etooeadclu rtis h etil rtoe of many 2007; proteomes identified al., has 2012) et The al., . Kilburn et cilium 2005; Ishikawa and al., 2011; 2012; al., et al., et Jakobsen 2009; et al., Keller 2004; et Hoh Keller al., 2004; 2008; al., et et al., McClintock Avidor-Reiss 2002; et al., Li et (Ostrowski 2011). approaches Dupuis-Williams, and (Fisch organization unique axoneme their the imparting supports on thus structure cilium, and the that of matrix microtubules axonemal polymerization (PCM) a material recruiting (Lu nucleation, by pericentriolar microtubule centrosome associated the of organize Peetades eateto oeua ilg n eeis o University, Koç Turkey. Genetics, 34450, and Biology Istanbul Molecular of Department address: *Present USA. lfN Firat-Karalar N. Elif new centrosome identifies the cells of sperm components mammalian of analysis Proteomic REPORT SHORT 4128 2014 July 23 Accepted 2014; June 23 Received § 2 1 of ninefold cylinder and unique a (doublets a in and has microtubules arranged radially centriole length compound are Carvalho- The which 2010; triplets) 2011). al., in et al., symmetry (Hodges et cilia make Santos in that conserved is eukaryotes that structure all microtubule-based a is centriole The INTRODUCTION a Sperm cilium, is Primary Centrosome, Centriole, it WORDS: KEY formation. satellite that cilium the primary in with functions found complex and PCM1, a highly and and in HAP1 is proteins the satellites, further centriolar examined of CCDC113 component We proteins. CCDC116 conserved and centrosome-associated CCDC81 CCDC146, as C7orf31, CCDC96, CCDC113, CCDC38, identified cells C4orf47, cultured subset in uncharacterized a candidates of these previously localization of of many Assessment proteins. and known centriole candidate identifies centrioles pericentriolar centrioles little of sperm but mammalian components of centrioles of of spectrometry pair Mass proteome a this material. have centriolar overcome which have the cells, we sperm determining study, this by In problem centrosome. material the pericentriolar up the of amount making of larger studies the of proteomic because centriole in centrosome, that under-represented essential microtubule-based hypothesized been are We have that cilia. proteins centrosome of conserved axoneme animal the the nucleating of for evolutionarily core the at structures are Centrioles ABSTRACT ae tnodSho fMdcn,Pl lo A934 USA. 94304, CA Alto, Palo Medicine, of School Stanford Care, uhrfrcrepnec ([email protected]) correspondence for Author 94305-5120, CA Stanford, USA. Medicine, of 94305-5020, School CA Stanford Stanford, Genetics, of University, Department Stanford Biology, of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,42–13doi:10.1242/jcs.157008 4128–4133 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. obnto fpoemc iifrai n genomic and bioinformatic proteomic, of combination A dr n tan,20) motnl,cnroe ulaethe nucleate centrioles Importantly, 2007). Stearns, and ¨ders , 7 mi imtr(Chre diameter in nm 275 ` rsn drs:Dvso fPloayadCritical and Pulmonary of Division address: Present 1, ,Jsu Sante Joshua *, ´ 1, ine l,1997). al., et tien ` aa Elliott Sarah , , 0 min nm 500 1 n i Stearns Tim and nw oeua ai.Freape rooorpyad3D- and of cryotomography example, studies For reconstruction basis. 2012; dictated molecular is (Go al., known SASS6 which of centrioles, et structure of and, the symmetry (Li by features, ninefold structural species the conserved than Morphological reveals several other 2013) reported. al., from et been centrioles Guichard not of of analysis has analysis proteomic direct centrioles but analyzed, mammalian been have 2007) al., et Chlamydomonas uui este soitdwt h ue fmicrotubule of them. between lumen linker the the with and with triplets, and associated doublets densities tubulin Trichonympha etilrstlie hti ihycnevdadfntosin functions and conserved highly of component is centrosome- formation. we a cilium that is which uncharacterized CCDC113, satellites these, of previously centriolar of spectrometry, One as proteins. mass localizing eight by proteins confirmed components 364 identifying formation. identified cilium sperm in for and of informative organization by analysis PCM be for egg Therefore, required the might centrosome 1994). into the Kirschner, centrioles form introduced to and once PCM vertebrates (Stearns recruit and most centrioles 1995), the in fertilization, al., inherited et paternally al., (Simerly et are distal (Manandhar degenerated Centrioles axoneme a flagellar 2000). the and nucleates and centriole head that proximal centriole between well-defined midpiece the typically a in sperm tail, centrioles mammalian orthogonal mature two A has 2005). PCM al., spermatogenesis associated during et microtubules most (Manandhar nucleate lost to have capacity the that and centrioles have which cells, fcnroe rmmdic iohnraadtebl fthe of bulk the and mitochondria midpiece Separation from 1976). al., increasing centrioles et tail of (Olson of The centrioles buffers for fraction). enrich with to (tail extracted stringency marker sequentially nuclear and were immunostaining the fraction) fractions for (heads by 1D) H3 (Fig. histone confirmed immunoblotting 1B). and (Fig. was 1C) supernatant (Fig. the fractionation were in a tails sperm through Successful sedimented the Bovine and isolating axoneme DAPI. tails, cushion, from glycerol stain the heads with DNA separate centrin, to stained the sonicated marker and cells the centriole tubulin for these marker the material shows distal starting against 1A the head and al., antibodies Fig. as et a enrichment; sperm (proximal (Palermo bovine of centriole (axoneme) chose midpiece tail consists We a a 1997). which and mitochondria) nucleus), cells, centrioles, sperm and specialized mature (acrosome the 1980). by Clark, of guided and was Kleve structure protocol 1976; enrichment al., centriole et based The (Olson centrioles sperm studies for previous enriching on for protocol a developed We DISCUSSION AND RESULTS oietf etil rtis eepotdmmainsperm mammalian exploited we proteins, centriole identify To nti td,w sltdcnroe rmbvn pr and sperm bovine from centrioles isolated we study, this In 1,2,§ etils(ucade l,21)so non- show 2013) al., et (Guichard centrioles Kle ta. 05 and 2005) al., et (Keller Chlamydomonas nz,21) hs etrshv no have features these 2012), ¨nczy, Tetrahymena L ta. 02 and 2012) al., et (Li (Kilburn

Journal of Cell Science HR REPORT SHORT n CC1 r osre nms iitdekroe;and eukaryotes; ciliated most in CCDC96 conserved systematic C4orf47, are more CCDC146, or CCDC113 cilia; one and and in of identified studies previously were and WDR65 C4orf47 LRRC48, LRRC34, criteria. CCDC81, several on CCDC113, based CCDC96, cells, human cultured in characterization an have not are proteins did these uncharacterized. dataset of previously our most and from fraction, compartment, proteins molecular centriole associated candidate the the of to for corresponded 19% Interestingly, proteins proteins. enrich cilium identified and the centrosome not identified of previously did 11% only which that revealed 2013), (Baker from proteome al., being data tail sperm the et mammalian them of the analysis of with studies our previous as contrast, the consistent annotated In 1F). compartment were (Fig. proteins contaminants molecular Few 2009). a al., having et previously (Keller 1999) been al., POC1 et had (Hart and 1 centrin proteins 1F; components centriole (Fig. 13 the Table including material S2), (supplementary and centrosome the cilia studies, to localize to these sperm shown one the and of in of identified more molecular previously 60% centrosomes or been of had total ontology proteins of candidate A centriole S2). Table studies material supplementary proteins genomic by bioinformatic 364 and proteomic, categorized with cross-referenced and of compartment, were proteome proteins centriole S1). sperm Table experiments material (supplementary yielded candidate independent (LC-MS) two spectrometry (third a from mass tubulin liquid-chromatography ext#2) centriole-enriched by marker the 1E, of (Fig. (second axoneme Analysis fractions 1E). centrin the (Fig. fraction) fraction), and extraction extraction fraction) (first extraction mitochondria MtHsp70, the for marker immunoblotting by confirmed was axoneme usto addt rtiswr eetdfrfurther for selected were proteins candidate of subset A 364 all candidates, proteome centriole sperm the assess To uui Fg ) oto h rtislreyclclzdwith colocalized largely proteins the of Most 2). as (Fig. centrosome, tubulin to the relative near localization or by at (supplementary assessed by localized 15 the proteins starvation determined of candidate Eight was tagged CCDC146. serum against localization antibody to by with CCDC146 immunostaining induced S1B). cilium were Fig. which the primary material cells, not RPE1 cloning a do and which form cilium, by cells, and primary HeLa a tags assayed in form GFP transfection C-terminal was transient and/or by expression N- cells with the to cDNAs somatic (supplementary localize corresponding most to testis proteins of in these the of centrosome distribution ability to The S1B). wide highly-restricted Fig. material from being proteins proteins. pattern, to these tissues expression uncharacterized that in showed the data were varied among expression C9ORF116 or and available from C7orf31 Publically domains above, centrosome randomly the with to contain chosen associated addition In proteins CCDC38, CCDC116 cilia. in or found TTC18, and motifs LRRC48, structural CCDC108 LRRC34, CCDC81, WDR65, EFCAB1, FrtKrlre l,21) n r mlctdi ciliopathies material in (supplementary proteins implicated centrosome-localizing other are the and 2014), (Ba duplication al., centriole et and (Firat-Karalar ciliogenesis trafficking, protein and centrosome centrosome cilium including linked processes the are centrosome-related satellites several to to Centriolar satellites. centriolar localize to localization not of did seven S1B). proteins 2), remaining Fig. (Fig. material tagged CEP164 The (supplementary centriole with 15 S1A). co-staining and one Fig. the by localized centriole material only satellites, mother (supplementary to which the centriolar as localized resembling CCDC113, identified which structures were CCDC146, punctate exceptions to the tubulin; ecoeCD13frfrhrsuy ae nisputative its on based study, further for CCDC113 chose We rn ta. 01.CD13i eae oCD9,oeof one CCDC96, to related is CCDC113 2011). al., et ¨renz ora fCl cec 21)17 1843 doi:10.1242/jcs.157008 4128–4133 127, (2014) Science Cell of Journal ed n al mgdb hs-otatmicroscopy. 10 phase-contrast bar: by Scale imaged tails and 5 heads bar: Scale DAPI. with and centrin against antibodies bovine from cells. protocol sperm enrichment Centriole 1. Fig. eti and centrin eaiet h animage. 4 main at the shown to are relative insets All ontology compartment. gene molecular the to candidate according centriole and proteins sperm tubulin of centrin, Categorization for (F) blotted MtHsp70. were step extraction each for and blotted H3 tails histone and centrioles, heads isolated sperm, intact a tbln cl a:5 bar: Scale -tubulin. A oiesem ie n tie with stained and fixed sperm, Bovine (A) m .()Ioae al ie n tie for stained and fixed tails Isolated (C) m. c m tblno polyglutamylated or -tubulin .()Itc pr,isolated sperm, Intact (B) m. a tbln E xrcsfrom Extracts (E) -tubulin. a tbln N a stained was DNA -tubulin. m .()Etat of Extracts (D) m. 6 4129 c -

Journal of Cell Science HR REPORT SHORT 4130 of component new a as satellites. CCDC113–GFP centriolar 2012). identify al., et as results (Stowe centrosome, components These the satellite was to other some protein satellites for the observed to of localization be localization CCDC113–GFP by In accompanied of would 3D). loss (Fig. PCM1 cases localization as satellite of both CCDC113–GFP depletion of 3C), Moreover, loss upon (Fig. satellites. caused these centriolar dispersed microtubules and for of expected puncta 3B,C), of (Fig. subset PCM1-positive a cells depolymerization to and RPE1 2008; localized al., in CCDC113-GFP- CCDC113–GFP et puncta Kim 2011). 2004; PCM1-positive al., centrosomally al., a et et and (Kim Lopes PCM1 array protein microtubule satellite focused the on and dependent HeLa is and 3A) U2OS (Fig. RPE1, 3B) in cells (Fig. expressed CCDC113 is cells. RPE1 against CCDC113 that antibody from showed an RNA with immunoblotting using experiments in not chordates, but in present are schistosomes, proteins both deuterostomes, of orthologs and S2A), Fig. h ucaeprcnrsmldsrbto fstlieproteins satellite of distribution pericentrosomal punctate The .elegans C. splmnaymtra i.SA.RT-PCR S2A). Fig. material (supplementary Trichoplax and Chlamydomonas , eltdclshdsmlrcl yl rflsa nlzdb flow by analyzed CCDC113- as CCDC113 and profiles control cycle cell mouse both defect similar a because had to cells GFP-tagged progression due depleted cycle not was of cell phenotype in ciliogenesis cilium expression The was 4C,D). phenotype reduced (Fig. this by and substantially cells, rescued Fig. RPE1 CCDC113 serum-starved material in of formation (supplementary by Depletion demonstrated CCDC113 as S2D). infection, for after d immunoblotting short 7 expressing observed of lentivirus was depletion Efficient CCDC113 a CCDC113. with targeting (shRNA) infection RNA CCDC113 hairpin by whether depleted cells was tested CCDC113 RPE1 We ciliogenesis. (Engelender from efficient 2011). for al., with ciliogenesis required et is complex regulates also Keryer a 1997; and forms al., huntingtin et HAP1 4B). and (Fig. material PCM1 satellites supplementary HAP1 with centriolar 4A; colocalized (Fig. at CCDC113–GFP interacted PCM1 Moreover, CCDC113 S2B,C). and which Fig. HAP1 in al., both cells co-immunoprecipitation et with HEK293T in Keryer interaction in 1997; this experiments al., centriolar validated et the We (Engelender 2011). and HAP1 CCDC113 protein between satellite interaction an identified ihtruhu w-yrdsre Sez ta. 2005) al., et (Stelzl screen two-hybrid high-throughput A ora fCl cec 21)17 1843 doi:10.1242/jcs.157008 4128–4133 127, (2014) Science Cell of Journal nioy etooe eesandwt anti- with stained anti-CCDC146 were with Centrosomes and or antibody. fixed antibody were anti-GFP cells with transfection, stained indicated after the h with 24 transfected constructs. were proteins. cells centrosome HeLa new (A) of Localization 2. Fig. uui)adDAwssandwt AI cl bar: image. Scale DAPI. with stained 10 was (glut. DNA tubulin and polyglutamylated tubulin) and for cilium stained primary were antibody The centrioles anti-GFP antibody. with anti-CCDC146 stained with and or fixed h, serum- 24 constructs, for cells indicated starved RPE1 the (B) with DAPI. transfected with were stained was DNA and antibody m .Alist r hw t4 at shown are insets All m. 6 eaiet h main the to relative c -tubulin

Journal of Cell Science HR REPORT SHORT ora fCl cec 21)17 1843 doi:10.1242/jcs.157008 4128–4133 127, (2014) Science Cell of Journal C1(iC1 iN,adsandfrGP C1and PCM1 GFP, or for (siCtl) stained c control and with siRNA, transfection (siPCM1) after PCM1 h 48 fixed were GFP and PCM1 10 with or treated DMSO were control). CCDC113-GFP expressing (loading cells p38 RPE1 cells and (C) U2OS CCDC113 and for HeLa blotted these RPE1, were with from gene Extracts CCDC113 (B) of primers. amplification PCR or is (+) control with primers CCDC113-specific ( of without pair a with cells cultured lines. in cell localization human and expression CCDC113 3. Fig. tbln cl as 10 bars: Scale -tubulin. 2 h diino ees rncits.Positive transcriptase. reverse of addition the ) c tbln D P1clsepesn CCDC113– expressing cells RPE1 (D) -tubulin. m /lncdzl,fxd tie o GFP, for stained fixed, nocodazole, g/ml hw t4 at shown AI cl as 10 with bars: stained Scale was DAPI. DNA PCM1. and for fixed, stained were cells RPE1 CCDC113-depleted and Control (E) (ANOVA). C; of Quantification (D) and DAPI. tubulin) (glut. tubulin with polyglutamylated staining by determined ciliogenesis was displaying cells of percentage the and (shCCDC113), shRNA or CCDC113 (shControl) virus vector with empty infection expressing after were d cells 7 RPE1 fixed (C) DAPI. was with DNA stained antibodies. anti-GFP and anti- Myc with stained were CCDC113–GFP and HAP1–Myc RPE1 expressing (B) cells antibodies. anti-PCM1 and anti- Myc anti-GFP, with detected were proteins (IP) co-immunoprecipitated and antibody anti-GFP with immunoprecipitated were Complexes cells. HEK293T in co- expression after HAP1–Myc and PCM1) (td- tomato–PCM1 CCDC113–GFP, of in ciliogenesis. functions CCDC113 4. Fig. e xeiet ** experiment. per A TPRaayi fRAfo RPE1 from RNA of analysis RT-PCR (A) m m. 6 eaiet h animage. main the to relative A Co-immunoprecipitation (A) P , .1 * 0.01, m .Alist are insets All m. P , n § 0.05 0 cells 100 4131

Journal of Cell Science opnnso h etooei eea.Tecmiaino the of combination The the general. in centrosome putative around the identify of to components sperm, focused mammalian the type, were cell differentiated satellites which to 4E). relative (Fig. in centrosome cytoplasm cells of the dispersal HAP1 control throughout caused with CCDC113 satellites of As depletion PCM1-positive 2011), S2E). al., Fig. et (Keryer material (supplementary cytometry REPORT SHORT eim(MM upeetdwt 0 ea oiesrm(FBS; serum 4132 bovine fetal Eagle’s 10% modified with Dulbecco’s in supplemented grown (DMEM) were medium cells HEK293T and HeLa transfection and culture Cell METHODS AND MATERIALS with association of somatic by cells. and regulated class somatic is cells a in function sperm that satellites represent which in for might (Hall functions but AZI1 similar sperm cells, cells have and sperm for that CCDC113 in required proteins 2013). trafficking is satellite al., protein CEP131) the et regulating that as by demonstrated known 2013) motility a (also but al., AZI1 cells, et somatic protein (Hall most of study lack typical recent Sperm satellites that centrosome. of suggesting the complement the centrosome, from CCDC113 the in sequester results to we satellites depletion CCDC113 hypothesis, PCM1 of by this satellites relocalization with of Consistent elimination rather that satellites, satellites. found by of is CCDC113 satellites function of with than modulation CCDC113 2010). represent al., of et to interaction (Hodges likely PCM1 the for that that suggests the to CEP290 not and This to but 2004) that 2008), al., similar al., et note (Kim et is BBS4 (Kim We proteins CCDC113 satellite of the ciliogenesis. for conservation that efficient cell evolutionary for satellites of centriolar pattern cycling important of component is new and that a as centrioles CCDC113 identified sperm between centrosome the centrosomes. at common with suggests interacts centrosome it cell that is structure somatic or the protein to testis the ability localize that the for to proteins, specific protein such the highly For of S1B). are Fig. that material profiles (supplementary expression expressed-sequence have (EST) proteins to tag these specific highly of proteins a several include Indeed, the in also the sperm. that would Given result expect proteome we centriole sperm, can proteins. the sperm of which function centriolar and proteins, structure be of specialized might the or distribution of 1997), broader al., overexpression centrosome et the to (Brohmann at midpiece due sperm structures the protein distinct in the morphologically which and in of ODF2, part with the as is such situations, with marker reflect centrosome PCM might larger the common a with to overlapping CCDC146, mostly localized domain, protein in proteins GFP-tagged centriole the mother components of the most for as the have Except cells that centrosome. cycling assumption centrioles in original the centrosome sperm validating the proteins, near fusion or GFP at localize study this al., et proteins. Guichard 2012; specific al., and to et 2013) respect (Li with structure known between is relationship little for the which useful particularly about be centriole, to the likely of well is studies dataset most this Thus, the 2011). of al., of et (Jakobsen Many dataset components PCM centrosome. are specific proteins the centrosome characterized to particularly localize a that centrioleproteins yielded sperm the protocol with centrioles enrichment sperm the of properties unique ih fte1 rvosyucaatrzdpoen etdin tested proteins uncharacterized previously 15 the of Eight a from centrioles of analysis proteomic used we report, this In ouigo n ftesemcnroecniae,we candidates, centriole sperm the of one on Focusing c tbln This -tubulin. hN agtn CC1.Clswr eu tre o 8h ie and fixed assays. h, ciliogenesis 48 subsequent the for for starved 7 serum day (RPE- were on CCDC113–GFP stained Cells CCDC113. mouse or targeting vector empty or shRNA expressing lentivirus with (RPE-1::GFP) infected were GFP 1::mCCDC113-GFP) resc expressing For 2014). stably previously al., described as et (siRNA) RNA shRNA- (Firat-Karalar interfering with small an cells using depleted p of was described infection as by lentivirus out expressing carried was depletion CCDC113 ATAGCAGAGATGTCCTTAAA-3 were cells 2014). al., et HEK293T (Firat-Karalar previously described using as performed experiments Co-immunoprecipitation Ivtoe) E23 el eetasetduig1 using transfected LTX Lipofectamine were MO). Louis, using St. cells (Sigma-Aldrich, transfected kDa) sperm (25 were polyethylenimine HEK293T Bovine cells enrichment. centriole HeLa FBS. for (Invitrogen). used in and 10% were grown TX) RPE1 Station, with were College Elite, cells supplemented (Bovine RPE1 (50:50) GA). DMEM/F12 Lawrenceville, Biologicals, Atlanta cN-ET7wr sdt eeaeMc n/rGFP-tagged and/or and Myc- generate DEST to pCS2+6xMyc were used using proteins. were remaining reactions Subsequent pcDNA-DEST47 the (Invitrogen). recombination pDONR221 of into ORFs cloned Gateway and The PCR PCR by material peGFP-N1. by amplified supplementary amplified was into in (ORF) frame cloned described reading all are and open of CCDC96 study The sources this S1. and in Fig. numbers used accession cDNAs DDBJ the and EMBL GenBank, The Plasmids were fractions tail The pellet. the in heads 4 at in the extracted tails sequentially the from isolate to supernatant cushion from glycerol heads the 30% separate a to through sonicated sedimented and and PBS tails, with rinsed were sperm Bovine enrichment centriole Sperm ie nmtao,poesdfridrc muoloecneand immunofluorescence indirect for processed coverslips, on methanol, grown in were cells fixed experiments, immunofluorescence a For with microscopy matches and Immunofluorescence peptide analysis. Blast two reciprocal by least identified were available at not was of annotation basis the Proteins probability Information. on Biotechnology for identified Spectrometry Center were National Mass the Stanford in of mass archived composed the LCq-Deca-XPPlus was database an at The Laboratory. using Scientific) analyzed (Thermo was spectrometer fraction centriole The spectrometry Mass S1C. Fig. material supplementary antibodies in Other described blotting. western are for used 1:5000 at used was Inc.) Biotechnology, and (1–461) 1– 0.5 MBP–CCDC146 at (Cocalico against acids used purified (amino affinity rabbits was CCDC146 antibody immunizing The GST-tagged 461). with by PA) obtained Reamstown, Biologicals, was antibody Anti-CCDC146 Antibodies 5 300–321, (nucleotides oligonucleotides shRNA CCDC113 experiments rescue and shRNA 5 were used Primers CA). synthesis Diego, cDNA a (Qiagen, San CTCTTATTCACTGC-3 using kit performed RNeasy (Invitrogen, an was kit using RT-PCR cells and RPE1 CA) from Chatsworth, isolated was RNA Total experiments immunoprecipitation and RT-PCR ufr3(0m rsHlp ,4Mue,ihbtr) h supernatant The inhibitors). (TCA). urea, acid trichloroacetic extraction M with 4 and precipitated inhibitors) 8, was extraction pH DTT, each Tris-HCl mM after mM 2 (50 KSCN, 0.1% 3 mM NP-40, buffer 1% 600 NaCl, 8, mM pH 500 Tris-HCl EDTA, mM 2 EGTA, mM b 2 8, pH HCl mratehnl MDT niios,etato ufr2(0mM (50 2 buffer extraction inhibitors), DTT, mM 1 -mercaptoethanol, ora fCl cec 21)17 1843 doi:10.1242/jcs.157008 4128–4133 127, (2014) Science Cell of Journal m . /lfrimnfursec.Rbi nip8(-0 at Cruz Santa (C-20; anti-p38 Rabbit immunofluorescence. for g/ml 5.Tehmnotooso the of orthologs human The 95%. 9 ˚ o ihetato ufr1(0m Tris- mM (50 1 buffer extraction with h 1 for C n 5 and 9 eiul Mhobe l,21) PCM1 2010). al., et (Mahjoub reviously -AGCAAGCTTCACAAGGCAAT-3 eeprmns ol fRE1cells RPE-1 of pools experiments, ue 9 eecoe nopLKO.1. into cloned were ) o taurus Bos .taurus B. rtisfrwhich for proteins rti sequences protein 9 -CGGAGC- m g/ m 9 9 - l .

Journal of Cell Science ucad . aht . auu . ee,A,Dmra,D,Oirc N., Olieric, D., Demurtas, A., Neves, N., Majubu, V., Hachet, P., Guichard, Go o,R . tw,T . uk .adSers T. Stearns, and E. Turk, K. R., T. Gull, Stowe, and A., R. A. Hoh, J. Langdale, B., Wickstead, N., Scheumann, E., L. M. J. Salisbury, Hodges, and M. G. Poynter, D., B., J. L. Orth, Smith, N., P., J. Glantz, A. E., P. Jarman, T., Hart, Davey, J., M. Ford, M., Keighren, A., E. Hall, P. T. Dupuis-Williams, Stearns, and and C. 3rd Fisch, R., J. Yates, N., Rauniyar, N., E. Firat-Karalar, Worley, A., Lanahan, K., M. Tokito, V., Colomer, H., A. Sharp, S., Engelender, Chre rhan . ince .adHyrFne,S. Hoyer-Fender, and S. Pinnecke, H., Brohmann, ..-. ..adSE efre h xeiet;ENFK n ..interpreted T.S. and E.N.F-K. experiments; the performed S.E. and J.S. E.N.F-K., contributions Author interests. competing no declare authors The as immunofluorescence interests Competing for processed and lines. cells cell coverslips Sperm for 2014). described on al., dried et (Firat-Karalar were previously described as imaged REPORT SHORT avloSno,Z,Aizdh . eer-el .B n Bettencourt-Dias, and B. J. Pereira-Leal, J., Azimzadeh, Z., Carvalho-Santos, and T. Velkov, Ba A., Weinberg, L., Hetherington, N., Naumovski, T., A., M. Keil, Baker, A., Polyanovsky, E., Koundakjian, M., A. Maer, T., Avidor-Reiss, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.157008/-/DC1 online available material Supplementary months. 12 material after Supplementary release for PMC number in [grant Deposited Health T.S.]. of number to Institutes [grant GM52022 National Award the R01 Service by and Research E.N.F.]; National to a 5F32GM106620 supported was work This Funding paper. the wrote T.S. and E.N.F-K. and results, the siaa . hmsn . ae,J . r n asal .F. W. Marshall, and 3rd R., J. Yates, J., Thompson, H., Ishikawa, aosn . aslw . kg,M,Tyd,Y,Lnbr,E,Psr I., Poser, E., Lundberg, Y., Toyoda, M., Skogs, K., Vanselow, L., Jakobsen, rn,F,Myl,D n rs,O J. O. Gruss, and D. Mayilo, F., ¨renz, cy P. nczy, ¨ lcie,I,Ymd,A,Khr,K,Nsia .e al. et Y. Nishida, 9-fold its underlying K., features reveals symmetry. Kihara, region radial proximal centriole A., the of Yamada, architecture I., Fluckiger, 21) eosrcigteeouinr itr ftecnroefo protein from centriole the of history evolutionary components. the Reconstructing (2010). protein. centrosome a encoding gene for a evidence of and characterization retroposition genomic Cetn1: centrin, murine Testis-specific phenotypes. ciliary P. distinct in Mill, results and Azi1/Cep131 J. I. Jackson, Biol. Cell Mol. Rev. future! the of to back regulators identify components centrosome duplication. centriole among interactions Proximity dynactin. of A. subunit p150Glued C. 2205-2212. the Ross, with and interacts L. (HAP1) E. 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