Ccdc181 is a microtubule-binding protein that interacts with Hook1 in haploid male germ cells and localizes to the sperm tail and motile cilia

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European Journal of Cell Biology

j ou rn a l h o m epa g e :w w w . e l s e v i e r . c o m / l o c a t e / e j c b

Research paper

Ccdc181 is a microtubule-binding protein that interacts with Hook1 in haploid male germ cells and localizes to the sperm tail and motile cilia

Thomas Schwarz

^a,∗

, Barbara Prieler

^a

, Johannes A. Schmid

^b

, Pawel Grzmil

^c

, Juergen Neesen

^a

aInstituteforMedicalGenetics,MedicalUniversityofVienna,1090,Vienna,Austria

bCenterforPhysiologyandPharmacology,MedicalUniversityofVienna,1090,Vienna,Austria

cDepartmentofGeneticsandEvolution,InstituteofZoology,JagiellonianUniversity,Gronostajowa9,30-387,Krakow,Poland

a r t i c l e i n f o

Articlehistory:

Received13September2016

Receivedinrevisedform20January2017 Accepted16February2017

Keywords:

Ccdc181 Cilia Manchette Microtubules Motility Hook1 Spermatogenesis

a b s t r a c t

DisruptionofmurineHook1resultsinadisturbedspermatogenesisandconsequentlyleadstomale infertilityinmice.Withinthesemiceabnormalspermdevelopmentstartswithadisorganizationofthe microtubularmanchetteinelongatingspermatidsthatleadstoanabnormalheadshapeaswellasto distinctivestructuralchangesintheflagellaofthesperm.ToelucidateHook1functioninmalegermcell differentiationayeasttwo-hybridscreenwasperformedusingamurinetesticularlibrary,whichleads totheidentificationofseveralputativeHook1interactingproteins.OneoftheisolatedcDNAfragments encodesforthecoiled-coildomaincontainingprotein181(Ccdc181).TheputativeinteractionofCcdc181 withHook1wasverifiedbyFRETanalysisandinteractingregionswereidentifiedusingyeasttwo-hybrid assays.Furthermore,Ccdc181seemstointeractdirectlywithmicrotubulesandlocalizestothemicro- tubularmanchetteofelongatingspermatids,resemblingthepreviouslyreportedlocalizationofHook1.

AccordingtotheobservedimmunostainingpatterntheRNAexpressionofCcdc181islessprominent inpre-meioticstagesofspermdevelopmentbutincreasesinthehaploidphaseofspermatogenesisand seemstoberestrictedtomalegermcells.However,Ccdc181expressionisalsoobservedtoalowerextent insomatictissues,particularly,intissuescontainingciliatedepithelia.Additionally,Ccdc181proteinis foundtolocalizetothespermﬂagellaandtothebasalhalfofmotilecilia,whereasCcdc181wasnot detectedinprimarynon-motilecilia.Furthermore,weshowedthatCcdc181isaputativeinteracting partnerofthedifferentcatalyticsubunitsofPp1,raisingthehypothesisthatCcdc181playsarolein mediatingciliarymotility.

1. Introduction

Spermatogenesisischaracterizedbysuccessiveperiodsofreg- ulatedcellproliferation,meioticdivisionsandcomplexphasesof differentiation of the newly formed haploid spermatids during spermiogenesis.Differentiationischaracterizedbytheshapingand condensationofthenucleusaswellastheformationoftheacro- someandthedevelopmentofthespermtail.Theformationofthe acrosome-acroplaxome-manchettecomplexisessentialforproper spermatiddevelopment(KierszenbaumandTres,2004).Whilethe acroplaxomeanchorsthedevelopingacrosometotheelongating spermatidheadandprovidesamechanicalscaffoldfortheshap-

∗ Correspondingauthorat:InstituteforMedicalGenetics,MedicalUniversityof Vienna,Vienna1090,Austria.Fax:+43140160956531.

E-mailaddress:thomas.schwarz@meduniwien.ac.at(T.Schwarz).

ingofthespermatidnucleus(KierszenbaumandTres,2004),the manchetteisatransientmicrotubular/actin-containingstructure thatoccursinelongatingspermatidsanddisappearsbeforesper- matozoaarereleasedtotheepididymis.Asmotorproteinsthat mightbeinvolvedinintraflagellartransporthavealsobeenlocal- izedto themanchette (Hayasaka etal., 2008), this structure is thoughttoserveinthetransportofvesiclesandmacromolecules tothecentrosomes aswell astothe growingspermatid tail, a processcomparabletotheintraflagellartransportfoundineukary- oticflagellaandmotilecilia(Kierszenbaum,2002).Disruptionof themanchettestructureresultsinadisturbedtransportalongthe microtubulesofthemanchetteandultimatelyleadstomaleinfer- tility.Inspermatidsofazh-mutantmicethemicrotubulesofthe manchetteareoftenectopicallypositioned,leadingtoabnormal sperm head shape and malformed sperm tails. This phenotype is caused by a deletion of two exonsof theHook1 gene, sug- gestingthatHook1seemstobeessentialfortheproperfunction http://dx.doi.org/10.1016/j.ejcb.2017.02.003

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of the manchette during sperm development(Meistrich et al., 1990; Mendoza-Lujambio etal., 2002; Mochidaet al., 1999).It has been shown that Hook1 can bind to the microtubules of themanchette and therefore it seemstoplay an essential role fortheformationand maintenanceof thisorganelle (Mendoza- Lujambio et al.,2002).In mammalsHook1 belongsto a family ofHookproteins,consistingofHook1,Hook2andHook3.Hook2 has been shown to be involved in primary cilia morphogene- sis (Baron Gaillard et al., 2011) while Hook3 has been shown toparticipateinthelocalizationoftheGolgicomplex(Walenta et al., 2001). Hook polypeptides consist of a highly conserved NH2-domain mediating attachment to microtubules, a central coiled-coildomaincapableofhomodimerizationandamorediver- gentCOOH-terminalorganellebindingdomainputativelyinvolved inbindingofspeciﬁcorganellesorcargos(Mendoza-Lujambioetal., 2002;Walentaetal.,2001).InDrosophilahookispredominantly expressedintestisbutisalsodetectedinsomaticcells,whereit wasfoundtoplay a role in endocytosisof transmembranelig- ands(KramerandPhistry,1999)andhumanHook1functionsas amicrotubule-dependent cargotransporterfor endosomalsort- ing(Maldonado-Baezetal.,2013).SinceHook1wasnotdetected in protein extractsof mature mouse spermsit is unlikely that Hook1actsasastructuralcomponentofthespermatozoa,rather Hook1mightbeinvolvedinintra-manchettetransportprocesses, sortingnewsynthesizedpolypeptidesintheelongatingspermatid tothecorrectcompartment(Mendoza-Lujambioetal.,2002).To identifyputativeHook1cargoesinmalegermcelldevelopmenta yeasttwo-hybridscreenusingatesticularlibrarywasperformed.

Bythis approach themousecoiled-coildomaincontainingpro- tein181(Ccdc181)formerlyknownas4930455F23rikenprotein wasisolated.Ccdc181belongstoagroupofpolypeptideswhose commonpatternisthecoiled-coildomain.However,lessisknown concerningthemolecularfunctionofCcdc181withtheexception ofitbeingpredictedasacilia-relatedgeneandhumanCcdc181 beingreportedasa putativeprognosticfactorfor prostatecan- cer(Haldrupetal.,2013;McClintocketal.,2008).Moreover,the murineCcdc181 wasidentiﬁedasaputativeinteractingpartner ofCCNB1IP1,aRINGdomain-containingproteinthatisrequired formeioticcrossingoverprocesses,andofproteinphosphatase1 subunit␣(Pp1␣),aubiquitousexpressedserine/threoninephos- phataseinvolvedinvariouscellularpathways(Doerksetal.,2002;

StrongandSchimenti,2010).ThehighlysimilarPp1␥2subunithas beenshowntoplayanimportantroleinspermmotilitysincethe knockoutofPp1␥2resultedinmaleinfertilityduetonon-motile sperms.Additionally,itwasshownthatintestisPp1␥2function couldnotbesubstitutedbyotherPp1catalytic subunitslikein contrasttosomatictissues(Chakrabartietal.,2007;Fardilhaetal., 2011;Sinhaetal.,2013).Inthisstudy,wereportthemolecular characterizationofmurineCcdc181withinmalegermcelldevel- opment.WedemonstratethatCcdc181isabletobindtoHook1 supportingtheideathatCcdc181isacargoofHook1.Moreover, ourresultsindicatethatCcdc181caninteractwithPp1␥2andbinds tomicrotubulesofmotileciliaandspermﬂagellasuggestingthat Ccdc181mightplayaroleinthegenerationofmotilityinthese organelles.

2. Materialsandmethods 2.1. Cellculture

Human HEK 293T and mouse NIH 3T3 cells were cultured in DMEM (GIBCO) supplemented with glutamine, 4.5mg/ml glucose, 10% fetal bovine serum (FBS) and 100␮g/ml peni- cillin/streptomycinat37^◦Cand5%CO₂.HEK293TorNIH3T3cells weretransfectedwiththejetPRIMEtransfectionreagent(Peqlab)

accordingtothemanufacturer’sprotocol.Brieﬂy,HEK293Tcells grownon10cmplatesweretransfectedusing10␮gDNAand30␮l transfectionreagent,whereasNIH3T3cellsgrownon6cmplates weretransfectedusing6␮gDNAand18␮ltransfectionreagent.

FortheinductionofciliaformationinNIH3T3cellsconﬂuentcul- tureswereserumstarvedfor24hpriorfurtheranalysis(Heetal., 2014).

2.2. Preparationofexpressionconstructs

Expressionconstructswerepreparedusingprimerswithunique restrictionsites.

Hook1full-length(NM030014,aa1–728)andHook1-AZH(aa 1–263),aswellasthetruncationsHook1-D1(aa1–276),Hook1-D2 (aa167–458)andHook1-D3(aa459–728)andthedeletionsHook1-

CC1(aa1–728,173–228),Hook1-CC2(aa1–728,254–443) andHook1-SSF2(aa1–728,185–283)weregenerated.Ccdc181 full-length (NM029115, aa 1–509) as well as the truncations Ccdc181-D1(aa1–254),Ccdc181-D2(aa248–448)andCcdc181-D3 (aa393–509)weregenerated.ForFACS-FRETHook1andCcdc181 wereclonedintopEGFP-N1andDsRed-C1(Clontech).Additionally, afusionconstructofGFPandDsRedwasgeneratedandafusion constructoftubulinandGFPwaskindlyprovidedbyJ.Schmid.For yeasttwo-hybridindicatedHook1andCcdc181constructsaswell asHook2(NM133255,aa1–716),Hook3(NM207659,aa1–718) andcatalyticsubunitsofPp1(Pp1␣(NM031868,aa1–330),Pp1␤ (NM172707,aa1–327),Pp1␥1(NM013636,aa1–323)andPp1␥2 (aa1–337))wereclonedintopGBKT7-BD(Clontech)andindicated Ccdc181constructswerealsoclonedintopGADT7-AD(Clontech).

Formicrotubule-bindingassay,indicatedCcdc181constructswere clonedintopEXPR-IBA43(IBA).

2.3. FACS-FRETanalysis

TransfectedNIH3T3cellswereharvested24hposttransfection bytrypsinization(0.5ml0.05%Trypsin-EDTA,5min),resuspended inPBSandsubjectedtoFACS-FRETanalysiswithin1hafterprepa- ration. FACS-FRET measurements were performed using a LSR Fortessa flow cytometer(BDBioscience)or a CytoFlex S(Beck- manCoulter)equippedwith405,488,561and640nmlasersas describedpreviously(Banningetal.,2010).Theprotocolwasmod- ifiedinregardsofthesetoffluorophoresused.TomeasureGFPand FRETsignals,cellswereexcitedwiththe488nmlaserandtheflu- orescencewascollectedusingastandard525/50filter,whilethe FRETsignalwasmeasuredwitha610/20filter.TomeasureDsRed signals,cellswereexcitedwiththe561nmlaserwhileemission wastakenwitha575/26or610/20filter.Tosetupthegatescells expressingeitherGFPorDsRed,wereusedtoexcludesingletrans- fectedcells.Additionally,cellsco-expressingGFPandDsRed,aswell ascellexpressingGFP-DsRedfusionproteinwereusedtosetupa gatetoexcludeFRETnegativecells(Fig.S1).Foreachsampleamin- imumof8000cellsthatfellwithinthebackgroundadjustedgate wereevaluated.

2.4. Yeasttwo-hybridassay

Yeasttwo-hybridassayswereperformedaccordingtotheman- ufacturer’sprotocol(MatchmakerGoldYeastTwo-HybridManual, Clontech).ConstructsweretransformedintotheAH109yeaststrain by the lithium acetate method (Gietz, 2014).Co-expression of transformedplasmidswastestedondoubledrop-outplateslacking theaminoacidsleucineandtryptophan(DDO,SD/-Leu/-Trp).Inter- actionbetweentwopolypeptideswasinvestigatedonquadruple drop-outplateslackingtheaminoacidsadenine,histidine,leucine

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andtryptophan(QDO,SD/-Ade/-His/-Leu/-Trp).Plateswereincu- batedat30^◦Cforuptosevendaysuntilcoloniesbecamevisible.

2.5. ReversetranscriptionPCRandquantitativeRT-PCRanalysis

TotalRNAwasextractedfrommousetissuesusingQIAshredder (Qiagen)andtheMicroarrayTissuesMiniKit(Qiagen).Forquan- titative expression analysis approximately 750ng of extracted RNAwerereversetranscribedusingtheQuantiTectReverseTran- scriptionKit (Qiagen),accordingtothemanufacturer’sprotocol.

QuantitativeRT-PCRwasperformedusingtheCFXConnectReal- TimePCRDetectionSystem(Bio-Rad)withtheGoTaqqPCRMaster Mix(Promega)accordingtothemanufacturer’sprotocol.Reactions wereperformedintriplicateswithasinglereal-timePCRthermal profile(95^◦Cfor10min;40cyclesof95^◦Cfor15sand60^◦Cfor 30s). For the amplification the following primer combinations were used: qrtCcdc181-fwd 5-CAAGAGGGAGGAAGAACAGC- 3 and qrtCcdc181-rev 5-CGCATTTCTATGACCTGCTC-3; qrtB2m-fwd 5-GTCAGCATGGCTCGCTCGGT-3 and qrtB2m- rev 5-AGGCGGGTGGAACTGTGTTACG-3; and qrtGadph-fwd 5-CATCACCATCTTCCAGGAGC-3 and qrtGadph-rev 5- ATGACCTTGCCCACAGCCTT-3.ExpressionlevelsofCcdc181were normalizedtotheexpressionofB2mandGapdhandtherelative quantificationofgeneexpressionwasanalyzedbythe2-C(t) method (Livak and Schmittgen, 2001). For non-quantitative expressionanalysisextractedRNAwasreversetranscribedusing theAMVFirstStrandcDNASynthesisKit(NewEnglandBiolabs)and amplifiedusingtheDreamTaqGreenPCR2xMasterMix(Fisher Scientific) according to the manufacturer’s protocols. For the amplificationthefollowingprimercombinationswereusedinthe samereaction: rtCcdc181-fwd 5-CTCAAGAGGGAGGAAGAACAG- 3 and rtCcdc181-rev 5-CCTTCTGAGCCATTGTCG-3; and rtGapdh-fwd 5-AGGCCGGTGCTGAGTATGTC-3 and rtGapdh-rev 5-TGCCTGCTTCACCACCTTCT-3.

2.6. Northernblotting

TotalRNAwasextractedfromdifferentmousetissuesandtestes ofdifferentmousemutantsusingtheRNeasyPlusMiniKit(Qia- gen).Approximately6␮gRNAwasseparatedonanagarosegel underdenaturingconditions.Separatedsamplesweretransferred ontoaHybond-N+membrane(Amersham)usingtheTurboBlotter (Whatman)andcross-linkedat80^◦C for120min.Hybridization andsignaldetectionwasperformedusingDIGDNALabelingand DetectionKit (Roche) according to the manufacturer’s instruc- tions. Brieﬂy, hybridization was performed overnight at 50^◦C in DIG Easy Hyb buffer (Roche), mixed with denatured dig- labeledCcdc181cDNAprobe.Hybridizedprobeswerelabeledwith alkaline phosphatase-coupled digoxigenine antibody (1:10,000) and detected using CDP Star Reagent (New England Biolabs) accordingtomanufacturer’sprotocol.TheCcdc181 cDNA probe was ampliﬁed with the following primers: nbCcdc181-for (5- GATGAAGATAAAGATATTGATTCAAAAGAGAG-3)and nbCcdc181- rev(5-GTTGTAGTGGTCGGTGAAGC-3)anddig-labeledusingthe PCRDIGProbeSynthesisKit(Roche)accordingtomanufacturer’s protocol.

2.7. Preparationoftestislysates

Testesof5–50-day-oldmicewerehomogenizedwith1:20(w/v) T-PER (Fisher Scientiﬁc) supplemented with 1×HALT protease inhibitorcocktail(FisherScientiﬁc)usingthe“TissueRuptor”(Qia- gen)andclearedbycentrifugation(20,000g,10min,4^◦C).Samples wereanalyzedbySDS-PAGEandWesternblotting.

2.8. SDS-PAGEandWesternblotting

Samplesweredenaturedat95^◦Cfor5min,separatedbySDS- PAGEand transferred onto nitrocellulosemembranes usingthe iBlotdryblottingsystem(Invitrogen).Forimmunodetection,rabbit polyclonalanti-Ccdc181(1:1,000,Sigma-Aldrich)andanti-Gapdh (1:10,000,CellSignaling),andmousemonoclonalanti-StrepTagII (1:10,000,IBA)andanti-␣-tubulin(1:1,000,Sigma-Aldrich)were used.AntibodiesweredetectedusingHRP-linkedpolyclonalgoat anti-rabbitandanti-mouseantibodies(1:10,000Bethyl).Signals weredetectedusingClarityWesternECLSubstrate(Bio-Rad)and capturedwiththeFluorChemHD2imager(ProteinSimple).

2.9. Preparationoftesticulargermcellsandtrachealepithelial cells

Mouse testicular germ cells were prepared as previously described (Bellve et al., 1977; Mendoza-Lujambio et al., 2002) with minor modiﬁcations. In brief, testes harvested from a C57BL/6 mouse were decapsulated, immersed in M2 medium (Sigma-Aldrich)supplementedwith0.5mg/mlcollagenase(Sigma- Aldrich) and incubated for 15min at 33^◦C. The dispersed seminiferoustubuleswereallowedtosedimentfor3minandwere washed withM2 mediafor three times. Afterthat the tubules weretakenupinM2mediasupplementedwith0.5mg/mltrypsin EDTAand1␮g/mlDNase-I(NewEnglandBiolabs)andincubated for15minat33^◦C. Remaining cellaggregates wereshearedby gentlepipetting.CellswerewashedtwotimesinM2media,cen- trifuged(200g,5min),resuspendedinM2mediacontaining0.5%

BSAandﬁlteredthrougha40␮mnylonmesh.Finally,twodrops ofthesolutionwereplacedonaslideandallowedtodryatroom temperature.

ThetracheaofanadultC57BL/6mousewasisolatedandsingle trachealcartilageswerecutusingaﬁnescissor,placed indrops ofPBSonslides,dissectedwithﬁneforcepsandallowedtodryat roomtemperature.

2.10. Immunostainingoftissuesections

Mousetestis,lung,tracheaandbrainwerefixedusingBouin’s solution(Sigma-Aldrich),dehydratedandembeddedinParablast- Xtra (Sigma-Aldrich). Sections of theses tissues as well as of commercially available mouse reproductive tissues (MAP-400, Amsbio) were deparaffinated using Roticlear (Carl Roth) and hydrated by descending alcohol concentrations. Antigens were unmaskedbyincubationofslidesinmodifiedcitratebuffer,pH6.1 (Dako)for10minat120^◦C.Afterthat,sectionswerepermeabilized for10minwith0.2%TritonX-100inPBS.Forchemiluminescence stainingslideswereincubatedwithBloxAllsolution(Vectorlabs) for10min,blockedwithIB-solution(10%Roti-Immunoblock,5%

normalgoatserumand3%BSAinPBS)for2handincubatedwith rabbitpolyclonal anti-Ccdc181(1:100,Sigma-Aldrich) overnight at 4^◦C. The next day, slides were incubated with polyclonal goatanti-rabbitAP-conjugatedantibody(1:50,Sigma-Aldrich)for 90minandstainedusingtheVectorRedAPSubstrateKit (Vec- torlabs)accordingtothemanufacturer’sprotocol,counterstained withMayer’sHematoxylin(Sigma-Aldrich),mountedwithRoti- Mount(CarlRoth)andexaminedusingtheZeissTissueFAXs.For immunoﬂuorescencestainingslideswereincubatedwithImageIT- FX(Invitrogen)for30minand withM.O.M.(Vectorlabs)for 1h.

AfterblockingwithIB-Solutionfor2h,slideswereincubatedwith rabbitpolyclonalanti-Ccdc181(1:100,Sigma-Aldrich)andmouse monoclonal anti-acetylated ␣-tubulin (1:2,000, Sigma-Aldrich) antibodiesovernight.The nextday,slides wereincubated with goatanti-mouseAlexa488-andgoatanti-rabbitAlexa568-coupled antibodies(1:500,Invitrogen)for90min,mountedwithProlong

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DiamondAntifadewithDAPI(Invitrogen)andexaminedusingthe ZeissLSM780laserscanningmicroscope.

2.11. Immunoﬂuorescencestainingofcells

Cells were ﬁxed with 4% paraformaldehyde in PBS (Sigma- Aldrich) for 15min and permeabilized with 0.5% Triton X-100 in PBS for15min.Slides were blockedwith IB-solutionfor 2h andincubatedeitherwithrabbitpolyclonalanti-Ccdc181(1:100, Sigma-Aldrich) and mouse monoclonal anti-␣-tubulin (1:500, Sigma-Aldrich) or mouse monoclonal anti-acetylated ␣-tubulin (1:2,000,Sigma-Aldrich),orwithrabbitpolyclonalanti-Ccdc181 (1:100,Bioss Antibodies) and anti-Hook1 (1:100,BiosAntibod- ies)directlycoupledtoAlexa488orAlexa555,respectively,over nightat4^◦C.Thenextday,slides incubatedwithunlabeledpri- maryantibodieswereincubatedwithgoatanti-mouseAlexa488- andgoatanti-rabbitAlexa568-coupledantibodies(1:500,Invitro- gen)for90min.Additionally,tovisualizetheacrosomeofisolated germcellsallslideswereincubatedwithAlexa647-conjugatedPNA (1:100,Invitrogen)inPBSfor30min.Allslidesweremountedusing ProLongDiamondAntifadewithDAPI(Invitrogen)andexamined usingtheZeissLSM780laserscanningmicroscope.

2.12. Microtubule-bindingassay

TransfectedHEK293Tcellswereharvested48hposttransfec- tionwithice-coldmicrotubule-bindingbuffer(80mMPipes,5mM MgCl₂,0.5mM EGTA,5%glycerol)supplemented with1×HALT ProteaseInhibitorCocktail(THPMedicalProducts)andlysedusing thePrecellys-24(PeqLabBiotechnology)at4500rpmfor1min.Pro- teinlysateswereclearedbyultracentrifugation(100,000g,40min) andstoredoniceuntilfurtheruse.

Invitro microtubule-binding assaywasperformed usingthe microtubule-bindingproteinspindownassaykit (Cytoskeleton) accordingtothemanufacturer’sprotocol.Brieﬂy,20␮linvitropoly- merizedmicrotubulesweremixedwith30␮lclearedproteinlysate andincubatedfor30minatroomtemperature.Asacontrol,lysates wereincubatedintheabsenceofpolymerizedmicrotubules.Micro- tubulesandbindingproteinsweresedimentedthrough100␮lof a40%glycerolcushionbyultracentrifugation(100,000g,40min).

PelletandsupernatantsampleswereanalyzedbySDS-PAGEand Westernblotting.

2.13. Statisticalanalysis

GraphPad Prism was used for statistical analysisand graph preparation.Errorbarsindicatestandarderrorofthemean(SEM).

Differenceswereconsideredsigniﬁcantwhenpwas0.05orlessin anunpairedstudentt-test.Signiﬁcantdifferenceswere***p<0.001,

****p<0.0001.

3. Results

3.1. IdentiﬁcationofmurineCcdc181cDNAusingtheyeast two-hybridsystem

ToidentifyputativeHook1interactingproteinsayeast two- hybridscreenusingamurinetesticularlibrarywasperformedand resultedintheisolationofmorethan20differentputativeHook1 interactionpartners.Oneoftheidentiﬁedclonescontaineda417 base pairfragment corresponding tothe Mus musculusRIKEN cDNA 4930455F23 gene (GenBank BC048086.1;NCBI Reference Sequence:NM029115.3),which waslater renamedtoCcdc181.

The Ccdc181gene is locatedat mouse chromosome 1, consists ofsixexonsand encodesa1909basepairlargetranscriptwith 509 amino acid residues and a predicted molecular weight of

59.25kDa.Ccdc181belongstothecoiled-coildomaincontaining proteinfamily,whichincludesvarioussofaruncharacterizedpro- teins,containingatleastonecoiled-coildomain.However,only lowsimilaritiesbetweenCcdc181andothermembersofthisfam- ilywerefoundbymulti-alignmentanalysis(datanotshown).To assess thelevel ofconservation ofCcdc181, we compared protein sequencesoftheCcdc181 homologsin human, mouseand xenopususingClustalOmega(Goujonetal.,2010;Sieversetal., 2011).Thisrevealedevolutionaryhighlyconservedregionsatthe N-andC-terminusoftheCcdc181proteinsequence(Fig.1).More- over,bioinformaticsanalysisofmouseCcdc181usingtheExPASy coilsserver(Lupasetal.,1991)identiﬁedtwoputativecoiled-coil domainsattheC-terminalpartofCcdc181ataminoacidresidues 333–385and458–488(Fig.1,greyboxes).

3.2. Ccdc181interactswithHook1

ToconfirmtheputativeinteractionofCcdc181andHook1found byyeast-twohybrid,weperformedaFACS(fluorescence-activated cellsorting)basedFRET(fluorescenceresonanceenergytransfer) analysis.Forthis,gatesweresetuptoexcludesingletransfected andFRETnegativecells.Therefore,cellsexpressingeitherGFP-only orDsRed-onlywereanalyzedtogetherwithcellsco-expressingGFP andDsRedandcellsexpressingaGFP-DsRedfusionprotein(Fig.

S1).Fortheanalysis,cellsco-expressingGFP-taggedCcdc181and DsRed-taggedHook1weretestedforFRETpositivecells.Because ofHook1’sknownabilitytoformhomodimers(Mendoza-Lujambio et al., 2002), cells co-expressing GFP-taggedand DsRed-tagged Hook1 wereused asan internal positive control,whereas cells co-expressing GFP-only and DsRed-only were used as a nega- tivecontrol.Insamplesofcellsco-expressingCcdc181andHook1 71.17%(±1.05%SEM)ofthecellswereFRETpositive,comparable tosamplesofthepositivecontrolwith76.12%(±5.36%SEM)FRET positivecellsand signiﬁcantlyhigherthansamplesofthenega- tivecontrolwith0.64%(±0.16%SEM)FRETpositivecells,strongly supportingtheproposedinteractionofCcdc181andHook1(Fig.2).

To further elucidate the interacting regions of Hook1 and Ccdc181,weprepareddifferenttruncationanddeletionconstructs for usein yeast two-hybridassays (Fig.3A). Theco-expression of these constructs was veriﬁed by yeast growth on double dropoutplates(SD/-Leu/-Trp),whereasinteractingconstructswere identiﬁed by yeast growth on quadruple dropout plates (SD/- Ade/-His/-Leu/-Trp).AsshowninFig.3B,thetruncationmutants Hook1-D1 (amino acids 1–276) and Hook1-D2 (amino acids 167–458),aswellasHook1-AZH(aminoacids1–263)wereable tointeractwithCcdc181,whereasnointeractionwasobservedfor Hook1-D3.Sincetheinteractingregionseemstoberestrictedtothe overlappingpartofHook1-D1andHook1-D2,wenextusedthetwo coiled-coildomaindeletionconstructsHook1-CC1andHook1-

CC2lacking amino acids 173–228 and 254–443, respectively, aswellastheSSF46579domaindeletionconstructHook1-SSF2 lackingaminoacids185–283.InteractionofCcdc181andHook1 was still observed in constructs lacking one of the coiled-coil domains(Hook1-CC1,Hook1-CC2)whereasthedeletionofthe SSFdomain(Hook1-SSF2)preventedinteraction(Fig.3B).There- fore,aminoacids229–253ofHook1seemtobemostcriticalfor itsinteractionwithCcdc181.ForCcdc181thetruncationmutants Ccdc181-D2(aminoacids248–448)andCcdc181-D3(aminoacids 393–509)wereidentiﬁedtointeractwithHook1,whereasnointer- actionwasobservedforCcdc181-D1(aminoacids1–254)(Fig.3B).

Therefore,theaminoacids393–448ofCcdc181, resemblingthe overlapping regionof Ccdc181-D2and Ccdc181-D3 seemto be mostcriticalforitsinteractionwithHook1.BecauseHook1shares highsimilaritywithHook2and Hook3, wenextaskedwhether Ccdc181isalsoabletointeractwiththetwo otherHookfamily members.Therefore,interactionofCcdc181withfulllengthHook2

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Fig.1. Ccdc181containsevolutionaryconservedregions.Proteinalignmentofhuman(NP001287898,Q5TID7),mouse(NP083391,Q80ZU5)andxenopus(F6UGJ0)Ccdc181 orthologuesusingClustalOmega.Symbolsforidentical(*),conserved(:)andsemi-conserved(.)aminoacidsaredisplayedbelowthealignments.Grayhighlightedamino acidsindicateputativecoiled-coilmotifsaspredictedbytheExPASycoilsserver.

andHook3wastestedbyayeasttwo-hybridassay.AsshowninFig.

S2AinteractionofCcdc181withHook2andHook3wasobserved.

Moreover,theinteractingregionofCcdc181wasidentiﬁedtobe thesameforallHookfamilymembers.Usingtheyeasttwo-hybrid approachwealsotestedthecapabilityofCcdc181toformhomod- imers, by using combinations of the three different truncation constructstogetherwiththefulllengthCcdc181 construct.The observedinteractionbetweenCcdc181-D1witheitherCcdc181- D2orCcdc181-D3suggeststhatCcdc181isatleastinyeastableto formhomodimers(Fig.S2B).

3.3. ExpressionanalysisofthemurineCcdc181gene

AftertheinteractionbetweenHook1andCcdc181wasveriﬁed wenextanalyzedtheexpressionpatternofCcdc181intestisand

differentsomatictissues.Therefore,RNAfromdifferenttissuesof adultmicewasextractedandanalyzedusingquantitativeRT-PCR (Fig.4A),RT-PCR(Fig.S3A)andNorthernblot(Fig.S3B).Thehighest levelofCcdc181expressionwasobservedintestis,whileinbrain, eye,tracheaandlung lowerexpressionlevelsweredetected.In tongue,heart,liver,kidney,spleenandmuscleCcdc181transcripts werebarelydetectable(Fig.4A).ThedifferenceinCcdc181expres- sionlevelsbetweentestisandsomatictissuesweresupportedby RT-PCRandNorthernblotresults(Fig.S3AandB).

Tofurtherinvestigatetheexpression ofCcdc181 in testisin more detail, testes of different mouse mutants were analyzed byquantitativeRT-PCR(Fig.4B).Inazh/azhmutantmice,where Hook1-deletionleadstomalformed sperms(Mendoza-Lujambio etal.,2002),aswellasinquaking(qk/qk)andoligotriche(olt/olt) mutantmice,in whichspermtailsareeitherabnormallydevel-

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Fig.2.Ccdc181isaHook1interactingprotein.InteractionbetweenCcdc181andHook1wasveriﬁedusingaFACS-basedFRETanalysis.(A)RepresentativeFACS-plotsshowing theamountofFRETpositivecellsinlivingNIH3T3cellsco-expressingtheindicatedGFPandDsRedfusionproteins.FRETpositivecellswithinthegates(orangebox)are displayedingreen,whileFRETnegativecellsoutsidethegatesaredisplayedinred.NumbersgivetotalpercentagesofcellswithintheFRETgate.Thenon-interactingproteins GFPandDsRedwereusedasanegativecontrol,whereasHook1’sabilitytoformhomodimerswasusedasapositivecontrol.(B)QuantiﬁcationofFRETpositivecellsusing meanvaluesandstandarderrorofthemeanforthetotalamountofFRETpositivecellsfromthreeindependentexperiments,thatwereanalyzedasdepictedin(A).p≤0.0001.

Fig.3.IdentiﬁcationofCcdc181andHook1interactingdomains.(A)SchemesofHook1andCcdc181truncationanddeletionconstructsshowingputativecoiled-coildomains (lightgrey)andtheSSF46579domain(darkgrey).FL,full-lengthprotein;MT,microtubule-bindingdomain;CC,coiled-coilrichregion;OB,putativeorganellebindingdomain;

aa,aminoacids.(B)YeastcellsharboringindicatedcombinationsofCcdc181constructsinpGADT7oremptypGADT7andHook1constructsinpGBKT7oremptypGBKT7 weregrownonmediumlackingleucineandtryptophan(doubledropout)toverifyco-transformation.Interactionwasassessedbyanalyzingyeastgrowthonmediumlacking adenine,histidine,leucineandtryptophan(quadrupledropout).DeletionofSSF2inhibitstheinteractionbetweenCcdc181,Ccdc181-D2andCcdc181-D3withHook1.

opedorabsent(Chubb,1992),nodifferenceinCcdc181expression wasobserved.However,intesticularfeminization(Tfm/y)andKit (W/Wv)mutantmice,whereeitherspermatogenesis isarrested inﬁrstmeiotic division(Lyonetal.,1975)or nogermcells are presentwithinthetestis(Ohtaetal.,2003),noCcdc181expres- sionwasdetected(Fig.4B).Theseresultswerealsosupportedby RT-PCRandNorthernblotexperiments(Fig.S3CandD).Therefore,

Ccdc181expressioninmaturemousetestisseemstobepredom- inantlyrestrictedtopostmeioticgermcells.Tofurtherelucidate Ccdc181 expressionduringtestisdevelopment,quantitativeRT- PCRanalysisoftestesof7–50-day-oldmicewasperformed(Fig.4C).

ObservedexpressionlevelsofCcdc181remainedlowuntilday20, whenroundspermatidsappearfortheﬁrsttime.Afterday20,a sharpandconstantincreaseinCcdc181expressionwasobserved.

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Fig.4. ExpressionanalysisofCcdc181inmousetissues,mutantsanddevelopmentalmalegermcellstages.Ccdc181expressionwasanalyzedbyquantitativeRT-PCRand expressionlevelsnormalizedtoB2mandGapdharedepictedasmeans±SEM.(A)ExpressionofCcdc181inmousetissuesrevealedelevatedexpressionintestis.(B)Indifferent mousemutants,noCcdc181expressionwasobservedinmutantslackingeithergermcells(W/Wv)orhaploidspermatids(Tfm/y).(C)AnalysisofCcdc181expressionduring postnatalmurinetesticulardevelopment,revealedasharpincreaseinCcdc181expressionwiththeﬁrstoccurrenceofelongatingspermatids.Sg,spermatogonia;ESc,early spermatocytes;LSc,latespermatocytes;RS,roundspermatids;ES,elongatingspermatids;Sp,maturespermatozoa.

AperformedRT-PCRsupportedtheobservedelevatedexpression ofCcdc181inlaterstagesofspermatogenesis(Fig.S3E).Thisfur- therconﬁrmsthatCcdc181 expressionpredominantly occursin spermiogenesis.

3.4. ExpressionandlocalizationanalysisoftheCcdc181protein

SinceCcdc181RNAexpressionoccurredmostlyinspermiogen- esis,wenextwantedtoinvestigateCcdc181proteinexpression during testis development. Therefore, testicular proteins were extracted from different aged male mice and probed withthe Ccdc181 antibody, while the amount and integrity of protein extractswereanalyzed using an␣-tubulin antibody.Similarto theRNAexpressionpatternofCcdc181,onlylowCcdc181protein levelswere observed until the ﬁrst appearance of roundspermatids.However,Ccdc181proteinlevelssharplyincreasearound day25withtheonsetofspermatidelongation(Fig.5A).Tofur- therinvestigatethelocalizationofCcdc181withindifferentiating germcells,immunohistochemicalexperimentswereperformed.

Therefore,parafﬁnsectionsofadultmurinetestes wereprobed withCcdc181antibody.StrongCcdc181expressionwasfoundin elongatingspermatids,lyingcloselytothelumenofthetubules, whereasroundspermatidsshowedweakerlabeling.Furtheranal- ysisrevealedthatCcdc181stainsthemicrotubularmanchetteof thesecellsaswellasthespermtail(Fig.5B).

Inorder todeterminethelocalizationof Ccdc181more pre- cisely,germcellsuspensionpreparationswerestainedforCcdc181 (red)and␣-tubulin(green).Additionally,theacrosomeofdevelop-

ingspermatidsandthenucleiwerevisualizedusingpeanut-lectin (white) andDAPI (blue),respectively (Fig.6).In earlystages of malegerm celldevelopmentonlya diffusestainingof Ccdc181 inspermatocyteswasdetected(Fig.6a–d).Duringmeioticdivi- siona weak accumulationof Ccdc181 atthe spindleapparatus wasobserved (Fig.6e–h).Inmoredifferentiated spermatidsthe manchettesandtheelongatingﬂagellaarestainedby␣-tubulin andtheCcdc181antibodies,whereasintheremainingcytoplasm ofthespermatidsCcdc181 isbarelydetectable(Fig.6i–p).Dur- ingfurtherspermatiddevelopmentwhenthecytoplasmelongates Ccdc181 remains localized to the manchette (Fig. 6q–x). As a similarlocalizationpattern waspreviouslydescribed for Hook1 (Mendoza-Lujambioetal.,2002)andourinvitroexperimentshave identiﬁedHook1andCcdc181asinteractingproteinswenextana- lyzedwhetherCcdc181andHook1co-localizeinmalegermcells.

Therefore,germcellpreparationswereanalyzedforlocalizationof Ccdc181(green)andHook1(red)andrevealedaco-localizationof bothpolypeptidesinspermatocytesandelongatinghaploidmale germcells(Fig.S4).NextthelocalizationofCcdc181inthehead regionofanearlyelongatingspermatidwasinvestigatedinmore detail.Ccdc181notonlylocalizestothemicrotubularmanchette, but alsodecorates thenuclearring andthe mostdistal partof themicrotubularmanchette,whereasCcdc181signalsshowedno overlapwithacrosomestaining(Fig.7A).Measurementoftherel- ativeﬂuorescenceintensitiesalongtheelongatingspermatidhead supportedtheobservedoverlappingstainingpatternoftubulinand Ccdc181,whereasthemostintenseCcdc181stainingwasfound atthedistaltipofthemanchettethatelongatesduringdevelop-

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Fig.5.ExpressionandlocalizationofCcdc181protein.(A)Proteinexpressionof Ccdc181duringpostnatalmurinetesticulardevelopmentwasanalyzedbyWestern blotusingtesticularproteinextractsof5–50-day-oldmice.Ccdc181expressionlev- elssharplyincreasearoundday25withtheonsetofspermatidelongation.Staining of␣-tubulinservedasaloadingcontrol.Sg,spermatogonia;ESc,earlyspermato- cytes;LSc,latespermatocytes;RS,roundspermatids;ES,elongatingspermatids;

Sp,maturespermatozoa.(B)LocalizationofCcdc181wasanalyzedbyimmuno- histochemicalstainingofadultmousetestissections(a).Ccdc181stainingofthe microtubularmanchetteinelongatingspermatids(b,arrows)andthespermtail (c,arrows)wasobserved.AsacontrolstainingwithoutCcdc181antibodywasper- formed(d).Scalebars:50␮m.

mentandsurroundsthegrowingspermatidtail(Fig.7B).Taken together,theseresultssuggestthatCcdc181associateswithmicro- tubularstructures,likethemicrotubularmanchetteandthesperm ﬂagellum.

3.5. LocalizationanalysisofCcdc181incilia

BecauseoftheobservedlocalizationofCcdc181inspermflag- ella,wenextaskedwhetherCcdc181alsolocalizestootherciliary structures.Therefore,paraffinsectionsofoviduct,brain,trachea andlungwereanalyzedforCcdc181localization.Inthesetissues Ccdc181stainingwasexclusivelyobservedinciliaoftheepithe- liallayer(Fig.S5).Inaddition,testisflagella,ciliaofthetracheal epitheliallayerandprimaryciliaofNIH3T3cellswerestainedfor Ccdc181andacetylated␣-tubulin.Interestingly,Ccdc181staining wasobservedinflagellaandmotilecilia,butnotinnon-motilepri- maryciliaofNIH 3T3cells.Moreover,Ccdc181wasobservedto accumulateatthemidpieceofspermatozoontailandthebasalhalf oftrachealcilia(Fig.8A).Relativefluorescenceintensityanalysis ofasingletrachealmotileciliumconfirmedourpreviousfinding ofCcdc181’slocalization attheproximalpartofmotilecilia, in

Fig.6.DistributionofCcdc181indevelopingmalegermcells.Testiculargerm cellswerepreparedfromadultC57BL/6miceandindirectimmunoﬂuorescence staining was performedusing polyclonalanti-Ccdc181 (red)and monoclonal anti-␣-tubulin(green) antibodies. Additionally,acrosomes werevisualized by peanut-lectin(white)andnucleiwerestainedwithDAPI(blue).(a–d)Inpremeiotic malegermcellsonlyadiffusestainingofCcdc181wasobserved.(e–h)Duringmei- oticdivisionCcdc181faintlyaccumulatesatthespindleapparatus.(i–x)Inhaploid malegermcellstagesCcdc181stainingisconcentratedatmicrotubularmanchettes andspermﬂagella.Scalebars:5␮m.

contrasttoacetylated␣-tubulinwhichstainedthewholecilium.

ThisindicatesthatCcdc181mightbeacomponentoftheappara- tusnecessaryforthegenerationofciliarymotility,ratherthana componentoftheciliarymicrotubuleskeletalstructurewhichis foundtobesimilarinmotileandnon-motilecilia.

3.6. Ccdc181isamicrotubule-bindingprotein.

AspreviousresultsindicatedalocalizationofCcdc181atmicro- tubular structures such as the manchette or the axoneme of motilecilia,wewereinterestedifCcdc181isalsoabletodirectly bindtomicrotubules.Therefore,aFACS-basedFRETanalysiswas performed withcells co-expressingDsRed-tagged Ccdc181 and GFP-taggedtubulin.Controlexperimentswithpositiveandneg- ativeFRETcontrolswereperformedasdescribedbefore(Fig.S1).

Approximately29.42%(±2.92%SEM)ofCcdc181andtubulinco- expressingcellswerefoundtobeFRET-positive,indicatingadirect interaction of Ccdc181 and tubulin (Fig. 9A and B). Addition- ally,aninvitromicrotubule-bindingproteinspin-downassaywas

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Fig.7. Ccdc181localizestodistinctstructuresoftheelongatingspermatid.(A)Indirectimmunofluorescencestainingofanelongatingspermatidwasperformedusing polyclonalanti-Ccdc181(red)andmonoclonalanti-␣-tubulin(green)antibodies.Additionally,acrosomeswerevisualizedbypeanut-lectin(white)(d–f)andnucleiwere stainedwithDAPI(blue)(f).Ccdc181stainingoverlapswiththemicrotubularmanchette(a–c),howeveradditionalCcdc181signalsweredetectedatthenuclearring(c, arrowheads)andthedistalendofthemanchette(c,arrows).NooverlapbetweenCcdc181andtubulinstainingwiththeacrosomewasobserved(d–f).(B)Thedistribution oftherelativefluorescenceintensitiesofthedifferentsignalsalongthespermatid-head(a–f,alongthearrow)supporttheCcdc181specificstainingatthedistalendofthe microtubularmanchette(blackarrow)andthenuclearring(blackarrowhead).Scalebar:5␮m.

performedto further strengthen these ﬁndings and toinvesti- gatetheresponsiblemicrotubuleinteractingdomainofCcdc181.

Therefore, purified bovine brain tubulin was polymerized into microtubulesandstabilizedwithtaxol.Pre-clearedlysatesofcells expressingStrepTag-conjugated Ccdc181 wereeither incubated withorwithoutpolymerizedmicrotubulesbeforesedimentation byultracentrifugationwasperformed.Insamplesincubatedwith polymerizedmicrotubules, Ccdc181as wellasCcdc181-D2 and Ccdc181-D3proteinswerefoundinthepellet,whereasCcdc181- D1remainedinthesupernatant.Incontrast,insampleslacking polymerizedmicrotubulesallCcdc181proteinvariantsremainedin thesupernatant,indicatingaspecificinteractionbetweenCcdc181 andmicrotubules.Gapdhandtubulinstainingwereusedtover- ifythatonlymicrotubulesandmicrotubule-bindingproteinswere sedimentedtothe pellet(Fig.9C). Taken together,ourfindings stronglysuggest that Ccdc181is a microtubule-binding protein andthatthisinteractionismediatedviatheoverlapping partof Ccdc181-D2andCcdc181-D3,previouslyidentifiedastheneces- saryregionforHook1interaction.

3.7. Ccdc181isaputativeinteractionpartnerofcatalytic subunitsofPp1

Previously, interaction of human Ccdc181 with the protein phosphatase1catalyticsubunitalpha(Pp1␣)hasbeensuggested (Doerksetal.,2002)andmurinePp1␥2hasbeenidentiﬁedtoplay acrucialroleinthemorphogenesisandmotilityofadultsperma- tozoa(Chakrabartietal.,2007;Fardilhaetal.,2011).Therefore,we nextwantedtoinvestigate,ifCcdc181alsointeractswithothercat- alyticsubunitsofPp1,especiallywithPp1␥2,byyeasttwo-hybrid.

AsshowninFig.10,Ccdc181andCcdc181-D1wereabletointeract withPp1␣,Pp1␤,Pp1␥1andPp1␥2.Incontrastnointeractionwith thedifferentcatalyticsubunitsofPp1wasobservedforCcdc181- D2andCcdc181-D3.ThisinteractionofCcdc181andPp1␥2further strengthensourhypothesisthatCcdc181mightplayanimportant roleinciliarymotility.

4. Discussion

WepreviouslydemonstratedthatthedisruptionofHook1isthe underlyingcauseoftheazh(abnormalspermatozoonheadshape) phenotypeinmice.Inmicecarryingthemutationazhinahomozy- gousstatethespermatozoa displayabnormalheadmorphology

andoftentailabnormalitiesresultingincoiledspermtailsorin thedecapitationofthespermheadfromtheﬂagellum.However, itseemsunlikelythatHook1actsasastructuralcomponentofthe spermatozoa,asitcannotbedetectedinmaturemousesperma- tozoa(Mendoza-Lujambioetal.,2002).Ratheritcanbeassumed thattheseabnormalitiesinmaturespermsareindirectlycaused bythe lossofHook1 function causing a dislocationof proteins duringspermatiddifferentiation.Oneofthemultiplespecialized organellesproposedtoensurecorrectmaturationofspermatozoais themanchette.Thisspecializedstructureconsistsoflaterallyasso- ciatedmicrotubulesappearingtransientlyduringspermiogenesis andisthoughttoplayaroleinnuclearshapingandintramanchette transport(IMT)processes,aputativelyaffectedprocessbytheazh mutation(Kierszenbaum,2002;ToshimichiYoshidaetal.,1994).

We assume that Hook1 mediates cargo transport along the microtubulesofthemanchette,asithasalreadybeenshownthat Hook1interactswithmicrotubulesofthemanchettethroughits N-terminalmicrotubule-bindingdomainandcontainsaputative organelle-bindingdomainatitsC-terminus(Mendoza-Lujambio etal.,2002;Walentaetal.,2001).ToidentifyputativeHook1inter- actingproteinsthatmightserveasHook1cargoproteinsinmurine spermiogenesisweperformedayeasttwo-hybridscreenbywhich severaldifferent putativeinteracting proteinswere found.One oftheidentiﬁedcDNAfragmentsrevealedastrongsimilarityto thecoiled-coildomaincontainingprotein181(Ccdc181).Ccdc181 belongstothelargefamilyofpolypeptidesdisplayingacoiled-coil domain.However,computationalsearchrevealed nosigniﬁcant similaritytootherproteinsofthecoiled-coildomaincontaining proteinfamily,butshowedthatCcdc181isevolutionaryconserved.

Additionally, Ccdc181 was shown to be a putative ciliary protein(McClintocketal.,2008).Ourresultssupportthisfindingand indicatethatCcdc181isastructuralcomponentoftheaxonemal structureofspermflagellaaswellasmotilecilia.Expressionanaly- sesidentifiedlowlevelsofubiquitouslyexpressedCcdc181mRNA, althoughthestrongestexpressionwasobservedintestis.Further- more,elevatedlevelsofCcdc181transcriptscouldbedetectedin severaltissuesexpressingmotileciliasupportingitsputativefunc- tionasa ciliaryprotein.We usedspecificantibodiestolocalize Ccdc181inciliaryandflagellarstructuresandfoundCcdc181dis- tributedalongthespermflagellumwhereaslocalizationofCcdc181 wasrestrictedtothebasal partofmotilecilia.Interestingly,we couldnotdetectCcdc181inthenon-motilesingleciliumofNIH3T3 cellsindicatingthatCcdc181isnotacomponentofthebasicaxone-

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Fig.8. Ccdc181localizestomotileciliabutnottonon-motileprimarycilia.(A)IndirectimmunoﬂuorescenceofCcdc181insectionsofadultmousetestis(a–c),indissected trachealcartilages(d–f)andinNIH3T3cellsinducedforprimaryciliaformation(g–i)wasperformedusingpolyclonalanti-Ccdc181(red)andmonoclonalanti-acetylated

␣-tubulin(green)antibodies.Additionally,nucleiwerestainedwithDAPI(blue)(c,f,i).Ccdc181predominantlylocalizestospermﬂagella,withamoreintensestainingat themidpiece(a–c).Intrachealepithelialcells,Ccdc181wasobservedtolocalizetothebasalpartofmotilecilia(d–f).Incontrast,inNIH3T3cellsCcdc181stainingwasabsent innon-motileprimarycilia,butwasdiffuselydistributedinthecytosol(g-i).(B)ThedistributionoftherelativeﬂuorescenceintensitiesofCcdc181andacetylated␣-tubulin alongasingletrachealcilium(a-c,carrow)isshown.Incontrasttoacetylated␣-tubulin,Ccdc181predominantlylocalizestothebasalpartofthetrachealcilium.Scalebar:

5␮m.

malstructurethatishighlysimilarinmotileaswellasinnon-motile cilia(SatirandChristensen,2007).RatheritseemsthatCcdc181is involvedinthegenerationofciliaryandﬂagellarmotilityalthough itsfunctioninthisprocessisunclear.Thisassumptionissupported bytheobservationthatCcdc181caninteractwithdifferentiso- formsofthecatalyticsubunitsofthephosphoproteinphosphatase 1(Pp1).Pp1isamajorserine-threoninephosphatasethatisubiq- uitouslydistributed andisinvolved ina broadrangeof cellular functions,includingcellcycleprogression(Korrodi-Gregorioetal., 2014).ThePp1familyconsistsoffourmainlyhomologuescatalytic subunits,thePp1␣,Pp1␤,Pp1␥1andPp1␥2.Previousﬁndingssug- gestedthatPp1␥2ispredominantlyexpressedintesticulartissues andplaysanimportantroleinregulatingthemotilityofsperma- tozoa(Fardilhaetal.,2011).Interestingly,aknock-downofPp1␥2 leadstomaleinfertilityduetoimmotilespermatozoaandcannotbe rescuedbytheothermembersofthePp1family,whereasinother tissuesthelossofaPp1subunitcanbesubstitutedbytheremaining

Pp1subunits(Fardilhaetal.,2011;Sinhaetal.,2013).AsPp1␣has beenidentifiedasaputativeCcdc181bindingprotein(Doerksetal., 2002)andthehighoverallsimilarityofthePp1catalyticsubunits, wetestedwhetherCcdc181isalsocapableofbindingtotheother Pp1catalyticsubunits.OurresultsindicatethattheN-terminalpart ofCcdc181 interactswithPp1␣,Pp1␤,Pp1␥1 andPp1␥2.Addi- tionallyweobservedthattheC-terminalregion(AA400–450)of Ccdc181interactswithmicrotubulesandHook1.Thisraisesthe hypothesisthatCcdc181 actsasalinkerproteinforPp1totack thephosphatasetotheaxonemalstructureofciliaandflagella.Pp1 doesnotonlycontrolmotilityinspermatozoabuthasbeenrecently identifiedasaputativeregulatorofmotilityinmammalianairway cilia(Price etal.,2015).Takentogetherourresultssuggestthat Ccdc181viaitsinteractionwithPp1mightplayanimportantrolein thegenerationandregulationofflagellaraswellasciliarymotility.

WefoundthattheC-terminalregionofCcdc181isresponsiblefor thebindingtomicrotubules.Interestingly,thisdomainisalsoused

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Fig.9. Ccdc181isamicrotubule-bindingprotein.(A)RepresentativeFACS-plotsshowingtheamountofFRETpositivecellsinlivingNIH3T3cellsco-expressingeitherGFP andDsRedorCcdc181-GFPandtubulin-DsRedproteins.FRETpositivecellswithinthegates(orangebox)aredisplayedingreen,whileFRETnegativecellsoutsidethegates aredisplayedinred.NumbersgivetotalpercentagesofcellswithintheFRETgate.(B)QuantiﬁcationofFRETpositivecellsusingmeanvaluesandstandarderrorofthe meanforthetotalamountofFRETpositivecellsfromthreeindependentexperiments,thatwereanalyzedasdepictedin(A).p≤0.001.(C)Amicrotubuleinvitrobinding assaywasperformedusingtotalproteinlysatesofHEK293TcellsoverexpressingindicatedStrep-taggedconstructswhichwereincubatedwith(+MT)orwithout(−MT) stabilizedmicrotubules.Aftersedimentationbyultracentrifugationproteinsofthesupernatant(S)andthepellet(P)wereseparatedbySDS-PAGE.Distributionofdifferent Ccdc181proteinsbetweensupernatantandpelletfractionwasanalyzedusinganti-Strepantibody.Gapdhandtubulindistributionwasusedasnegativeandpositivecontrol, respectively.AninteractionwithmicrotubuleswasobservedforCcdc181,Ccdc181-D2andCcdc181-D3.

fortheinteractionwithHook1suggestingthatCcdc181exclusively bindstoeitherHook1ormicrotubules.Incontrast,thenecessary aminoacidsofHook1neededforinteractionwithCcdc181donot overlapwiththeputativemicrotubule-bindingdomainofHook1.

AlthoughnotyetprovenexperimentallyweassumethatCcdc181 doesnot binddirectlytothemicrotubules ofthemanchette in spermatidsbutratherislinkedtothemanchettebyHook1forintra- manchettetransportationtotheaxonemeofthegrowingsperm ﬂagellaandinthiscontextavoidanyunwantedbindingofCcdc181 tothemicrotubulesof themanchette.Additionally,we demon- stratedinvitrothatCcdc42isaputativeHook1interactingprotein (Fig. S6). Interestingly,Ccdc42 has recentlybeen reported as a ﬂagellarproteinwhichplaysacrucialroleinmalegermcelldevel- opment(Pasek et al., 2016), strengthening our hypothesis that Hook1actsasacargolinkerinintra-manchettetransportprocesses fordifferentproteinssuchasCcdc42andCcdc181.Inmeioticstages ofmalegermcelldifferentiationwedetectedCcdc181stainingin

thecytoplasmaswellasatthemicrotubulesofthespindleappara- tusindicatinganinvolvementofCcdc181intheprocessofmeiotic division.Recently,Ccdc181wasidentiﬁedasinteractingpartnerof theRINGdomain-containingproteinCCNB1IP1(CyclinB1Interact- ingProtein1),whichisaputativeubiquitinE3ligasethatseemsto beessentialforchiasmataformationduringmeiosis(Strongand Schimenti,2010).Moreover,CCNB1IP1hasalsobeenidentiﬁedas aHook1interactingprotein(StrongandSchimenti,2010).Further experimentshavetobeperformedtoelucidatetheimpactofthese differentinteractionsoneachotherandtheirroleinspermatoge- nesis.

5. Conclusions

We reportthemolecularcharacterization of a newmember ofthecoiled-coil domaincontaining proteinfamilyencoded by themurineCcdc181gene.Ccdc181isaHook1interactingprotein,