary detectable centlein region no material and supplementary was sufficient a right; There bottom was S3D). 2C, whereas Fig. Cep68 top (Fig. of centlein 2C, to S3B), 509–757 (Fig. bind to residues centlein Fig. acid to amino material Cep68 binding spanning its supplementary to for Two 1838– right; binding necessary S3C). and 608–1221 were its Fig. residues 2442, material encompassing left; for C-Nap1, supplementary of top required spanning left; regions region 2C, bottom was (Fig. a 2C, 1–600 that (Fig. C-Nap1 reciprocal and acids bind residues S3A), in amino acid Fig. to amino other material spanning sufficient supplementary each region was a analyses with that 151–600 Deletion showed interact centlein HEK293T 2B). of (Fig. to in experiments expressed able and immunoprecipitation and tagged were Cep68 epitope cells right). were 2A, that (Fig. 2A, (Fig. centlein cells the immunoprecipitate in detected HEK293T was Myc–centlein C-Nap1 transfected endogenous Conversely, left). transiently bottom from C- results co- endogenous Nap1 by with verified centlein was Myc-tagged The which of left), with immunoprecipitation top 2A, co-immunoprecipitated performed. (Fig. centlein C-Nap1 endogenous endogenous were co- that reciprocal revealed proteins, assays the between immunoprecipitation interactions the Cep68 confirm and To C-Nap1 with interacts Centlein cells not but Cep68 C-Nap1-depleted and C-Nap1 the rootletin. with interacts indicate physically in results centlein These S2F). that greatly Fig. material supplementary 1H; reduced by (Fig. diminished unaffected was centlein was centrosomal whereas centlein but significantly, silencing centrosomes 2007a) at summary, staining al., Cep68 In et Graser S2E; depletion. Fig. 2005; centlein material 1F; al., supplementary (Fig. 1G; et (Fig. Bahe C-Nap1 Cep68 or depletion of al., C-Nap1 upon et centrosomes of Graser 2007a), from localization displaced 2005; was al., al., the Rootletin et 2007a). Bahe et in S2D; Fig. Graser produce material change not supplementary 2005; supplementary did visible rootletin al., or 1E; Cep68 any (Fig. et centlein, diminished of Bahe centrosomes silencing of or whereas S2C; the one displaced Fig. at any either material of staining rootletin 1D; (Fig. Depletion or Cep68 centrosomes S2B). C-Nap1 from centlein Fig. centlein, of C- material loss of a knockdown supplementary whereas in Cep68, resulted or Nap1 rootletin unaltered of was protein centrosomes depletion siRNA-mediated the upon at using localization by Centlein Cep68 depletion. and rootletin C-Nap1, centrioles. parental for important of rootletin particularly tethering 2007a), are the or al., rootletin et and C-Nap1 Graser C-Nap1 1C; that either (Fig. indicating ratio of splitting highest depletion the produced studies, previous accordance In with S2A). Fig. material supplementary 1C; (Fig. controls enx ogtt eemn h nedpnec fcentlein, of interdependence the determine to sought next We ofrhretbihteascaino etenwt ohC- both with centlein of association the establish further To noeoscnli yuigsRA n bevdta the that observed and siRNAs depleted using first by We centlein experiments. endogenous knockdown-rescue performed etenat samlclrln ihnteC-Nap1–centlein– the within cohesion. link centrosome mediate molecular to that complex a Cep68 notion as the acts substantiate centlein also its but through centrosome domain to only the by C-Nap1-interaction to not recruited localize induced experiments is centlein these to splitting that Thus, demonstrate centrosome 3D–F). able (Fig. rescued were depletion for centlein and Cep68 required centrosomes regions and the to the C-Nap1 construct comprising with each mutants splitting. interaction centrosome centlein of on effect ability the its Only the and centrosomes examined the to and localize centlein cells of mutants 3B,C; those deletion (Fig. various into transfected centrosomes then We split 1C). Fig. displayed cells centlein-depleted oe aes.Teedt eosrt htcnli,indeed, centlein, that Cep68. and and demonstrate middle C-Nap1 both 2D, data with (Fig. These interacts filaments Graser centlein panels). 2005; to lower resulted al., recruitment Cep68 et or their C-Nap1 produce Bahe either in whereas of not panels; Overexpression did panel), 2007a). top al., Cep68 2D, et filaments left or (Fig. of C-Nap1 top structures either such formation 2D, of (Fig. the expression cytoplasm ectopic to the led throughout centlein of overexpression e6 hog h needn ietitrcin fboth of interactions with complexes direct C-Nap1 independent centlein. that with the suggests proteins through 2007a), shown; Cep68 al., not (data et Cep68 with and along C-Nap1 Graser data, between This interaction 3A). of lack (Fig. a Flag–Cep68 extracts cell expressed in transiently GFP–C-Nap1 and both bound sepharose on selectively of a immobilized beads, 89–437), fragment an in residues GST-tagged acid Therefore, A exists (amino centlein Cep68. performed. centlein was and assay that C-Nap1 direct-binding suggest both with above complex described results The for Cep68 cohesion and centrosome C-Nap1 with complexes Centlein xmndwehrNkAbudt n phosphorylated we and First, to substrates. bound Nek2A Cep68 Nek2A and are centlein whether Cep68 examined partner binding the of to substrate 2000). similar well-characterized al., a et very (Mayor – is on Nek2A C-Nap1 which reduced for 4A,B), were observed (Fig. pattern but poles been centrosomes spindle had interphase mitotic were that at centlein by high cells of examined relatively Levels U2OS were microscopy. 2007a). (Mayor immunofluorescence populations al., using manner stage-specific et for cell-cycle-dependent Graser synchronized 2000; associated a Cep68, al., in and et C-Nap1 centrosomes complex, like protein centlein, with a whether form Cep68 asked and we centlein C-Nap1, substrates that Nek2A Given are Cep68 and Centlein om fcnli n e6 rmHK9Tclsexpressing cells migrating HEK293T slowly from antibodies more Cep68 detected Furthermore, and tags centlein Flag right). of or forms Myc Flag–Cep68 4C, (Fig. the with from against co-transfected prepared GFP–Nek2A been Flag lysates had the and that against from cells antibody GFP–Nek2A an HEK293T this, co-precipitated to centlein tag Similar immunoprecipitate endogenous GFP–Nek2A middle). the the 4C, in (Fig. experiment, detected readily reciprocal be a could in 4C, Nek2A and, (Fig. that left), Myc–centlein demonstrated with we co-immunoprecipitated cells, specifically HEK293T in Myc–centlein oeuiaean elucidate To hs esuh odtriewehrcnli n its and centlein whether determine to sought we Thus, ora fCl cec 21)17 6113 doi:10.1242/jcs.139451 1631–1639 127, (2014) Science Cell of Journal nvivo in nvivo in ycepesn F–e2 and GFP–Nek2A coexpressing By . ucinlrl ftecmlx we complex, the of role functional nvitro in 1633

Journal of Cell Science HR REPORT SHORT ramn fteHK9Tcl xrcswt afintestinal calf 4D). with (Fig. 1634 extracts GFP–Nek2A cell HEK293T (K37R), the inactive of 1 bars: but Scale Treatment (WT), microscopy. catalytically immunofluorescence wild-type by with monitored not together was in proteins Flag–Cep68 red, of +, or distribution residues. Myc–centlein the acid amino and represent panel), numbers (bottom The Cep68 right). (bottom and betwe were Cep68 sites centlein Myc–centlein and Interaction or with left) (C) panel) Flag–Cep68 (bottom reactions. (rig (middle centlein coexpressed reciprocal between Myc that in as or detected cells well left) HEK293T were as 2 (bottom of proteins right), Both C-Nap1 lysates (top C-Nap1 tags. The against and respective (B) antibody left) the an antibodies. against either indicated antibodies with the with (IP) with immunoprecipitated (IB) immunoprecipitated blotted Myc–centlein, western with then transfected Cep68. were and cells C-Nap1 HEK293T with C-Nap1. interacts Centlein 2. Fig. lc,n neato D 2Sclswr rnfce ihtge eten -a1o e6 ln tppnl,wt agdcnli n C-Nap1 and centlein tagged with panel), (top alone Cep68 or C-Nap1 centlein, tagged with transfected were cells U2OS (D) interaction no black, , A etr ltigo noeoscnli tplf)atrimnpeiiainwt natbd against antibody an with immunoprecipitation after left) (top centlein endogenous of blotting Western (A) hnei h e oiiyo h rtiswsdet protein to the due was that proteins confirming the of slower bands, mobility these gel Cep68 of the in and loss change in centlein resulted migrating (CIAP) phosphatase alkaline ora fCl cec 21)17 6113 doi:10.1242/jcs.139451 1631–1639 127, (2014) Science Cell of Journal ncnli (top centlein en teraction; t and ht) 0 m m.

Journal of Cell Science HR REPORT SHORT pitn nUO el Fg E.W hndtrie whether determined centrosome Cep68 then and to centlein We phosphorylate leading 4E). directly (Fig. could turn, cells Nek2A centlein in U2OS of centrosomes, in dissociation splitting from the Cep68 caused K37R, and WT, GFP–Nek2A GFP-Nek2A not of but Overexpression 4D). (Fig. phosphorylation 10 bars: and (green) antibodies indicated the with co-stained were siRNA b centlein GST–centlein western or GST, by NS analyzed with with were incubated transfected beads were glutathione–Sepharose-4B cells from U2OS Flag–Cep68 extracted (B,C) and proteins antibodies. washing, GFP–C-Nap1 indicated extensive coexpressed After the that aa. with 901–1191 cells GST–centlein HEK293T or cohesion. from aa centrosome extracted 89–437 for Protein Cep68 89–437). and C-Nap1 residues with acid complexes Centlein 3. Fig. ecnaeo etooe htsltuo xrsino h ullnt rtevrostuctdfrso F–eteni 2Sclsdpee fcent of depleted cells U2OS in GFP–centlein of forms truncated and various localized the – or 151–600 full-length and the vario 1–600 by of the 1–898, (indicated antibodie or expression acids splitting indicated upon full-length amino centrosome the split the ** to and rescued that with corresponding (green) also centrosomes transfected GFP fragments and of then against and centrosomes and percentage antibody protein the centlein an full-length to of with the Cep68 depleted analyses including recruited immunostaining first – to were centlein subjected cells GFP-tagged being U2OS The before (D,E) GFP–centlein centrosomes. of of forms views truncated enlarged show insets The P , .1cmae ihGP mean GFP, with compared 0.01 m BE;2 (B–E); m m BEinsets). (B–E m 6 .. prxmtl 0 oiieytasetdclswr one nec ftetreidpneteprmns Scale experiments. independent three the of each in counted were cells transfected positively 100 approximately s.d., nvitro in . muorcpttdfo E23 el hthdbeen was had protein that K37R. GFP–Nek2A Nek2A or cells WT substrates. GFP–Nek2A either HEK293T centlein with as transfected residues from acid of purified (amino immunoprecipitated Cep68 were fragments and 89–437) 1–282) GST-tagged residues acid (amino expressed Bacterially (A) nvitro In c ora fCl cec 21)17 6113 doi:10.1242/jcs.139451 1631–1639 127, (2014) Science Cell of Journal tblnsann) nesso nagdcnrsms F The (F) centrosomes. enlarged show Insets staining). -tubulin idn fCNp n e6 ihafamn fcnli (amino centlein of fragment a with Cep68 and C-Nap1 of binding c tbln(red). -tubulin (red). s us lein. 1635 lotting

Journal of Cell Science HR REPORT SHORT 1636 4. Fig. e etpg o legend. for page next See ora fCl cec 21)17 6113 doi:10.1242/jcs.139451 1631–1639 127, (2014) Science Cell of Journal

Journal of Cell Science bto w os.Ist hwelre mgso etooe.Saebars: Scale centrosomes. of images 10 enlarged show Insets rows). antibodies two with (bottom stained with and transfected K37R were against GFP–Nek2A cells or WT U2OS GFP–Nek2A (E) antibodies. either immunoblot indicated to the subjected with were analysis (CIAP) phosphatase alkaline intestinal anandmil yCNp–otei Bh ta. 2005; al., thus, C-Nap1–centlein– et is, by 4G). supported (Fig. (Bahe and cohesion Cep68 2012) C-Nap1–rootletin Schiebel, Centrosome and to by Mardin 4G). (Fig. mainly leads proximal the centrioles maintained at turn, and Cep68 of and interacts in C-Nap1 ends centlein with complex and, cells, protein a interphase forms centrosomes In splitting. from centrosome centlein Cep68 in of Knockdown centrosomes 4G). displaces with (Fig. associated manner cell-cycle-dependent and a substrate Nek2A identified are Cep68 and centlein substrates. that Nek2A conclude thus novel threonine We using and 4F). serine by (Fig. phosphorylated Nek2A residues the inactive to not specific antibodies but the active by phosphorylated were nso etilsadbnswt -a1adCp8t atcpt in centrosomes. participate from to dissociates Cep68 centlein and mitosis, C-Nap1 In with cohesion. link binds proximal centrosome molecular the and a at centrioles localizes as of centlein acting interphase, ends centlein In Cep68. for and model C-Nap1 A between (G) phosphorylated Cep68. indicate or arrows centlein and The serine residues. phosphorylated threonine against phosphorylated by antibodies revealed with was blotting phosphorylation GST–Cep68 western Substrate the aa). or (1–282 aa) fragment (89–437 N-terminal fragment N-terminal GST–centlein the ( without or inactive (+) catalytically with or treated (WT) lysates wild-type HEK293T Flag–Cep68 with GFP–Nek2A. or together (K37R) panel) or HEK293T (left alone (D) Myc–centlein panel) panel). either (right (right with antibody transfected western GFP were and against cells tag antibody Flag an the with against blotted then antibody were an lysates were with Flag–Cep68, cells immunoprecipitated HEK293T and (middle). GFP–Nek2A western centlein with were against co-transfected immunoprecipitates antibody and an GFP with against blotted antibody (left HEK293T an antibody transfected with GFP from against cells immunoprecipitated antibody was an GFP–Nek2A with (WB) panel). blotted western immunoprecipitates been then The had were Myc–centlein. was that and tag cells GFP–Nek2A Myc HEK293T with The of co-transfected Cep68. lysates and from centlein (IP) to immunoprecipitated binds Nek2A (C) experiments. h etooei 2Sclsa ifrn tgso h elcce ** cycle. cell the mean of stage, stages G1 different with at compared cells at U2OS the rootletin in of and centrosome C-Nap1 Quantification the Cep68, (B) centlein, of proteins. of intensity indicated images fluorescence the enlarged normalized for show stained Insets indicated centrosomes (bottom). the distribution the analysis with cycle FACS co-stained Cell by and (red). monitored (G1) centrin2 h was 0 against 18 antibody for and and released (M) (green) then 12 antibodies (G2), and 9 block (S), thymidine 6 double (G1/S), a poles, with spindle substrates. Nek2A mitotic synchronized novel on were are Cep68 drops and Cep68, centlein like both and staining, Centlein 4. Fig. REPORT SHORT n a ucoe notep3 the 2005) into al., et subcloned (Bahe Nigg A. truncated was Erich from The gift pEGFP- and a and into was pCMV-Myc pCMV-Myc. C-Nap1 vectors cloned Full-length the and into C1. and cloned were pEGFP-N1 cDNA centlein of HeLa pEGFP-C1, mutants from vectors obtained the was centlein proteins Full-length recombinant and construction Plasmid METHODS AND MATERIALS The rmEihA ig(aee l,20) eunigwspromdfrall for gifted performed was was Sequencing 2005). Nek2A al., cloned et (K37R) (Bahe and Nigg inactive A. cDNA Erich catalytically HeLa from The from al., p3 et pEGFP-C1. obtained the (Graser was into Nigg into A. cDNA) Nek2A Erich cloned Wild-type from HeLa gift were 2007a). a from was Cep68 rootletin (obtained GFP-tagged of vector. Cep68 mutants truncated full-length and C-Nap1, of mutants nsmay eten oehrwt e6,i newly a is Cep68, with together centlein, summary, In m ;2 m; nvitro in c tbln(o w os,cnli mdl w os n Cep68 and rows) two (middle centlein rows), two (top -tubulin m ,ist.(F) insets. m, iaeasysoe htbt etenadCep68 and centlein both that showed assay kinase nvitro In 6 iaeasyuigW rK7 e2 and Nek2A K37R or WT using assay kinase s.d., 6 n lgCVvco.Tetruncated The vector. Flag-CMV . 0prgopi he independent three in group per 50 A 2Scells U2OS (A) 6 Flag-CMV 2 P calf ) , 0.01 rtiswr eltduigsRAdpe lgncetdstargeting oligonucleotides duplex siRNA using depleted were proteins AGGGCATCTCGGTA-3 GTACACGCAA-3 eeaqie sn iia aeaLiaDC5 n Leica and C DFC450 Leica camera 4.0.0). digital (version software a Suite using Application (Leica, acquired 100 Immunofluorescence microscope a were DM600B 20H5, with Invitrogen). Leica equipped a (1:100, APEX Germany) using (A10474, an performed antibody using was Kit 594, microscopy anti- Fluor Labeling Alexa mouse to anti-mouse Antibody coupled a goat covalently was centrin, 555 centlein Millipore) visualize Fluor and simultaneously To Alexa (11A4) Invitrogen). anti-rabbit A21424, and (1:1500, goat IgG Invitrogen) IgG anti-mouse 594 goat A11029, 488 Fluor Fluor (1:500, Alexa Alexa Invitrogen), used. A11012, Invitrogen), anti-rabbit (1:1200, donkey were IgG A21206, 488 Fluor International) (1:1500, Alexa were MBL IgG used M047-3, antibodies secondary and (1:10000, The ProteinTech) 20543-1-AP, anti-Myc (1:5000, (1:5000, anti-Flag anti-Cep68 mouse rabbit rabbit Nigg), Nigg), ProteinTech), A. A. Erich 50430-2-AP, Erich anti-Cep135 14498-1- (1:1000, (1:3000, anti-rootletin (1:800, anti-GFP rabbit rabbit anti-C-Nap1 rabbit Korea), rabbit Proteintech), South ), Kunsoo AP, Blue (1:500; University, A02C0240, anti-CP110 National (1:3000, rabbit Seoul abgent), Rhee, anti- AP7481c. rabbit (1:500, Abcam), ab4448, PCM-1 (1:1000, anti-pericentrin rabbit Signaling), anti-acetylated anti- rabbit Biotechnology), mouse Sigma-Aldrich), Ivtoe)acrigt h auatrrspooo.Clswere Cells protocol. manufacturer’s transfection. post the h U2OS reagent 12–24 to h. 2000 analyzed 48 Lipofectamine according for using cells transfected (Invitrogen) RPE-1 were for cells HEK293T medium and serum-free with replaced was el eegono oesis ahdi B n ie in fixed and PBS in washed coverslips, on grown were Cells microscopy Immunofluorescence and respectively, (Hyclone), IU/ml F12 100 DMEM (Hyclone), Ham’s serum 100 bovine and and in fetal penicillin (v/v) Hyclone) cultured 10% with Ham’s were supplemented (1:1, with (DMEM) cells medium F12 Eagle’s HEK293T modified Dulbecco’s and (Hyclone), RPE-1 901–1191, U2OS, transfections used. and (Beijing, and were culture Ltd Cell 7H2 89–437 Clone Biotechnology rat residues and Absea 11A4 C-terminal acid clone by and Mouse generated China). N- (amino the were centlein against respectively) antibodies of monoclonal regions rat and Mouse production Antibody L002-2 agtn h olwn eune:5 sequences: ON-TARGETplus following Dharmacon using transfection. from the post obtained transfected targeting were h centlein 72 (L-020720-02) against were at siRNAs harvested SMARTpool cells manufacturer’s and the to protocol according interference, (Invitrogen) RNAiMAX Lipofectamine RNA-mediated For experiments siRNA otiigDP osanDA h rmr nioismouse antibodies primary P36931) anti- The (Invitrogen, rabbit DNA. Gold 11A4), stain ProLong and (1:500, PBS with to in anti-centlein again slides DAPI rinsed on were containing Coverslips mounted min. 30 PBS) then in for Tween-20 0.1% performed BSA, (2% were AbDil in diluted antibodies secondary n netdit h xrsinvco GX4-.GST-tagged pGEX-4T-1. vector expression in the expressed amplified were were into Cep68 of fragments 1–846 inserted pairs base 265–1311 or and pairs centlein base of proteins, 2701–3573 recombinant and of expression For constructs. ne %CO 5% under oiesrmabmn(S)fr3 i tro eprtr,then temperature, room overnight PBS) at 3% in BSA min in 4 3% 30 blocked in at (diluted for and antibodies primary washed (BSA) with were incubated albumin coverslips serum Then bovine min. 8 for methanol uiidwt lttin–ehrs-B(705-1 EHealthcare GE (17-0757-01, Sciences). glutathione–Sepharose-4B Life with purified ˚ .Atrtrewse nPSfr5mnec,icbtoswith incubations each, min 5 for PBS in washes three After C. ora fCl cec 21)17 6113 doi:10.1242/jcs.139451 1631–1639 127, (2014) Science Cell of Journal 2 oidc rmr iimfrain h rwhmedium growth the formation, cilium primary induce To . 9 m ,5 /lsrpoyi.Teclswr rw t37 at grown were cells The streptomycin. g/ml 9 -GTTGAAGTATCACAGAGTA-3 9 n 5 and shrci coli Escherichia c 6 tbln(:00 -1 at Cruz Santa C-11, (1:1000, -tubulin 9 -ACGGAAAATTGCAGT-3 i meso betv,adimages and objective, immersion oil a tbln(:20 2G,Cell D20G3, (1:1200, -tubulin c tbln(:00 T5192, (1:1000, -tubulin tanB2 D3 and (DE3) BL21 strain 9 -GAGCTGAA- 9 ,5 9 -GCTAG- 9 Several . 2 1637 20 ˚ ˚ C C

Journal of Cell Science a de orato ufr(0 MNC,5 MTi C,1 mM 10 HCl, Tris mM 50 NaCl, mM (100 buffer MgCl reaction to added was transiently was been mixture had The that Flag–Cep68. cells HEK293T and 4 of at GFP–C-Nap1 incubated extract with GST of glutathione– or transfected mg GST with (20 1 beads. beads chromatography the with Sepharose to to bound affinity proteins crosslinked fusion by then and purified Sepharose-4B and (DE3) o t37 at Biolabs) h England 2 New (M0290S, for (CIAP) phosphatase alkaline intestinal ufradsbetdt muoltiganalysis. immunoblotting to subjected and buffer D-AEsml ufradwr hnaaye ywsenblotting western by in analysed boiling 2000). then by al., were et beads and (Rountree Sepharose buffer the sample from SDS-PAGE extracted were proteins buffer, ae ihteidctdDAcntut sn ioetmn 2000 Lipofectamine using constructs h DNA 72 indicated cells the (Invitrogen). siRNA-treated transfecting with by later Rescue performed Dharmacon). were D-001810-10, experiments pool, Non-Targeting TARGETplus ACAAGGA-3 5 C-Nap1, CCTA-3 sequences: TGAT-3 following the REPORT SHORT To F–e2 3R t2 fe rnfcin h oa cell total the transfection, after GFP–Nek2A 1638 h with 24 transfected At K37R. were GFP–Nek2A dishes or 10-cm WT in cells HEK293T vitro In lysates, cellular of treatments phosphatase For treatment Phosphatase in expressed were proteins fusion GST and GST vitro In were membranes The Millipore). 354206, 4 at (1:2000, incubated anti-GST anti-Cep68 rabbit and rabbit International), ProteinTech) 50430-2-AP, ProteinTech), (1:1000, anti-GFP rabbit Nigg), 20543-1-AP, MBL A. Erich (1:1000, (1:1000, M047-3, 14498-1-AP, anti-Flag mouse (1:1000, (1:800, rabbit anti-Myc 7H2), anti-C-Nap1 mouse (1:1000, rabbit ProteinTech), anti-centlein 11A4), rat at (1:1000, used concentrations: anti-centlein and milk following non-fat 1% containing the TBS-T onto in diluted Primary 0.1% h. and were transferred NaCl antibodies 1 for mM temperature membranes room 150 at and 7.4, milk the non-fat pH 5% containing Tris-HCl Tween-20) SDS-PAGE mM Then (10 TBS-T (Millipore). 10% in blocked were membranes by difluoride separated polyvinylidene were Proteins Immunoblotting at incubated been 50 had mixtures with the 4 After incubation Biotech). Cowin by (CW0012A, precleared (Roche)]. 12,000 PMSF cocktail at was mM centrifuged cold inhibitor 1 were of protease NP-40, lysates ml 0.1% EDTA-free The 1 NaCl, complete mM with and 250 lysed HEPES, (Sigma) were mM dishes [50 10-cm buffer ELB in cultured cells HEK293T Immunoprecipitation ltigdtcinraet(P 22 EHatcr ieSine)and Sciences) (Kodak). Life western film Healthcare X-ray prime GE to chemiluminescence exposure 2232, (RPN enhanced reagent membranes using detection the blotting TBS-T, by with washes developed final After were h. 1 for at temperature milk non-fat room 1% containing TBS-T in diluted peroxidase Beyotime) A0192, (1:2000, IgG horseradish anti-rat goat with or DakoCytomation) P0399, (1:5000, Liankebio), incubated IgG anti-rabbit GAM0072, swine (1:10,000 then IgG and anti-mouse goat TBS-T (HRP)-conjugated with times three 20 of addition by followed nSSPG apebfe n muoltiganalysis. material immunoblotting precipitated and the buffer of sample heating SDS-PAGE by followed in buffer, ELB using times hmgnosnra g scnrl t4 at control) as IgG normal (homogeneous nirgn n nuae t4 at incubated and Invitrogen) ˚ o ,tespraatwsicbtdwt 2 with incubated was supernatant the h, 3 for C 2 MDT.Temxuewsicbtdwt 0 nt fcalf of units 200 with incubated was mixture The DTT). mM 1 , 9 9 iaeassays kinase assays binding Gae ta. 07)adroltn 5 rootletin, and 2007a) al., et (Graser Bh ta. 05;Cp8 5 Cep68, 2005); al., et (Bahe ˚ ˚ 9 vrih.Atricbto,temmrnswr washed were membranes the incubation, After overnight. C ˚ .Temxuewste etdi D-AEsample SDS-PAGE in heated then was mixture The C. vrih ihaiain fe orwse ihELB with washes four After agitation. with overnight C Bh ta. 05.CnrlsRA eeas sd(ON- used also were siRNAs Control 2005). al., et (Bahe ˚ vrih.Baswr ahdfour washed were Beads overnight. C m yaed–rti- (10004D, Dynabeads–protein-G l m g )wsicbtdi L buffer ELB in incubated was g) 9 9 o 0mnadtesupernatant the and min 10 for -CTGGAAGAGCGTCTAAC- -ACCGAAGATGATCCATC- m fprotein-G–Sepharose of l ˚ o nadtoa h, 3 additional an for C , 9 -AAGCCAGTCTAG- go oa protein total of mg 1 .coli E. m tanBL21 strain antibody g Go and L. M. J. A. Fuchs, Fry, J., and Seemann, J. E., A. Faragher, R. Hammer, R., L. A. E. Kao, Nigg, A., and J. F. Barrera, Leiss, J., C. Wilkinson, D., Y. Stierhof, S., Bahe, aioai eius8–3 fcnli)o S uinN-terminal fusion GST (500 Cep68) centlein or of of 1–282 centlein) residues fragment acid of (amino N-terminal Cep68 89–437 of fusion fragment residues GST and acid buffer the ELB (amino with in times incubated four 2 then washed with were kinases incubated immunoprecipitated was lysate ifrnebtentomaswsdtrie sn two-tailed a the using of determined significance was statistical means The two 2010). and between al., intensity et difference fluorescence detailed the (Mardin are measurements measure of elsewhere methods to detailed The used distance. centrosomal was software ImageJ analysis statistical and Measurements in suspended trypsinized, were cells The then h. and 18 block and 12 thymidine 100 9, double 6, a 0, for with released synchronized were cells analysis FACS U2OS and synchronization cycle Cell rsr . tehf .D,Lvi,S . ase,O . al,S,L Clech, Le S., Lamla, S., O. A. Gassner, B., E. S. Lavoie, Nigg, D., Y. and Stierhof, S., D. Graser, Y. Stierhof, S., Graser, A. E. Nigg, and D. Y. Stierhof, F., K. Sonnen, C., Arquint, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.139451/-/DC1 online available material Supplementary material Supplementary number [grant Program 973 X.B.]. China, the to L.Y.]; of 2010CB934004 to Program Y15102GN00 Research Chinese 095102GN00, Basic of numbers National University China [grant X.B.]; of Sciences and Foundation L.Y. of Science to Academy Natural 31271480 National 31071182, the numbers by [grant supported was work This Funding manuscript. the wrote G.F. and and L.Y. idea the research. conceived the X.B. L.Y., planned experiments. performed L.Z. and H.Y. D.Z., G.F., contributions Author interests. competing no declare authors The interests of assistance. Competing (Institute technical Qin for Zhihai Beijing) of Sciences, members of the Academy thank Chinese also Rhee Biophysics, We Kunsoo antibody. to CP110 grateful GFP-C- very the of for are for plasmids We Switzerland) K37R. and Basel, GFP-Nek2A Cep68, and of and GFP-rootletin (University Nap1, rootletin Nigg against Elena antibodies and providing Nigg kindly A. Erich thank We Acknowledgements Student’s okalad200 and cocktail 0 e n hnimnbotduigrbi nioisagainst antibodies rabbit (1 using (1 residues a immunoblotted residues threonine on phosphorylated SDS-PAGE then serine by separated and phosphorylated were proteins gel The buffer min. 10% sample 5 SDS-PAGE for adding boiling by and terminated was reaction The 2013). iaebfe 5 MTi C H77 0 MNC,2 mM 25 NaCl, mM 100 7.7, pH MgCl HCl mM Tris 25 mM glycerophosphate, (50 buffer kinase 0m/lpoiimidd P)fr1h h el eete analyzed then were Biosciences). cells (BD with The cytometer incubated h. flow and 1 C6 for PBS Accuri (PI) an with using iodide washed propidium were mg/ml cells 10 fixed The overnight. cy P. nczy, ¨ ijnto n srqie o omto fbplrmttcspindles. mitotic bipolar of formation for required Cell is and disjunction mice. in cohesion centrosome in Megraw,T.L. functions and filaments centriole-associated cohesion. forms Rootletin eurdfrpiayclu formation. cilium A. primary E. for required Nigg, and M. cohesion. centrosome for required are (Cdk5rap2) Biol. Cell Mol. Rev. cells. human in number centriole controls Sci. STIL of expression regulated m B n ie yadn lo od7%ehnladincubating and ethanol 70% cold of ml 3 adding by fixed and PBS l 125 14 2876-2889. , ora fCl cec 21)17 6113 doi:10.1242/jcs.139451 1631–1639 127, (2014) Science Cell of Journal 1342-1352. , t 21) oad oeua rhtcueo etil assembly. centriole of architecture molecular a Towards (2012). ts.Dfeecswr osdrdsgiiatwhen significant considered were Differences -test. .Cl Biol. Cell J. 21) D5A2rgltscnroeeggmn and engagement centriole regulates CDK5RAP2 (2010). m e.Cell Dev. T)fr3 i t30 at min 30 for ATP) M 13 20b.Cp6,anvlcnroeapnaeprotein appendage centriole novel a Cep164, (2007b). 171 425-435. , 27-33. , 18 913-926. , 20) e2 iaesiuae centrosome stimulates kinase Nek2A (2003). m fa nioyaantGP The GFP. against antibody an of g 2 .Cl Biol. Cell J. MDT rtaeinhibitor protease DTT, mM 1 , m m /l 180,Invitrogen). 71-8200, g/ml, /l 180,Ivtoe)or Invitrogen) 61-8100, g/ml, 20a.Cp8adCep215 and Cep68 (2007a). 179 .Cl Sci. Cell J. ˚ Sniakme al., et (Sundivakkam C 321-330. , 21) Cell-cycle- (2012). 120 4321-4331. , m P o.Biol. Mol. /l in g/ml) , (2005). .Cell J. 0.01. Nat. b -

Journal of Cell Science oe,C . rse,S . oi,L,Hrt .A,OClahn . Woolf, C., O’Callaghan, A., R. Hirst, L., Romio, L., S. Prosser, A., C. Lopes, D. B. Dynlacht, and T. Kobayashi, K. Rhee, and S. Kim, J. Chen, and J. Teng, H., Zhou, Y., Bao, N., Huang, R., He, REPORT SHORT adn .R,Lne . atr .E,Hry . coz .R,Fy .M. A. Fry, R., S. Scholz, T., Hardy, E., J. Baxter, C., Lange, R., B. Mardin, E. and Schiebel, and H. R. B. Ihn, Mardin, T., Sakamoto, Y., Hiragami, A., Uezu, K., Umeda, K., Makino, n cibl E. Schiebel, and biology. centrosome in 962. centlein. protein centrosomal H. Nakanishi, 1. syndrome oral-facial-digital including Sci. ciliopathies, human in M. implicated A. Fry, and S. A. body. basal to cells. interphase in Sci. proteins Cell material J. pericentriolar of accumulation centrosomal cohesion. centrosome for required 1100-1107. component linker centrosome 124 600-612. , 124 .Cl Biol. Cell J. 3760-3770. , 20) dniiainadcaatrzto ftenovel the of characterization and Identification (2008). 21) etilrstlie r sebypit o proteins for points assembly are satellites Centriolar (2011). 21) opnnso h ip aha cooperate pathway Hippo the of Components (2010). 21) E7i seta o etil ulcto and duplication centriole for essential is NEK7 (2011). .Cl Biol. Cell J. 193 21) raigtete htbn:nwadvances new bind: that ties the Breaking (2012). 435-444. , ice.Bohs e.Commun. Res. Biophys. Biochem. 21) euaigtetasto rmcentriole from transition the Regulating (2011). 197 11-18. , 21) RC5i a is LRRC45 (2013). elRep. Cell 366 958- , .Cell J. 4 , ig .A n af .W. J. Raff, A. and E. A. Nigg, E. and Nigg, M. A. Fry, K., Tanaka, D., Y. Stierhof, T., Mayor, udvka,P . aaaa,V,Mlk .B n iupti C. Tiruppathi, and B. A. Malik, V., Natarajan, C., P. K., Umeda, Sundivakkam, K., Makino, T., Kuramoto, S., B. Kawauchi, A., S. Uezu, T., Baylin, Sakamoto, and E. K. Bachman, R., M. Rountree, T. Stearns, and A. E. Nigg, etooa rti -a1i eurdfrcl yl-euae centrosome cycle-regulated cell for required is cohesion. C-Nap1 protein centrosomal disjunction. centrosome regulate 1176. to kinase Nek2 with hog M-ciae rti iaeadp38 cells and endothelial in kinase SOCE protein inhibit kinase. receptor-1 to AMP-activated protease-activated phosphorylation through by protein induced STIM1 (SOCE) mediates entry Ca2+ Store-operated brain. rat from H. proteins Nakanishi, co-sedimented and microtubule H. Baba, foci. N., replication Araki, at complex a form to DMAP1, Genet. co-repressor, Nat. new a and HDAC2 asymmetries. inherent and duplication disease. and ora fCl cec 21)17 6113 doi:10.1242/jcs.139451 1631–1639 127, (2014) Science Cell of Journal .Bo.Chem. Biol. J. .Cl Biol. Cell J. 25 Cell 269-277. , 139 663-678. , 151 288 837-846. , 21) h etooecce etil biogenesis, Centriole cycle: centrosome The (2011). 17030-17041. , 20) etils etooe,adclai health in cilia and centrosomes, Centrioles, (2009). a.Cl Biol. Cell Nat. 20) assetoercaayi of analysis spectrometric Mass (2008). b ee Cells ioe-ciae protein mitogen-activated 13 a.Cl Biol. Cell Nat. 1154-1160. , 20) NT binds DNMT1 (2000). 13 20) The (2000). 295-312. , 12 (2013). 1166- , 1639

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