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Methods in
Molecular Biology 2563
Huan-Xiang Zhou
Jan-Hendrik Spille
Priya R. Banerjee Editors
Phase-Separated
Biomolecular
Condensates
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
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-Xiang Zhou Jan-Hendrik Spille
Department of Chemistry Department of Physics
University of Illinois at Chicago University of Illinois at Chicago
Chicago, IL, USA Chicago, IL, USA
Priya R. Banerjee
Department of Physics
University at Buffalo, State University
Buffalo, NY, USA
This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Preface
v
vi Preface
Table 1
Summary of chapters
(continued)
Preface vii
Table 1
(continued)
large number of mixtures and then a high-content analysis system to rapidly select mixtures
that form condensates. Wang and Hayer-Hartl (Chap. 14) review their studies on conden-
sates involved in the biogenesis of carboxysomes, which are cytosolic bodies for photosyn-
thetic CO2 fixation. Interestingly, these condensates are formed by folded domains,
including Rubisco large and small subunits and multiple Rubisco small subunit-like domains
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
in vitro and in cell to analyze how the phase behaviors of ribonucleoproteins (RNPs) can be
modulated by post-translational modifications (PTMs) such as phosphorylation. Given that
many signaling proteins harbor multiple sites for PTMs, these assays provide simple yet
useful tools to study such processes. Reinkemeier and Lemke (Chap. 17) describe an elegant
method to generate synthetic organelles in live cells that perform genetic code expansion
(GCE), protein-selective non-canonical amino-acid incorporation, and subsequent labeling
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
condensates in live cells. Kim and Shin (Chap. 19) describe the optodroplet assay and show
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
the heterochromatin protein HP1 in the nucleus of live cells. Together, these two chapters
provide a detailed recipe for using optogenetic approaches to study condensates in live cells,
from plasmid preparation to image acquisition and analysis. Giesler et al. (Chap. 21) review
mass balance imaging (MBI) for determining the phase behaviors and dissecting the kinetic
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
parameters at length scales inaccessible by conventional assays.
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
made continuously in this burgeoning field, these chapters by no means provide a complete
list of methods that are applicable in condensate studies. Rather they represent a collection
of well-established tools that can be readily applied to existing as well as newly discovered
condensate systems. We expect that this collection will catalyze quantitative studies of both
biophysical properties and biological functions of complex biomolecular condensates.
We thank all the authors for their valuable contributions to this volume on phase-
separated biomolecular condensates.
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1 Calculating Binodals and Interfacial Tension of Phase-Separated
Condensates from Molecular Simulations with Finite-Size Corrections . . . . . . . . 1
Konstantinos Mazarakos, Sanbo Qin, and Huan-Xiang Zhou
2 Field-Theoretic Simulation Method to Study the Liquid–Liquid
Phase Separation of Polymers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Saeed Najafi, James McCarty, Kris T. Delaney,
Glenn H. Fredrickson, and Joan-Emma Shea
3 Numerical Techniques for Applications of Analytical Theories
to Sequence-Dependent Phase Separations of Intrinsically
Disordered Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Yi-Hsuan Lin, Jonas Wessén, Tanmoy Pal, Suman Das,
and Hue Sun Chan
4 An Introduction to the Stickers-and-Spacers Framework as Applied
to Biomolecular Condensates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Garrett M. Ginell and Alex S. Holehouse
5 Multiscale Modeling of Protein-RNA Condensation
in and Out of Equilibrium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Rabia Laghmach, Isha Malhotra, and Davit A. Potoyan
6 Fluorescence Lifetime Imaging Microscopy of Biomolecular
Condensates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
My Diem Quan, Shih-Chu Jeff Liao, Josephine C. Ferreon,
and Allan Chris M. Ferreon
7 Single-Molecule Fluorescence Methods to Study Protein-RNA
Interactions Underlying Biomolecular Condensates . . . . . . . . . . . . . . . . . . . . . . . . . 149
Laura R. Ganser, Yingda Ge, and Sua Myong
8 Fluorescence Correlation Spectroscopy and Phase Separation. . . . . . . . . . . . . . . . . 161
Juan Jeremı́as Incicco, Debjit Roy, Melissa D. Stuchell-Brereton,
and Andrea Soranno
9 Measurement of Protein and Nucleic Acid Diffusion Coefficients Within
Biomolecular Condensates Using In-Droplet Fluorescence
Correlation Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Ibraheem Alshareedah and Priya R. Banerjee
10 Single-Molecule Imaging of the Phase Separation-Modulated
DNA Compaction to Study Transcriptional Repression. . . . . . . . . . . . . . . . . . . . . . 215
Linyu Zuo, Luhua Lai, and Zhi Qi
11 Phase Separation-Based Biochemical Assays for Biomolecular
Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Gaofeng Pei, Min Zhou, Weifan Xu, Jing Wang, and Pilong Li
ix
x Contents
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
Contributors
xi
xii Contributors
SUA MYONG • Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA
SAEED NAJAFI • Department of Chemistry and Biochemistry, University of California, Santa
Barbara, CA, USA; Materials Research Laboratory, University of California, Santa
Barbara, CA, USA
ARJUN NARAYANAN • Max Planck Institute of Molecular Cell Biology and Genetics, Dresden,
Germany; Center for Systems Biology Dresden, Dresden, Germany; Max Planck Institute for
the Physics of Complex Systems, Dresden, Germany
TANMOY PAL • Department of Biochemistry, University of Toronto, Toronto, ON, Canada
GANESH PANDEY • Department of Physics, University of Illinois at Chicago, Chicago, IL, USA
BALJYOT SINGH PARMAR • Department of Physics, McGill University, QC, Canada
GAOFENG PEI • Beijing Advanced Innovation Center for Structural Biology & Frontier
Research Center for Biological Structure, School of Life Sciences, Tsinghua University,
Beijing, China; Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing,
China
DAVIT A. POTOYAN • Department of Chemistry, Iowa State University, Ames, IA, USA
ZHI QI • Center for Quantitative Biology and Peking-Tsinghua Center for Life Sciences,
Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
SANBO QIN • Department of Chemistry, University of Illinois at Chicago, Chicago, IL, USA
MY DIEM QUAN • Department of Pharmacology and Chemical Biology, Baylor College of
Medicine, Houston, TX, USA
ANNE RADEMACHER • Division of Chromatin Networks, German Cancer Research Center
(DKFZ) and Bioquant, Heidelberg, Germany
CHRISTOPHER D. REINKEMEIER • Biocentre, Departments of Biology and Chemistry, Johannes
Gutenberg University Mainz, Mainz, Germany; Institute of Molecular Biology gGmbH,
Mainz, Germany
KARSTEN RIPPE • Division of Chromatin Networks, German Cancer Research Center
(DKFZ) and Bioquant, Heidelberg, Germany
DEBJIT ROY • Department of Biochemistry and Molecular Biophysics, Washington University
in St Louis, St. Louis, MO, USA; Center for Science and Engineering of Living Systems
(CSELS), Washington University in St Louis, St. Louis, MO, USA
JENNY SACHWEH • Structural and Computational Biology Unit, European Molecular Biology
Laboratory (EMBL), Heidelberg, Germany; Max Planck Institute of Biophysics, Frankfurt
am Main, Germany
JOAN-EMMA SHEA • Department of Chemistry and Biochemistry, University of California,
Santa Barbara, CA, USA; Department of Physics, University of California Santa
Barbara, Santa Barbara, CA, USA
YONGDAE SHIN • Interdisciplinary Program in Bioengineering, Seoul National University,
Seoul, Republic of Korea; Department of Mechanical Engineering, Seoul National
University, Seoul, Republic of Korea; Institute of Advanced Machines and Design, Seoul
National University, Seoul, Republic of Korea
ANDREA SORANNO • Department of Biochemistry and Molecular Biophysics, Washington
University in St Louis, St. Louis, MO, USA; Center for Science and Engineering of Living
Systems (CSELS), Washington University in St Louis, St. Louis, MO, USA
JAN-HENDRIK SPILLE • Department of Physics, University of Illinois at Chicago, Chicago, IL,
USA
xiv Contributors
JULIEN.
La mort de Constance était un événement si imprévu
et si heureux, pour le nouvel empereur, que la An 361.
plupart des amis de Julien n'osaient la croire. C'était,
à leur avis, une fausse nouvelle, par laquelle on i. Julien arrive à
voulait endormir sa vigilance, et l'attirer dans un Constantinople.
piége. Pour vaincre leur défiance, Julien leur mit Amm. l. 22, c. 2.
sous les yeux une prédiction plus ancienne, qui lui Liban, or. 10, t.
promettait la victoire sans tirer l'épée. Cette 2, p. 289.
prétendue prophétie, qui pour des esprits Mamert. pan. c.
raisonnables aurait eu besoin d'être confirmée par le 27.
fait, y servit de preuve. Julien, exercé depuis long-
Idatius, chron.
temps à prendre toutes les formes convenables aux
circonstances, n'oublia pas de se faire honneur en Zos. l. 3, c. 11.
versant quelques larmes, que ses panégyristes ont Socr. l. 3, c. 1.
soigneusement recueillies: il recommanda qu'on Zon. l. 13, t. 2,
rendît au corps de Constance tous les honneurs dus p. 24.
aux empereurs; il prit l'habit de deuil; il reçut avec un
chagrin affecté les témoignages de joie de toutes ses légions, qui le
saluèrent de nouveau du titre d'Auguste. Il marcha aussitôt, traversa
sans obstacle le défilé de Sucques, passa par Philippopolis, et vint à
Héraclée[353]. Tous les corps de troupes envoyés pour lui disputer
les passages, se rangeaient sous ses enseignes; toutes les villes
ouvraient leurs portes et reconnaissaient leur nouveau souverain.
Les habitants de Constantinople vinrent en foule à sa rencontre. Il y
entra le 11 de décembre, au milieu des acclamations du peuple, qui
se mêlant parmi ses soldats le considérait avec des transports
d'admiration et de tendresse. On se rappelait qu'il avait reçu dans
cette ville la naissance et la première nourriture: on comparait avec
sa jeunesse, avec son extérieur qui n'annonçait rien de grand, tout
ce qu'avait publié de lui la renommée, tout ce qu'on voyait exécuté;
tant de batailles et de victoires; la rapidité d'une marche pénible,
semée de périls et d'obstacles qui n'avaient fait qu'accroître ses
forces; la protection divine qui le mettait en possession de l'empire
sans qu'il en coûtât une goutte de sang. Le concours de tant de
circonstances extraordinaires frappait tous les esprits[354]: on formait
les plus heureux présages d'un règne qui s'était annoncé par tant de
merveilles.
[353] Cette ville, située sur la Propontide, actuellement mer de
Marmara, était à une vingtaine de lieues à l'ouest de
Constantinople. Elle avait porté antérieurement le nom de
Périnthe. On trouve souvent les deux noms réunis dans les
auteurs. Elle avait été long-temps la métropole de la portion de la
Thrace, qui se nommait Europe. Du temps de Procope (de ædif.
L. 4, c. 9), elle tenait le premier rang après Constantinople.—S.-
M.
[354] Paulò ante in laceratis Galliæ provinciis lapsus, inimicorum
capitalium apertis armis, et occultis insidiis petebatur; in pauculis
mensibus, divino munere, Libyæ, Europæ, Asiæque regnator est.
Mamert. Pan. c. 27.—S.-M.