METHODS
IN
MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651
The Nucleus
Second Edition
Edited by
Ronald Hancock
Laval University Cancer Research Centre-CRCHUQ Oncology, Québec, QC, Canada; Systems Biology Group,
Biotechnology Centre, Silesian University of Technology, Gliwice, Poland
Editor
Ronald Hancock
Laval University Cancer Research
Centre-CRCHUQ Oncology
Québec, QC, Canada
Systems Biology Group
Biotechnology Centre
Silesian University of Technology
Gliwice, Poland
ISSN 1064-3745
ISSN 1940-6029 (electronic)
ISBN 978-1-4939-1679-5
ISBN 978-1-4939-1680-1 (eBook)
DOI 10.1007/978-1-4939-1680-1
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2014951833
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Cover Image Caption: Water content in the nucleus of a HeLa cell; pixels containing 0 to 50 % water are yellow and
those containing 51 to 100 % water as a linear scale from light to dark blue (see Chapter 12).
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Preface
This volume presents detailed recently developed protocols ranging from isolation of nuclei
to purification of chromatin regions containing single genes, with a particular focus on
some less well-explored aspects of the nucleus.
The methods described include new strategies for isolation of nuclei, for purification of
cell type-specific nuclei from a mixture, and for rapid isolation and fractionation of nucleoli.
For gene delivery into and expression in nuclei, a novel gentle approach using gold nanowires is presented. The developing interest in analysis of specific regions of chromatin is
illustrated by protocols for the isolation and structural and proteomic analysis of chromatin
containing a single gene or containing newly synthesized DNA. A widely used method to
purify chromatin regions is immunoprecipitation (ChIP), but during isolation chromatin
structure may be modified by DNA damage response systems, and conditions which allow
these artifacts to be avoided are described.
The concentration and localization of water and ions are crucial for macromolecular
interactions in the nucleus, and a new approach to measure these parameters by correlative
optical and cryo-electron microscopy is described. Similarly, redox conditions in the nucleus
have been little explored, and a method to follow the redox dynamics of nuclear glutathione is an important step in this direction.
An important aspect of analyzing images of nuclear structures is the extraction of quantitative information, and this volume presents methods and software for high-throughput
quantitative analysis of 3D fluorescence microscopy images, for quantification of the formation of amyloid fibrils in the nucleus, and for quantitative analysis of chromosome territory
localization.
The friendly and timely collaboration of the contributors to this volume is greatly
appreciated.
Québec, QC, Canada
Ronald Hancock
v
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PART I
ISOLATION OF NUCLEI
1 Cell Type-Specific Affinity Purification of Nuclei for Chromatin
Profiling in Whole Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Florian A. Steiner and Steven Henikoff
2 Lysis Gradient Centrifugation: A Flexible Method
for the Isolation of Nuclei from Primary Cells . . . . . . . . . . . . . . . . . . . . . . . . .
Karl Katholnig, Marko Poglitsch, Markus Hengstschläger,
and Thomas Weichhart
3 Isolation of Nuclei in Media Containing an Inert Polymer
to Mimic the Crowded Cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ronald Hancock and Yasmina Hadj-Sahraoui
PART II
3
15
25
NUCLEOLI
4 A New Rapid Method for Isolating Nucleoli . . . . . . . . . . . . . . . . . . . . . . . . . .
Zhou Fang Li and Yun Wah Lam
5 Sequential Recovery of Macromolecular Components of the Nucleolus . . . . . .
Baoyan Bai and Marikki Laiho
PART III
v
ix
35
43
GENES AND CHROMATIN
6 Au Nanoinjectors for Electrotriggered Gene Delivery
into the Cell Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mijeong Kang and Bongsoo Kim
7 Improving Chromatin Immunoprecipitation (ChIP)
by Suppression of Method-Induced DNA-Damage Signaling . . . . . . . . . . . . .
Sascha Beneke
8 Purification of Specific Chromatin Loci for Proteomic Analysis . . . . . . . . . . . .
Stephanie D. Byrum, Sean D. Taverna, and Alan J. Tackett
9 Chromatin Structure Analysis of Single Gene Molecules
by Psoralen Cross-Linking and Electron Microscopy . . . . . . . . . . . . . . . . . . . .
Christopher R. Brown, Julian A. Eskin, Stephan Hamperl,
Joachim Griesenbeck, Melissa S. Jurica, and Hinrich Boeger
10 Purification of Proteins on Newly Synthesized DNA Using iPOND . . . . . . . .
Huzefa Dungrawala and David Cortez
vii
55
67
83
93
123
viii
Contents
11 Applying the Ribopuromycylation Method to Detect Nuclear Translation . . . .
Alexandre David and Jonathan W. Yewdell
PART IV
THE INTRANUCLEAR MILIEU
12 Targeted Nano Analysis of Water and Ions in the Nucleus
Using Cryo-Correlative Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Frédérique Nolin, Dominique Ploton, Laurence Wortham,
Pavel Tchelidze, Hélène Bobichon, Vincent Banchet,
Nathalie Lalun, Christine Terryn, and Jean Michel
13 A Redox-Sensitive Yellow Fluorescent Protein Sensor
for Monitoring Nuclear Glutathione Redox Dynamics. . . . . . . . . . . . . . . . . . .
Agata Banach-Latapy, Michèle Dardalhon, and Meng-Er Huang
PART V
133
145
159
IMAGING NUCLEAR STRUCTURES
14 Determination of the Dissociation Constant of the NFκB p50/p65
Heterodimer in Living Cells Using Fluorescence
Cross-Correlation Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Manisha Tiwari and Masataka Kinjo
15 Imaging and Quantification of Amyloid Fibrillation in the Cell Nucleus . . . . .
Florian Arnhold, Andrea Scharf, and Anna von Mikecz
16 Analysis of Nuclear Organization with TANGO,
Software for High-Throughput Quantitative Analysis
of 3D Fluorescence Microscopy Images. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Jean Ollion, Julien Cochennec, François Loll, Christophe Escudé,
and Thomas Boudier
17 Quantitative Analysis of Chromosome Localization in the Nucleus . . . . . . . . .
Sandeep Chakraborty, Ishita Mehta, Mugdha Kulashreshtha,
and B. J. Rao
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
173
187
203
223
235
Contributors
FLORIAN ARNHOLD • IUF – Leibniz Research Institute of Environmental Medicine at
Heinrich- Heine-University Duesseldorf, Duesseldorf, Germany
BAOYAN BAI • Department of Radiation Oncology and Molecular Radiation Sciences,
The Johns Hopkins University School of Medicine, Baltimore, MD, USA;
Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School
of Medicine, Baltimore, MD, USA
AGATA BANACH-LATAPY • UMR3348 “Genotoxic Stress and Cancer,” Centre National de
la Recherche Scientifique, Institut Curie, Orsay, France
VINCENT BANCHET • Laboratoire de recherche en Nanosciences EA 4682, UFR Sciences
Exactes et Naturelles, Université de Reims Champagne Ardenne, Reims, France
SASCHA BENEKE • Institute of Veterinary Pharmacology and Toxicology/Vetsuisse,
University of Zurich, Zurich, Switzerland
HÉLÈNE BOBICHON • CNRS UMR 7369, UFR Médecine, Université de Reims
Champagne Ardenne and CHU de Reims, Reims, France
HINRICH BOEGER • Department of Molecular, Cell and Developmental Biology,
University of California, Santa Cruz, CA, USA
THOMAS BOUDIER • Sorbonne Universités, UPMC Université Paris 06, Paris, France
CHRISTOPHER R. BROWN • Department of Molecular, Cell and Developmental Biology,
University of California, Santa Cruz, CA, USA
STEPHANIE D. BYRUM • Department of Biochemistry and Molecular Biology,
University of Arkansas for Medical Sciences, Little Rock, AR, USA
SANDEEP CHAKRABORTY • Department of Biological Sciences, Tata Institute
of Fundamental Research, Mumbai, Maharashtra, India; Plant Sciences
Department,University of California, Davis, CA, USA
JULIEN COCHENNEC • CNRS UMR7196, INSERM U565, Muséum National d’Histoire
Naturelle, Paris, France
DAVID CORTEZ • Department of Biochemistry, Vanderbilt University School of Medicine,
Nashville, TN, USA
MICHÈLE DARDALHON • UMR3348 “Genotoxic Stress and Cancer,” Centre National de la
Recherche Scientifique, Institut Curie, Orsay, France
ALEXANDRE DAVID • CNRS UMR-5203; INSERM U661; UM1; UM2, Institut de
Génomique Fonctionnelle, Montpellier, France
HUZEFA DUNGRAWALA • Department of Biochemistry, Vanderbilt University School
of Medicine, Nashville, TN, USA
CHRISTOPHE ESCUDÉ • CNRS UMR7196, INSERM U565, Muséum National d’Histoire
Naturelle, Paris, France
JULIAN A. ESKIN • Department of Biology and Rosenstiel Basic Medical Science Research
Center, Brandeis University, Waltham, MA, USA
JOACHIM GRIESENBECK • Lehrstuhl fürBiochemie III, Biochemie-Zentrum Regensburg
(BZR), Universität Regensburg, Regensburg, Germany
ix
x
Contributors
YASMINA HADJ-SAHRAOUI • Laval University Cancer Research Centre-CRCHUQ
Oncology, Québec, QC, Canada
STEPHAN HAMPERL • Lehrstuhl für Biochemie III, Biochemie-Zentrum Regensburg (BZR),
Universität Regensburg, Regensburg, Germany
RONALD HANCOCK • Laval University Cancer Research Centre-CRCHUQ Oncology,
Québec, QC, Canada; Systems Biology Group, Biotechnology Centre, Silesian University
of Technology, Gliwice, Poland
MARKUS HENGSTSCHLÄGER • Institute for Medical Genetics, Medical University of Vienna,
Vienna, Austria
STEVEN HENIKOFF • Basic Sciences Division, Howard Hughes Medical Institute, Seattle,
WA, USA; Fred Hutchinson Cancer Research Center, Seattle, WA, USA
MENG-ER HUANG • UMR3348 “Genotoxic Stress and Cancer,” Centre National de la
Recherche Scientifique, Institut Curie, Orsay, France
MELISSA S. JURICA • Department of Molecular, Cell and Developmental Biology, University
of California, Santa Cruz, CA, USA
MIJEONG KANG • Department of Chemistry, KAIST, Daejeon, South Korea
KARL KATHOLNIG • Institute for Medical Genetics, Medical University of Vienna,
Vienna, Austria
BONGSOO KIM • Department of Chemistry, KAIST, Daejeon, South Korea
MASATAKA KINJO • Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life
Science, Hokkaido University, Sapporo, Japan
MUGDHA KULASHRESHTHA • Department of Biological Sciences, Tata Institute
of Fundamental Research, Mumbai, Maharashtra, India
MARIKKI LAIHO • Department of Radiation Oncology and Molecular Radiation Sciences,
The Johns Hopkins University School of Medicine, Baltimore, MD, USA
NATHALIE LALUN • CNRS UMR 7369, UFR Médecine, Université de Reims Champagne
Ardenne and CHU de Reims, Reims, Cedex, France
YUN WAH LAM • Department of Biology and Chemistry, City University of Hong Kong,
Kowloon, Hong Kong
ZHOU FANG LI • Department of Biology, South University of Science and Technology of
China, Shenzhen, Guangdong, P.R. China
FRANÇOIS LOLL • CNRS UMR7196, INSERM U565, Muséum National d’Histoire
Naturelle, Paris, France
ISHITA MEHTA • Department of Biological Sciences, Tata Institute of Fundamental
Research, Mumbai, Maharashtra, India; UM-DAE-Centre for Excellence
in Basic Sciences, Biological Sciences, Mumbai, Maharashtra, India
JEAN MICHEL • Laboratoire de Recherche en Nanosciences EA 4682, UFR Sciences Exactes
et Naturelles, Université de Reims Champagne Ardenne, Reims, France
ANNA VON MIKECZ • IUF – Leibniz Research Institute of Environmental Medicine at
Heinrich-Heine-University Duesseldorf Duesseldorf, Germany
FRÉDÉRIQUE NOLIN • Laboratoire de Recherche en Nanosciences EA 4682, UFR Sciences
Exactes et Naturelles, Université de Reims Champagne Ardenne, Reims, France
JEAN OLLION • CNRS UMR7196, INSERM U565, Muséum National d’Histoire
Naturelle, Paris, France
DOMINIQUE PLOTON • CNRS UMR 7369, UFR Médecine, Université de Reims
Champagne Ardenne and CHU de Reims, Reims, France
MARKO POGLITSCH • Attoquant Diagnostics GmbH, Vienna, Austria
Contributors
xi
B. J. RAO • Department of Biological Sciences, Tata Institute of Fundamental Research,
Mumbai, Maharashtra, India
ANDREA SCHARF • IUF – Leibniz Research Institute of Environmental Medicine at
Heinrich- Heine-University Duesseldorf, Duesseldorf, Germany
FLORIAN A. STEINER • Basic Sciences Division, Howard Hughes Medical Institute, Seattle,
WA, USA; Fred Hutchinson Cancer Research Center, Seattle, WA, USA
ALAN J. TACKETT • Department of Biochemistry and Molecular Biology,
University of Arkansas for Medical Sciences, Little Rock, AR, USA
SEAN D. TAVERNA • Department of Pharmacology and Molecular Sciences,
Johns Hopkins University School of Medicine, Baltimore, MD, USA
PAVEL TCHELIDZE • CNRS UMR 7369, UFR Médecine, Université de Reims Champagne
Ardenne and CHU de Reims, Reims, France
CHRISTINE TERRYN • Plate-forme IBISA, Université de Reims Champagne Ardenne,
Reims, France
MANISHA TIWARI • Laboratory for Nano-Bio Probes, Quantitative Biology Center, OLABB,
Osaka University, Suita, Japan
THOMAS WEICHHART • Institute for Medical Genetics, Medical University of Vienna,
Vienna, Austria
LAURENCE WORTHAM • Laboratoire de Recherche en Nanosciences EA 4682, UFR Sciences
Exactes et Naturelles, Université de Reims Champagne Ardenne, Reims, France
JONATHAN W. YEWDELL • Laboratory of Viral Diseases, National Institute of Allergy and
Infectious Diseases, Bethesda, MD, USA