Table Of ContentMethods in
Molecular Biology 2175
Ronald Hancock Editor
The Nucleus
Third Edition
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire, UK
Forfurther volumes:
http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas
thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-
by-stepfashion,openingwithanintroductoryoverview,alistofthematerialsandreagents
neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexedinPubMed.
The Nucleus
Third Edition
Edited by
Ronald Hancock
Biosystems Group, Biotechnology Center, Silesian University of Technology, Gliwice, Poland
Editor
RonaldHancock
BiosystemsGroup,Biotechnology
Center
SilesianUniversityofTechnology
Gliwice,Poland
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-0716-0762-6 ISBN978-1-0716-0763-3 (eBook)
https://doi.org/10.1007/978-1-0716-0763-3
©SpringerScience+BusinessMedia,LLC,partofSpringerNature2020
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computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped.
Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply,
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CoverIllustrationCaption:SinglemoleculelocalizationimageofasectionofanucleuslabeledwithanALU-specific
oligonucleotide(red)andanantibodyagainstH3K9me3(heterochromatin,green)(seeChapter6byHausmannetal).
ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer
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Preface
This third edition of The Nucleus presents recently developed and detailed protocols for
thoseworkingonthecellnucleus,includingsomelesswell-exploredaspects.Thevolumesin
thisseriesaredesignedprimarilyforresearchers,butwhenrecallingmyownexperienceasa
PhDstudentwhichincludedvisitstoalltheneighboringlaboratoriestoseethemostrecent
methods at work, they should also help students and postdocs to be aware of available
methodologieswhenplanningtheirprojects.
Ithanktheauthorswhocontributedtothisbookfor their friendlyandtimelycollabo-
ration, and Joanna Rzeszowska and Andrzej S´wierniak for their support during its
preparation.
Gliwice,Poland RonaldHancock
v
Contents
Preface ..................................................................... v
Contributors................................................................. ix
1 Live-CellImagingandAnalysisofNuclearBodyMobility.................... 1
DmitryV.Sorokin,EugeneA.Arifulin,YegorS.Vassetzky,
andEugeneV.Sheval
2 LaserTargetedOligoLigation(LTOL)toIdentifyDNASequences
intheVicinityofaSingleSubnuclearStructureinaSingleCell............... 11
DavidAnchelandReaganW.Ching
3 VisualizingChromatinModificationsinIsolatedNuclei ..................... 23
YuanLi,ZacharyKlase,andLucaSardo
4 Dual-ColorMetal-InducedEnergyTransfer(MIET)Imaging
forThree-DimensionalReconstructionofNuclearEnvelopeArchitecture...... 33
AlexeyI.Chizhik,AnnaM.Chizhik,DajaRuhlandt,JaninePfaff,
NarainKaredla,IngoGregor,RalphH.Kehlenbach,andJ¨orgEnderlein
5 StudyingProtonGradientsAcrosstheNuclearEnvelope .................... 47
RaulMartı´nez-Zaguila´nandSouadR.Sennoune
6 COMBinatorialOligonucleotideFISH(COMBO-FISH)
withUniquelyBindingRepetitiveDNAProbes............................. 65
MichaelHausmann,Jin-HoLee,AaronSievers,MatthiasKrufczik,
andGeorgHildenbrand
7 Genome-WideMappingofUV-InducedDNADamagewithCPD-Seq........ 79
PengMaoandJohnJ.Wyrick
8 AP-Seq:AMethodtoMeasureApurinicSitesandSmallBaseAdducts
Genome-Wide.......................................................... 95
AnnaR.Poetsch
9 Locus-SpecificChromatinProteomeRevealedbyMass
Spectrometry-BasedCasID............................................... 109
EnesUgur,MichaelD.Bartoschek,andHeinrichLeonhardt
10 MethylAdenineIdentification(MadID):High-ResolutionDetection
ofProtein-DNAInteractions............................................. 123
DavidUmlauf,MichalSobecki,andLaureCrabbe
11 OptimizedDetectionofProtein-ProteinandProtein-DNAInteractions,
withParticularApplicationtoPlantTelomeres ............................. 139
Sˇa´rkaSchorˇova´,Jirˇı´ Fajkus,andPetraProcha´zkova´ Schrumpfova´
12 MacromolecularCrowdingMeasurementswithGeneticallyEncoded
ProbesBasedonFo¨rsterResonanceEnergyTransferinLivingCells .......... 169
SaraN.Mouton,LiesbethM.Veenhoff,andArnoldJ.Boersma
13 AnalysisofaNuclearIntrinsicallyDisorderedProteome ..................... 181
BozenaSkupien-Rabian,UrszulaJankowska,andSylwiaKedracka-Krok
vii
viii Contents
14 TimingofCytosineMethylationonNewlySynthesizedRNA
byElectronMicroscopy ................................................. 197
LorenaZannino,StellaSiciliani,andMarcoBiggiogera
15 TheNucleusofIntestinalCellsoftheBacterivoreNematode
CaenorhabditiselegansasaSensitiveSensorofEnvironmentalPollutants ...... 207
AnnettePiechulek,LutzBerwanger,PeterHemmerich,
andAnnavonMikecz
Index ...................................................................... 219
Contributors
DAVIDANCHEL • GeneticsandGenomeBiologyProgram,TheHospitalforSickChildren,
Toronto,ON,Canada
EUGENEA.ARIFULIN • BelozerskyInstituteofPhysico-ChemicalBiology,LomonosovMoscow
StateUniversity,Moscow,Russia
MICHAELD.BARTOSCHEK • DepartmentofBiologyIIandCenter forIntegratedProtein
ScienceMunich(CIPSM),HumanBiologyandBioImaging,Ludwig-Maximilians-
Universita€tMu¨nchen,Munich,Germany
LUTZBERWANGER • IUF-LeibnizResearchInstituteofEnvironmentalMedicineatHeinrich-
Heine-UniversityDuesseldorf,Duesseldorf,Germany
MARCOBIGGIOGERA • LaboratoryofCellBiologyandNeurobiology,DepartmentofBiology
andBiotechnology,UniversityofPavia,Pavia,Italy
ARNOLD J.BOERSMA • DWI-LeibnizInstituteforInteractiveMaterials,Aachen,Germany
REAGANW.CHING • GeneticsandGenomeBiologyProgram,TheHospitalforSickChildren,
Toronto,ON,Canada;DepartmentofEpigenetics,MaxPlanckInstituteof
ImmunobiologyandEpigenetics,Freiburg,Germany
ALEXEYI.CHIZHIK • ThirdInstituteofPhysics–Biophysics,GeorgAugustUniversity,
Go¨ttingen,Germany
ANNAM.CHIZHIK • ThirdInstituteofPhysics–Biophysics,GeorgAugustUniversity,
Go¨ttingen,Germany
LAURE CRABBE • LBCMCP,CentredeBiologieIntegrative(CBI),CNRS,Universite´de
Toulouse,Toulouse,France
JO¨RGENDERLEIN • ThirdInstituteofPhysics–Biophysics,GeorgAugustUniversity,
Go¨ttingen,Germany;MultiscaleBioimaging:fromMolecularMachinestoNetworksofEx-
citableCells(MBExC),GeorgAugustUniversity,Go¨ttingen,Germany
JIRˇI´ FAJKUS • LaboratoryofFunctionalGenomicsandProteomics,NationalCentrefor
BiomolecularResearch,FacultyofScience,MasarykUniversity,Brno,CzechRepublic;
MendelCentreforPlantGenomicsandProteomics,CentralEuropeanInstituteof
Technology,MasarykUniversity,Brno,CzechRepublic;InstituteofBiophysics,Academyof
SciencesoftheCzechRepublic,v.v.i.,Brno,CzechRepublic
INGO GREGOR • ThirdInstituteofPhysics–Biophysics,GeorgAugustUniversity,Go¨ttingen,
Germany
MICHAELHAUSMANN • KirchhoffInstituteforPhysics,UniversityofHeidelberg,Heidelberg,
Germany
PETERHEMMERICH • LeibnizInstituteonAging-FritzLipmannInstitute,Jena,Germany
GEORGHILDENBRAND • KirchhoffInstituteforPhysics,UniversityofHeidelberg,Heidelberg,
Germany
URSZULA JANKOWSKA • LaboratoryofProteomicsandMassSpectrometry,MalopolskaCentre
ofBiotechnology,JagiellonianUniversity,Krakow,Poland
NARAINKAREDLA • ThirdInstituteofPhysics–Biophysics,GeorgAugustUniversity,
Go¨ttingen,Germany
SYLWIA KEDRACKA-KROK • DepartmentofPhysicalBiochemistry,FacultyofBiochemistry,
BiophysicsandBiotechnology,JagiellonianUniversity,Krakow,Poland
ix
x Contributors
RALPHH.KEHLENBACH • DepartmentofMolecularBiology,GZMB,UniversityMedical
CenterofGo¨ttingen,GeorgAugustUniversity,Go¨ttingen,Germany
ZACHARYKLASE • DepartmentofPharmacologyandPhysiology,DrexelUniversityCollegeof
Medicine,Philadelphia,PA,USA
MATTHIASKRUFCZIK • KirchhoffInstituteforPhysics,UniversityofHeidelberg,Heidelberg,
Germany
JIN-HOLEE • KirchhoffInstituteforPhysics,UniversityofHeidelberg,Heidelberg,Germany
HEINRICHLEONHARDT • DepartmentofBiologyIIandCenterforIntegratedProteinScience
Munich(CIPSM),HumanBiologyandBioImaging,Ludwig-Maximilians-Universita€t
Mu¨nchen,Munich,Germany
YUANLI • DepartmentofInfectiousDiseasesandVaccines,MRL,Merck&Co.Inc.,West
Point,PA,USA
PENG MAO • SchoolofMolecularBiosciences,WashingtonStateUniversity,Pullman,WA,
USA
RAULMARTI´NEZ-ZAGUILA´N • DepartmentofCellPhysiologyandMolecularBiophysics,Texas
TechUniversityHealthSciencesCenter,Lubbock,TX,USA
SARAN.MOUTON • EuropeanResearchInstitutefor theBiologyofAgeing,Universityof
Groningen,UniversityMedicalCenterGroningen,Groningen,TheNetherlands
JANINE PFAFF • DepartmentofMolecularBiology,GZMB,UniversityMedicalCenterof
Go¨ttingen,GeorgAugustUniversity,Go¨ttingen,Germany
ANNETTEPIECHULEK • IUF-LeibnizResearchInstituteofEnvironmentalMedicineat
Heinrich-Heine-UniversityDuesseldorf,Duesseldorf,Germany
ANNAR.POETSCH • St.AnnaChildren’sCancerResearchInstitute,Vienna,Austria
DAJARUHLANDT • ThirdInstituteofPhysics–Biophysics,GeorgAugustUniversity,
Go¨ttingen,Germany
LUCA SARDO • DepartmentofInfectiousDiseasesandVaccines,MRL,Merck&Co.Inc.,
WestPoint,PA,USA
SˇA´RKASCHORˇOVA´ • LaboratoryofFunctionalGenomicsandProteomics,NationalCentrefor
BiomolecularResearch,FacultyofScience,MasarykUniversity,Brno,CzechRepublic
PETRA PROCHA´ZKOVA´ SCHRUMPFOVA´ • LaboratoryofFunctionalGenomicsandProteomics,
NationalCentreforBiomolecularResearch,FacultyofScience,MasarykUniversity,Brno,
CzechRepublic;MendelCentreforPlantGenomicsandProteomics,CentralEuropean
InstituteofTechnology,MasarykUniversity,Brno,CzechRepublic
SOUADR.SENNOUNE • DepartmentofCellPhysiologyandMolecularBiophysics,TexasTech
UniversityHealthSciencesCenter,Lubbock,TX,USA
EUGENEV.SHEVAL • BelozerskyInstituteofPhysico-ChemicalBiology,LomonosovMoscow
StateUniversity,Moscow,Russia;DepartmentofCellBiologyandHistology,Facultyof
Biology,LomonosovMoscowStateUniversity,Moscow,Russia
STELLA SICILIANI • LaboratoryofCellBiologyandNeurobiology,DepartmentofBiologyand
Biotechnology,UniversityofPavia,Pavia,Italy
AARONSIEVERS • KirchhoffInstituteforPhysics,UniversityofHeidelberg,Heidelberg,
Germany
BOZENASKUPIEN-RABIAN • DepartmentofPhysicalBiochemistry,FacultyofBiochemistry,
BiophysicsandBiotechnology,JagiellonianUniversity,Krakow,Poland
MICHAL SOBECKI • InstituteofAnatomy,UniversityofZurich,Zurich,Switzerland
DMITRYV.SOROKIN • LaboratoryofMathematicalMethodsofImageProcessing,Facultyof
ComputationalMathematicsandCybernetics,LomonosovMoscowStateUniversity,
Moscow,Russia
