Background: recognized as an important integral membrane protein, responsible for establishing and

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1 Background: Sinceitsdiscovery50yearsago[1],Na + /K + ATPasewasquickly recognized as an important integral membrane protein, responsible for establishing and maintainingtheelectrochemicalgradientsofsodiumandpotassiumionsacrossthecellular plasma membrane. Utilizing the energy of ATP hydrolysis, it transports cytoplasmic Na + (out) and extracellular K + (in) in a 3:2 ratio and maintains the gradient of these cations acrossthemembrane[2].thus,itcontrolstheelectrolyticandfluidbalanceinthecellsand organsthroughoutthebodyofallorganisms[3].amongitsotherfunctions,na + /K + ATPase maintainstherestingpotentialofcells,cellularvolumeandisalsoinvolvedintransportof solutesinandoutofthecell.sinceitisresponsibleformajorityoftheenergydemandsof the living cell, Na + /K + ATPase is arguably the most crucial cellular pump across all biologicalsystems. Ionichomoeostasisofmostbiologicalsystemsincludingtheauditorysystemdepends onthenapump.thelocalizationandtheroleofthispumphasbeenlinkedtothe maintenanceoftheinnerearosmoticbalance.theinnerearisfilledwithak + rich extracellularfluidknownasendolymph,whichisessentialforpreservingthesensory structurespresentinthecochlea.lossofendolymphaticbalancecausescollapseofthe endolymphaticcompartment,leadingtohearinglossinmammals[4].maintainingthe endolymphhomeostasisisofcriticalimportancetosustainauditoryfunction.k + channels andpumpsincludingna + /K + ATPaseensurepropercyclingandsecretionofK + ionsinthe supportcellsinthestriavascularisofthecochlea.thena + pumphasalsobeenlinkedto agerelatedhearingloss[5]andmeniere sdisease[6].adetailedfunctionalanalysisofthis pumpisthereforenecessarytogaininsightintothemolecularphysiologyofhearingloss 1

2 duetolossofionichomeostasisintheauditoryorgan.weareusingtheauditory mechanosensorysystemofdrosophilamelanogasteraswellasthemoleculargeneticand electrophysiologicaltechniquesatourdisposalintheflymodelsystem,tounderstandthe roleofna + /K + ATPaseinmaintainingionichomoeostasisinhearing. Animalhearingorgansaremadeofspecialsensoryneuronscalledmechanosensory neurons.althoughvertebrateandinvertebrateauditorysystemsaredifferentstructurally andtheyhaveevolvedindependentlyfromthesameprimitivemechanosensors[7],[8], therearestrikingsimilaritiesbetweenthetwolineagesbothatthegeneticlevelandthe molecularlevel.theflyauditoryorgan,johnston sorgan(jo)ishousedinthesecond segmentoftheflyantennawhichhasthreesegments(fig1a).thejocomprisesofanarray of~250auditoryunitsorscolopidia.eachscolopidiumcomprisesof2 3ciliatedsensory neurons(chordotonalneurons)associatedwithseveralsupportcells.thesebipolar neuronsaremonodendriticwithasingledistalciliumandaproximalaxon(fig1b).the scolopalecell,whichisoneoftheprinciplesupportcells,enclosestheauditoryneuronsina fluidfilledlumen,whichisbelievedtoberichink + ions.thisfluidiscalledthereceptor lymphwhichissimilartothecochlearendolymphandliketheendolymph,isbelievedtobe richink + ions.auditorymechanosensationinvolvesthetransductionofthemechanical soundstimulusintoaneuronalimpulse.usingelectrophysiologicaltechniqueswecan recordthesesoundevokedpotentialsfromtheauditorynerveofthefly. Na + /K + ATPasehastwoprincipalproteinsubunits,namelythealpha(α)andbeta (β)subunits.theα subunitpossessesthecatalyticporethroughwhichtheionicexchange takesplace.theβ subunitisessentialfortheproperna + /K + ATPasefunctioning,asit controlstheaffinityofα subunitforna + andk + ions.β subunitmayalsoplayacriticalrole 2

3 inthetraffickingofα subunittotheplasmamembrane[9]afteritsassemblyinthe endoplasmicreticulum[10].thereisgrowingevidencethatathirdna + /K + ATPasesubunit thegamma(γ)subunitalsoknownasfxyd2,hasregulatoryrolesinna + pumpactivity [11],[12]. TheplasmamembranelocalizedDrosophilaNa + /K + ATPasealsohasαandβasits2 principalsubunits.therehasbeennoreportofaflyhomologoftheγsubunit.bothαandβ subunitshavemultipleisoforms.drosophilahastwoknownα subunitgenes,atpαand CG17923[13],whichcodefortheα subunits.theatpαhasatleast10knownα subunit mrnaisoforms[14].cg17923appearstohaveminimalexpressionasitstranscriptsare poorlyrepresentedintheexistingcdnalibraries[13].sofar2βsubunitshavebeen characterizedindetail,nervana1andnervana2,bothexpressedbroadlyinthenervous systemandepithelia.insituhybridizationinembryosshowthatathirdcomponent, nervana3(nrv3),isexpressedinasubsetofcentralnervoussystem(cns)neuronsand peripherally,inchordotonal(cho)organneurons.inaddition,nrv3proteinexpressionin someoftheadultperipheralnervoussystem(pns),includingchoorgans,suggestthat nrv3hasthepotentialtoplayaroleinflyhearing.thealphasubunitappearstocolocalize withnrv3inthejoneuronalsomamembrane,butnotinthecytoplasm.inadditionthereis stronger,moreapicalalphasubunitproteinexpression,thatmaybepresentinthe scolopalecells(fig2). WehypothesisthatNa + /K + ATPaseinDrosophilamelanogasterplaysafunctionalrolein establishingandmaintainingtheionicgradientofthescolopalecellofthejo.scolopale spaceislikelytobek + richinordertodrivethereceptorpotentialandsustainthesound 3

4 evokedpotential.wealsopredictthatthena + /K + ATPasepumpispresentpreferentiallyin theablumenalplasmamembraneofthescolopalecellwhereitperformsitsfunction. Methodology: Geneticstrainsused NompAGal4;Sp/CyOandAtonalGal4weregiftsfromSokolTodiandB.Hassan.TheGFPproteinfusionlineATP wasdonatedbyw.chia.uas nrv3 RNAiandUAS ATPα RNAiwere orderedfromvdrcstockcentre.thenp6215gal4wasorderedfromthedgrcstock centre. Electrophysiology Auditoryrecordingswereconductedonexperimentalandcontrolflies.Theanimalwas mountedona200µlpipettewiththetipcutoffsuchthatonlyitsheadprotrudesout.the neckwasimmobilizedwithplasticine.thepulsecomponentofthedrosophilacourtship songwasplayedthroughaspeakerandthesoundwastransportedthroughatygontube placedatadistanceof1mmfromtheheadofthefly.twotungstenelectrodeswereused forelectrophysiologicalrecordings.therecordingelectrodewasinsertedatthejoint betweenthefirstandsecondantennalsegmentandthereferenceelectrodewasinsertedin thehead,penetratingthecuticlefromadorsofrontaldirection.itwasascertainedthatthe antennarotatedfreelyandspontaneouslyduringtheelectrophysiologicalrecordingto ensuregoodresponse.thesignalswereamplified,digitizedandnormalizedusing Superscope3.0software.Detailsabouttheauditoryelectrophysiologyassayareavailable in[15] ElectronMicroscopy 4

5 TheheadsofNompAGal4;RNAi αanditssiblingswerefixed,processedandfinally embeddedineponresinaccordingtotheprotocoldescribedin[16]..transmission electronmicroscopywasconductedonthemwithhitachih ImmunohistochemistryandMicroscopy: DrosophilaantennaweredissectedinPBS,fixedin4%paraformaldehydefor30min, embeddedino.c.t.andthencutinto25µsectionsinacryostat.theantennaewerewashed severaltimesin1xpbsbeforefixation.theantennaewerestainedwithprimarypolyclonal antibodynrv3,agenerousgiftfromg.bietelandα5,monoclonalantibodyfromuniversity ofiowadevelopmentalstudieshybridomabank.secondaryantibodiesusedwerealexa Fluor 488,TRITC.TexasRedPhalloidindyewasalsousedtostainthescolopalerodsin ATPαflystrain.AllimagedweretakenusingaLeicaConfocalMicroscopeexceptforthe ATPαfusionproteinimages,whichweretakenusingDeltaVisionDeconvolution microscope. Statistics: Statisticalanalysesoftheelectrophysiologydatawereperformedusingstudent sttestand one wayanova. Results/Discussion: A)Na + K + ATPasesubunitsareexpressedinthenervoussystemingeneraland specificallyinthejoscolopidia: WewantedtoseewhichNa + K + ATPasesubunitsarepresentintheDrosophila mechanosensoryneurons,specificallythechoneuronsofthejoinlatestageembryo. Immunohistochemistryinembryos,usingamonoclonalantibody(α 5)againstthealpha 5

6 subunitshowedexpressioninplasmamembraneofbothcnsneuronsandperipheral epithelialcells(fig2:c&d).usingapolyclonalantibodyagainstthenrv3protein,wehave shownthatitexpressedinasubsetofthecnsneuronsandperipherallyinthecho neurons,specificallythelateralpentascolopidialorganlch5andthev ch(embryoniccho neurons)ineachabdominalsegmentoftheembryo(fig2:e&f).insituhybridization studieswithnrv3cdnaandwithnp6215gal4reportershowedsimilarembryonic expression(datanotshown)suggestingthatnrv3hasthepotentialtoplayaroleinfly hearing.np6215gal4reporterdrivesexpressioninchoneuronsofbothembryoandadult PNS.TheexpressionincludesembryonicLch5,salivaryglandsandadultChoneuronsofthe leg,flightmusclesandspermtails,inadditiontothejochoneurons(datanotshown). Nextwelookedattheexpressionpatternofboththesesubunitsinadultanimals.In adultanimals,alphasubunitispresentintheeyeandbrain,consistentwithotherstudies [17]andtheJOchordotonalarray(Fig2:A&H).IntheJO,αisexpressedinthecellbody plasmamembraneoftheneuronsandmuchmoreabundantlyinthescolopalecells (Fig2H).Thisindicatesthatthealphasubunitlikelyhasascolopalecellfunction.Nrv3on theotherhandispresentinasubsetofthebrain,eyeandalsothejoneurons(fig2:b, G&H).IntheJOneuronsitisspecificallyfoundinthecellbodyandtheinnerdendritic segmentofthechoneuronsbutnotinthescolopalecells.counterstainingwiththeα 5 showssimilarlocalizationofthealphasubunitandthenrv3intheplasmamembraneof thejochoneurons(fig2handfig3a).alltheseexpressiondatasuggestthatthealpha subunitandthenrv3arepotentiallyimportantforflyhearing.bothsubunitsseemto colocalizeattheplasmamembraneofthechoneurons,whichlendssupporttotheplasma membranespecificfunctionofthena + pump. 6

7 B)BothATPαandnrv3arefunctionallyrequiredinthescolopidiumtosustain auditorytransductioninthejo WewantedtoseeiftheNa + /K + ATPasesubunitsarefunctionallyimportantinthe scolopidiaofthejo.allavailablemutantsofatpαandthenrv3genearehomozygous lethalintheearlylarvalstages(datanotshown)whichisnotsurprising,giventhe importantphysiologicalroleofthena + /K + ATPaseinmostanimalcells.Thereforewecould notconducthearingstudiesinthepumpmutants.tocircumventtheselimitations,weused RNA interference(rnai)toknockdownthesubunitgeneswiththehelpofyeastuas/gal4 system.weusedtheatonalgal4drivertodriveexpressionofdoublestrandedrnaunder UAScontrolinthesenseorganprecursorcellsoftheJOtoknockdownATPαonlyinthese cellsandtheirprogeny.ourimmunohistochemicaldataindicatethatatpαproteinis presentinboththejochoneuronsandthesupportingcells,specificallythescolopalecells (Fig2I).ThereforeusingRNAi,weshouldbeabletoknockdownthealphasubunitfrom boththeneuronandthescolopalecellsinthejo(fig3a).auditoryelectrophysiological recordingsfromtheknockdownanimalsshowcompletehearingloss(fig3b).confocal imagesofstainedknockdownadultanimalsshowthatatpαproteinispresentatnearwild typelevelintheadultjobutthechordotonalorgansaremalformed(datanotshown).the presenceofatpαproteininatpαknockdownconditionindicatesthatatonalgal4islikely tobeactivelydrivingrnaibrieflyduringdevelopment,afterwhichalphasubunit expressionresumes.theseveredamagetothejomorphologyalongwiththehearing phenotypeindicatesthatatpαexpressionisessentialduringthedevelopmentofthe auditoryorgans.theshorttimeperiodduringdevelopmentwhentheatpαproteinwas 7

8 knockeddownwaslikelytocausesufficientstructuraldamagetoimpairthehearing function. NextweusedUAS RNAiagainstnrv3andknockeddownthisparticularbetasubunit inthejounitsusingthesamegal4driver.removalofnrv3fromthesenseorganprecursor cellsalsoresultedinanimalswithnearcompletelossofhearing(fig3c). C)ATPαisnecessaryforscolopalecellfunctioninhearing. Toinvestigateifthepresenceofthealphasubunitaffectshearing,wewantedto removeitfromthescolopalecells.weusedthenompagal4(whichisspecifictoscolopale cells)todrivernaiexpressioninthescolopalecellsofthejo,toknockdownatpαonlyin thesecells(fig4a).toconfirmintheseanimalsthatwehavespecificknockdown,we stainedforboththeatpαandthenrv3proteins.antibodystainingforatpαshowedthat thernaiknocksdownalphasubunitexpressionalmostcompletelyinthescolopalespace ofthechoneurons(fig5b).however,alphasubunitexpressionisretainedinthecapcells andintheneuronalcellbodyatwildtypelevel.electrophysiologicalrecordingsrevealed NompAGal4drivenRNAi αknockdownanimalstobedeaf(fig4b). UltrastructuralanalysisusingElectronMicroscopyasconductedontheknockdown animalsindicatedabnormalaccumulationofelectrondensematerialinthescolopalespace, whichincludeddifferenttypesofmembrane boundorganellessuchasmitochondria.the ciliaoftheneuronsappeartobedistendedsuggestingionicimbalance(fig5:d&e).wealso wantedtoseeifsuchmorphologicaldefectsextendedtoextracellularproteinsknowntobe presentinthescolopalespace.welookedatlocalizationandexpressionofspam/eyeshut protein[18],whichlocalizestothescolopalespaceatthelevelofciliarydilationaswellas distaltothebasalbodies(bothstructuresspecifictothejochoneuronsasindicatedin 8

9 Fig1B).WefoundthattheEyeshutproteinexpressionwasnotaffectedbyalphasubunit knockdown(datanotshown). RNAimediatedknockdownofthenrv3inscolopalecellwithNompAGal4causesonly amildhearingdefect(fig4c),seeninthepresenceofuas Dicer2enhancementofRNAi. SuchfindingsindicatethatalthoughATPαisfunctionallyimportantinthescolopalecellfor morphologicalintegrityofthescolopalespace,nrv3mayhaveonlyasmallsupportingrole inthescolopalecell.thefactthatknockingdownnrv3inthesenseorganprecursors resultedinnearcompletehearinglossindicatesthatnrv3isfunctionallyimportantinthe neurons. D)ThefunctionalimportanceofATP α: Inthelastsectionwehaveshownthatthealphasubunitisimportantinthe scolopalecellofjoforauditorymechanosensationindrosophila.butthesubcellular locationatwhichitisfunctionallyimportantinthescolopalecellisunclear.campaniform sensillaandbristleorganreceptorlymphisthoughttoberichink +,notunlikethe vertebrateauditoryhaircellendolymph[7].therefore,itislikelythatthena + /K + ATPaseis activelypumpingk + ionsintothescolopalespaceofjounitstohelpmaintaintheionic potentialofthescolopalespace. HereweproposeamodelofNa + /K + ATPasebeingpresentpreferentiallyinthe ablumenal(outer)plasmamembraneofthescolopalecellwhere,byitsactiveionexchange mechanism,itconstantlypumpsk + ionsintothescolopalecellcytoplasminexchangefor Na + ions.thispositionofthepumpwillensurethek + enrichmentofthescolopalecell cytoplasmandtherebythescolopalespacereceptorlymph.weusedatransgenicflyline whichhasachimericatp αgfpfusionproteinexpressedinthepatternofthenative 9

10 protein[19].imagingusingthedeltavisiondeconvolutionmicroscopeshowthattheatp α GFPfusionproteinlocalizespreferentiallytotheablumenalplasmamembraneofthe scolopalecell(fig:6a&b).theproteinseemstobemoreabundantattheapicalandbasal endsofthescolopalecell,atthejunctionbetweenthecapcellandscolopalecellandalsoat thejunctionbetweenthescolopalecellandneuronalcellbodies.proteinexpressionisalso observedalongtheplasmamembraneofboththescolopalecellandthechordotonal neuronalcellbodies.thepumpproteinexpressionpatternsupportsourhypothesisofthe asymmetricdistributionofthenapumptotheablumenalplasmamembrane.itisalso possiblethatthepumphassomecelljunctionfunctions,asseenintheotherdevelopmental contextsinvolvinglumenformationssuchassalivaryglandsandtrachea[13] OurresultssupportourhypothesisthattheNa + /K + ATPaseisimportantforthe auditorymechanosensationindrosophila.thecatalyticαsubunitispresentinthe ablumenalplasmamembraneofthescolopalecellwhereitisactivelypumpinginthek + ionsintothescolopalespaceandensuringthattheauditorytransductionissustained throughthechordotonalunitsandfurtherdownstream.itislikelythattheremightbe otherionchannelsorpumpsthatworkinconjugationwiththena + /K + ATPaseand maintaintheionichomeostasisofthesystem.nrv1andnrv2inadditiontonrv3,mayhavea functionalroleinthejo,especiallyinthescolopalecellswherenrv3isnotexpressed.our understandinginthisfieldisfarfromcompleteanddetailedanalysisofallthesemolecular playersisnecessary.suchknowledgewouldenableustounderstandtheeffectsoftargeted drugdeliveryagainstthesepumpsandmakeclinicallyrelevantcontributionsadefinite possibility. 10

11 Figures: Figure1:Schematicdiagramsshowing(A)Drosophila antennaandthejohnston sorgan(jo).(b)asingle scolopidialunit. Figure2:ATP αandnrv3areexpressedinthenervous systemofdrosophila,especiallyinthechordotonalorgans. (A)ATP αexpressedmorediffuselyinthebrain,labeledwith anti ATP αmonoclonal(green FITC).(B)nrv3expressedina subsetofthenervoussysteminbrain,labeledwithanti nrv3 polyclonal(red TRITC).(C)and(D)ATP αexpressedprofusely inthecnsofembryo,stage16,especiallyintheplasma membraneofcells,labeledwithanti ATP αmonoclonal(green FITC).(E)and(F)nrv3isexpressedinasubsetofthenervous systeminthecnsandspecificallyinthepentascolopidial organsinthepnsofembryos,labeledwithanti nrv3polyclonal (green FITC).(G).ExpressionofNrv3inadultJOlabeledwith FITC(green)(H)ExpressionofATP α(red TRITC)andnrv3 (green FITC)intheJO. Figure3:KnockingdownNa + /K + ATPasesubunitsinthe chordotonalorganprecursorswithatonalgal4causes almostcompletedeafness.(a)schematicdiagramshowinga singlechounitandthegreenrectangleshowingtheextentof RNAiexpression.(B)and(C)Histogramsofauditoryresponse amplitudesinatonalgal4mediatedknockdownofatp (B)or thenrv3(c)geneandrespectiveparentalcontrolfliesare shown.thedifferencesbetweenthecontrolandthe experimentalgroupsinbothcasesarestatisticallysignificant. 11

12 12 Figure5:AlphasubunitknockdownintheJOremoveαsubunitexpressionfromscolopalecellsandcauses morphologicaldefects. A)Confocalimageshowingalphasubunit(green)andNrv3 (red)expressioninadultantennaincontrolanimals B)Confocalimageshowingalphasubunit(green)andNrv3 (red)expressioninadultantennainnompagal4;uas RNAialphaanimalswherealphaexpressionisreducedin scolopalecells C)EMimageofcross sectionalviewofthecontroladult antenna D)EMcross sectionalviewofnompagal4;uas RNAi alpha showingaccumulationofelectrondensesubstanceinside thescolopalespace. E)EMshowingthelongitudinalsectionalviewofthesame alphasubunitknockdownanimalasd. Figure6:ATP GFPfusionproteinlocalizationtothe ablumenalplasmamembraneofthescolopalecell whereitisrequiredforhearing. (A)LongitudinalviewofJOscolopidiashowingpresenceof ATP protein(green)onoutsidethescolopidialrods(red) andtheplasmamembraneofthescolopalecell,more abundantattheapicalandbasalendsofthecell(b)crosssectionalviewofthesameshowingthatthetexasred Phalloidin,whichstainsthescolopalecellrods,hasbeen usedasamarkerfortheboundarybetweenthelumenal andablumenalplasmamembranesofthescolopalecell(c) PredictedmodelforthesubcellularlocalizationofATP Figure4:KnockingdowntheNa + /K + ATPasesubunitsinthe scolopalecellswithnompagal4affectshearing. (A)SchematicdiagramshowingasingleChounitandthegreen rectangleshowingtheextentofrnaiexpression.(b)histogram showingtheauditoryamplitudeofnompagal4mediated knockdownofatp andtheparentalcontrolsandthe experimentalanimalsarecompletelydeafandthisdifferenceis highlystatisticallysignificant. (C)HistogramshowingtheauditoryamplitudeofNompAGal4 mediatedknockdownofnrv3andtheparentalcontrolandthe experimentalanimalshavepartiallossofhearing,evenwith additionaldicertoenhancernaieffect.thedifferenceinthe auditoryresponseinthiscaseisalsostatisticallysignificant.

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