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The Journal of Cell Biology
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T Hoi YeungLi, Introduction production onthechromatin invivo A mechanism ofcouplingRCC1mobilitytoRanGTP and Englmeier,1998).Recentstudiesusing envelope andonthecondensedmitoticchromosomes(Mattaj RanGTP concentrationgradientacrosstheinterphasenuclear ization oftheRanregulatorsstronglysuggeststhatthereis a RanGAP1 andRanBP1,arecytoplasmic.Thisuniquelocal- the proteinsthatstimulateRanGTPaseactivity,such as chromatin boundbothininterphaseandmitosis,whereas Renault etal.,2001).PreviousstudiesshowedthatRCC1is et al.,1992;Seki1996;Nemergut2001; factor (GEF)forRan(BischoffandPonstingl,1991;Seino (RCC1)* istheonlyknownguaninenucleotideexchange as amolecularswitch.Regulatorofchromosomecondensation Ran exitsaseitheraGDP-orGTP-boundstateandfunctions spindle assembly(Dasso,2002).LikemanysmallGTPases, and Englmeier,1998),nuclearenvelopeformation, functions includingnucleocytoplasmictransport(Mattaj The smallGTPaseRanplaysakeyroleindiversecellular 2 1 this mobilityisregulatedduringthecellcycle. Our kinetic phase andmitoticchromosomes inliving cells,andthat (RCC1) rapidly associates anddissociateswithbothinter- Here, wereportthatregulatorofchromosome condensation maintaining theRanGTPgradient invivo remainsunknown. for RanGTPproduction,themechanism ofgenerating and the chromosome-associated enzymeRCC1isresponsible nucleocytoplasmic transport andspindle assembly. Although intensity fluorescence nucleotideexchange; Key words: Ran;nuclear;chromatin; some condensation. GEF, guaninenucleotideexchange factor;RCC1,regulatorofchromo- *Abbreviations usedinthispaper:FLIP, fluorescencelossinphot (410) 243-6311.E-mail:[email protected] University Pkwy.,Baltimore,MD21210.Tel.:(410)554-1232.Fax: tute, Address correspondenceto The onlineversionofthisarticleincludessupplementalmaterial. http://www.jcb.org/cgi/doi/10.1083/jcb.200211004 The Journal ofCellBiology
Department ofChemicalEngineering,Department The Johns Hopkins University, Baltimore, MD21210 Howard Hughes Medical Institute, ofEmbryology, Department CarnegieInstitution of Washington, Baltimore, MD21210 TheRockefeller University Press, 0021-9525 Report
Dept. ofEmbryology,CarnegieInstitutionWashington,115W.
is essentialformany cellularprocesses,including envelope andonthecondensedmitoticchromosomes he RanGTPgradient acrosstheinterphasenuclear
1 Denis Wirtz, , Volume 160, Number 5,March 3,2003 635–644 Yixian Zheng,HowardHughesMedicalInsti- 2 and Yixian Zheng /2003/03/635/10 $8.00 Xenopus eggextract obleaching; 1 Results anddiscussion chromatin surface. from thechromatin andtheproductionofRanGTPon complex, permittingthereleaseofRCC1andRanGTP Successful nucleotideexchange dissociatesthebinary binding ofthebinarycomplexRCC1–Rantochromatin. exchangeon Raninvivo. The couplingisduetothestable of RCC1withthechromatin iscoupledtothenucleotide we have demonstrated experimentallythattheinteraction chromosome bindingtogenerate aRanGTPgradient. Indeed, modeling suggeststhatRCC1couplesitscatalyticactivity to phase andmitosis(Fig.1A). Interestingly,ininterphase, RCC1-GFP wasidenticalto endogenousRCC1ininter- minus (RCC1-GFP).Wefound thatthelocalizationof expresses humanRCC1fused totheGFPatitsCOOHter- somes invivo,weestablished a stableSwiss3T3celllinethat and maintainstheRanGTPgradientondynamicchromo- To understandthemechanismbywhichRCC1generates Establishing thecelllinetostudyRCC1functioninvivo in livingcellsisessentialtounderstandtheRansystem. understanding themechanismsregulatingRanGTPproduction of smallGTPasessuchasRas,Rho,andArfinvivo.Clearly, mechanisms havebeencrucialindecipheringthefunctions tide exchangecatalyzedbyGEFs,andunderstandingthese a plethoraofmechanismsareinvolvedinregulatingnucleo- production. StudiesofothersmallGTPaseshaveshownthat result, littleisknownabouthowthecellregulatesRanGTP maintains RanGTPinlivingcellsremainsunknown.Asa importantly, themechanismbywhichRCC1catalyzesand not clearwhetherthegradientalsoexistsinlivingcells.Most (Kalab etal.,2002). in vitro,thereforelendingsupportfortheideaofagradient in theinterphasenucleiandoncondensedchromosomes revealed thatthereisindeedahighRanGTPconcentration Although RanGTPgradientappearstoexistinvitro,itis brought toyouby provided byPubMedCentral 635 CORE The Journal of Cell Biology 636 of stablecelllines abletogrowat39.5 nonpermissive temperature.The samecellstransfectedwithRCC1-GFP,andthecontrolBHK-21 cellssurvived,leadingtoestablis inactivate endogenousRCC1in tsBN2 cellsandwerecountedevery24h.Thetransfected withGFPdidnotsurviveat in fiverandomfieldswitha20 temperature-sensitive mutationin RCC1,andthewild-typeparentalcells,BHK-21.Thecellsexpressing RCC1-GFPorGFPwerecou of saltandanalyzedbyWestern blotting.(D)PlasmidsexpressingRCC1-GFPorGFPweretransfected intotsBN2cells,whichharb cells duringmitosis.Bar,10 and endogenousRCC1colocalized withDAPIininterphaseandmitosis.Bar,10 to visualizeDNA.Thenonexpressingcellswerealsostainedwith anti-RCC1antibodytovisualizeendogenousRCC1.BothRCC1-GFP Figure 1. The JournalofCellBiology
Characterization ofRCC1-GFP.
| m. (C)RCC1-GFPandendogenous RCC1wereextractedfromisolatednucleiwithincreasingconcentrations
Volume 160,Number5,2003 objective24haftertransfection. Cellswerethenshiftedtothenonpermissivetemperature(39.5 C. (A)RCC1-GFP–expressingor–nonexpressingSwiss3T3cellswere fixedandstainedwithDAPI m. (B)Time-lapsemicroscopyof RCC1-GFP–expressing C) to hment the or a nted
The Journal of Cell Biology from atleastfive differentcells. methanol-fixed cells. (D)GFPandhistoneH2B-GFP FRAPkineticsintheinterphase nucleus.ValuesinCandDrepresent means the chromosome.Bar,10 enlargement oftheindicatedarea (whitedashedsquare)inpseudocolor.(B)Selectedimagesofamitotic cellduringFRAPofan Figure 2. trol vectorintoeitherwild-type(BHK-21)orRCC1mutant the endogenousRCC1.WetransfectedRCC1-GFPorcon- dogenous RCC1,itshouldbeabletoreplacethefunctionof they bindtothechromatinwithsameaffinity(Fig.1C). phase nucleiatthesamesaltconcentrations,suggestingthat GFP andendogenousRCC1areextractedfromtheinter- at allstagesofmitosis(Fig.1B).Furthermore,bothRCC1- RCC1-GFP showedthatitislocalizedonthechromosomes gions wherethereisstrongDAPIstaining.Liveimagingof both endogenousRCC1andRCC1-GFPareenrichedinre- We reasonedthatifRCC1-GFPisfullyactiveastheen- FRAP. (A)Selectedimagesofaninterphase cellduringFRAPofanareainthenucleus(redcircle).Bottom panelsshowthe
m. (C)RCC1-GFPFRAPkineticsin theinterphasenucleus,oncondensedchromosomes,andin nucleiof
(tsBN2) CHOcelllines.1daftertransfection,thecellswere cultured them(monthssofar).Clearly,RCC1-GFPisfully ture likethewild-typecounterpartforaslongwehave expressing tsBN2cellscansurviveattherestrictivetempera- whereas GFPalonedidnot(Fig.1D).StableRCC1-GFP shifted torestrictivetemperature(39.5 the temperature-sensitivemutationinRCC1at39.5 RCC1-GFP intsBN2cellsrescuedthelethalitycausedby was countedevery24hfor7d.Wefoundthatexpressionof (Nishitani etal.,1991).ThenumberofGFP-expressingcells endogenous RCC1(throughdegradation)inthetsBN2cells A mechanismofRanGTPproductioninvivo| C) toinactivatethe areaon Lietal. SD 637 C, The Journal of Cell Biology 638 tsBN2 cellsexpressingRCC1-GFP at39.5 have performedtheFRAP and FLIPexperimentsusing dence timesofRCC1onchromatin, computedasthein- times fasterinmitosisthaninterphase.Themeanresi- RCC1 fromthechromosome,notthatdiffusesfive efficient ofRCC1inmitosisreflectsthefasterdissociation developed kineticmodelsforthemovementofRCC1inin- RCC1 releasefromchromosomes.Basedonthesedata,we that transportinmitosisismainlyregulatedbytherateof the distancefrombleachspot(Fig.3D).Thissuggests decline offluorescencesignalinmitosiswasindependent non-negligible diffusiontransportlimitations.However,the bleach spotincreased(Fig.3C).Thisispresumablydueto rate offluorescencelossdecreasedasthedistancefrom both interphaseandmitoticcells.Innuclei,the signal atvariousdistancesfromthebleachspotovertimein (Fig. 3B).Then,wemeasuredthedeclineoffluorescence that ofinterphasecells,consistentwiththeFRAPanalyses The overallfluorescencelosswasfasterinmitoticcellsthan cell wasimagedaftereachroundofbleaching(Fig.3A). sol wasbleachedrepeatedlywithhighlaserpower,andthe nucleus, onthemitoticchromosome,orincyto- verse ofthedissociationrate ofRCC1,are50 0.71 in interphaseandmitoticcellsare0.51 We foundthattheeffectivediffusioncoefficientsofRCC1 http://www.jcb.org/cgi/content/full/jcb.200211004/DC1). at terphase andmitosis(seesupplementalmaterial,available chromatin. Aspot(1- to determinethedissociationkineticsofRCC1from (Fig. 2D),whichfreelydiffusedthroughoutthenucleus. 2001), butslowerthantheGFPmoleculeinnucleus immobileduringourobservations(KimuraandCook, was was muchfasterthanthatofhistoneH2B-GFP,which ing (Fig.2,AandB).Afterphotobleaching, bility oftheRCC1-GFP,wasmeasuredbysequentialimag- pulse of500ms.Thekineticsrecovery,reflectingthemo- condensed mitoticchromosomeswasbleachedwithalaser RCC1-GFP. Adefinedregionofthe3T3cellnucleusor Therefore, wefirstusedFRAPtostudythedynamicsof interacts withthechromatinininterphaseandmitosis. duction invivo,itisimportanttounderstandhowRCC1 To decipherthemechanismofRanGTPgradientpro- and mitosis RCC1 isahighlymobileenzymeininterphase hamster cells. report theendogenousRCC1functioninmouseand functional. Therefore,RCC1-GFPcanbeusedtofaithfully RCC1-GFP expressedinSwiss3T3cellsshouldalsobefully 97% identicaltothehamsterRCC1,weconcludethat functional inthehamstercells.BecausemouseRCC1isover vealed thatRCC1 associateswiththechromatin transiently similar results(unpublisheddata). Together,theseworksre- cells, respectively(Fig.2C).TherecoveryofRCC1-GFP with ahalftimeof RCC1 fluorescencesignalwasrecoveredwithin55and10s, 20 Next, weusedfluorescencelossinphotobleaching(FLIP) The JournalofCellBiology 5sininterphaseandmitosis, respectively.Also,we m 2 /s, respectively.Thesmallereffectivediffusionco- 5 and m diam)inthe3T3cellinterphase
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Volume 160,Number5,2003
3 sininterphaseandmitotic
0.11and2.86
C andobtained
90% ofthe
12and differently ininterphaseandmitosisvivo. during thecellcycle,andthatassociationisregulated chromosomes (Fig. 4A).Asexpected,most Rh-RanGDPis calized withRCC1-GFPininterphase nucleiandonmitotic ing liveimaging,wefoundthat Rh-RanT24Nclearlycolo- totic cytoplasmofthe3T3cells expressingRCC1-GFP.Us- Rh-RanGTP, orRh-RanT24N intointerphasenucleiormi- we injectedrhodamine-labeled (Rh)wild-typeRanGDP, tin inbothinterphaseandmitosis. Totestthispossibility, colocalization ofRanT24NandRCC1-GFPonthechroma- nary complexischromosomebound,weshouldseeaclear 1994; Klebeetal.,1995a;Lounsbury1996).Ifthebi- cleotide exchange(Dassoetal.,1994;Kornbluth forms astablebinarycomplexwithRCC1duetolackofnu- for nucleotidecomparedwithwild-typeRan;therefore, it bound inthecell.RanT24Nhasagreatlyreducedaffinity whether thebinarycomplexofRanandRCC1ischromatin find thebinarycomplexonchromatin. change onRanoccurredthechromatininvivo,wemight Renault etal.,2001).Wereasonedthatifnucleotideex- RCC1– andRan–nucleotide(Klebeetal.,1995a,1995b; centration ishigh,theternarycomplexdissociatesinto plex ofRCC1–Ran.However,whenthefreenucleotidecon- nucleotide, theternarycomplexrelaxesintobinarycom- 1995a, 1995b;Renaultetal.,2001).Whenthereisnofree is oflowaffinity,whichdissociatesquickly(Klebeetal., otide toRan,theternarycomplexofRCC1–Ran–nucleotide al., 2001).BecauseRCC1destabilizesthebindingofnucle- guanine nucleotides(Klebeetal.,1995a,1995b;Renault mation ofternaryandbinarycomplexesRCC1,Ran, catalyzed exchangeisamulti-stepprocess,involvingthefor- Studies usingpurifiedRCC1andRanshowedthatRCC1- vitro nucleotideexchangereactioniswelldocumented. catalyzes nucleotideexchangeonRaninlivingcells,the change onRan.AlthoughlittleisknownabouthowRCC1 ple itsbindingtothechromosomesnucleotideex- Fig. S3). change onRan(supplementalmaterialand couple itsbindingtothechromosomenucleotideex- steep RanGTPgradientonthechromosomes,RCC1may gests thatinorderforthehighlymobileRCC1togeneratea dient onthechromatin.Indeed,ourkineticmodelingsug- prevent theformationofasteepRanGTPconcentrationgra- as thoseboundtothechromatininvivo,itcouldpotentially RCC1 cancatalyzenucleotideexchangealmostasefficiently tion oftime(Nemergutetal.,2001).Ifthepoolfree by thepercentageofnucleotidereleasedfromRanasafunc- stimulated theGEFactivityofRCC1by tones H2A/H2Borisolatedchromatin(RCC1free)only a strongGEFactivitytowardRaninvitro.Furthermore,his- showed thatRCC1incelllysatesorasapurifiedproteinhas RanGTP concentrationgradientinvivo.Previousstudies regarding whetherandhowRCC1couldgenerateasteep mosome duringthecellcycleraisesanimportantquestion The findingthatRCC1transientlyassociateswiththechro- to thechromatininvivo The binarycomplexofRCC1andRanbindsstably We usedamutantalleleofRan(RanT24N)totest We hopetodeterminewhetherandhowRCC1maycou- 20% asmeasured The Journal of Cell Biology
of RCC1-GFPFLIPininterphase(C) andmitotic(D)cells. FLIP ininterphaseandmitoticcells. (CandD)Kinetics circles). Bars,10 mitotic cellsduringFLIPoftheindicated area(yellow Figure 3. A mechanismofRanGTPproductioninvivo| FLIP. (A)Selectedimagesofinterphaseor m. (B)KineticsofoverallRCC1-GFP Lietal. 639 The Journal of Cell Biology 640 five cells.(E)FRAP kineticsofinjectedmitoticcells. Valuesrepresentmeans into interphasenucleiormitoticcytoplasm ofRCC1-GFP–expressingcells,followedbyliveimagingusing fluorescencemicroscop Figure 4. area (whitedashed square)inpseudocolor.Bar,10 images beforeandafterthebleach pulses (redcircledenotesthebleachedspot)wereshown.Coloredpanels showtheenlargemen (C) RanGDPorRanT24Nwasinjected intotheinterphasenucleiormitoticcytosolat1mg/mlfollowed byFRAPanalysisofRCC1-G complex bindstothechromatinstably. TheternarycomplexesofRCC1–Ran–GDP(orGTP)areomitted fromthedrawingforsimplici (B) Themodel.RCC1,RanGDP,and RanGTPinteractwiththechromatinreversiblyduetolowaffinity binding,whereastheRCC1–Ra The JournalofCellBiology The binarycomplexofRan–RCC1 bindsstablytothechromatininvivo.
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Volume 160,Number5,2003 m. (D)FRAPkinetics ofinjectedinterphasecells.Values representmeans
SDfromatleast threecellsandfiveindependent FRAPs. (A) Rh-RanGDP,Rh-RanT24N,or Rh-RanGTP wasinjected t oftheindicated SDfromatleast y. Bar,10 FP. Selected n binary ty. m. The Journal of Cell Biology GFP onthecondensed chromosomes,whereas RanGDP we foundthat RanT24Ncompletelyimmobilized RCC1- RanT24N orRanGDPwas injected intothemitoticcells, bleached spot(Fig.4,Cand D). Whenthesameamountof completely blockedtherecovery ofRCC1-GFPinthe the next110s(Fig.4D). However, RanT24Ninjection the fluorescencesignalto levelofcontrolinjectionsin RCC1-GFP inthefirst10s, followed byaslowrecoveryof RanGDP injectionallowedtherecoveryof55% cules inthecell(seeMaterialsandmethods).Wefoundthat which issimilartotheestimatednumberofRCC1mole- cessful nucleotideexchange,whichdissociatesRCC1from exchange tothechromatin(Fig.4B).Weproposethatsuc- the binarycomplexcouldallowcouplingofnucleotide than RCC1andRanalone,thatthestablebindingof an increasedaffinitytothecorehistonesonchromatin that thebinarycomplexofRCC1–Ranshouldhavemore gut etal.,2001;Bilbao-Cortes2002).Wereasoned 2002). Thesebindingsareoflowaffinityinnature(Nemer- et al.,1992;Nemergut2001;Bilbao-Cortes to histonesH2AandH2B,RanbindsH3H4(Seino al., 2001;Bilbao-Corteset2002).AlthoughRCC1binds the chromatinviahistones(Seinoetal.,1992;Nemergut could deliver We estimatedthatinjectingatthisconcentrationofRan higher concentration(1mg/ml)intotheinterphasenuclei. significantly reducedthemobilityofRCC1-GFP(Fig.4D). controls. However,injectingthesameamountofRanT24N nuclei hadnoeffectonFRAPofRCC1-GFPcomparedwith larly, injectingRanGDPat0.1mg/mlintotheinterphase with cytoplasm-injectedoruninjectedcells(Fig.4D).Simi- small effectonthemobilityofRCC1-GFPwhencompared bation bycontrolmicroinjectionintothenucleushadonlya RanT24N orRanGDP(seeMaterialsandmethods).Pertur- GFP inthe3T3cellsaftermicroinjectionofpurified Therefore, weusedFRAPtoprobethemobilityofRCC1- tion ofthebinarycomplexthatallowsnucleotideexchange. down themobilityofRCC1becauseincreasedforma- the otherhand,excesswild-typeRanGDPshouldonlyslow stable binarycomplexthatresistsnucleotideexchange.On mobilize RCC1onthechromatinduetoformationofa generation ofRanGTPonthechromatinsurface(Fig.4B). RanGTP candissociatefromthechromatin,allowing the RanGTP, isessentialforthetwoproteinstoreturn complex ischromosomeboundinvivo. 4 A).Basedonthesestudies,weconcludethatthebinary RanGTP isdiffuselydistributedinthemitoticcytosol(Fig. is localizedintheinterphasenucleus,mitosis,Rh- sistent withthisidea,wefoundthatalthoughRh-RanGTP RCC1, whichbindstothecondensedchromosomes.Con- cleotide exchangebyformingthebinarycomplexwith condensed chromosomesisbecauseRanGDPundergoesnu- A). WebelievethatthelocalizationofRh-RanGDPto plasm, resultinginasomewhatdiffuseddistribution(Fig.4 was foundoncondensedchromosomesandinthecyto- found inthenucleus.Interestingly,mitosis,Rh-RanGDP Recent studiesshowedthatRanandRCC1interactwith Next, RanT24NorRanGDPwasinjectedat10-fold If theabovemodeliscorrect,excessRanT24Nshouldim- low affinitybindingstates.Consequently,RCC1and 4 10 6 moleculesofRanintothecells, sociate fromthechromatin. RanGTP, whichinturnallowsRCC1andRanGTPtodis- change isrequiredforthedissociationofRCC1from stably withthechromatininvivo.Successfulnucleotideex- suggest thatthebinarycomplexofRCC1–Ranassociates the chromatininbothinterphaseandmitosis.Theyfurther Ran isrequiredforbothRCC1andtodissociatefrom data). Theseresultsshowedthatnucleotideexchangeon though RanGDPismobile,RanT24Nnot(unpublished RanT24N thatwereinjectedintocells,andfoundal- also performedFRAPoffluorescentlylabeledRanGDPor only reducedthemobilityofRCC1(Fig.4,CandE).We in theformofbinarycomplexeggextracts.Weadded mine whetherRanandRCC1bindtothechromatintightly the chromosomes.Next,weusedcompetitionassaystodeter- complex ofRCC1–RanenhancesthebindingRCC1to consistent withtheideathatformationofbinary 5 B)tothechromatinassembledfromsperm,whichis enous RCC1(Fig.5A)andtheexogenousRCC1-GFP RanGDP stronglystimulatedthebindingofbothendog- We foundthattheadditionofexogenousRanT24Nand RCC1-GFP inthepresenceofeitherRanT24NorRanGDP. were addedtotheeggextractssupplementedwithorwithout using matin. Wehopetobiochemicallyconfirmthesefindingsby otide exchangeisrequiredfortheirdissociationfromchro- Ran–RCC1 bindstochromosomestightlyandthatnucle- The aboveinvivostudiesrevealedthatthebinarycomplexof from thechromatinrequiresnucleotideexchange The dissociationofthebinarycomplexRan–RCC1 ciently, evenin thepresenceofexcessGTP or GDP(Fig.5, RCC1-GFP did notdissociatefromthe chromatin effi- sence, offreeGTPorGDP.As expected,Rh-RanT24Nand from thechromatinonlyin presence,butnotintheab- extracted withbuffer,Rh-Ran andRCC1-GFPdissociated 1995b; Renaultetal.,2001). Indeed,whenthespermwas fore, shouldbindstablyto the chromatin(Klebeetal., the binarycomplexduetonucleotide depletion,andthere- bound RCC1-GFPandwild-typeRh-Ranisintheformof GDP. Undertheseconditions,afractionofthechromatin- coverslips throughaglycerolcushioncontainingnoGTPor RanT24N, orRCC1-GFP.Thespermwerethenspunonto the eggextractssupplementedwith Rh-RanGDP,Rh- in plex fromthechromatin.Weassembledspermchromatin quired forthedissociationofRan–RCC1binarycom- unlabeled RanT24NandRCC1onlyshowed Xenopus for Rh-RanGDPandRCC1-GFP,respectively.However, found thatunlabeledRanGDPandRCC1readilycompeted RanT24N, RanGDP,orRCC1wasusedascompetitors.We RanT24N, Rh-RanGD,orRCC1-GFP.Excessunlabeled chromatin structuresfrom plex intheformofRCC1–RanT24N. tin whentheyarelockedintotheRCC1–Ranbinarycom- This suggeststhatRCC1andRanbindstablytothechroma- excess ofthelabeledproteins,respectively(Fig.5,CandD). when thecompetitorconcentrationsreached20-and10-fold 20% competitionofRh-RanT24NandRCC1-GFP,even Next, weexaminedwhetherguaninenucleotideisre-
Xenopus
spermtotheextractssupplementedwithRh-
A mechanismofRanGTPproductioninvivo|
eggextractsthatsupporttheformationof
Xenopus sperminvitro.Sperm Lietal. 40 and 641 The Journal of Cell Biology 642 unlabeled RanGDP orRanT24Nattheindicatedconcentrations, respectively.Theamount oflabeledRanboundtothechromatin was to thechromatin was quantified.(D)Rancompetition. Spermwasincubatedwithegg extractscontainingRh-RanGDP orRh-RanT24N GFP andeitherRanGDP orRanT24Ninthepresence ofunlabeledRCC1attheindicated concentrations.Theamount ofRCC1-GFPboun chromatin wasquantifiedasarbitrary unit(AU).(C)RCC1competition.Thespermwasincubatedwith eggextractssupplementedw sperm wasaddedtotheeggextract supplementedwithpurifiedRanGDPorRanT24N.Thefluorescence intensityofRCC1-GFPonthe Figure 5. binding oftheendogenousRCC1 tothechromatinisdetectedbyimmunofluorescenceusingananti-RCC1 antibody.(B)RCC1-GFPand stimulate thebindingofendogenous RCC1intheeggextracttomitoticchromosomesassembled fromthespermchromatin. The JournalofCellBiology Biochemical characterizationof the bindingofRanandRCC1tochromosomes.
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Volume 160,Number5,2003
(A)BothRanGDPandRanT24N strongly
quantified. ith RCC1- The and sperm
d The Journal of Cell Biology chromatin viathecorehistones,whicharepresentin RanGTP onthechromatin.RanandRCC1bindto plex dissociatesthecomplex,liberatesRCC1,andgenerates nucleotide exchangeonthechromatin-boundbinarycom- RCC1–Ran binarycomplextochromosomes.Successful The couplingisestablishedthroughthestablebindingof with chromosomedockingtogenerateRanGTPinvivo. mobile enzyme,RCC1couplesnucleotideexchangeonRan subjected tocellcycleregulation.Moreimportantly,asa and thebindingofRCC1tochromatinappearsbe quired foritsdissociation. stably tothechromatinandthatnucleotideexchangeisre- and methods).Thisshowsthatthebinarycomplexbinds found intheeggextract(Fig.5,GandH;seealsoMaterials absence offreeGTP. Theamountofchromatin-bound RCC1-GFP(G)orRh-Ran(H) wasquantifiedafterincubation. Errorbarsrepre chromatin asinE andFwereincubatedwithbuffer containingindicatedconcentrations ofunlabeledRanandRCC1 ascompetitors amount oflabeled proteinsonthespermchromatin wasquantifiedeitherbefore(c, control)orafterincubation.(Gand H)The (E andF)Buffercontaining0–5mM GTPwasincubatedwithspermchromatinboundRh-Ranor RCC1-GFPoncoverslipsfor20mi (Roche). Stablecelllinesexpressing RCC1-GFPwereselectedandmain- and transfectedintoSwiss3T3,tsBN2, orBHK21cellsusingFuGENE™6 Human RCC1wasclonedintopEGFP-N3 (CLONTECHLaboratories,Inc.) Plasmids andcelltransfection mechanisms towardtheproductionofRanGTPgradient. tant tounderstandtherelativecontributionsofdifferent RCC1 invivomaybenegativelyregulated.Itwillimpor- though freeRCC1incelllysatesisactivevitro,the and onthecondensedchromosomesinmitosis.Finally,al- RanGTP gradientacrossthenuclearenvelopeininterphase RanGDP,andtherefore,shouldfurthersharpenthe into and RanBP1presentinthecytosolhydrolyzeRanGTP production onthechromosomes.Inaddition,RanGAP1 some-coupled exchange,shouldfurtherenhanceRanGTP 2001). Thisstimulation,incombinationwiththechromo- the GEFactivityofRCC1modestly(Nemergutetal., ent. Forexample,thechromosomeswereshowntostimulate other mechanismsthatmayalsoinfluenceRanGTPgradi- the productionofRanGTPgradient,itdoesnotexclude by RCC1describedinthispapercansignificantlyinfluence RanGTP gradientduringthecellcycle. namic chromatinreorganizationwiththeproductionof change mechanismmaybenecessarytocoordinatethedy- cell cycle.AmobileRCC1andachromosome-coupledex- dynamic structuresthatundergoremodelingthroughoutthe mechanism.Eukaryoticchromosomesarehighly exchange binary complexestosupportthechromosome-coupling matin hassufficientcapacitytobindallRCC1–Ran Ran (40 the chromatin,evenwhenconcentrationsofunlabeled of wild-typeRh-RanandRCC1-GFPremainedboundto vealed that,intheabsenceoffreeGTPorGDP,over80% labeled RanandRCC1ascompetitors.Quantificationre- GTP orGDP.Werepeatedtheaboveexperimentsusingun- the binarycomplextochromatininabsenceoffree E andF).Next,weassessedthestabilityofbinding Materials andmethods fold molarexcessofRCC1inthecell.Therefore,chro- We haveshownthatRCC1isahighlymobileenzyme, Although thechromosome-couplednucleotideexchange M) andRCC1(2 M) werehigherthanthose 100- estimated that our injectionconditionandthemanufacturer’scalibration(Eppendorf),we as describedpreviously(Mistelietal.,2000;PhairandMisteli,2000). ized tothechangeintotalfluorescencecausedbybleachingandimaging est, thefluorescenceintensityinregionateachtimepointwasnormal- FRAP. Todeterminetherelativefluorescenceintensityinaregionofinter- each bleachingfor250msandwithimagingidenticaltothoseusedin ments, cellswererepeatedlyimagedandbleachedatintervalsof1swith (Murray andKirschner,1989).ForRancompetition,spermwasincubated Therefore, at1mg/mlofRan,wedelivered for thenext10images,and10-sintervalsfinalimageswith collected at1-sintervalsforthefirst10images,followedby5-s laser powerattenuatedto laser lineofanargonat37and23 performed onaconfocalmicroscope(TCS-SP2;Leica)usingthe488-nm streptomycin, and25mMHepeswithoutphenolred).FRAPFLIPwere pression containingculturemedium(DME,10%FBS,10U/mlpenicillin/ Cells wereplatedonacoverslipandmountedontoglassslidewithde- Microscopy ofFRAPandFLIP era equippedwiththeMetaMorph (MBL InternationalCorporation).ImageswereobtainedwithaCCDcam- previously (Zhangetal.,2000)usingacommercialmAbagainstRCC1 Immunofluorescence microscopyoncellswasperformedasdescribed Fluorescence microscopyandanalysisofsalt-extractedRCC1 with eggextractssupplemented 2 Xenopus In vitroassaysusing mitotic cellswithanInjectMan lecular Probes,Inc.)intothenucleiofinterphasecellsorcytoplasm in PBSwascoinjectedwithtetramethyl-rhodaminesuccinididylester(Mo- rhodamine andfluorescein,respectively.UnlabeledRanGDPorRanT24N fied usingNi-agarose.RanandRCC1werelabeledtetramethyl- 1994; WildeandZheng,1999).6his-RCC16his-RCC1-GFPwerepuri- Ni-agarose adoptingthemethodsasdescribedpreviously(Dassoetal., 6his-Ran and6his-RanT24Nwereexpressedinbacteriapurifiedusing Recombinant proteinandmicroinjection mobilization. zation ofRCC1-GFP,whereasinjectingat1mg/mlleadtoacompleteim- we observedthatinjectingRanT24Nat0.1mg/mlleadtopartialimmobili- should leadtoacompleteimmobilization.Consistentwiththisprediction, lead topartialRCC1immobilization,whereasinjectingRanat1mg/ml RanT24N canimmobilizeoneRCC1,injectingRanat0.1mg/mlshould whereas at0.1mg/mlofRan,only 500 msusingaspot1 periments, cellswerescannedtwice,followedbyasinglebleachpulseof RCC1-GFP expressedinthe3T3cellsshouldbe Bischoff andPonstingl(1995),thenumberofbothendogenousRCC1 pressed ataboutthesamelevelasendogenousRCC1.Accordingto plasm followedbyliveimagingandmicroscopy. salted intoPBSbeforeinjectingtheinterphasenucleusormitoticcyto- tetramethyl-rhodamine succinimidylester(MolecularProbes,Inc.)andde- rhodamine alone.Forlocalizationstudies,bothproteinswerelabeledwith lowed byFRAP.Controlinjectionswereperformedwithtetramethyl- 30a (forRanandRanT24N)orpET-15aRCC1;Novagen). wild-type Ran,RanT24N,RCC1,andRCC1-GFPweresubclonedintopET- and 0.6mg/mlGeneticin,allpurchasedfromGIBCOBRL.6His-tagged tained inMEM(GIBCOBRL)with10%FBS,1%penicillin/streptomycin, the presenceof0–40 anti-RCC1 antibody. subjected toSDS-PAGEfollowedbyWesternblottingandprobingwiththe ously (Ohtsuboetal.,1989).Thesupernatantandpelletfractionswere GFP andextractedwithincreasingsaltconcentrationsasdescribedprevi- Corp.). ToextractRCC1,nucleiwereisolatedfromcellsexpressingRCC1- through aglycerolcushion(80mM Pipes,pH6.8,1mMEGTA, 40 minatRT.Afterincubation,the spermwasspunontocoverslips According toourWesternblottinganalysis(Fig. 1C),RCC1-GFPisex- eggextractsandspermwerepreparedasdescribedpreviously A mechanismofRanGTPproductioninvivo| 0.2 plofRanwasdeliveredintothecellperinjection. M unlabeledRanGDPorRanT24N,respectively, for Xenopus m indiameter.Singlesectionimageswerethen 9% ofthebleachintensity.InFLIPexperi-
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643
The Journal of Cell Biology 644 of RCC1andRantothechromatinissensitivefreeGTP.Forassaying labeled RCC1and0–40 slips throughtheglycerolcushionwithoutGTP.250 Rh-RanT24N. Aftera40-minincubation,thespermwasspunontocover- plemented with0.5 ment. ForRCC1competition,spermwasincubatedwitheggextractssup- MetaMorph the backgroundwassubtractedfromareanexttoeachspermusing the camera.Thefluorescenceintensityofeachspermwasmeasured,and CCD cameraatthesameexposuretimethatisbelowsaturationlimitof to thespermchromatinwasquantifiedbytakingimagesusingacooled (Wilde andZheng,1999).TheamountoflabeledRanthatremainedbound RCC1-GFP and40 Bilbao-Cortes, D.,M.Hetzer,G.Langst,P.B.Becker,andI.W.Mattaj.2002.Ran References Accepted: 21January2003 Revised: 17January2003 Submitted: 1November2002 Wirtz). Zheng) andbytheNationalScienceFoundation(NSE/NIRT 0210718 toD. critical commentsonthemanuscript. JudithYanowitz,MaxGuo,andmembersoftheZhenglabfor Koshland, nana KarpovaforadviceonFRAPmicroscopy,andJoeGall,Doug Kimura andPeterCookforH2B-GFP,TomMisteli,JimMcNally,Tati- reading ofthemanuscript,OnaMartinfortechnicalassistance,Hiroshi We thankBobPhairforadviceonFRAPandFLIPanalysescritical www.jcb.org/cgi/content/full/jcb.200211004/DC1. the chromatin.Onlinesupplementalmaterialavailableathttp:// on analyses. Fig.S3showsthesimulationofRanGTPconcentrationprofile and RanGTPgradient.Figs.S1S2showcurvefitsofFRAPFLIP Online supplementalmaterialincludeskineticmodelingofRCC1mobility Online supplementalmaterial Hepes, pH7.7,100mMKCl,2MgCl RCC1-GFP binding,spermwasaddedtoextractssupplementedwith0.5 MgCl amounts ofpurifiedRanandRCC1,respectively. tract wereestimatedusingquantitativeWesternblottingwithknown coverslips, thespermwasincubatedwithXBbuffercontaining0–2 performed. AfterspinningthespermlabeledwithRCC1-GFPorRh-Ranonto chromatin intheabsenceoffreeGTP,sameexperimentsasabovewere plex. Totestthestabilityofbindingbinarycomplextosperm remain boundtothechromatinaftercentrifugationformedbinarycom- taining noGTP.ThisobservationsuggeststhatmostofRh-RanandRCC-GFP mained boundtothechromatinbeforeandafterextractionwithbuffercon- were quantifiedasabove.SimilaramountsofRh-RanandRCC1-GFPre- amount ofRCC1-GFPorRh-Ranthatremainedonthespermchromatin and incubatedfor20minatRTfollowedbyfixationDAPIstaining.The and 5mMEGTA)containing0–5GTPwasaddedontothecoverslips RanT24N inthepresenceof0–5 ing, spermwasaddedtoextractssupplementedwith2 tracts. TheconcentrationsofRan(20 Ran competitorswereusedatthesimilarratioasthatpresentineggex- bound tothespermchromatinwerequantified.TheunlabeledRCC1and lowed byfixationandDAPIstaining.Rh-RanRCC1-GFPthatremained The followingexperimentswereusedtodeterminewhetherthebinding This workwassupportedbytheHowardHughesMedicalInstitute(toY. The JournalofCellBiology 2 , and30%glycerol),fixedwithmethanol,stainedDAPI ® software.Atleast20spermwerequantifiedforeachexperi- M ofRanGDPorRanT24N.ForassayingRh-Ranbind- M RCC1-GFPand40 M unlabeledRanascompetitorsfor20minfol-
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M unlabeledRCC1ascompetitors. Volume 160,Number5,2003 M) andRCC1(1.3 2 , 0.1mMCaCl M unlabeledRanGDPor l ofbuffer(10mM 2 M Rh-RanGDPor , 50mMsucrose, M) intheeggex- M un- M Zhang, L.,T.J.Keating,A.Wilde,G.G.Borisy,andY.Zheng.2000.Therole of Wilde, A.,andY.Zheng.1999.Stimulationofmicrotubuleasterformationspindle Seki, T.,N.Hayashi,andT.Nishimoto.1996.RCC1intheRanpathway. Murray, A.W.,andM.W.Kirschner.1989.Cyclinsynthesisdrivestheearlyem- Seino, H.,S.Hisamoto,T.Uzawa,Sekiguchi,andNishimoto.1992.DNA- Renault, L.,J.Kuhlmann,A.Henkel,andWittinghofer.2001.Structuralbasis Phair, R.,andT.Misteli.2000.Highmobilityofproteinsinthemammaliancell Ohtsubo, M.,H.Okazaki,andT.Nishimoto.1989.TheRCC1protein,aregula- Nishitani, H.,M.Ohtsubo,K.Yamashita,H.Iida,J.Pines,Yasudo,Y.Shibata, Nemergut, M.,C.A.Mizzen,T.Stukenberg,C.D.Allis,andI.G.Macara.2001. Kalab, P.,K.Weis,andR.Heald.2002.VisualizationofaRan-GTPgradientin Dasso, M.,S.T.Azuma,T.Ohba,andNishimoto.1994.Amutantformofthe Dasso, M.2002.TheRanGTPase:themeandvariations. Kimura, H.,andP.R.Cook.2001.Kineticsofcorehistonesinlivingcells:littleex- Misteli, T.,A.Gunjan,R.Hock,B.Michael,andD.Brown.2000.Dynamicbind- Mattaj, I.,andL.Englmeier.1998.Nucleocytoplasmictransport:thesoluble Lounsbury, K.M.,S.A.Richards,K.L.Carey,andI.G.Macara.1996.Mutations Kornbluth, S.,M.Dasso,andJ.Newport.1994.EvidenceforadualroleTC4 Klebe, C.,H.Prinz,A.Wittinghofer,andR.S.Goody.1995b.Thekineticmecha- Klebe, C.,F.R.Bischoff,H.Ponstingl,andA.Wittinghofer.1995a.Interactionof Bischoff, F.R.,andH.Ponstingl.1995.Catalysisofguaninenucleotideexchange Bischoff, F.R.,andH.Ponstingl.1991.Catalysisofguaninenucleotideexchange ment. Xgrip210 ingammatubulinringcomplexassemblyandcentrosomerecruit- assembly bythesmallGTPaseRan. chem (Tokyo). bryonic cellcycle. DNA replication. binding domainofRCC1proteinisnotessentialforcouplingmitosiswith condensation (RCC1). for guaninenucleotideexchangeonRanbytheregulatorofchromosome nucleus. binds toDNA. tor fortheonsetofchromosomecondensationlocatesinnucleusand tion ofcdc2proteinkinaseandmitosis. ing protein,uncouplesthecompletionofDNAreplicationfromactiva- T. Hunter,andNishimoto.1991.LossofRCC1,anuclearDNA-bind- tones H2AandH2B. Chromatin dockingandexchangeactivityenhancementofRCC1byhis- interphase andmitotic EMBO J. inhibiting theRCC1protein,aregulatorofchromosomecondensation. Ran/TC4 proteindisruptsnuclearfunctionin R508. change ofH3andH4somerapidexchangeH2B. ing ofhistoneH1tochromatininlivingcells. phase. with theRan/TC4GTPase. 125:705–719. protein inregulatingnuclearstructureandcellcycleprogression. 12543–12552. nism ofRan-nucleotideexchangecatalyzedbyRCC1. and RanGAP1. the nuclearGTP-bindingproteinRanwithitsregulatoryproteinsRCC1 1341–1354. GAP1. of RanbyRCC1andstimulationhydrolysisRan-boundGTPRan- on RanbythemitoticregulatorRCC1. binds tochromatinbytwodistinctmechanisms. J. CellBiol. Annu. Rev.Biochem. Methods Enzymol. Nature. 13:5732–5744. 120:207–214. J. CellBiol. Biochemistry. 404:604–609. 151:1525–1535. J. CellSci. Nature. Science. Xenopus Cell. 257:135–144. 339:275–280. 109:1389–1397. 67:265–306. 105:245–255. 102:393–400. J. Biol.Chem. 34:639–647. 292:1540–1543. eggextracts. Science. EMBO J. Nature. 284:1359–1362(seecomment). 271:32834–32841. Science. Nature. Xenopus laevis 354:80–82. Curr. Biol 10:1555–1564. 295:2452–2456. 408:877–881. Curr. Biol. . 12:1151–1156. Biochemistry. J. CellBiol. eggextractsby J. CellBiol. 12:R502–
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