Phase-Separated Biomolecular Condensates: Methods and Protocols (Methods in Molecular Biology, 2563)

454 Pages·2022·19.681 MB·English

by  Huan-Xiang Zhou (editor), Jan-Hendrik Spille (editor), Priya R. Banerjee (editor)

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
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School of Lifeand MedicalSciences
University ofHertfordshire
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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
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https://doi.org/10.1007/978-1-0716-2663-4
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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