CorrespondencemaysimerahmailnihgovLaboratoryofCochlearDevelopmentNationalInstituteonDeafnessandOtherCommunicationDisordersNIH35ConventDriveBethesdaMD20892USAMaySimeraandKelleyhttpwwwci ID: 327648
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REVIEWOpenAccessCilia,Wntsignaling,andthecytoskeletonHelenLMay-SimeraandMatthewWKelleyAbstractPrimaryciliahaverecentlybeenhighlightedaskeyregulatorsindevelopmentanddisease.Thisreviewfocusesoncurrentworkdemonstratingthebroadroleofcilia-relatedproteinsindevelopmentalsignalingsystems.Ofparticularconsiderationistheimportanceofthebasalbodyregion,locatedatthebaseofthecilium,initsroleasafocalpointformanysignalingpathwaysandasamicrotubuleorganizingcenter.Astheciliumiseffectivelyamicrotubularextensionofthecytoskeleton,investigatingconnectionsbetweentheciliumandthecytoskeletonprovidesgreaterinsightintosignalingandcellfunction.Ofthemanysignalingpathwaysassociatedwithprimarycilia,themostextensivelystudiedinassociationwiththecytoskeletonandcytoskeletalrearrangementsarebothcanonicalandnon-canonicalWntpathways.Oneofthekeyconceptscurrentlyemergingisapossibleadditionalroleforthetraditionallycilia-relatedproteinsinotheraspectsofcellularprocesses.Inmanycases,disruptionofsuchprocessesmanifestsatthelevelofthecilium.Whiletheinvolvementofciliaandcilia-relatedproteinsinsignalingpathwaysiscurrentlybeingunraveled,thereisagrowingbodyofevidencetosupportthenotionthatciliaryproteinsarerequirednotonlyforregulationofWntsignaling,butalsoasdownstreameffectorsofWntsignaling.ThisreviewsummarizesrecentadvancesinourunderstandingoftheinvolvementofciliaandbasalbodyproteinsinWntsignalingpathways.Keywords:Cilia,basalbody,Wntsignaling,PCP,-catenin,microtubules,actin,cytoskeleton,kinesins,GTPases *Correspondence:maysimerah@mail.nih.govLaboratoryofCochlearDevelopment,NationalInstituteonDeafnessandOtherCommunicationDisorders,NIH,35ConventDrive.Bethesda,MD20892,USAMay-SimeraandKelleyhttp://www.ciliajournal.com/content/1/1/7 ©2012May-SimeraandKelley;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited. trafficking.Ourunderstandingofhowmutationsincilia-associatedproteinsleadtodisruptionsinciliafunc-tionandsubsequentcellularpathologyremainslimited,butvaluableinsightsarebeginningtoemerge[5-7].Thisreviewwillhighlightourcurrentunderstandingoftheciliumandbasalbodyasoneofthemicrotubuleorganizingcentersofthecellandhowtheyfunctionasacriticalsignalingcenter.Theciliumhasbeenimpli-catedintheregulationofagrowingnumberofsignalingpathways,andwhilemuchrecentresearchhasfocusedonsonichedgehog(Shh)-signaling,thisreviewwillmorecloselyexaminetheroleoftheciliumandbasalbodyinregardtoWntsignaling,anasyetlessexploredavenueofresearch.Tobegin,wewilltakeacloserlookattheciliumandrelatedproteins,aswellas,thepossiblerolesofthoseproteinsbeyondthecilium.Then,theroleofciliaandbasalbodiesinbothcanonicalandnon-canoni-calWntsignalingwillbeexamined;focusingmorespe-cificallyontheirassociationwithcytoskeletalregulation.TheCilium-StructureandFunctionCiliaaremicrotubule-basedorganelles,extendingfromtheplasmamembraneandcoupledtothecytoskeleton.Theyareanchoredtothecellandemanatefromthebasalbody,abarrelstructureofninetripletmicrotu-bulesthatelongatetoformthemicrotubuledoubletsoftheciliaryaxoneme.Thebasalbodyisderivedfromthemothercentriole,andisalsoconsideredamicrotubule-organizingcentre(MTOC).CentriolesaremorethanjusttheprimaryMTOCofacell;theyalsoserveasimportantregionsforproteininteractions,andasdock-ingstationsforproteinsonthewaytothecilium[8,9].Theregionatthebaseoftheciliumishighlycomplex.Distaltothebasalbodyisthetransitionzone,whichstartswherethetripletmicrotubulesendandextendstothebasalplate,uponwhichthecentralpairofmicrotu-bules(inthecaseofamotilecilium)arenucleated(Fig-ure1).Ineukaryoticciliathetransitionzoneiscomposedofseveraldistinctstructuresincludingtransitionalfibers,Y-linkers,andtheciliarynecklace(ontheciliamem-brane),allflankedbytheterminalandbasalplates.Whiletheirappearancesvaryamongspecies,thebasiccomponentsareconserved[10].Thoughtheexactpro-teincompositionofthisregionoftheciliumiscurrentlybeingelucidated[11],thetransitionzoneisemergingasthekeysiteforregulationofciliaryfunction.Transi-tionalfibersarethoughttoberequirednotonlyfordockingandanchoringthebasalbodytothemembrane,butalsoasdockingsitesforIFTparticlesandassociatedmolecularmotors,bothofwhicharedestinedfortheciliarycompartment.Ithasbeenfurtherproposedthatthefibersformpartofaporecomplex(comparabletonuclearpores),regulatingtrafficintoandoutofthecilium[12].Similartothesefibers,theY-linkersandciliarynecklaceareproposedtoregulatetraffickingintoandoutofthecilium[10,13].Manyoftheproteinsassociatedwithciliafunctionappeartointeractdynami-cally[14-16],anddisruptionsoftheseinteractionscouldplayasubstantialroleinthephenotypicvariabilityamongthevariousciliopathiesaswellasamongindivi-dualswiththesamecondition[17].Morespecifically,interactionsbetweenproteins,whichwhendefectivecauseciliopathiessuchasMeckelsyndrome,nephro-nophthisis(NPHP)andJoubertsyndrome,appeartoberequiredforbasalbodyanchoringandestablishmentofaciliarygateway[11,18].Disruptionsoftheseproteinnetworks,particularlyattheciliarytransitionzone,resultindisruptionofciliogenesisandalsoindefectivesignaltransduction[11,19].Sensoryciliaareadditionallyspecializedbycelltypetotransducethespecificfunctionormodalityofthatparti-cularcell.Forexample,olfactoryciliadetectodorantsviaspecificreceptorsontheciliarymembrane,thecon-nectingciliainmammalianphotoreceptorscontinuouslytransportvariousproteinstosupporttheouterseg-ments,andsensorycilialiningthekidneytubulessensefluidflowthroughthenephron.Initialidentificationofdiseasecausinggenesincilio-pathiessuchasBardet-Biedlsyndrome(BBS)orNPHP,suggestedthateachciliopathyhadadistinctsetofdis-ease-causinggenes.However,inrecentyearsithasbecomeapparentthatthereisconsiderableoverlapbetweenciliopathygenes,suggestingthatphenotypicvariabilityamongciliopathiesiscausedbyacombinationofthenatureofthemutationandthecontributionofotherallelicvariations.MutationsingenessuchasCEP290cancausenearlyallciliopathies[20];otherssuchasBBS4arerestrictedtotwo[21].Ingeneral,genescausingBBSandMeckelsyndromeover-lapasdothosecausingSeniorLokensyndromeandNPHP[11,18,21].Thetheorythat9+2ciliasolelymediatemotilefunc-tions,whilethe9+0ciliahaveastrictlysensoryfunctionisnowconsideredanoversimplification.Non-motileolfactorysensoryciliahavea9+2structure[22],whilethe9+0ciliaattheembryonicnodearemotileandgen-erateleftwardflow,requiredforestablishmentofasym-metry[23,24].Further,ependymalciliainthebrainventriclesareproposedtohaveasensoryfunctioninadditiontotheirwell-characterizedmotilefunction[5].Intriguingly,motileciliaonhumanairwayepithelialcellswererecentlyfoundtoexpressbittertasterecep-tors.Activationofthesereceptorsincreasesintracellularcalciumlevels,thusstimulatingbeatfrequencyandsoinitiatingadefensivemechanismagainstnoxioussub-stances[25].Theseexampleswouldsuggestthataddi-tionalmotileciliawithsensoryfunctionareprobablyMay-SimeraandKelleyhttp://www.ciliajournal.com/content/1/1/7Page2of16 awaitingdiscoveryandthatthesimplisticclassification ofciliawillneedtobereconsidered.Infact,ciliawitha 9+3microtubuletriplethavealsobeenidentified[26], althoughthespecificfunctionofthisconfigurationis stillunknown. Asthestructuralbasisofallciliaisaringofnine microtubules,ciliopathiescouldalternativelybeviewed asdiseasesofmicrotubulefunction,withthecilium representingaspecializedextensionofthecytoskeleton. Whereasmicrotubuledefectsinsidethecellmightbe mitigatedduetocompensatoryrolesofotherproteins, thehighlyspecializedciliarytransportmachinerymay beparticularlysensitivetoperturbationsinmicrotubule function/transportduetoitsremotelocationandlack ofcompensatorysubunits.Manyoftheciliopathygenes codeforproteinsthatlocalizetothebasalbodyand/or transitionzoneand,assuch,mayexertaregulatoryrole onciliaryfunction.Asmicrotubulesarehighlyabundant inthisregion,theirassociationandinteractionwithcili- aryproteinsareessentialforciliaryfunction.Inaddi- tion,wemustconsiderthepossibilitythatproteins importantforcilia-relatedfunctionsmayalsobe requiredfornon-ciliarymicrotubuleandcytoskeletal associatedprocesses[27-29]. CiliaryProteins-BeyondtheCilium Iftheciliumisregardedasaspecializedextensionofthe cytoskeleton,itisnotsurprisingthatmanyciliarypro- teinsarealsobeginningtoemergeaskeyplayersin othercellularprocessesthatinvolvethecytoskeleton. Theseincludecellcycleprogressionandvesiculartrans- port,bothofwhichareheavilydependentontheconfig- urationofthemicrotubulearchitecturewithinacell. Thebasalbody,whichanchorsthecilium,isoneof themicrotubule-organizingcentersofthecell,and recentworkhasidentifiedac ruciallinkbetweencilio- genesisandcellcycleprog ression(reviewedinKim andTsiokas[30]).Incellswithasingleprimarycilium, thebasalbodyisderivedfromthedistalendofthe mothercentriolewhichbecomesencapsulatedby Golgi-derivedvesiclesinadirectionalmanner[31]. Priortociliogenesis,themothercentrioledocksatthe plasmamembrane[32].Furtherfusionofadditional vesiclesenablestheciliumtoprotrudeawayfromthe cellbodyandintotheextracellularmatrix.Hence, growthandmaintenanceofciliafunctionisheavily dependentnotonlyonIFTbutalsooninternaltraf- fickingofcomponentstothebasalbody,whichalso utilizesIFTproteins[33-36]. Figure1 Ultrastructuraldepictionofth ebaseofagenericmammaliancilium .Thebasalbody(BB),derivedfromthemothercentriole, nucleatesthemicrotubuleaxonemeofthecilium.Thebasalbodyendsattheterminalplate(tp)andthetransitionfibers.Thetransitionfibers areelectrondensefibersatthebaseoftheciliumconnectingtheciliaryaxonemetotheplasmamembrane.Thetransitionzone(TZ)is characterizedbythepresenceoftheciliarynecklaceontheciliarymembrane(notdepicted)andY-linkers,theendofwhichcorrespondwith thebasalplate(bp).Inciliathatcontaincentralmicrotubules,theseemanatefromthebasalplate.Thetransitionfibersandtransitionzone encompasstheso-called ciliarygate ,whichpossiblyregulatesproteinentranceandexit.Thefibersactasdockingsitesforintraflagellar transportparticlesandtheirmotors,andcouldformpartofaporecomplexsimilartothenuclearpores.Thedaughtercentriole,connectedto thebasalbodyviaaninterconnectingfiber,andstriatedrootletarealsodepicted.Theciliarypocketisaninvaginationofspecializedcell membraneatthebaseoftheciliumlikelytobeimportantforregulationofciliacomposition. May-SimeraandKelley Cilia 2012, 1 :7 http://www.ciliajournal.com/content/1/1/7 Page3of16 Asthebasalbodyderivesfromthemothercentriole, thetimingofciliogenesisiscloselyrelatedtomitosis, mitoticspindledeconstruction,andcentrioleduplica- tion.Ciliacustomarilynucleateandgrowduringthe G1-G0phaseofthecellcycleandthenretractduring mitosis[6].ThecentrosomeinG1cellsiscomprisedof twocentrioles,termedthemotheranddaughtercen- triole.Eachofthetwocentriolesinthecentrosomeself- duplicatesonceduringthecellcycle,buttheirrateof maturationisasymmetric[3 7].Uponself-duplication, bothcentriolesbecomemot hercentrioleswithnewly associateddaughtercentrioles.Consequently,thetwo centrosomesinaG2cellhaveeithertheolder(original) mothercentrioleoranewermothercentriole[38].As therateofmaturationisasymmetric,thisresultsintwo differentiallyagedcentrosomesthatsegregateuponcell division(Figure2).Eachnewcellobtainsacentrosome witheithertheoldermothercentrioleortheyounger mothercentriole.Segregationofdifferentiallyaged mothercentriolesaffectssubsequentcellfatesandattri- butes,includingthegrowthofaprimarycilium[39]. Additionalaspectsofhowcentriolesandcentrosomes transitiontobecomebasalbodies,whichisthoroughly discussedinarecentreviewbyKobayashiandDynlacht [40],involvesproteinsrequiredforbothcentriolemigra- tionaswellasciliaassemblyanddisassembly. Asymmetriccentrosomalinheritancehasbeenimpli- catedinsubsequentcellfatedecisionsin Drosophila neuroblastsandmalegermlinecells[41-43].Inmouse corticalneuroepithelium,inheritanceofthemothercen- trosomecorrelatedwithaprogenitorcellfate,whereas inheritanceofthedaughtercentrosomewasassociated withdifferentiatedprogenycells[43].Asymmetriccen- trosomalinheritanceaftercelldivisionalsoaffectsthe timingofciliagrowth[39]. Cellsinheritingtheolder mothercentrosomesappeartogenerateciliasooner thanthosewithyoungerdaughtercentrosomes.Differ- encesincompositionofbothpericentriolarmaterial (PCM)andpericentriolarsatellites(sub-distalanddistal appendages)betweenthetwocentriolesmightcontri- butetotheasymmetricrateofdivisionandsubsequent cellfatedetermination.Thedifferentiallocalizationof varioussignalingproteinstotheolder vs .youngercen- trioleofdividingsistercellsaddsafurtherlayerof Figure2 Centrosomeduplicationduringthecellcycle . (A) Centrosomesarecomprisedoftwocentrioles(motheranddaughter)connected viaaninterconnectingfiber.Themothercentriolehasadditionaldistalandsubdistalappendages.Thecentriolesaresurroundedbyamatrixof proteins,thepericentriolarmaterial(PCM). (B) Duringthecellcycle,eachcentriole(theoriginalmotheranddaughtercentriole)duplicatesonce, growinganewdaughtercentriolefromtheirsides.Theoriginalmothercentrioleduplicatesatafasterratethantheoriginaldaughtercentriole. Theoriginaldaughtercentrioleacquiresadditionalappendagesandthusbecomesanewmothercentriole.Mitosisseparatesthetwo centrosomes(duplicatedcentrioles)resultingintwocellseachwithadifferentiallyagedmothercentriole.Differencesbetweenthesecells regardingcellfateandregulationarebeginningtoemerge. May-SimeraandKelley Cilia 2012, 1 :7 http://www.ciliajournal.com/content/1/1/7 Page4of16 complexitytosignalingregulation[39].Asymmetriclocalizationofproteinstodividingcentriolescouldalsoallowforpotentialestablishmentofapical-basalcellpolaritywithinthatcell.Thesefindings,namelythatasymmetricdistributionofproteinsaroundthecentrioleisalsoimportantduringbothcellcycleprogressionandciliogenesis,supporttheconceptthatthebasalbodyregionplaysacriticalregu-latoryrole.Othercontributorstosuchfunctionsincludetheciliary-relatedproteinsCEP290,CP110,andCep97,whicharehighlyinvolvedinciliaassemblyandregula-tionandarealsoasymmetricallylocalizedtothebasalbodyregion[13,44,45].Ataregulatorylevel,thecentrio-larkinesinKif24(seesectiononkinesins),whichprefer-entiallylocalizestomothercentrioles,hasbeenfoundtopreventciliaassemblyviarecruitingand/orstabilizingCP110atthedistalendofmothercentrioles[46].Evenmaturebasalbodies,asymmetricdistributionofpro-teinssuchasDishevelledhavebeenshowntoberequiredforcellularpolarization[47].Intheemergingassociationofciliaandcellcyclepro-gression,twodyneinbindingproteins,Ned1andTctex-1,havebeenshowntoregulatecellcycleprogressionviaeffectsoncilialength[48,49].Boththeseproteinsloca-lizetothebaseofthecilium(Ned1atthemothercen-trioleandTctex-1atthetransitionzone)andarethoughttocontrolcilialengthregulation,whichinturndeterminescellcycleprogression.Thisobservationcouldexplainthepresenceofonlyasingleprimaryciliumpercellduringtissuemorphogenesisanddevel-opmentwhencellscontinuetodivide.Oncecellsterm-inallydifferentiate,theyareabletoformmultiplecilia,whichisnotpossiblepriortoterminaldifferentiationasthesingleciliummustretractduringcelldivision[6].ItwouldbeinterestingtoknowiftheexpressionofNed1orTctex-1wasdown-regulatedindifferentiated,post-mitoticcells.AsIFTproteinshavebeenshowntoberequiredforintracellulartraffickingtothecilium,notjustalongthecilium,itisplausiblethatthesecomponentsmightalsoberequiredforadditionalmicrotubulerelatedtransport.Intriguingly,theintraflagellartransportproteinIFT88,requiredforciliaryanterogradetransport,hasalsobeenshowntoberequiredforspindleorientationduringmitosis[29,50],thuscouplingcellcycleprogression,centrioledivision,andmicrotubuletransport.Aspartofadynein-1drivencomplex,IFT88appearstotransportmicrotubule-nucleatingproteinstothespindlepoles,whicharerequiredforformationofastralmicrotubulearraysandsubsequentcorrectspindleformationandorientation.ThissuggestspossiblerolesforIFTproteinsincytoplasmiccellularmechanisms.Additionally,IFT20,anotherIFT-relatedprotein,isknowntofacilitatetrans-portofproteinsfromtheGolgitothebaseofthecilium[34].IFT20alsointeractswithIFT57andIFT88attheimmunesynapseinnon-ciliatedT-lymphocytes[28].Similarly,innon-ciliatedretinalneurons,IFT20,IFT57,andIFT52havebeenshowntotransportcargovesiclestothedendriticpost-synapticterminal[36].Thesenon-ciliarylocalizationscouldhoweverrepresentpossibleciliaryremnantsformedinthesecells.Additionally,itshouldbenotedthattheplasmamembraneoverthecentrosomecouldbefunctionallyequivalenttothecili-arymembraneandsosharesimilarproteincomposition.Whileinvitroevidenceismountingthatmanyciliaryproteinshaveadditional,alternaterolesawayfromthecilium,itisimportanttonotethatinvivogeneticevi-denceisasyetlacking.Manymouseandzebrafishmutantswhichlackciliaappeartosurvivewellintodevelopment[51,52].Nevertheless,thiscouldreflectapotentiallyhighdegreeoffunctionalredundancyand/ordevelopmentalstageandtissuespecificity.Aswillbediscussedinsubsequentsections,thedistinctionbetweenciliaversusbasalbodyproteinsmayalsobeafactorinfurtherdissectingtherolesforindividualciliaproteins.Primaryciliabiologyisstillinitsinfancy,soitisnotnaïvetoassumethatwemayfindmanymoresec-ondary/additionalrolesofciliaryproteins,whichwillgreatlyadvanceourunderstandingofciliaryfunctionandgeneralcellbiology.CiliainWntSignaling-theImportanceoftheBasalBodyInrecentyears,manydifferentsignalingpathways,suchascanonicalandnon-canonicalWnt[7],Shh[53-57],FGF[58,59],Notch[60,61],mTor[62],PDGF[63],andHippo[64]signaling,havebeenshowntobeassociatedwiththeprimarycilium.However,ciliarysignalingandthecytoskeletonappeartoplayparticularlyimportantrolesinthemodulationoftheWntpathways.IntegralWntsignalingiscrucialinmodulatingthecytoskeletonand,reciprocally,Wntsignalingrequiresanintactcytos-keleton.Downstreameffectsofnon-canonicalWntsig-naling(alsoreferredtoasplanarcellpolarity[PCP])resultincytoskeletal-actinrearrangementswhilecompo-nentsofthecanonicalWntsignalingpathwayassociateheavilywithmicrotubuleandcytoskeletalcomponents.Whilebothcanonicalandnon-canonicalWntsignal-ingareactivatedbyWntmoleculesbindingtodistinctmembrane-boundreceptors,downstreamstepsdiverge.CanonicalWntsignalingresultsinstabilizationofcyto--catenin,whichsubsequentlyentersthenucleusandinitiatestranscriptionofWnttargetgenes.Incontrast,downstreameventsofPCPsignalingresultinchangesincellmorphology(actindynamics,cellpolarity,cellshape,andcellchange)ratherthantran-scription.ThisprocessultimatelyresultsinthedirectedorientationofcellsrelativetoaplanaraxiswithintheMay-SimeraandKelleyhttp://www.ciliajournal.com/content/1/1/7Page5of16 epithelium.WhilethepreciseroleofciliainWntsignal-ingiscurrentlybeingdissected[7],akeystepwillbetodistinguishtheroleofthebasalbodyandtransitionzone,whichmaybeindependentofthecilium.Alsoofconsequencewillbethedistinctionbetweentheroleofciliaryproteinsintheciliumversusinthecellbody,wheretheymayplayadditionaldownstreamrolesinsig-naltransmission.OneofthefirstindicationsthatciliamightbeinvolvedinWntsignalingwastheidentificationoftwokeyPCPcomponents,Inversin(Nephrocystin2)andDishevelled(Dvl),atthebasalbody(Table1)[47,65,66].MutationsinInversincausesoneoftheciliopathiesNPHPaswellassitusinversus[67].ThiswasfollowedbyobservationsthatknockdownofseveralBBSandotherciliary-associatedgenes(Kif3aIft88resultedinhyperactivecanonicalWntresponses[68-70].Mutationsinotherciliary-associatedproteinsalsosug-gestedaroleforciliainrestrainingWntsignaling(Chibby,Seahorse,SREBP1c)[71-75].Thesedifferencesmightbeattributedtotissue-andtime-dependentvari-ables,butalsotothevaryinglocalizationandfunctionoftheseproteins.ItisalsoimportanttonotethattherehavebeensomestudiesthatsuggestthereisnoconnectionbetweenciliaandWntsignaling.Maternal-zygoticift88zebrafish Table1Summaryofproteinsassociatedwithcanonical/non-canonicalWntsignalingandciliaCiliaorWntassociationReferenceWntproteinsInv(NPHP2)CiliarylocalizationandphysicalinteractionwithDvl[65,66]DvlDockingandpolarizationofbasalbodies,basalbodylocalization,targetedfordegradationbyInv[47,66]Cilia-associatedproteinsHyperactiveWntresponseinknockoutcelllines[68]UpregulationofcellularWntresponseinknockoutcelllinesandmutantmicemiceIft88aUpregulationofcellularWntresponseinmutantmicemiceIft40IncreaseinexpressionofcanonicalWntpathwaygenesinkidneyofmutantmicemiceIft20Increaseinnuclearbeta-cateninandexpressionofWnttargetgenesinkidneyofmutantmicemiceOfd1UpregulationofcellularWntresponseinmutantmicemiceChibbyBindsbeta-cateninpreventingnuclearentrynegativelyregulatingWntsignalingsignalingSeahorseBindstoDvl,constrainsWntsignalinginzebrafishzebrafishSREBP1cOverexpressiondisruptsciliogenesisandincreasescanonicalWntsignalinginXenopus.Xenopus.Ahi1/JbnAbrogatedWntsignalinginkidneyandcerebellumofmutantmice,facilitatesbeta-cateninentryintonucleus[19,106,143]Non-canonicalWntPCPeffectorsInturnedHighlyexpressedinciliatedtissue,requiredforciliogenesis.Actinassembly,Rholocalization,dockingofbasal[100,144-146,47,147]FuzzyRequiredforaxonemeelongation,predictedroleinvesiculartraffickingtraffickingFritzExpressedinciliatedtissue,requiredforciliogenesis,mutationsidentifiedinhumanciliopathiesciliopathiesCorePCPDubroyaRegulatesciliogenesis,ApicalActinassemblyassemblyFrizzledDefectiveciliogenesisatzebrafishkupffersVesicleVesicleDvlRegulatesciliogenesis,Actinassembly,Rholocalization,dockingofbasalbodies,associatedwithhumanciliopathyproteinsTMEM216andTMEM67TMEM67Celsr2/Celsr3Regulatesciliogenesisinmulticiliatedependymalcellsviabasalbodydockingatapicalplasmamembrane[150]PrickleRegulatesciliumlengthinzebrafishzebrafishVangl2aLocalizestosomecilia,xenopusbasalbodylocalizationandciliogenesis,zebrafishconflictingdatadataaFurtherdescribedinthelegendTheprimaryciliumwasfirstimplicatedinsuppressionofcanonical,-catenin-dependentWntsignaling,whilebeingrequiredfornon-canonicalWntsignaling(PCP).Oneofthefirststudiestomakethissuggestionimplicatedinversin(Inv),abasalbodyproteinassociatedwithcystickidneydisease,asamolecularswitchbetweenthetwoWntsignalingpathways.ItwassuggestedthatthisoccurredviatargetingofcytoplasmicDishevelled(Dvl)fordegradationatthebasalbody.Anothercilia-associatedprotein,thekinesin-likeproteinKif3a,isthoughttorestraincanonicalWntsignalingbyrestrictingtheCK1-dependentphosphorylationofDvl,whichresultsinanactivebeta-catenindestructioncomplex,limitingbeta-catenininducedtranscription.Itispossiblethatdefectsinproteininteractionsatthebasalbody,ratherthaninintraflagellartransport(IFT),couldberesponsiblefordysregulationofWntsignalinginciliamutants.SuchasuggestionisconsistentwiththefindingsthatbothIFT-mutantmiceandmutantzebrafishdonotshowdisruptionofcanonicalWntsignaling.Axin2(Wnttargetgene)andtransgenicWntreporternormalinift88,andmouseembryos[51].NoWntphenotypeinift88zebrafish[52].Conflictingzebrafishvangl2datacouldbeduetovaryinganimalmutantsandanalysismethods.May-SimeraandKelleyhttp://www.ciliajournal.com/content/1/1/7Page6of16 mutantslackallcilia,yetdisplaynormalcanonicalandnon-canonicalWntsignaling[52].Atmidgestation,andmutantmouseembryos,allofwhichlackprimarycilia,shownormalexpressionofthecano-nicalWnttargetgene,,andnormalactivationofatransgeniccanonicalWntreporter[51].Mouseembryo-nicfibroblastsderivedfromthesemutantsalsorespondnormallytoWntligands[51].However,thesefindingsdonotexcludethepossibilitythattherearetissue-andtime-dependentdifferencesinciliaryassociationwithWntsignaling.OneexplanationforthenormalWntresponsivenessshownbythesezebrafishandmouseciliamutantsmightbethatthebasalbodiesandciliarytran-sitionzonesintheseorganismsarenotdisrupted.Itispossiblethatiftheciliumismissingbutthebasalbodyandsurroundingstructuresremain,thisallowssomedegreeofWntsignalingtocontinue.ThisstrengthenstheargumentforthebasalbodyortransitionzoneregionbeingakeymodulatoroftheWntresponse.Non-canonicalWnt(PCP)signalingThePCPsignalingpathwaywasinitiallydescribedinandincludespolarizedlocalizationofcorePCPproteins,whichultimatelyleadstotheestablish-mentofpolarizedcellmorphology[76,77].Inverte-brates,PCPsignalinghasbeenshowntoberequiredforconvergentextension[78],defectsinwhichresultindis-ruptedneuraltubeclosures,aswellasmisorientationofhaircellsinthemammaliancochlea[79]andhairfolli-clesintheepidermis[80].OneoftheearlyindicationsthatciliaryproteinsmaybelinkedtoPCPregulationwastheobservationofPCPdefectsinciliarymutantcochlea,linkingbasalbodypolarizationwithPCPregu-lation[81,82].InrecentyearsPCPsignalinghasalsobeenfoundtoberequiredfortheorientationofbasalbodiesinseveralothertissues,includingpositioningofmotilemonociliaatthenode,gastrocoel,andKupffervesicle,whichisinvolvedintheestablishmentofleft-rightasymmetryinfish[47,83-86].Asdiscussed,initialobservationsofPCPdefectsinciliarymutants(mice,zebrafish)[66,82]suggestedaroleforciliainPCPsignaling.Concurrently,theemergingroleofPCPsignalinginrenalcystdevelopmentstrengthenedthishypothesisasmostoftheciliopathiesareassociatedwithkidneydiseases.Inversinhasbeensuggestedasamolecularswitchbetweenthecanonicalandnon-canonicalsignalingpathwaysbytargetingDishevelledfordegradationviatheAPCcomplexatthebasalbody[66].Inthemammaliankidney,cilia/basalbodydysfunctionhasalsobeensuggestedtoaffecttheorientationofmitoticspindles,associatedwithdefectivetubuledivi-sionandresultantpolycystickidneydisease[87].Defec-tiveciliogenesis,centrosomalamplification,centrosomepositioning,andmitoticspindleorientationhavebeenobservedinthekidneysofcentrosome/basalbodymutants(MKS1,MKS3,IFT20)[88-90].Interestingly,amorerecentstudyinwhichIFT40,anIFTcomplexAprotein(asopposedtoIFT20whichisanIFTcomplexBprotein),wasdeletedfrommousecollectingductsshowedthatdeletionofIFT40causescystickidneyswithoutalteringcentrosomeormitoticspindleposition[91].Similarly,datafrommicewithmutationsinpoly-cystickidneydiseasegenesPkd1Pkd2,andPkhd1conflictingandsuggestthatlossoforientedcelldivisionalonedoesnotinitiatecystformation.Whileorientedcelldivisionisdisruptedinmutants,thisaloneappearsnottobesufficienttoproducekidneycysts.IncontrastmutationsinPkd1andPkd2doresultincystformationdespitenormalorientedcelldivision,suggest-ingthatlossofPCP-relatedorientedcelldivisionisnotsufficienttogeneratekidneycysts[92].Therefore,whilebasalbodypositioning,orevenearliercentrosomeposi-tioning,appearstorequiresomedegreeofintactPCPregulation,itisunclearwhetherciliaorbasalbodyintegritycanaffectPCPsignaling.Ifso,howandwhatisrequiredfirst;intactPCPforcentrosomepositioningorintactcentrosomesforsubsequentpolarizingevents?CiliarylocalizationofseveralcorePCPproteinsaddsafurtherlayerofcomplexitytodissectingthisrelation-ship.TheprotocadherinFat4wasshowntolocalizetothecilium,andmicedisplayseverePCPpheno-typesthoughttobecausedbyabnormalorientationofmitoticspindles[93].TwoothercorePCPproteins,Vangl2andDvl,havealsobeenreportedtoassociatewiththeprimarycilium.WhereasDvl,whichisdegradedbyinversin,docksbasalbodiestotheapicalmembraneprecedingciliogenesis[94],Vangl2isrespon-sibleforasymmetricpositioningofmotilecilia[95].Vangl2hasbeenshowntolocalizealongtheciliaryaxo-nemeinolfactoryneurons[82]andinciliaofmultici-liatedependymalcells[96].Inaddition,Vangl2hasalsobeenshowntointeractbiochemicallyandgeneticallywiththeciliaryproteinBBS8inzebrafish,resultinginlateralitydefectsduetodefectivefluidflowattheKupf-fersvesicle,theequivalentoftheembryonicmousenode[97].Fluidflowinfluencescentriolarmovementandcontributestoorientationofmotileciliainconjunc-tionwithPCP[96-98].Itispossiblethatcorrectbasalbodyandcentrosomepositioningcouldberequiredforsubsequentregulationofcytoskeletalarchitecture.Thebasalbody,asaMTOC,couldregulatethenucleationandoutgrowthofmicrotubules;indeed,severaloftheBBSproteinshavebeenshowntoparticipateinmicrotubulenucleationandgrowth[27,99].EvidenceforciliaandPCPregula-tionoftheactincytoskeletonisemerging.ManyPCPproteinsareinvolvedinRho-mediatedapicalactinMay-SimeraandKelleyhttp://www.ciliajournal.com/content/1/1/7Page7of16 assembly,Rholocalization,andbasalbodydocking,whichincludestetheringofmembraneboundvesicles(Table1).XenopusandDrosophilaPCPeffectorsInturnedandFuzzyareresponsibleforciliogenesisandhavebeenshowntoaffectapicalactinassemblyandmicrotubulepolaritybutnotnucleation.Defectsmayoccurincytoskeletalassemblythatimpairmovementofbasalbodiesandcentrioles[100].ThePCPcomponentFritzcontrolslocalizationofthecytoskeletalproteinSeptin,crucialforcollectivecellmovementandciliogen-esisinembryos.Arecentstudyfoundanenrichmentofnon-synonymouscodingchangesinintwociliopathies:Bardet-BiedlandMeckel-Grubersyndrome[101].Ofnote,theidentifica-tionofthepolycystickidneydiseaseproteinPC1atcelladherensjunctionsanddesmosomes,andnotjustciliaandbasalbodies,indicatesapossibleadditionalroleforPC1incontrolofcytoskeletalrearrangementpotentiallydownstreamofPCPsignaling[102].Aswillbediscussedinsubsequentsections,regulationofvarioussignalingmolecules(includingRhoAandDvl)couldoccuratthebasalbody.ExamplesofthisincludetheSmurfubiquitinligases,whichtargetpro-teinsfordegradationbyproteasomes.TheseproteinsregulatePCPsignalingvialocalizedPrickledegradationbythePar6-Dvl2-Smurfcomplex[103].Incochlea,polarityproteins(Prickle1)arenotasymmetri-callydegradedattheregionofthebasalbodyasinwild-typeanimals.Thisfurtherimplicatesthebasalbodyasaregionalsignalingcenterassistingtheactivationand/ordegradationofsignalingproteins.Arecentpro-teomicscreenfoundseveralPCP-associatedproteinsinthehumancentrosomesuchasPRICKLE3,SCRIB,CCDC66,andAlbatross[104].Thisscreenalsoidenti-fiedasymmetricallylocalizingcentriolarproteinsanddifferentiallyagedcentriolesincelldivision(Figure2),whichappeartobeimportantincellpolarizationandstemcellfatedeterminationviaasynchronousmicrotu-bulespindleorientationandprimaryciliagrowthwthSimilartothepreviousquandaryregardingPCPandcentrosome/basalbodypositioning,akeyquestionthatremainstobeelucidatediswhichcomesfirst:isintactPCPsignalingrequiredforciliogenesis,orisanintactciliumnecessaryforcorrectPCPsignaling?Asdown-streameffectsofPCPsignalingareassociatedwithactinandmicrotubules,onecouldenvisageapossiblefeed-backloopwhereintactcytoskeletalarchitectureandfunctionisinturnnecessaryforciliaformationorfunc-tion.AnotherkeyquestioniswhethertheciliumitselfisresponsibleforthedefectsobservedinsomePCPmutants.Manyofthecilia-relatedproteinsarenowbeingidentifiedinothercellularlocationsandcouldpossiblyberequiredforadditionalintracellularfunctions.Assuggestedbefore,theciliumisheavilydependentonciliaryproteins,anddefectsintheirfunc-tionmaybemorereadilyobservableatthisorganelle.CanonicalWntsignalingAtfirstglance,the-catenin-dependentcanonicalWntsignalingpathwayappearsrelativelystraightforward.WntligandsbindandactivatetheFrizzledclassofreceptors,therebystabilizing-catenin,whichthenentersthenucleusandsubsequentlyactivatesWnttar-getgenes[105].Thispathwayrequiresalargenumberofregulatorycomponents,whichdelicatelybalancemul-tiplefeedbackmechanisms.IntheabsenceofWnt,cateninissequesteredanddegradedbyadegradationcomplexconsistingofAxin,Adenomatosispolyposis(APC),Glycogensynthase3beta(Gsk3),andCaseinkinase1(CK1).UponWntstimulation,thisdegradationcomplextranslocatestothemembranewhereitisinac-tivatedviaphosphorylation,subsequentlystabilizingcateninandreleasingitforentryintothenucleus.Jou-berin,mutationsofwhichcausetheciliopathyJoubertsyndrome,hasbeenshowntofacilitate-catenintrans-locationtothenucleus[106].Aswillbefurtherdis-cussed,itispossiblethatthebasalbodyisthesiteofregulationoftheAPCcomplex,localizationofwhichcouldinturnaffectitsregulatoryactivity.Manycompo-nentsofcanonicalWntsignalinglocalizeorassociatewiththecilium(Table1,Figure3).Similartotheques-tionastowhatregulateswhatwithregardtociliaandPCPsignaling,isthedependenceofcanonicalWntsig-nalingoncytoskeletalarchitecture.Asdiscussedbelow,cilia/basalbodyassociationsbetweenAPCandcanonicalWnt-relatedkinesinsarebeginningtoemerge.Thesefindingsfurtherenhanceourunderstandingoftheroleofthecilium/basalbodyinWntregulationorviceversa.AswefurtherdiscussthevariouscomponentsrequiredforWntsignalingandtheirassociationwithciliaandbasalbodies,onefactortoconsideriswhichismoredependentontheother:isintactcytoskeletalarchitecture(includingciliaandbasalbodies)requiredforWntsignaling,orisWntsignalingrequiredfortheestablishmentofafullyfunctionalcytoskeletonencom-passingaciliumandbasalbody?ThetumorsuppressorproteinAPCisoneofthekeycomponentsinthecanonicalWntsignalingpathway.Mostofthescientificliteratureisfocusedarounditsabilitytobindandinducedegradationof-catenin.However,thereareadditionalrolesforthisproteininvolvingtheorganizationofcytoskeletalnetworks[107].APChasbeenidentifiedatanumberofintracel-lularsites(plasmamembrane,cytoplasm,centrosomes,kinetochores,nucleus)[108],aswellasatthedistalMay-SimeraandKelleyhttp://www.ciliajournal.com/content/1/1/7Page8of16 endsofmicrotubulesattheedgesofmigratingepithe- lialcells[109-111].APCa ssociateswiththeplasma membraneinanactin-dependentmannerandregu- latesactincytoskeletalnetworksrequiredforcell polarizationanddirectionalmigration(reviewedin AkiyamaandKawasaki[107]).TheAPCproteinalso bindsandstabilizesmicrotubulesubsets,associating bothdirectlyandindirectlywithmicrotubulesviaits C-terminaldomain.Directinteractionwiththemicro- tubulebindingproteinEB 1[112]enhancesmicrotu- buleassemblyandstabilization[113],whiletransport ofAPCtowardsmicrotubuleplus-endsinvolvesseveral kinesins(kinesin-1,KIF3andKIF17,kinesin-2) [109,114].TransportofAPCtoitsvariouscellular locationsbykinesinmotors playsavitalroleinmicro- tubulestabilization,activationofproteinkinases,and cellpolarizationandthereforekinesinsareofpara- mountimportancetotheseprocesses.Importantly,in regardtociliogenesis,APChasbeenshowntobeone ofthefewproteinsthatlo calizesasymmetricallyto mother vs .daughtercentriolesandmayplayarolein primaryciliaformationand function[104].Thismost likelyoccursviaregulationo factinormicrotubulesat thebasalbodyregion.Onlyasmallnumberofproteins displaythisdistinctivefeatureofasymmetricalcen- trioledistributioninacellcycle-dependentmanner; otherknownexamplesincludemicrotubule-related proteinssuchasEB1,whic hinteractsdirectlywith APC,aswellascilia-relatedproteinssuchasCP110 [104].TheEBproteins,EB1andEB3(butnotEB2), alsodirectlypromoteciliabiogenesisviastabilizing minus-endmicrotubulesatthecentrosomeandbasal body[115].Interestingly,lackofEB1andEB3results indefectivemicrotubuleanchoringatthecentrosome/ basalbodyandvesiculartraffickingatthebaseofthe cilium.TheauthorssuggestthatEB1and/orEB3dis- ruptciliogenesisthrougheffectsatthebasalbodyor cilium,notthroughalterationsofcytoplasmicmicrotu- buleplus-enddynamics.BothEB1andEB3interact withproteinsimplicatedin centrosomalmicrotubule minus-endanchoringandciliaryvesicletransportsuch asPCM1,Ninein,Cep290,Cep170[115].Studiesin bothmammaliancellsand Chlamydomonasreinhardtii haveidentifiedEB1localizingtosubdistalappendages ofthemothercentriole/basalbody[116,117],which areinvolvedinmicrotubuleminus-endanchoring [115,118]. Asthecentrosomeandbasalbodyregionatthebase oftheciliumisbeginningtoemergeasacellularsignal- ingcentre,itisnotdifficulttoimaginethatAPC,inits roleaspartofthe b -catenindestructioncomplex,might beanchoredtothisregion.I nsupportofthistheory, Figure3 DiagrammaticoverviewofWntinvolvementwiththecytoskeleton .TransportofAPCbykinesinmotorsplaysanimportantrolein microtubulestabilization,activationofproteinkinasesandcellpolarization,whichcouldallberegulatedviatheGsk3 b kinase.Phosphorylationof kinesinsviakinasescontrolstheirsub-cellularlocalizationandactivity.Gsk3 b isoneofthecentralkinasespredominantlyassociatedwithWnt signalingandmayinfluencemanyfunctionsofkinesinsthroughregulatingcargobinding.PhospohrylationofAPCbyGsk3 b decreasesthe interactionofAPCwithmicrotubules,subsequentlydecreasingmicrotubulestability.Gsk3 b couldbeconsideredamasterregulatorofkinesin controloverMTdynamics,duetoitsabilitytoregulatearangeofkinesins. May-SimeraandKelley Cilia 2012, 1 :7 http://www.ciliajournal.com/content/1/1/7 Page9of16 otherrelatedcomponentsarealsobeingidentifiedinthisareasuchasmembersofthekinesinclassofmotorThekinesins,aclassofmotorproteins,exertaregula-toryeffectonWntsignaling.Manyproteinsthatbindtomicrotubulesparticipateintheregulationofmicrotu-buledynamics.Theseincludethekinesins,whicharepoweredbythehydrolysisofATP.Althoughkinesinssharesimilarmotordomains,theyexhibitavarietyoffunctions,includingregulationofmicrotubuledynamics.Kinesinsareknowntobecrucialforciliogenesisandintraflagellartransport[15,119].Recently,acentriolarkinesinKif24wasshowntoremodelacellularsubsetofmicrotubulesregulatingciliaryassembly.Kif24preferen-tiallylocalizestothemothercentrioleandisrequiredforcentrosomallocalizationofCP110[46].N-kinesinsarecommonlyassociatedwithanterogradetransport,whileC-kinesinsareinvolvedwithretrogradetransport.M-kinesins,bycontrast,donotpossessamotorfunc-tion,butplayaroleinmicrotubulestabilization[120].Theabilityofkinesinstocontrolmicrotubuledynamicsenablesthesemoleculestoexertregulatoryfunction.Suchregulatorymechanismsarecoupledtovarioussig-nalingpathways,whichcould,inturn,controltheirassociationswithmicrotubulesorregulatemicrotubulesand/ormicrotubuledynamicsdirectly.Askinesinscanbebothsignalingtargetsandsignalingintermediates,kinesincontrolovermicrotubuledynamicsiscomplex.Wntsignalingatthecentrosomeisoneofthewaysinwhichkinesinsexertregulatoryfunctions.NotonlyarekinesinsrequiredforlocalizationofAPCtothecen-triolesandcentrosome,butanotherkinesin,MCAK(alsoreferredtoasKIF2C),alsoassociatespredomi-nantlywiththesestructures.Asamemberofthekine-sin-13family(othermembersofwhichincludeKIF2AandKIF2B),MCAKhasbeenshowntobecriticalformicrotubuledynamicsandorganization[120].Thispro-miscuouskinesin,localizesdynamicallytocentrosomes,kinetochores,thespindlemid-zoneduringcelldivision,andtomicrotubuletips[121-123].MCAKdimersbindtomicrotubuleends(plusandminus)andareabletoinduceaconformationalchangecausingthetubulintobendandsubsequentlydestabilize.AsMCAKhasbeenshowntoassociatedirectlywithAPC,MCAKdysfunc-tioncouldhindercorrectlocalizationofAPC,therebydisruptingWntsignaling.MCAKhasalsobeenshowntoassociatewiththemicrotubule-associatedproteinEB1viaitsC-terminusatgrowingendsofmicrotubules[124,125].Whenasso-ciatedwithEB1,MCAKappearstobeinaninactiveconformation.Thiscouldprovideamechanismtofacili-tatefastswitchesbetweengrowthanddepolymerizationofmicrotubules,whichisofparticularimportancedur-ingciliogenesis.AsEB1alsoassociatesdirectlywithAPCandplaysanimportantroleinmicrotubulestabili-zation,itispossiblethatthesethreemoleculesareinvolvedinaregulatoryfeedbackloop(Figure3).Insummary,kinesinsarenotonlyrequiredfortraf-fickingofstructuralorsignalingcomponentstoandalongthecilium,butarealsorequiredforstabilizationandregulationofthecytoskeletalinfrastructurealongwhichthesemoleculestravel.Inthesecapacitiesthekinesinsareabletoexertsignificantcontrolovervariouscellularmechanisms.Factorsthatregulatebothcanonicalandnon-canonicalWntsignalingIdentificationofciliaryproteinsthataffectbothcanoni-calandnon-canonicalWntsignalingarenotsurprising,consideringthattheciliumcanpotentiallybeseenasabranchpointbisectingthetwopathways.Theseincludeafamilyofproteins,theGTPases.TheGTPasesarestructurallyrelatedtokinesins,however,theyhydrolyzeGTPinsteadofATP.GTPasesareinvolvedwithcellulartrafficking,andseveralmembersoftheARF-likefamilyofsmallGTPaseshavebeenimplicatedinciliaryfunc-tion[126,127].AsthissubsetofGTPasesisgenerallyassociatedwithmembranetraffickingandmicrotubule-associatedprocesses,itismostlikelythattheyarerequiredforthetransitionbetweenpost-Golgivesiculartraffickingtothebasalbodyandkinesin-dependentIFTwithinthecilium,atthetransitionzone.Cdc42,aRhofamilyGTPase,isakeyplayerinWnt-dependentcytoskeletalrearrangementsandakeyregula-torofcellpolarity.Cdc42actsasamolecularswitch,modulatingawiderangeofsignalingpathways.Incellu-lartrafficking,Cdc42functionsatseveralstepsofendo-cytosis,recycling,andbiosynthesis,influencingpolarizedtraffickinginepithelialcells[128].Initsroleasapolar-ityregulator,Cdc42hasbeenshowntoorchestratepolarityofmicrotubuleandactincytoskeletonsviatwodifferentsignaltransductionpathways[129](Figure4).First,directinteractionwithPar6leadstoactivationofaPKCandsubsequentaccumulationofAPCattheplus-endtipsofmicrotubulespromotingpolarizationofthemicrotubulecytoskeleton.TherequirementofDvl,Axin,andtheWnt5aligandinthisprocessimplicatestheCdc42/Par6complexinestablishingmicrotubulepolarityviatranscription-independentnon-canonicalWntsignaling[130].Second,Cdc42associationwithPAKresultsinactivationoftheRacguaninenucleotideexchangefactorPIXandsubsequentdownstreamRac-dependentpolarizationoftheactincytoskeleton[129,131].IthasbeensuggestedthattheParpolaritycomplexcanalsoaffecttrafficking,furtherlinkingthedualactionsofCdc42[132].DvlhasalsobeenshowntoMay-SimeraandKelleyhttp://www.ciliajournal.com/content/1/1/7Page10of16 directlyregulateaPKCstabilizationandactivationpro- motingaxondifferentiationmediatedbythePar6com- plexinculturedhippocampalneurons[133]. Importantly,theCdc42/Par6/aPKCcomplexhasbeen localizedtotheprimaryciliumandhasbeenshownto benecessaryforciliogenesisinmammaliancells [134,135].Whatdoesthismeanforcilia?Asseveral othercomponents(forexample,Dvl,kinases,GTPases) requiredforsubsequentsignalingeventsalsolocalize nearthecilium(basalbody),itispossiblethatCdc42is functioningunderthecontr olofapotentialbasalbody signalingcenter .Cdc42canberegardedasamolecular switch,anditsciliarylocalizationputsitattheputative siteofregulationbetweencanonicalandnon-canonical signalingatthebaseofthecilium.Amorecomprehen- siveroleforCdc42attheciliumcouldencompassvesi- cletransportanddocking(fusionwiththeplasma membrane)inthedeliveryofproteinsnecessaryfor ciliogenesis.Suchaviewissupportedbyrecentfindings thatCdc42localizestheexocyst(ahighlyconserved eight-proteinmembranetra ffickingcomplex),tothe baseofthecilium,whereitispossiblystabilizedby bindingtotheParcomplexviaPar6[136].Oncethe exocystcomplexisstabilized,itcanthentargetand dockfurthercilia-boundvesicles.Amoreupstreamrole forCdc42involvementinciliogenesiscouldinvolve basalbodypositioning,centrosomeregulation,spindle orientation,andcellularpolarity,allviatheabilityof Cdc42toregulatethecytoskeleton(Figure4). Cdc42isnottheonlyGTPaseassociatedwiththe cilium.OtherGTPasesthatalsolocalizetothecilium couldfunctioninasimilarwayto,orinconjunction with,Cdc42.OnesuchGTPaseisARL6/BBS3,oneof theBBSproteinsnotassociatedwiththecoregroupof BBSproteinscomprisingtheIFT-participating BBSome ,astableproteincomplexwhichfunctionsin primaryciliabiogenesis[14] .Thissuggestsamoredis- tinctfunctionforARL6/BBS3,whichmayberequired forregulatingmembranetraffickingatthebaseofthe ciliumandconsequentlyaf fectingciliaassemblyand subsequentciliarysignalingevents[137].BBS3loca- lizestodistalendsofthebasalbodiesakintotransi- tionfiberswheretheciliary-boundvesiclesdockand fuseatthebaseofthecilium.AnadditionalBBSpro- tein,BBS1(anothercomponentoftheBBSome),is knowntointeractwithRabin8,aGDP-GTPnucleotide exchangefactor(GEF)forthesmallGTPaseRAB8 [14].Rabin8co-localizeswiththeBBSomeatthebasal bodywhileRAB8enterstheciliuminitsGTP-bound stateandisnecessaryforciliogenesis.InturnRAB8 interactswithRabaptin5,whichalsolocalizestothe basalbodyandisassociatedwiththeIFTprotein Ey11/DYF11.Itisthoughtthatthistrio(RAB8/Rabap- tin5/DYF11)maybecriticalforthetransitionbetween vesiculartraffickingtotheciliarymembraneandIFT. Furthermorein C.elegans ,thesmallGTPaseRAB5has beenshowntolocalizetothebaseoftheciliumand regulatepolycystin1and2 removalfromthecilium [138]. Otherproteinsthatappeartointerceptthetwosignal- ingpathwaysincludepVHL.ThepVHLproteinhas beenproposedtocontrolciliogenesisbyorienting microtubulegrowth.ThisproteinisacargoofKif3and hasbeenshowntointeractwiththepolarizingPAR3- PAR6-aPKCcomplex,suggestingthattheseproteins mayoperateinthesamepathwaytoregulatecortical growthofmicrotubulesandformationofcilia[139]. PreciselyhowpVHLsignalsviakinesinmotorsor microtubulestocontrolcellularprocessessuchaspolar- ityaswellasciliogenesisre mainstobeinvestigated. Intriguingly,KIF17hasrecentlybeenshowntostabilize microtubulesandcontribut etoestablishingepithelial cellpolarity,purportedlybycontributingtoAPClocali- zationatmicrotubuleplusends[114].Anotheraspectto consideristheassociationofbothkinesinsandIFTpro- teinswithmitoticspindleassembly[29].Itisthought thatwhileallofthekinesin-13sarerequiredforspindle assembly,itislikelythatvariousmembersmight Figure4 DiagramofCdc42regulationofcytoskeletalrearrangement .Cdc42cancontrolpolarityviatwoseparatepathways.Activationof Par6/aPKCinhibitsGSK-3,whichresultsinpolarizationofmicrotubules(MT).Rac-dependentpolarizationoftheactincytoskeletonisduetoPak activated b PIX. May-SimeraandKelley Cilia 2012, 1 :7 http://www.ciliajournal.com/content/1/1/7 Page11of16 contributetospindlepolarityanddynamicsthroughdif-ferentpathways.Identificationofmoleculesthatarealreadyknowntofunctionasmolecularswitchesbetweensignalingpath-ways,suggeststhattherearepossiblymoreawaitingdis-covery.ThesemayalsohavethecapacitytocontrolorfinetuneWntsignalingdecisions.Thebasalbody,anemergingsignalinghotspotperfectlylocatedatthebaseofthesensory-loadedciliummightbeanidealcellularlocationfortheswitch.ConclusionsWeareonlybeginningtounderstandtheroleoftheciliumanditsbiologicalinfluence.Theciliumismorethanjustamicrotubularappendageanchoredtothecellviathebasalbody.Themultiplepartsoftheciliumfromthebasalbodytothedistaltip(Figure1)arejustbeginningtobedefined.Theciliummayserveasareceptivedeviceforsignalsinthecellsenvironment,helpingtoregulatedevelopmentandothercellfunc-tions.Onekeyquestionthatremainstobeaddressediswhethertheciliumcouldpotentiallyreleasesignalingfactorsforcommunicationwithitsenvironment.Thebaseoftheciliumisthemostcrucialpartofthiscommunicationdevice,astheincomingsignalsneedtobeorderedandregulated.Itisperhapsnotacoinci-dencethatthebasalbodydoublesastheMTOCofthecell.Thebasalbodyismostlikelyregulatingotherinter-nalsignalingandtraffickingeventsaswell.Regulationofthisregionisahugelycriticalandasyetunderexploredarea.However,manykinases,phosphatases,kinesins,andproteosomalproteinsthatlocalizetothisregionarebeingidentified.Ofparticularconsiderationinregardtocilia,twoofthekinasesknowntoaffectWntsignaling,CKIDandPPPIC,havebeenidentifiedintheciliarypro-teome[68].Thebaseoftheciliumisnotjustasignalingcenter,butalsoasortingcenter.Anemergingconceptisthatofscaffoldproteinsactingashubsforcontrollingtheflowofcellularinformation(spatialandtemporalorga-nizationofmolecules)withinacell[140].Severalbasalbodyproteinscouldbeseenassuchpotentialscaffoldproteins.Forexample,Cep290isanenormousproteinandappearstohavewide-rangingeffectsonbothbasalbody/ciliarystructureandregulation[141].Thisconceptfitswiththenotionofaciliaryporeakintothenuclearpore,regulatingciliaryproteincomposition[7].Isthisgate-keepingfunctionrestrictedtociliaryproteins?Mightthebasalbodyberegulatingothertraffickinginthecell?AsanMTOCfortheentirecell,thebasalbodycouldpossiblyregulatemuchmorethanjustwhatishappeningatthecilium.Perhapsitcanbecomparedtoabusterminalforcilia-relatedproteins:wherecargoisloadedandunloadedpriortobeingsentalongitswayandbusroutescanberegulatedandestablished.Importantly,arecentpaperdemonstratedthatWntsig-nalingregulatesspindleasymmetrytogenerateasym-metricdistributionof-catenininC.elegans[142].Thisisoneofthefirstexamplesofnuclearproteinlocaliza-tionbeingcontrolledviamicrotubulesandsuggeststhatmicrotubuleorganizationmayhavedirectimpactongeneexpressioninthenucleus.ItalsosuggeststhatWntsignalingmaybebothup-anddownstreamofmicrotubulemodulation.Theconceptoftheciliumasapotentialregulator(molecularswitch)betweencanonicalandnon-canoni-calWntsignalinghasbeensuggested[66]andcouldexplainwhythereisconflictingdataontheassociation(whetherpositiveornegative)betweenciliaandWntsignaling.TheinterplaybetweenregulationofWntsig-nalingandthebasalbody/MTOCcouldexpandthemechanismsbywhichciliaareinvolvedinthemolecu-larswitchbetweencanonicalandnon-canonicalsignal-ing,asallofthesemolecularpathwaysconvergeatthebasalbody.Itispossiblethatdefectsinproteininter-actionsatthebasalbody,ratherthaninIFT,couldberesponsiblefordysregulationofWntsignalinginciliamutants.Itwouldcertainlybeanoversimplificationtoassumethatasingletypeofciliumwouldsufficefortheneedsofeverycell.Thekeytoourunderstandingofciliafunc-tionwillbetodistinguishthecharacteristicsofciliaindifferingcellsandtissues.Therecentexplosionofciliaryresearchislikelytocontinuefortheforeseeablefutureasthislongforgottenorganellerevealsitsinnersecrets.APC:adenomatosispolyposis;CK1:Caseinkinase1;BBS:Bardet-Biedlsyndrome;GSK3:glycogensynthase3b;IFT:intraflagellartransport;NPHP:nephronophthisis;PCM:pericentriolarmaterial;PCP:planarcellpolarity;Shh:Sonichedgehog.AcknowledgementsTheauthorswouldliketothankRRachel,MKai,ATadenev,andAColesforhelpfuldiscussionsandreadingofthemanuscript.WewouldalsoliketoacknowledgetheeditorialassistanceoftheNIHFellowsEditorialBoard.HMSpreparedthemanuscript,MKeditedthemanuscript.Bothauthorsreadandapprovedthefinalmanuscript.CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.Received:11October2011Accepted:2May2012Published:2May20121.BakerK,BealesPL:Makingsenseofciliaindisease:thehumanAmJMedGenetCSeminMedGenet2.WareSM,AygunMG,HildebrandtF:Spectrumofclinicaldiseasescausedbydisordersofprimarycilia.ProcAmThoracSoc3.LeeL:Mechanismsofmammalianciliarymotility:Insightsfromprimaryciliarydyskinesiagenetics.May-SimeraandKelleyhttp://www.ciliajournal.com/content/1/1/7Page12of16 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