Buffersandsolutions(AllsolutionsaresupplementedwithCompleteProteaseinhibitortablet(Roche,Catno:1-873-580)atthefinalconcentrationof1tablet/50ml):S1solution:0.25Msucrose,10mMMgCl2S2solution:0.35Msucrose,0.5mMMgCl2S3solution:0.5Msucrose,25mMTris-HClpH9.0SP1buffer:1Msucrose,34.2%Percoll,22.2mMTris-HClpH7.4,1.11mMMgCl2SP2buffer:20%Percoll,10mMTris-HClpH7.4,1%TritonX100,0.5mg/mlheparinHTbuffer:10mMTris-HClpH7.4,1%TritonX100,0.5mg/mlheparin(See\"Notes”belowonmakingsucrosestocksolution)

Procedure(Allproceduresshouldbeperformedeitheroniceorat4°C)

A.SONICATION1.Preparethestartingmaterialof2.5x109HeLanuclei.SuitableHeLanucleicanbepurchasedfrom4CBiotech(Seneffe,Belgium;Catno:CC-01-30-25).AddS1solutiontothethawednucleitoafinalvolumeof30ml.Mixthoroughlybutavoidvigorousvortexing.2.Dividethedilutednucleiinto2x15mlportions.Overlayeachportiononto15mlofS2solutionina50mlFalcontube.Makesuretheinterfaceofthetwolayersissharp(Figure1).

Figure1:Steps2-3oftheprocedure.NotetheclearboundarybetweentheS1andS2layersbeforecentrifugation.3.Centrifugeat1430g(2500rpm,BeckmanGS-6centrifuge,GH-3.8rotor)for5minat4°C.

4.Carefullydecantthesupernatant.Thepelletsshouldbesolidandfirm(Figure1).ResUSPendeachpelletwithS2solutionsothatthefinalvolumeis30ml.Thepelletsshouldresuspendeasily.Apelletthatcannotberesuspendedcompletelyindicatesthepresenceoflysednucleiandshouldbediscarded.5.Dividetheresuspendednucleiinto10x3mlportions,eachina15mlFalcontube.Inourexperience,sonicationismoreeffectivewiththissamplesize.6.Sonicateeachaliquotusingatunedsonicator(MisonixXL2020)for3x6sec,witha6secintermissionbetweeneachburst.See\"Notes”belowonsonication.7.Examinethesonicatednucleiunderaphasecontrastmicroscope.Themajorityofthenucleishouldhavebeenlysed,whilethenucleolishouldbeclearlyvisIBLe.Ifthepercentageofunlysednucleiistoohigh(over5%),sonicateagainfor2-3sec,thenre-examineunderthemicroscope.Stopatthepointwhenmostnucleiarelysed.OversonicationdisruptsCajalbodies.TheconditionsusedtosonicateHeLanucleinotonlymaintainsthestructureofCBs,butalsothatofnucleoli,PMLbodiesandsplicingspeckles.Thisindicatesthat,undersuitableconditions,manynucleardomainsremainintactevenaftertheoverallnuclearstructureisdestroyed.

B.REMOVALOFNUCLEOLI8.Poolthe10aliquotsbacktogether.Measurethevolume.Add0.42Xvolumeof2.55Msucrosetothesonicatednucleisothatthefinalsucroseconcentrationisnow1M.Mixthoroughlybutdon’tvortex.Dividethemixtureintotwoportions,approx.20mleach,intwo50mlFalcontubes(Figure2).9.Centrifugeat3000g(3500rpm,BeckmanGS-6centrifuge,GH-3.8rotor)for10minat4°C.

Figure2:Steps8-10oftheprocedure.Notethesmallpelletaftercentrifugation.Thepanelsontherightshowthemicroscopeimagesofthesupernatantandpellet.Anti-coilinantibodies(5P10,green)andPyroninY(red)wereusedtolabelCBsandnucleolirespectively.Bar:10µm.

10.Asmallpelletshouldbevisibleineachtube(Figure2).Thispelletcontainsnucleoliandunlysednuclei,whilethesupernatantisnucleoplasmcontainingCBs.UsingaPipette,carefullycollectthesupernatant.Donotdecant,asthepelletsarenotfirm.Stopwhenyouareabout5mmabovethepellet.Thisistheregionwherethedistinctionbetweenthepelletandthesupernatentisblurred.Thispartofthesupernatantshouldnotbeincludedinthesubsequentsteps.11.Tocollectnucleoliforotherexperiments,resuspendthepelletwith3mlS2.Sonicatethemixturefor3x10sec.Proceedfromstep7ofthenucleolarisolationprotocol.

C.GRADIENTONE12.Measurethevolumeofthesupernatant.Add0.82XvolumeofSP1.Forexample,inatypicalexperiment,mix37mlofthesupernatantwith30.27mlSP1.Add0.05Xvolumeof20%TritonX100(3.36mlintheaboveexample).Mixthoroughlybutdonotvortex.13.Dividethemixtureintopre-cooledSW41ultracentrifugetubes(Figure3).Thevolumeofthesampleshouldfit6tubesperfectly.Balancethetubescarefully.Loadthetubesintoapre-cooledSW41rotor.Transfertherotortoapre-cooledultracentrifuge.14.Ultracentrifuge37,000rpm,2hours.

Figure3:Steps13-15oftheprocedure.Notetheloosepellet(1P)aftercentrifugation.Immunofluorescence(panelsontheright)showsthatmostCBsarein1P.Bar:10µm.

15.Aloosepelletcanbeseenrestingonthepercollprecipitateatthebottomofeachtube(Figure3).ThispelletcontainsmostoftheCajalbodies.Carefullypipetteawaytheturbidsupernatant.Donotdecant.Stopwhenyouare5-6mmabovethepellet.KeepthesupernatantlabeledasFraction1S.UseaP1000pipetteandresuspendthepelletsintheremainingsupernant.Transfertheresuspendedpelletstoa15mlFalcontube.ThisisFraction1P.MeasurethefinalvolumeofFraction1P.Inatypicalexperiment,thefinalvolumeisabout5ml.InFraction1P,theCBspresentweremostlyentangledwithlargepiecesofchromatin,asrevealedbyDAPIstaining.Nucleolithathadnotbeenremovedinthefirststepwerealsoasignificantcontaminant.

D.GRADIENTTWO16.Add600unitsofDNase1(SigmaCatno:D4527;20,000units/ml)toFraction1P.Mixwitharotatingwheelfor1houratRT.17.Add0.05xvolumeofheparin(10mg/ml).Themixtureshouldbecomemoretransparentonmixing.18.Add1xvolumeofSP2.Mixgentlywitharotatingwheelfor1min.19.Loadthemixtureinpre-cooledtubessuitableforSW55rotor(Figure4).Inatypicalexperiment,themixturefits2tubesperfectly.Ultracentrifuge,45,000rpm,1hour.

Figure4:Steps19-20oftheprocedure.Notethethinband(2.2)inthemiddleofthegradientaftercentrifugation.Bar:10µm.

20.Threebandsshouldbevisible(Figure4).Abroadwhitebandfloatsonthetop,athinwhitebandatthemiddle,andapelletrestingonthePercollprecipitateatthebottom.UsingaP1000pipette,verycarefullyunloadthegradientfromthetop.Takethetopband,andgoonuntilyouare2-3mmabovethemiddleband.ThisisFraction2.1.Useanewpipettetip,andtakethemiddleband(Fraction2.2).Finally,resuspendthepelletusingtheremainingpartofthegradient.ThisisFraction2.3.Foreach5mlgradient,typicalvolumesofthe3fractionsare:Fraction2.1:2.5ml;Fraction2.2:1ml;Fraction2.3:1.5ml.MostoftheCBsarecontainedinFraction2.2inthemiddleofthegradient,wheretheresolutionisthegreatest.

E.CONCENTRATIONANDFINALENRICHMENTOFCAJALBODIES21.IntoFraction2.2,add10xvolumeofHTbuffer.Mixwellusingarotatingwheel.22.Either:Loadthedilutedfractioninto2xSW41tubes.Ultracentrifuge.12,000rpm,15min.Or:Dividethemixtureinto20x1.5mlEppendorftubes.CentrifugeinanEppendorfcentrifuge,14,000rpm,15min(Figure5).

Figure5:Step22-27oftheprocedure.MostCBsarefoundinFraction3S.Arrowsintheinsetindicatethepresenceofsmallamountofcoilin-negativematerialin3S.Bars:Biginset:25µm,smallinsets:10µm.

23.Carefullyremovethesupernatant.Resuspendthepelletin0.5mlHTbuffer.Poolalltheresuspendedpelletsintoasingletube,andcentrifugeagainasabove.Thefinalpelletshouldbevisible,butverysmall.Carefullyremovethesupernatant.24.Resuspendthepelletwith0.5mlS3solution.Thepelletdetachesfromthebottomofthetubeandisresuspendedonpipettingupanddown.25.SpindowntheresuspendedpelletinanEppendorfcentrifuge,8000rpm,5min.Avisiblepelletshouldbeseen.CarefullytakethesupernatantandtransferittoanewEppendorftube.Repeatspinningthesupernatantat8000rpm,5min.CarefullytakethesupernatantandtransferittoanewEppendorftube.26.Combinethepellets.LabelthisasFraction3P.27.Dilutethesupernatantwith10xvolumesof25mMTris-HClpH9.0.Dividethedilutedsupernatantinto3x1.5mlEppendorftubes.Centrifugeat14,000rpm,15min.Carefullyremovethesupernatants,leavingabout0.1mlatthebottomofeachtube.Pipettetheremainingsupernatantsupanddown,resuspendingthesmallpellet.Mixthecontentofthe3tubestogether.Fillupthetubewith25mMTris-HClpH9.0,andcentrifugeagainat14,000rpm,15min.Carefullyremovethesupernatant,leavingabout20µlinthetube.Pipettetheremainingsupernatantsupanddowntoresuspendthesmallpellet.LabelthisasFraction3S.ThisfractioncontainsenrichedCBs.

Notes(1)Making2.55MsucrosestockHereisaprotocolforpreparingasucrosestocksolution(ClineandRyel,l1971)suitableforthenucleolarisolationprotocol.Theresultingsolutionis2.55M,or66%byweight.Itsdensityis1.3224g/cm3at20oC,andrefractiveindexis1.4558.Thestocksolutionisstableindefinitelyat4oC.ThisprocedurecanbecarriedoutatRT.Thereisnoneedtoheatupthesolutiontohelpdissolvingthesucrose.Heatingupanincompletelydissolvedsucrosesolutioncanleadtocharringofsucroseandaffectthequalityofthesucrosesolution.1.Weighout1710gsucrose(BDH).Keepitasideinacleancontainer.2.Putexactly900mlwaterandamagneticbarina5litrebeaker.Putthebeakeronastirrerandstartstirring.3.Add1/3ofthesucroseintothebeaker.Makesurethemagneticbarisrotatingfreely.Stirfor1hour.4.Addanother1/3ofthesucroseintothesolution.Againmakesuretherotationofthestirbarisnotimpaired.Stirforanother1hour.5.Addtheremainingsucrose.Stirforanother1hour,oruntilallthesucrosehasgoneintosolution.Thefinalvolumeshouldbeexactly2litres.(2)SonicationWeuseaMisonix2020sonicatorfittedwithamicrotipatpowersetting5.Toensurereproduciblesoncationthesepointsshouldbefollowed:-Itisnecessarytotunethesonicatoreverytimeafteryouchangetheprobe.Followthemanufacturer’smanualforthetuningprocedure.-Sonicationproducesintenseandlocalizedheatinyoursolution.Ifyouareconcernedabouttheheating,thecorrectwaytoreduceheatingistoshortenthesonicationtimeandtoincreasetheintermissionbetweenbursts.Keepingthetubeoniceorperformingthesonicationinthecoldroomishelpful,butisnotthemosteffectivewayofheatcontrol.-Iftheprobeistooclosetotheliquidsurface,itproducesafoamandreducestheefficiencyofsonication.Makesuretheprobeiswellsubmergedinthesolution,about5mmabovethebottomofthetube.Donot,however,touchthebottomorthewallofthetubewiththeprobe.-Sonicatorprobethathasbeenusedrepeatedlydevelopspitsonitsend.Thesonicationefficiencygraduallydecreasesastimegoeson.Therefore,thesonicationtimerecommendedherecanonlybeusedasaguideline.Alwaysmonitortheoutcomeofsonicationusingaphasecontrastmicroscope.Youmayneedtoadjustthesonicationtimetomaintaintheefficiency,especiallyiftheprobeisgettingold.Changetheprobewhentheefficiencyisnoticeablydown.

(3)AnalysisoftheenrichedCajalbodyfraction-ToimmunolabeltheisolatedCBs,spotabout5µlofFraction3Sontoapolylysine-coatedslide(BDHCatno:406/0178/00),andairdrythespot.RehydratetheslideinPBSfor5minbeforeusingstandardimmunostainingprocedure.-ToseparateandanalysetheproteinsbySDSPAGE,eitherresuspenddirectlyinLaemmliSDSsamplebuffer,orinyourpreferredbuffer.