Phase-Separated Biomolecular Condensates: Methods and Protocols (Methods in Molecular Biology, 2563) PDF
Preview Phase-Separated Biomolecular Condensates: Methods and Protocols (Methods in Molecular Biology, 2563)
Methods in Molecular Biology 2563 Huan-Xiang Zhou Jan-Hendrik Spille Priya R. Banerjee Editors Phase-Separated Biomolecular Condensates Methods and Protocols 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 biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents 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. Phase-Separated Biomolecular Condensates Methods and Protocols Edited by Huan-Xiang Zhou Department of Chemistry, University of Illinois at Chicago, Chicago, IL, USA Jan-Hendrik Spille Department of Physics, University of Illinois at Chicago, Chicago, IL, USA Priya R. Banerjee Department of Physics, University at Buffalo, State University, Buffalo, NY, USA Editors Huan-XiangZhou Jan-HendrikSpille DepartmentofChemistry DepartmentofPhysics UniversityofIllinoisatChicago UniversityofIllinoisatChicago Chicago,IL,USA Chicago,IL,USA PriyaR.Banerjee DepartmentofPhysics UniversityatBuffalo,StateUniversity Buffalo,NY,USA ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2662-7 ISBN978-1-0716-2663-4 (eBook) https://doi.org/10.1007/978-1-0716-2663-4 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface Thisvolumeonphase-separatedbiomolecularcondensatescomesatanopportunetime.As stated in the chapter by Mazarakos et al. (Chap. 1), “Phase-separated biomolecular con- densates are revolutionizing our understanding of biology. Although phase separation is a decidedlyphysicalphenomenon,therearegrowinggapsbetweenthebiologyandphysicsof condensates.” The methods presented in this volume have contributed significantly to narrowing these gaps, and it is thus timely to take stock of these contributions. Moreover, as these methods have become established, we hope that this volume will help popularize them and lead to broader applications. Lastly, many of the chapters take a forward look to assesshowthesemethodswillevolveinthefuture.Wethushopethatthisvolumewillalso spur thedevelopmentofnewmethods. Table1givesaquicksummaryofthe22chaptersinthisvolume.Thefirstfivechapters presenttheoreticalandcomputationalmethods.ThechaptersbyMazarakosetal.(Chap.1), Najafi et al. (Chap. 2), and Lin et al. (Chap. 3) all describe methods for determining binodals,thatis,theequilibriumconcentrationsofthediluteanddensephasesasafunction oftemperature.Mazarakosetal.illustratetwotraditionalmethods,Gibbs-ensembleMonte Carlo and slab-geometry molecular dynamics simulations, as well a more recent method called FMAP. In addition to binodals, this chapter also explains the calculation of the interfacial tensions of condensates. It specifically shows how the finite sizes of simulation systems affect binodals and interfacial tensions, and how these effects can be corrected. Najafietal.reviewfield-theoreticsimulations,whichprovideapowerfulmethodfordeter- mining phase equilibria, initially for polymers and now adapted for intrinsically disordered proteins (IDPs). Interestingly, field-theoretic simulations have been implemented in the Gibbs ensemble, thereby reducing the computational cost for binodal determination. Lin et al. also cover field-theoretic simulations, as well as an analytic method called random- phase approximation and coarse-grained simulations. Illustrative results of the methods includesequenceeffectsonthebinodalandonthemultiphaseorganizationofcondensates. Ginell and Holehouse (Chap. 4) review the stickers-and-spacers polymer model and show how it can rationalize experimental observations on phase equilibria. Laghmach et al. (Chap. 5) give an overview of modeling protein-RNA condensates at different scales, from all-atom to ultra-coarse-grained. Modeling results include various morphologies of condensatesandnonequilibriumeffects. The next ten chapters present experimental methods for in vitro characterization of biomolecular condensates. Quan et al. (Chap. 6) illustrate the application of fluorescence lifetime imaging microscopy (FLIM) in characterizing the material states of condensates formedbytheTDP-43low-complexitydomain(TDP-43LCD).Aninterestingobservationis that trimethylamine N-oxide (TMAO), a chemical chaperone, enhances the phase separa- tion of TDP-43LCD but inhibits its aggregation, by compacting the protein. Ganser et al. (Chap.7)describesingle-moleculeFo¨rsterresonanceenergytransfer(smFRET)andsingle- molecule protein-induced fluorescence enhancement (smPIFE) experiments for probing RNA conformational dynamics that can be regulated by interactions with proteins. These methodspotentiallycanbeusedtostudyRNAconformationaldynamicsinsidecondensates. Two chapters, by Incicco et al. (Chap. 8) and by Alshareedah and Banerjee (Chap. 9), describe the applicationof fluorescence correlation spectroscopy (FCS)in determining the v vi Preface Table1 Summary of chapters Chapter Methods Applications Uniqueangle Theoryandcomputation Mazarakosetal. Threesimulation Binonalsand Correctionforfinitesystemsize methods interfacialtension Najafietal. Field-theoretic Binodalof Samplingofpolymerconfigurations; simulations polyampholytes greatpotentialforIDPs Linetal. Analyticaltheory;field- Binodalsand SequencedependenceofIDPsin theoreticandcoarse- multiphase phaseseparation grainedsimulations organization Ginelland Stickers-and-spacers Rationalizationofexperimentalobservationsonphase Holehouse model equilibrium Laghmachetal. Multi-scalemodeling Protein-RNA Condensatemorphologyand condensates nonequilibriumeffects Opticalmicroscopy Quanetal. FLIM TDP-43LCD TMAOenhancesphaseseparation condensates butinhibitsaggregation,andcan thustunethematerialstate Ganseretal. smFRET/PIFE Protein-RNA Potentiallycanprobeinteractionsat interactions single-moleculelevelinside condensates Inciccoetal. FCS Concentrations; Theoreticalbackgroundand diffusion practicalconsiderations constants Alshareedahand FCS Diffusionofsingle Calibrationforaccuratelymeasuring Banerjee moleculesin diffusionconstants condensates Zuoetal. DNAcurtains Protein-DNA Real-timeobservationofDNA condensates compaction Peietal. Condensate-aided Identificationof High-throughputscreening enhancementof protein-protein interactions interaction partners OT Ghoshetal. Fluorescencemicroscopy Concentrations; OTisonlymethodformeasuring andOT interfacial viscoelasticityofcondensates tension; viscoelasticity Otherin-vitromethods Lietal. Crystallizationrobot; Conditionsfor High-throughputscreening high-contentanalysis phaseseparation (continued) Preface vii Table1 (continued) Chapter Methods Applications Uniqueangle Wangand Fluorescence Condensates Condensatesformedbyfolded Hayer-Hartl microscopy; involvedin proteins transmissionelectron biogenesisof microscopy carboxysome Tollerveyetal. Cryo-electron SPD-5;FUS; Identificationofinteraction tomography G3BP1 networks In-cellmethods GruijsdaSilva Sedimentation PTMsinRNP Invitroandincell andDormann condensates Reinkemeier Syntheticorganellesfor Reprogrammingof Selectiveproteinlabelinginlivecells andLemke GCE translation Parmarand Single-moleculetracking MobilityofRNA Sub-micronbiomolecular weber polymerase condensatesinlivecells KimandShin Optodroplets Plasmidpreparationfordeliveringoptodroplet componentsintoeukaryoticcells Rademacher Optodroplets ImageacquisitionandanalysisprotocolforassessingHP1 etal. phaseseparation Giesleretal. Massbalanceimaging N-WASP/F-actinin Growthkineticsofcondensates C.elegans Pandeyetal. Super-resolution Intracellularsingle-moleculeimagingmethodsforprobing microscopy;single- sub-diffractioncondensates moleculetracking concentrations and diffusion constants of condensate components. The former focuses on the theoretical background of FCS and practical considerations such as fluorophore selec- tion; the latter focuses on diffusion constants and the necessary calibration experiments. Combined, these two chapters provide the essential technical details, experimental proce- dure,anddataanalysisschemesfortheimplementationofFCSforstudyingphase-separated biomolecularcondensates.ThechapterbyZuoetal.(Chap.10)reviewstheapplicationofa methodcalledDNAcurtainsincharacterizingprotein-DNAcondensates.Thismethodcan detect the conformational change of a single DNA molecule induced by phase separation. Peietal.(Chap.11)illustratecondensate-aidedenrichmentofbiomolecularinteractionsin test tubes (CEBIT), whereby a protein of interest is fused to a scaffold protein that drives condensate formation, and client proteins that are interaction partners of the protein of interest are identified by their recruitment into the condensate. This method has the potentialforhigh-throughputscreeningofprotein-proteininteractions. Ghoshetal.(Chap.12)describetheapplicationoffluorescencemicroscopyandoptical tweezers(OT)inmeasuringconcentrations,interfacialtension,andviscoelasticity.Notably, OT is the only method for measuring the viscoelasticity of phase-separated biomolecular condensates.Lietal.(Chap.13)introduceahigh-throughputmethodforscreeningphase- separation conditions. In this method, the authors use a crystallization robot to prepare a viii Preface largenumberofmixturesandthenahigh-contentanalysissystemtorapidlyselectmixtures thatformcondensates.WangandHayer-Hartl(Chap.14)reviewtheirstudiesonconden- sates involved in the biogenesis of carboxysomes, which are cytosolic bodies for photosyn- thetic CO fixation. Interestingly, these condensates are formed by folded domains, 2 includingRubiscolargeandsmallsubunitsandmultipleRubiscosmallsubunit-likedomains connected by flexible linkers. The methods used include transmission electron microscopy in both negative-staining and cryo conditions. The chapter by Tollervey et al. (Chap. 15) describes the application of cryo-electron tomography in a variety of reconstituted biomo- lecular condensates. This method is capable of revealing interaction networks inside condensates. The last seven chapters present methods that enable in-cell characterization of biomo- lecular condensates.Gruijs da Silva and Dormann (Chap.16)outline sedimentation assays invitroandincelltoanalyzehowthephasebehaviorsofribonucleoproteins(RNPs)canbe modulatedbypost-translationalmodifications(PTMs)suchasphosphorylation.Giventhat many signaling proteins harbor multiple sites for PTMs, these assays provide simple yet usefultoolstostudysuchprocesses.ReinkemeierandLemke(Chap.17)describeanelegant method to generate synthetic organelles in live cells that perform genetic code expansion (GCE),protein-selectivenon-canonicalamino-acidincorporation,andsubsequentlabeling by small-molecule fluorophores. Parmar and Weber (Chap. 18) review single-molecule tracking for quantifying the mobility of a bacterial RNA polymerase in transcriptional condensates. This technique is particularly useful for studying sub-micron biomolecular condensatesinlivecells.KimandShin(Chap.19)describetheoptodropletassayandshow efficient ways to generate and deliver plasmids into eukaryotic cells. Rademacher et al. (Chap. 20) demonstrate how to use this assay to assess the phase-separation propensity of theheterochromatinproteinHP1inthenucleusoflivecells.Together,thesetwochapters provideadetailedrecipeforusingoptogeneticapproachestostudycondensatesinlivecells, fromplasmidpreparationtoimageacquisitionandanalysis.Giesleretal.(Chap.21)review massbalanceimaging(MBI)fordeterminingthephasebehaviorsanddissectingthekinetic properties of multi-component condensates. Finally, Pandey et al. (Chap. 22) discuss intracellular single-molecule imaging methods (super-resolution microscopy and single- molecule tracking). Carefully interpreted, these methods yield quantitative biophysical parametersatlengthscalesinaccessiblebyconventionalassays. The 22 chapters collectively provide a broad repertoire of theoretical, computational, and experimental methods to quantitatively interrogate the properties of phase-separated biomolecular condensates in diverse systems. As new discoveries of condensates are being madecontinuouslyinthisburgeoningfield,thesechaptersbynomeansprovideacomplete listofmethodsthatareapplicableincondensatestudies.Rather theyrepresentacollection of well-established tools that can be readily applied to existing as well as newly discovered condensatesystems.Weexpectthatthiscollectionwillcatalyzequantitativestudiesofboth biophysicalpropertiesandbiologicalfunctionsofcomplexbiomolecularcondensates. We thank all the authors for their valuable contributions to this volume on phase- separatedbiomolecularcondensates. Chicago,IL,USA Huan-XiangZhou Buffalo,NY,USA PriyaR.Banerjee Chicago,IL,USA Jan-HendrikSpille Contents Preface ..................................................................... v Contributors................................................................. xi 1 CalculatingBinodalsandInterfacialTensionofPhase-Separated CondensatesfromMolecularSimulationswithFinite-SizeCorrections ........ 1 KonstantinosMazarakos,SanboQin,andHuan-XiangZhou 2 Field-TheoreticSimulationMethodtoStudytheLiquid–Liquid PhaseSeparationofPolymers........ ....... ....... ........ ....... ........ 37 SaeedNajafi,JamesMcCarty,KrisT.Delaney, GlennH.Fredrickson,andJoan-EmmaShea 3 NumericalTechniquesforApplicationsofAnalyticalTheories toSequence-DependentPhaseSeparationsofIntrinsically DisorderedProteins........ ........ ....... ....... ........ ....... ........ 51 Yi-HsuanLin,JonasWesse´n,TanmoyPal,SumanDas, andHueSunChan 4 AnIntroductiontotheStickers-and-SpacersFrameworkasApplied toBiomolecularCondensates.... .... ...... ....... ......... ....... ........ 95 GarrettM.GinellandAlexS.Holehouse 5 MultiscaleModelingofProtein-RNACondensation inandOutofEquilibrium .......... ....... ....... ........ ....... ........ 117 RabiaLaghmach,IshaMalhotra,andDavitA.Potoyan 6 FluorescenceLifetimeImagingMicroscopyofBiomolecular Condensates........ ....... ........ ....... .... ... ...... .. ... .... ........ 135 MyDiemQuan,Shih-ChuJeffLiao,JosephineC.Ferreon, andAllanChrisM.Ferreon 7 Single-MoleculeFluorescenceMethodstoStudyProtein-RNA InteractionsUnderlyingBiomolecularCondensates.......... ....... ........ 149 LauraR.Ganser,YingdaGe,andSuaMyong 8 FluorescenceCorrelationSpectroscopyandPhaseSeparation......... ........ 161 JuanJeremı´asIncicco,DebjitRoy,MelissaD.Stuchell-Brereton, andAndreaSoranno 9 MeasurementofProteinandNucleicAcidDiffusionCoefficientsWithin BiomolecularCondensatesUsingIn-DropletFluorescence CorrelationSpectroscopy ........... ....... ....... ........ ....... ........ 199 IbraheemAlshareedahandPriyaR.Banerjee 10 Single-MoleculeImagingofthePhaseSeparation-Modulated DNACompactiontoStudyTranscriptionalRepression....... ....... ........ 215 LinyuZuo,LuhuaLai,andZhiQi 11 PhaseSeparation-BasedBiochemicalAssaysforBiomolecular Interactions ........ ....... ........ ....... ....... ........ ....... ........ 225 GaofengPei,MinZhou,WeifanXu,JingWang,andPilongLi ix
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