NuclearIncorporationofIronduringtheEukaryoticCellCycle
IanRobinsona,b,YangYangc,FucaiZhanga,b,ChristopheLyncha,bandYusuf
Mohammeda,b
aResearchComplexatHarwell,RutherfordAppletonLaboratory,Didcot,Oxon,OX11
0FA,UK
bLondonCentreforNanotechnology,UniversityCollegeLondon,WC1E6BT,UK
cEuropeanSynchrotronRadiationFacility,38043Grenoble,France
ScanningX-rayfluorescencemicroscopyhasbeenusedtoprobethedistributionof
S,PandFewithincellnuclei.Nuclei,whichmayhaveoriginatedatdifferentphases
ofthecellcycle,arefoundtoshowverydifferentlevelsofFepresentwithastrongly
inhomogeneousdistribution.PandSsignals,presumablyfromDNA,arehighand
relativelyuniformacrossallthenuclei;theseagreewithcoherentzoomholography
phasecontrastimagesofthesamesamples.WediscusspossiblereasonsfortheFe
incorporation.
1.Introduction
HumancellnucleiareappropriatetestsamplesfortheNanoscaleImaging(NI)
branchofthenew“NINA”beamline(ID16A)atESRF,describedbyMartinez-Criado
etal(2012).Theirsizesareintherangeof10µm,whichfallswithinthefieldof
viewofthepropagation-basedphasecontrastimagingcapabilityofID16A.In
addition,high-resolutionsubstructureisexpected,atleastifthenucleiarecloseto
themetaphasepointofthecellcyclewhentheparentcellispreparingfordivision.
ForthefluorescenceimagingcapabilitiesofID16A,knownquantitiesofDNAand(to
alesserextent)proteinsareexpectedtobepresentinacellnucleus,whichcanbe
usedinquantitativechemicalanalysisandtoverifythecalibrationofthesensitivity.
Nucleiclosetometaphaseweretargetedinthisstudybecauseofinterestinthe
higher-orderstructureoftheseparatedchromosomeslocatedwithinthem,butit
wasalsoappreciatedthatthisreallyneedsathree-dimensional(3D)imaging
capabilitytosegregatethem.Oursamplepreparationmethodsmakeuseofcell
cyclecheckpointinhibitorstosynchronizethecellsduringculture,butthisstill
allowssomenucleitoemergefromthepreparationsatotherpointsofthecellcycle.
Carefulfilteringisusedtoremovecytoplasmandmostoftheothercellcomponents
(Yusufetal,2014),soarelativelypurepreparationofwholenucleiandindividual
chromosomesisobtained,manywiththenuclearmembraneintact.Thisstrictly
excludesnucleiinmetaphaseproper,oncethenuclearmembranedissolves,but
doesincludeprophasejustbeforehand,whenthe46chromosomesarefullyformed
withinanucleus.IfthecellswereinG1phasewhenthesampleswereprepared,
theywouldcontaintwodouble-strandedcopiesofallthegenomicDNA;iftheywere
inG2phaseorthebeginningofmetaphase(M-phase),thereshouldbefourcopies;
inSphase,therewouldbesomewherebetweentwoandfourcopies.
Thefullhumangenomecontains3.2x109basepairs(bp)perdouble-strandofDNA,
whichisdividedintothe23chromosomes.Associatedwitheachbasepairaretwo
phosphates,oneoneachstrand.Thesearebyfarthelargestexpectedcontribution
tothePfluorescentX-raysignal,withsmalladditionalamountscomingfrom
buffers,thelipidsinthecellmembranesandanyresidualRNAorATP.Soacell
nucleusshouldhaveawell-definedsignalfromthese2.6x1010Patomsinits
fluorescentimagesifitisinthesecondhalfofthecellcycle(G2orMphase),or
1.3x1010Patomsinitsfluorescentimagesifitisinthefirsthalfofthecellcycle(G1
phase).
Similarly,theSsignalwouldbemostlyattributedtoCysteine(Cys)andMethionine
(Met)residuesinthenuclearproteins.Fortunately,muchisknownaboutthemakeupofthe(mostlystructural)chromosomalproteinsfoundinmetaphasefromthe
workofUchiyamaetal(2005):71%ofthetotalmassishistones,whicharethecore
proteinsaroundwhichtheDNAisspooledtomakenucleosomes.Thehistones
containmanybasicArginineandLysinegroups,whichhelpneutralizethenegative
chargecarriedbytheDNA.Onenucleosometypicallyoccupies170basepairsof
DNAand,sincemostoftheDNAcanbeassumedtohavecondensedinto
nucleosomes,wecanusethistoestimatetheexpectedtotalamountofproteinper
nucleus.Moreover,thehistonesequencesareallknown,sowecanexpectthereto
be14Satoms(2xCysand12xMet)per170bpofDNAassociatedwiththehistones
(Mariño-Ramírezetal,2011).Wethereforeexpectacellnucleustohave2.1x109S
atomsinitsfluorescentimagesinG2orMphaseand1.0x109SatomsinG1phase.
ThepresenceofFeinthecellnucleushasbeendiscussedrepeatedlyinthescientific
literature.Yagietal(1992)havesuggestedtheremaybeanevolutionary
connectionbetweenironandDNAbecauseofthepowerfulredoxpotentialofFe.Fe
isanessentialelementofproteins,oftenintheformofiron-sulfur(FeS)clusters
usedinelectrontransportenzymes(Johnsonetal,2005)orinhemecomplexesin
cytochromes(Dawson,1988).Ironcanbetoxictocellsviathegenerationoffree
radicals(Yagietal,1992).SincethepresenceofironcanleadtoDNAdamage
pathways,theremaybeevolutionaryadvantagetokeepingtheDNAinitsown
nuclearcompartment,awayfrommanyofthemetabolicprocesses.
DespitetheviewthatFe-containingenzymeswouldnotbewidelyusedwithinthe
nuclearcompartmentofthecell,therehavebeenrecentreportsofFeS-containing
enzymesdirectlyinvolvedwithDNAreplication.DNAprimasewasfoundtocontain
anFeSdomain(Klingeretal2007,Weineretal2007)alongwithDNAhelicase(Wu
andBrosh,2012)andDNArepairglycosylases(WuandBrosh,2012).Areviewby
Lilletal(2006)namedfiveassociationsofFeSproteinswiththecellnucleus:DNA
glycosylase(Ntg2),Histoneacetyltransferase(Elp3),P-loopATPase(Nbp35),Irononlyhydrogenase(Nar1)andABCprotein(Rli1).Allofthesesfunctionsare
believedtobeassociatedwithDNAreplicationandrepairsoshouldbeexpressed
onlyduringS-phaseofthecellcycleandshouldbeabsentduringotherphases.
Ferritin,theEukaryoticironstorageprotein,isnotexpectedtobecolocalizedwith
DNA,yetthiswasreportedinafewdiverseexamplesbyThompsonetal(2002).
NuclearferritinmightbeassociatedwiththeprotectionofDNAorconverselywith
oxidativeDNAdamage.Ifnuclearferritinispresent,itmightbeexpectedtobe
associatedwiththenuclearmembrane,ratherthanmixedinwiththeDNAcontainingchromatin.Toaddressthesequestions,FluorescentX-rayimagingof
humancellnucleiwasundertakenintheworkreportedhere.
2.Methods
2.1Samplepreparation
Nucleiwerepreparedaccordingtoapreviouslypublishedfiltration-basedprotocol
forchromosomes(Yusufetal,2014)withmodificationstopreservetheintact
nuclei.HumanLymphocytecellswerecultivatedinFetalBovineSerum(FBS)and
treatedwithColcemidtoarresttheminmetaphase.Followingextraction,thenuclei
werefixedin0.5%Glutaraldehydecontaining10mMHepes-KOHand5mMMgCl2.
Thesampleswerepipettedin5µLdropsonto200nmthicksiliconnitridewindows
andstainedwith150µMSybergolddye.Afterwashinginwater,thesampleswere
lefttodryinair.TheywereimagedusingaZeissAxioZ2microscope(using“Isis”
software)toobtainvisiblelightandopticalfluorescenceimagesforreferenceand
correlationwiththeX-rayresults.
ForX-rayimaging,severalmembraneswerepreparedwiththesamesample
material.Theresultingsampleswerefoundtocontainalargenumberofintact
nuclei,butalsochromosomespreadsandindividualchromosomesfromburst
nuclei.Someofthemembrane-boundsampleswerestainedwithplatinumblue
(WannerandFormanek,1995),at5mMfor30minutesandwashedinwater,butno
significantdifferenceswerefoundinthephasecontrastimagesandnoPtsignalwas
detectedinfluorescence.ResultsarereportedfromsamplespreparedwithoutPt
staining.AftertheX-rayexperiment,thesampleswerereimageswithanOlympus
LEXT4000confocalmicroscopetoobtainfurtherreferenceimagesoftherelevant
samples.
2.2X-raymeasurements
Samplesonmembraneswereclampedintothesampleinsertionstubsdesignedfor
ID16A.Thesewereload-lockedintotheUHVsamplechamberundervacuumat
roomtemperaturebydroppingthemfromamanipulatorintothepiezo-driven
samplestage.Thepiezodrivesystemwaskeptactiveduringthisoperationsothat
thecontactforcescouldbemonitoredinordertopreventoverloadingthestages.
TheID16AbeamlinehasamultilayercoatedKirkpatrick-Baez(KB)focusingmirror
pairlocatedat185mfromanundulatorsourceoperatingat17keV(Moraweetal,
2015).TheKBsystem,withextremedemagnificationdesignedfora14x14nm
focus,producedameasuredfocusof20x30nmwithveryhighfluxfromthebroad
bandpassofthemultilayer.
Forfluorescentimagingexperiments,thesamplewasraster-scannedacrossthe
beamatthefocuspositionin30nmsteps.Theforwardandbackwarddirected
fluorescencewasdetectedbyapairof6-elementsilicondriftdiodedetectorswitha
dwelltimeof100ms(CHECK).Spectraweredecomposedintopuresignalsfromthe
P-K,S-KandFe-Lemissionlinesat2014,2308and705eVrespectively(CHECK)by
principlecomponentanalysisandcalibratedwithstandardsamplesofknownmass
density(DETAILS).
Dataforthephasecontrastimageswereobtainedbymovingthesample
downstreamofthefocusbyarangeofdistances(DETAILS)andrecordingthe
projectedimageonalens-coupledFReLoNdetectorwith2048x2048pixels,giving
aneffectivepixelsizeof1.1µm.Thesedata,measuredcoherentlyatfourdifferent
magnifications,werecombinedusingtheholotomographymethod,nowcalled
“zoom”tomography,toobtainfull-fieldquantitativephasecontrastimagesofthe
sampleintransmission(Cloetensetal,1999).
3.ResultsandDiscussion
Figure1showsanoverviewopticalfluorescenceimagetakenundertheexcitation
conditionsforSybergolddye,whichbindsspecificallytoDNA.Whilethenucleiare
clearlywellisolatedonthemembrane,itisclearthatnotallofthemareequally
bright.Thissuggeststhateitherthedyeisunabletopenetratethesamples
uniformlyor,morelikely,thatsomenucleihavebecomedepletedintheirDNA
content.Thismighthaveoccurredduringthewashingstepsofthesample
preparation,orpossiblyduringhandlingofthesamples.Wenotethattheimage
wastakenshortlyaftersamplepreparation,beforetransportingthesamplesto
ESRF,sothisdoesnottakeintoaccounttheeffectsofthevacuumsampletransfer
intotheID16Ainstrument.
Figure2showscomparisonimagesofanisolatednucleusbybothavailableX-ray
imagingmethods:zoomtomographyphasecontrastandscanningX-ray
fluorescenceoftheP,SandFelines.Thetotalsignalsforthethreeelements,
integratedovertheimagesandcalibratedinunitsofnumbersofatoms,arelistedin
Table1.Thefieldofviewofthisimagealsocontainsoneortwoindividual
chromosomesinasmallclusterattheside.Thisnucleuscontainstheleastquantity
ofFeobserved,thelowestlevelofPandthehighestS.ThedistributionoftheP-and
S-signalsoverlaywellontopofeachotherandalsoagreewiththedistributionof
phaseshiftmeasured(QUANTITATIVEANALYSISOFPHASEVALUES??).Thedomeshapeddistributionofallthreeimagesisroughlywhatwouldbeexpectedfora
sphericalorhemisphericalnucleuswithauniformdensityofchromatinmatter
withinitsvolume.
ThetotalPsignalcomingfrom3.9x109Patomsisafactor-of-threebelowthelower
estimateof1.3x1010Patoms,givenabove,expectedforanucleusinthefirsthalfof
thecellcycle.ThissuggestseitheracalibrationerrororthatsomePhasbeenlost
duringthesamplepreparationandinsertionintovacuum.Wedonotattributethis
toradiationdamagebecausethesignallevelsintheimageswerefoundtobe
reproducibleuponrepeatedscanning.Ontheotherhand,thetotalfluorescentS
signalof4.3x109atomsissubstantiallyhigherthanthehigherestimateaboveof
2.1x109SatomsinG2orMphase.Sincethisappearstobehomogeneously
distributedwithinthechromatinfilledregionofthenucleus,thissuggeststhatthe
extrasignalmaybecomingfromthe29%non-histoneproteins,whichmayhave
higherrelativelevelsofCysandMetaminoacids(Uchiyamaetal,2005).
Correspondingimagesfromthreemorenuclei,aslabelledinFig1andshowninFig
3,gavetheintegratedsignalslistedinTable1.Thetrendissimilarwithan
underrepresentationofPandoverrepresentationofS.FortheSandPsignalsfrom
5nucleimeasured,therearefactorsof2variationsfromonenucleustoanother,
whichmightbeinherentmeasurementerrorsorvariationsofsamplepreparation.
ThehighestPsignaldoesjustreachthelowerestimatefortheamountofDNA
presentinG1phase,asdoesthelowestSsignalcrossovertheexpectedlevelfor
G2/M.
MuchgreatervariationwasfoundinboththemassesanddistributionsoftheFe
signal,forwhicha27xvariationwasfound.Fig2showsthenucleuswiththe
smallestlevelofFe,whilethatofFig3(a)hasthehighestlevel.UnlikeSandP,the
Fesignalsarestronglyclusteredandoftenseentobelocatedattheperipheryofthe
nuclei.ItisthereforeconcludedthemostoftheFesignaliscomingfromthenuclear
membranestructures,ratherthanthecentralregions,asdiscussedfurtherbelow.
WealsonotethattheseparatedchromosomestructureseenatthetopofFig2has
colocalisedPandSsignalscomingfromitsdistinctarmregionsandaseparateFe
signalinthecentre,whichisdepletedinPandS.Thisisappearstobean
agglomerationoftwochromosomesontheleftandrightsidesandmaybeapieceof
Fe-richnuclearmembraneinthecentre.
4.Conclusions
Forthehumanlymphocytecellnucleipresentedinthisstudy,thedistributionsofP
andSX-rayfluorescence,associatedwiththeDNA-proteincomplexofchromatin,
arefoundtoberelativelyuniformandstructureless.Thiscouldbebecausethecells
areininterphase(G1,S,orG2ofthecellcycle),whenthechromatinisdecondensed,
oritcouldbebecauseofinsufficientresolutiontoseetheindividualchromosomes.
AcertainamountofmodulatedstructurecanbedistinguishedinthePsignalsofFig
3(a)and3(c),whichresemblestheexpectedpatternofcondensedmetaphase
chromosomes,eventhoughtheyarenotfullyresolved.Ifso,thesenucleiarein
prophase,sincetheystillpossesstheirnuclearmembrane.
ThelevelsofbothPandSarerelativelyreproduciblefromnucleustonucleus,
withinafactorof2.ThelevelofPissystematicallylowerthanexpectedfromthe
numberofPatomscontainedintheDNAofthehumangenome.Notingthatsome
relativelyempty(deflated)nucleiwereobservedintheopticalfluorescenceimage
ofFig1,thismaybecausedbypartiallossofchromosomesduringthewashingstep
ofsamplepreparation.Itisunfortunatethatsuchlosseshavetakenplaceasa
reliablePlevelmeasurementcouldhavebeenausefuldeterminationofthephaseof
thecellcycle.BoththelevelsofSandtheS/Pratiosarefoundtobehigherthan
expectedfromthehistoneproteinsalone,whichcomprise71%ofthetotal
chromosomalprotein.Thissuggeststhatthenon-histoneproteinsmaybericherin
CysandMetresidues.
TheFeconcentration,while3ordersofmagnitudelowerthanPorS,ismuchmore
variedamongthesamplesexamined,by27-foldamongtheintegratedsignalsin
Table1.FeisnotexpectedtobeassociatedwithDNAingeneralforevolutionary
reasons(Yagietal,1992),yetsomeexceptions,particularlyduringDNAreplication
inSphase,arenotedabove.Feisseentoformsmallbrightspots,about100nmin
diameter,inthesamplesshowninthelow-concentrationcasesinFigs2and3(b).In
onecase,Fig3(b),FespotsarecolocalisedwithS,perhapssuggestingthepresence
ofFeSenzymes;intheothercases,Fig2,FeandSareseparatelylocalizedinspots.
Feisseentoformshell-likecrescent-shapedplaquesaroundtheedgesofthehighFeconcentrationnucleiinFigs3(a)and3(c).TheseareastrongsuggestionofFe
beinglocatedinthenuclearmembrane,ratherthanthechromatin-filledcentres.In
mostcasestheFesignalcanbeseentosurroundthatofthePandS.Thismay
thereforebeconsistentafterallwiththeevolutionaryhypothesisofYagietal
(1992).
Asfaraswecantell,thezoomtomographymeasurementsdidnonoticeabledamage
tothesamples,evenaftermultipleandlongerexposuresweretested.Thebeamis
substantiallyoutoffocushere,enlargedtomorethanthe20x20µmfieldofviewin
theclosest-distancecase.Howevertheraster-scanningfluorescencemeasurement
didcausevisiblechangestothesample,asrecordedinFig4.Thinningofthe
membraneovertheentirescannedareacanbedetectedintheconfocalheightmap
(greyscaleimage).Multipleoverlappingscannedareascanbeobservedforthe
uppernucleus,forwhichtheimagesappearinFig3(b).However,nomassloss
betweenthesesscanswasdetectedinthefluorescencesignal.
Acknowledgements
ThisworkwassupportedbyaBBSRCProfessorialFellowshipBB/H022597/1
"DiamondProfessorialFellowshipforimagingchromosomesbycoherentX-ray
diffraction".AdditionalsupportcamefromanEPSRCgrantEP/I022562/1“Phase
modulationtechnologyforX-rayimaging”.WethankESRFforbeamtimeand
hospitalityduringthemeasurements.
References
Cloetens,P.,Ludwig,W,Baruchel,J,VanDyck,D,VanLanduyt,J,Guigay,JPand
Schlenker,M(1999),Holotomography:Quantitativephasetomographywith
micrometerresolutionusinghardsynchrotronradiationxrays,Appl.Phys.Letts.75
2912-2914
Dawson,JH(1988)ProbingStructure-FunctionRelationsinHeme-Containing
OxygenasesandPeroxidases,Science240433-439
Johnson,DC,Dean,DR,Smith,ADandJohnson,MK(2005),Structure,function,and
formationofbiologicaliron-sulfurclusters,Ann.Rev.Biochemistry74247-281
Klinge,S.,Hirst,J.,MamanJD,KrudeT.andPellegrini,L.(2007),Iron-sulfurdomain
oftheeukaryoticprimaseisessentialforRNAprimersynthesis,NatureStructural&
MolecularBiology14875
Lill,R.,Dutkiewicz,R.,Elsässer,H-P,Hausmann,A.Netz,DJA,Pierik,AJ,Stehling,O.,
Urzica,E.andMühlenhoff,U.,(2006),Mechanismsofiron–sulfurproteinmaturation
inmitochondria,cytosolandnucleusofeukaryotes,BiochimicaetBiophysicaActa
1763652
Mariño-Ramírez,L.,Levine,K.M.,Morales,M.,Zhang,S.,Moreland,R.T.,Baxevanis,
A.D.,andLandsman,D.(2011)TheHistoneDatabase:anintegratedresourcefor
histonesandhistonefold-containingproteins.DatabaseVol.2011:articleIDbar048,
doi:10.1093/database/bar048.
Martinez-Criado,G,Tucoulou,R,Cloetens,P,Bleuet,P,Bohic,S,Cauzid,J,Kieffer,I,
Kosior,E,Laboure,S,Petitgirard,S,Rack,A,Sans,JA,Segura-Ruiz,J,Suhonen,H,
Susini,J.andVillanova,J(2012),StatusofthehardX-raymicroprobebeamlineID22
oftheEuropeanSynchrotronRadiationFacility,JournalofSynchrotronRadiation19
10-18
Morawe,C,Barrett,R,Cloetens,P,Lantelme,B,Peffen,JC,Vivo,A(2015),Graded
multilayersforfiguredKirkpatrick-BaezmirrorsonthenewESRFendstation
ID16A,AdvancesinX-Ray/EUVOpticsandComponentsX,ProceedingsofSPIE9588
958803-1
Thompson,KJ,Fried,MG,Ye,Z.,BoyerP.andConnor,JR,(2002),Regulation,
mechanismsandproposedfunctionofferritintranslocationtocellnuclei,Journalof
CellScience1152165
Wanner,G;Formanek,H(1995),ImagingofDNAinHumanandPlantChromosomes
byHigh-ResolutionScanningElectron-Microscopy,ChromosomeResearch3368374
Weiner,B.E.,HuangH.,Dattilo,BM,NilgeMJ,FanningE.andChazin,W.J.(2007),An
Iron-SulfurClusterintheC-terminalDomainofthep58SubunitofHumanDNA
Primase,J.BiologicalChemistry28233444
WuY.andBrosh,R.M.(2012),DNAhelicaseandhelicase–nucleaseenzymeswitha
conservediron–sulfurcluster,NucleicAcidsResearch404247
Yagi,K.,Ishida,N.,Komura,S.,Ohishi,N.,Kusai,M.andKohno,M,(1992),Generation
ofhydroxylradicalfromlinoleicacidhydroperoxideinthepresenceofepinephrine
andiron,Biochem.Biophys.Res.Commun.183945–951
Yusuf,M,N.Parmar,G.K.BhellaandI.K.Robinson(2014),Asimplefiltration
techniqueforobtainingpurifiedhumanchromosomesinsuspension,BioTechniques
56257-261
FiguresandTables
Figure1.Lowmagnificationopticalfluorescenceimagetakenundertheexcitation
conditionsforSybergolddyeusingaZeissAxioZ2microscope.Boxesandlabels
indicatethenucleithatareanalysedfurtherinthiswork.
Figure2.X-rayimagesofthenucleusoutlinedinFig1,withagroupofindividual
chromosomesontheupperside.Topleft:zoom-tomographyphasecontrastimage.
Otherpanels:elementaldistributionsfromraster-scannedfluorescencemapping.
Figure3.X-rayimagesofthreemorenucleioutlinedinFig1.Leftcolumn:zoomtomographyphasecontrastimage.Centrecolumns:elementaldistributionsfrom
raster-scannedfluorescencemapping,scaledtothemaximumpixelvalue.Right:
opticalconfocalheightmap,measuredaftertheX-rayexperiment.Thescalebar
appliestoallpanels.
Figure4.Grey-scaleconfocalimage
measuredwitha50xlensonan
OlympusLEXT4000microscope
aftertheX-rayexperiment.Nuclei
3(b)and3(c)canbeseen,alongwith
aclearmodificationtothemembrane
intheregionwherethefluorescence
mappinghadtakenplace.
Sample
Pcount
Scount
Fecount
9
9
Fig2
3.9x10 4.3x10 2.8x106
9
9
Fig3(a)
9.3x10 2.6x10 7.6x107
Fig3(b)
6.1x109
1.5x109
5.6x106
Fig3(c)
7.1x109
1.8x109
5.0x107
9
9
Nucleus7
12.8x10 1.4x10 1.1x107
Average
7.8x109
2.3x109
9
9
G1phase
13x10 1.0x10 G2orMphase
26x109
2.1x109
Table1.CalibratedX-rayfluorescentsignals,integratedoverthefivemostreliable
rasterscansofhumancellnuclei.Derivedmasseshavebeenconvertedinto
numbersofatomsfoundwithinthenuclearregionsofthesamplesmeasuredat
ID16A.Thelasttworowsgivethecountsexpectedfordifferentphasesofthecell
cycle,asdiscussedinthetext.