(Ebook) Cell Cycle Control: Methods and Protocols by

Anna Castro, Benjamin Lacroix ISBN 9781071635568,

1071635565 Pdf Download

https://ebooknice.com/product/cell-cycle-control-methods-and-
protocols-55851382

★★★★★
4.7 out of 5.0 (49 reviews )

Instant PDF Download

ebooknice.com
 (Ebook) Cell Cycle Control: Methods and Protocols by Anna
Castro, Benjamin Lacroix ISBN 9781071635568, 1071635565 Pdf
Download

EBOOK

Available Formats

■ PDF eBook Study Guide Ebook

EXCLUSIVE 2025 EDUCATIONAL COLLECTION - LIMITED TIME

INSTANT DOWNLOAD VIEW LIBRARY

 We believe these products will be a great fit for you. Click
the link to download now, or visit ebooknice.com
to discover even more!

(Ebook) Biota Grow 2C gather 2C cook by Loucas, Jason; Viles,

James ISBN 9781459699816, 9781743365571, 9781925268492,
1459699815, 1743365578, 1925268497

https://ebooknice.com/product/biota-grow-2c-gather-2c-cook-6661374

(Ebook) Cell Cycle Checkpoint Control Protocols by Howard B.

Lieberman ISBN 9781617373695, 1617373699

https://ebooknice.com/product/cell-cycle-checkpoint-control-
protocols-925826

(Ebook) Cell Cycle Control Mechanisms and Protocols by Tim

Humphrey, Gavin Brooks ISBN 9781588291448, 9781592598571,
1588291448, 1592598579

https://ebooknice.com/product/cell-cycle-control-mechanisms-and-
protocols-1019846

(Ebook) Cell Cycle Control: Mechanisms and Protocols by Eishi

Noguchi, Mariana C. Gadaleta (eds.) ISBN 9781493908875,
1493908871

https://ebooknice.com/product/cell-cycle-control-mechanisms-and-
protocols-4691382
(Ebook) Cambridge IGCSE and O Level History Workbook 2C - Depth
Study: the United States, 1919-41 2nd Edition by Benjamin
Harrison ISBN 9781398375147, 9781398375048, 1398375144,
1398375047
https://ebooknice.com/product/cambridge-igcse-and-o-level-history-
workbook-2c-depth-study-the-united-states-1919-41-2nd-edition-53538044

(Ebook) Stem Cell Transcriptional Networks: Methods and

Protocols by Benjamin L. Kidder (eds.) ISBN 9781493905119,
1493905112

https://ebooknice.com/product/stem-cell-transcriptional-networks-methods-
and-protocols-4667962

(Ebook) Cell Cycle Synchronization: Methods and Protocols by

Gaspar Banfalvi (auth.), Gaspar Banfalvi (eds.) ISBN
9781617791819, 9781617791826, 1617791814, 1617791822

https://ebooknice.com/product/cell-cycle-synchronization-methods-and-
protocols-2450058

(Ebook) Cell Cycle Oscillators: Methods and Protocols by Amanda

S. Coutts, Louise Weston (eds.) ISBN 9781493929566,
9781493929573, 1493929569, 1493929577

https://ebooknice.com/product/cell-cycle-oscillators-methods-and-
protocols-5353282

(Ebook) SAT II Success MATH 1C and 2C 2002 (Peterson's SAT II

Success) by Peterson's ISBN 9780768906677, 0768906679

https://ebooknice.com/product/sat-ii-success-math-1c-and-2c-2002-peterson-
s-sat-ii-success-1722018
Methods in
Molecular Biology 2740

Anna Castro
Benjamin Lacroix Editors

Cell Cycle
Control
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
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
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
 Cell Cycle Control

Methods and Protocols

Edited by

Anna Castro
Université de Montpellier, Centre de Recherche en Biologie cellulaire de Montpellier (CRBM),
CNRS UMR 5237, Montpellier Cedex 5, France; Programme équipes Labellisées Ligue Contre le Cancer,
Paris, France

Benjamin Lacroix
Université de Montpellier, Centre de Recherche en Biologie cellulaire de Montpellier (CRBM),
CNRS UMR 5237, Montpellier Cedex 5, France
Editors
Anna Castro Benjamin Lacroix
Université de Montpellier, Centre de Université de Montpellier, Centre de Recherche
Recherche en Biologie cellulaire de en Biologie cellulaire de Montpellier (CRBM)
Montpellier (CRBM) CNRS UMR 5237
CNRS UMR 5237 Montpellier Cedex 5, France
Montpellier Cedex 5, France
Programme équipes Labellisées
Ligue Contre le Cancer
Paris, France

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-3556-8 ISBN 978-1-0716-3557-5 (eBook)
https://doi.org/10.1007/978-1-0716-3557-5
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part
of Springer Nature 2024
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and
retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter
developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,
even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations
and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been
made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Paper in this product is recyclable.

Preface

Cell division is a crucial process by which one cell divides into two genetically identical
daughter cells. During this process, the genetic information has to be appropriately dupli-
cated and segregated to ensure the correct transmission of the genetic information to the
next generation. Because cell viability and therefore the sustainability of every form of life
depends on the correct duplication and partition of the DNA, the cell cycle is a highly
regulated process.
Cell proliferation is under the control of fine-tuned molecular cascades that ensure
correct chromatin duplication even under damaged DNA. Cycling cells are undergoing
crucial sequences of molecular and physical events including chromosomes duplication and
condensation, nuclear envelope reorganization, and assembly and disassembly of the mitotic
structures. Likewise, a tight regulation of protein phosphorylation/dephosphorylation
during mitotic division is required to timely and spatially orchestrate all these processes. In
this book, we put together several chapters dedicated to investigating these processes and
assessing how cells respond when these complicated pathways are simplified by using
synthetic biology and in vitro reconstitutions.
Besides the regular molecular mechanisms required to progress into the cell cycle, cells
ensure a safe division by additionally sensing and responding to environmental conditions.
Physical constraints and metabolic stress impact on cell cycle regulation. This is a new
essential and evolving research area for which we also decided to collect some protocol
chapters that will help the readers to address this field.
In the last part of the book, we report some new protocols in different model systems
and cellular types to visualize cellular architecture during cell division, and we emphasize
innovative single cell microscopy techniques to highlight the heterogeneity of the cell
population with respect to cell cycle progression. Our book finally provides a computer-
assisted protocol dedicated to the analysis and quantification of the cell cycle.
We would like to thank all the authors who contributed to this protocol book by sharing
their protocols supported by key advice for their development and practical application. We
are also indebted to the Editor-in-Chief of Methods in Molecular Biology, Dr. John Walker,
for his great support and guidance.

Montpellier, France Anna Castro

Benjamin Lacroix

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 Single-Molecule Approaches to Study DNA Condensation. . . . . . . . . . . . . . . . . . . 1

Stefan Golfier, Thomas Quail, and Jan Brugués
2 Studying Translesion DNA Synthesis Using Xenopus In Vitro Systems. . . . . . . . . 21
Antoine Aze, James R. A. Hutchins, and Domenico Maiorano
3 Cell Cycle–Specific Protein Phosphatase 1 (PP1) Substrates Identification
Using Genetically Modified Cell Lines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Dorothee C Kommer, Konstantinos Stamatiou, and Paola Vagnarelli
4 Dissecting the Multiple Functions of the Polo-Like Kinase 1
in the C. elegans Zygote . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Griselda Velez-Aguilera, Batool Ossareh-Nazari, and Lionel Pintard
5 Artificial Modulation and Rewiring of Cell Cycle Progression
Using Synthetic Circuits in Fission Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Akanksha Jain, Pei-Yun Jenny Wu, and Damien Coudreuse
6 Measuring Molecular Diffusion in Self-Organizing Xenopus Extracts
by Fluorescence Correlation Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
William Y. C. Huang, James E. Ferrell Jr., and Xianrui Cheng
7 Mechanical Characterization of Murine Oocytes by Atomic Force
Microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Rose Bulteau, Lucie Barbier, Guillaume Lamour, Tristan Piolot,
Elsa Labrune, Clément Campillo, and Marie-Emilie Terret
8 Manipulation of Embryonic Cleavage Geometry Using Magnetic Tweezers . . . . 125
Jing Xie, Daniel L. Levy, Nicolas Minc, and Jérémy Sallé
9 Cross Talk Between Metabolism and the Cell Division Cycle . . . . . . . . . . . . . . . . . 141
Diana Vara-Ciruelos and Marcos Malumbres
10 Give and Take: The Reciprocal Control of Metabolism and Cell Cycle . . . . . . . . 155
Romain Riscal, Blanche Riquier-Morcant, Gilles Gadea,
and Laetitia K. Linares
11 Preparation of Xenopus borealis and Xenopus tropicalis Egg Extracts
for Comparative Cell Biology and Evolutionary Studies . . . . . . . . . . . . . . . . . . . . . 169
Maiko Kitaoka, Gabriel Guilloux, Rebecca Heald, and Romain Gibeaux
12 Measuring Mitotic Spindle and Microtubule Dynamics in Marine Embryos
and Non-model Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Janet Chenevert, Morgane L. V. Robert, Jérémy Sallé, Sébastien Cacchia,
Thierry Lorca, Anna Castro, Alex McDougall, Nicolas Minc,
Stefania Castagnetti, Julien Dumont, and Benjamin Lacroix
13 Whole-Mount Immunofluorescence Staining to Visualize Cell Cycle
Progression in Mouse Oocyte Meiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Safia El Jailani, Katja Wassmann, and Sandra A. Touati

vii
viii Contents

14 Imaging and Analysis of Drosophila Neural Stem Cell Asymmetric Division. . . . 229
Anne-Marie Berisha, Gregory Eot-Houllier, and Régis Giet
15 Cell Cycle Mapping Using Multiplexed Immunofluorescence . . . . . . . . . . . . . . . . 243
Katarzyna M. Kedziora and Wayne Stallaert
16 Investigating Heterogeneous Cell-Cycle Progression Using Single-Cell
Imaging Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Hee Won Yang
17 MAARS Software for Automatic and Quantitative Analysis of Mitotic
Progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Tong Li, Yannick Gachet, and Sylvie Tournier

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Contributors

ANTOINE AZE • Genome Surveillance and Stability Laboratory, Institute of Human Genetics,
UMR9002, CNRS-University of Montpellier, Montpellier, France
LUCIE BARBIER • Center for Interdisciplinary Research in Biology (CIRB), Collège de France,
CNRS, INSERM, Université PSL, Paris, France
ANNE-MARIE BERISHA • Univ Rennes, CNRS, INSERM, IGDR (Institut de Génétique et Dé
veloppement de Rennes)-UMR6290-U1305, Rennes, France
JAN BRUGUÉS • Max Planck Institute of Molecular Cell Biology and Genetics, Dresden,
Germany; Max Planck Institute for the Physics of Complex Systems, Dresden, Germany;
Center for Systems Biology Dresden, Dresden, Germany; Physics of Life, TU Dresden,
Dresden, Germany
ROSE BULTEAU • Université Paris-Saclay, Univ Evry, CNRS, LAMBE, Paris, France; Center
for Interdisciplinary Research in Biology (CIRB), Collège de France, CNRS, INSERM,
Université PSL, Paris, France
SÉBASTIEN CACCHIA • Université de Montpellier, Centre de Recherche en Biologie cellulaire de
Montpellier (CRBM), CNRS UMR 5237, Montpellier Cedex 5, France
CLÉMENT CAMPILLO • Université Paris-Saclay, Univ Evry, CNRS, LAMBE, Paris, France;
Institut Universitaire de France (IUF), Paris, France
STEFANIA CASTAGNETTI • Sorbonne Universités, CNRS, Laboratoire de Biologie du Dé
veloppement de Villefranche-sur-mer (LBDV), Villefranche-sur-mer, France
ANNA CASTRO • Université de Montpellier, Centre de Recherche en Biologie cellulaire de
Montpellier (CRBM), CNRS UMR 5237, Montpellier Cedex 5, France; Programme é
quipes Labellisées Ligue Contre le Cancer, Paris, France
JANET CHENEVERT • Sorbonne Universités, CNRS, Laboratoire de Biologie du Développement
de Villefranche-sur-mer (LBDV), Villefranche-sur-mer, France
XIANRUI CHENG • Department of Biological Sciences, University of Southern California, Los
Angeles, CA, USA
DAMIEN COUDREUSE • Institute of Genetics and Development of Rennes, CNRS UMR 6290
and University of Rennes, Rennes, France; Institute of Biochemistry and Cellular Genetics,
CNRS UMR 5095 and University of Bordeaux, Bordeaux, France
JULIEN DUMONT • CNRS, Institut Jacques Monod, Université Paris Cité, Paris, France
GREGORY EOT-HOULLIER • Univ Rennes, CNRS, INSERM, IGDR (Institut de Génétique et
Développement de Rennes)-UMR6290-U1305, Rennes, France
JAMES E. FERRELL JR. • Department of Chemical and Systems Biology, Stanford University
School of Medicine, Stanford, CA, USA; Department of Biochemistry, Stanford University
School of Medicine, Stanford, CA, USA
YANNICK GACHET • MCD, Centre de Biologie Intégrative, Université de Toulouse, CNRS,
UPS, Toulouse Cedex, France
GILLES GADEA • INSERM U1194, IRCM, Institut de Recherche en Cancérologie de
Montpellier, Institut régional du Cancer de Montpellier, Université de Montpellier,
Montpellier, France
ROMAIN GIBEAUX • Univ Rennes, CNRS, IGDR (Institut de Génétique et Développement de
Rennes) – UMR 6290, Rennes, France

ix
x Contributors

RÉGIS GIET • Univ Rennes, CNRS, INSERM, IGDR (Institut de Génétique et Dé
veloppement de Rennes)-UMR6290-U1305, Rennes, France
STEFAN GOLFIER • Max Planck Institute of Molecular Cell Biology and Genetics, Dresden,
Germany; Max Planck Institute for the Physics of Complex Systems, Dresden, Germany;
Center for Systems Biology Dresden, Dresden, Germany; B CUBE, Center for Molecular
Bioengineering, TU Dresden, Dresden, Germany
GABRIEL GUILLOUX • Univ Rennes, CNRS, IGDR (Institut de Génétique et Développement
de Rennes) – UMR 6290, Rennes, France
REBECCA HEALD • Department of Molecular and Cell Biology, University of California,
Berkeley, CA, USA
WILLIAM Y. C. HUANG • Department of Chemical and Systems Biology, Stanford University
School of Medicine, Stanford, CA, USA
JAMES R. A. HUTCHINS • Genome Surveillance and Stability Laboratory, Institute of Human
Genetics, UMR9002, CNRS-University of Montpellier, Montpellier, France
SAFIA EL JAILANI • Université Paris Cité, CNRS, Institut Jacques Monod, Paris, France
AKANKSHA JAIN • Institute of Genetics and Development of Rennes, CNRS UMR 6290 and
University of Rennes, Rennes, France; Institute of Biochemistry and Cellular Genetics,
CNRS UMR 5095 and University of Bordeaux, Bordeaux, France
KATARZYNA M. KEDZIORA • Department of Cell Biology, Center for Biologic Imaging (CBI),
University of Pittsburgh, Pittsburgh, PA, USA
MAIKO KITAOKA • Department of Molecular and Cell Biology, University of California,
Berkeley, CA, USA; Whitehead Institute of Biomedical Research and Howard Hughes
Medical Institute, Cambridge, MA, USA
DOROTHEE C KOMMER • College of Health, Medicine and Life Science, Brunel University
London, London, UK
ELSA LABRUNE • Hospices Civils de Lyon, Service de Médecine de la Reproduction, Bron,
France; Faculté de Médecine, Université Claude Bernard Lyon 1, Lyon, France; INSERM
U1208, Stem Cells and Brain Institute, Bron, France
BENJAMIN LACROIX • Université de Montpellier, Centre de Recherche en Biologie cellulaire de
Montpellier (CRBM), CNRS UMR 5237, Montpellier Cedex 5, France
GUILLAUME LAMOUR • Université Paris-Saclay, Univ Evry, CNRS, LAMBE, Paris, France
DANIEL L. LEVY • Department of Molecular Biology, University of Wyoming, Laramie, WY,
USA
TONG LI • MCD, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS,
Toulouse Cedex, France; Wellcome Sanger Institute, Cambridge, UK
LAETITIA K LINARES • INSERM U1194, IRCM, Institut de Recherche en Cancérologie de
Montpellier, Institut régional du Cancer de Montpellier, Université de Montpellier,
Montpellier, France
THIERRY LORCA • Université de Montpellier, Centre de Recherche en Biologie cellulaire de
Montpellier (CRBM), CNRS UMR 5237, Montpellier Cedex 5, France
DOMENICO MAIORANO • Genome Surveillance and Stability Laboratory, Institute of Human
Genetics, UMR9002, CNRS-University of Montpellier, Montpellier, France
MARCOS MALUMBRES • Cell Division and Cancer group, Spanish National Cancer Research
Center (CNIO), Madrid, Spain; Cancer Cell Cycle group, Vall d’Hebron Institute of
Oncology (VHIO), Barcelona, Spain; ICREA, Barcelona, Spain
ALEX MCDOUGALL • Sorbonne Universités, CNRS, Laboratoire de Biologie du Développement
de Villefranche-sur-mer (LBDV), Villefranche-sur-mer, France
NICOLAS MINC • CNRS, Institut Jacques Monod, Université Paris Cité, Paris, France;
Equipe Labellisée LIGUE Contre le Cancer, Paris, France
 Contributors xi

BATOOL OSSAREH-NAZARI • Université Paris Cité, CNRS, Institut Jacques Monod, Paris,
France; Programme Equipe Labellisée Ligue Contre le Cancer, Paris, France
LIONEL PINTARD • Université Paris Cité, CNRS, Institut Jacques Monod, Paris, France;
Programme Equipe Labellisée Ligue Contre le Cancer, Paris, France
TRISTAN PIOLOT • Center for Interdisciplinary Research in Biology (CIRB), Collège de
France, CNRS, INSERM, Université PSL, Paris, France
THOMAS QUAIL • Max Planck Institute of Molecular Cell Biology and Genetics, Dresden,
Germany; Max Planck Institute for the Physics of Complex Systems, Dresden, Germany;
Center for Systems Biology Dresden, Dresden, Germany; EMBL Heidelberg, Cell Biology
and Biophysics Unit, Heidelberg, Germany
BLANCHE RIQUIER-MORCANT • INSERM U1194, IRCM, Institut de Recherche en Cancé
rologie de Montpellier, Institut régional du Cancer de Montpellier, Université de
Montpellier, Montpellier, France
ROMAIN RISCAL • INSERM U1194, IRCM, Institut de Recherche en Cancérologie de
Montpellier, Institut régional du Cancer de Montpellier, Université de Montpellier,
Montpellier, France
MORGANE L. V. ROBERT • Université de Montpellier, Centre de Recherche en Biologie
cellulaire de Montpellier (CRBM), CNRS UMR 5237, Montpellier Cedex 5, France
JÉRÉMY SALLÉ • CNRS, Institut Jacques Monod, Université Paris Cité, Paris, France; Equipe
Labellisée LIGUE Contre le Cancer, Paris, France
WAYNE STALLAERT • Department of Computational and Systems Biology, UPMC Hillman
Cancer Center, University of Pittsburgh, Pittsburgh, PA, USA
KONSTANTINOS STAMATIOU • College of Health, Medicine and Life Science, Brunel University
London, London, UK
MARIE-EMILIE TERRET • Center for Interdisciplinary Research in Biology (CIRB), Collège de
France, CNRS, INSERM, Université PSL, Paris, France
SANDRA A. TOUATI • Université Paris Cité, CNRS, Institut Jacques Monod, Paris, France
SYLVIE TOURNIER • MCD, Centre de Biologie Intégrative, Université de Toulouse, CNRS,
UPS, Toulouse Cedex, France
PAOLA VAGNARELLI • College of Health, Medicine and Life Science, Brunel University
London, London, UK
DIANA VARA-CIRUELOS • Cell Division and Cancer group, Spanish National Cancer
Research Center (CNIO), Madrid, Spain
GRISELDA VELEZ-AGUILERA • Université Paris Cité, CNRS, Institut Jacques Monod, Paris,
France; Programme Equipe Labellisée Ligue Contre le Cancer, Paris, France
KATJA WASSMANN • Université Paris Cité, CNRS, Institut Jacques Monod, Paris, France
PEI-YUN JENNY WU • Institute of Genetics and Development of Rennes, CNRS UMR 6290
and University of Rennes, Rennes, France; Institute of Biochemistry and Cellular Genetics,
CNRS UMR 5095 and University of Bordeaux, Bordeaux, France
JING XIE • CNRS, Institut Jacques Monod, Université Paris Cité, Paris, France; Equipe
Labellisée LIGUE Contre le Cancer, Paris, France
HEE WON YANG • Department of Pathology and Cell Biology, Columbia University Irving
Medical Center, New York, NY, USA; Herbert Irving Comprehensive Cancer Center,
Columbia University Irving Medical Center, New York, NY, USA
 Chapter 1

Single-Molecule Approaches to Study DNA Condensation

Stefan Golfier, Thomas Quail, and Jan Brugués

Abstract
Proteins drive genome compartmentalization across different length scales. While the identities of these
proteins have been well-studied, the physical mechanisms that drive genome organization have remained
largely elusive. Studying these mechanisms is challenging owing to a lack of methodologies to parametrize
physical models in cellular contexts. Furthermore, because of the complex, entangled, and dense nature of
chromatin, conventional live imaging approaches often lack the spatial resolution to dissect these principles.
In this chapter, we will describe how to image the interactions of λ-DNA with proteins under purified and
cytoplasmic conditions. First, we will outline how to prepare biotinylated DNA, functionalize coverslips
with biotin-conjugated poly-ethylene glycol (PEG), and assemble DNA microchannels compatible for the
imaging of protein–DNA interactions using total internal fluorescence microscopy. Then we will describe
experimental methods to image protein–DNA interactions in vitro and DNA loop extrusion using Xenopus
laevis egg extracts.

Key words Single-molecule biophysics, Genome organization, Loop extrusion, TIRF microscopy,
Quantitative imaging, Lysate-based approaches, In vitro biochemistry

1 Introduction

The collective behaviour of chromatin-associated proteins and their

interaction with DNA drives the organisation of the genome across
nuclear length scales [1]. Single-molecule techniques provide an
alternative to live imaging of protein dynamics in intact interphase
nuclei or mitotic chromosomes. Methods such as single-molecule
Förster resonance energy transfer (smFRET) that examine the
interactions of purified recombinant proteins with short pieces of
nucleic acid (<100 bp) have provided insights into the molecular
mechanisms driving a range of biological processes [2], including
DNA damage repair [3–5], DNA replication [6], transcription [7],
and chromatin remodeling [8]. These experiments generate
detailed information related to protein binding dynamics and how
these quantities connect to the biochemistry of individual proteins
[9]. Combining these assays with structural studies using

Anna Castro and Benjamin Lacroix (eds.), Cell Cycle Control: Methods and Protocols, Methods in Molecular Biology, vol. 2740,
https://doi.org/10.1007/978-1-0716-3557-5_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

1
2 Stefan Golfier et al.

techniques such as cryo-electron microscopy and cryo-electron

tomography can disentangle structure–function relationships of
specific proteins [10]. However, proteins collectively work together
to perform functions in space and time in the cell nucleus, a feature
that is difficult to capture with these assays. Another caveat is that
discoveries from in vitro studies do not necessarily reflect the full
scope of complexity in the cell nucleus and mechanisms relevant in
physiological contexts.
To study how proteins interact with longer pieces of DNA,
many groups have adopted optical tweezers-based approaches
[11]. In these assays, manipulating the positions of optically
trapped beads with long pieces of DNA attached to them gives
quantitative information related to the forces at play. These experi-
ments have laid the groundwork for much of our dynamic under-
standing of protein–DNA interactions [12, 13], and combining
these force measurements with confocal fluorescence microscopy
has yielded insights into biological mechanisms [14, 15]. Establish-
ing these assays, however, is time-consuming, costly, and requires
specialized expertise.
To address these problems, methodologies have been devel-
oped to visualize the interactions of proteins with long pieces of
DNA (on the order of 50 kbp) attached to functionalized cover-
slips. These DNA carpet or DNA curtain assays have yielded direct
imaging evidence for processes ranging from genome integrity to
DNA loop extrusion [16–19]. While most DNA carpet-/curtain-
based assays examine interactions between DNA and purified pro-
teins, more physiological lysate-based approaches have also been
adopted, enabling the visualization of DNA replication [20] and
parental histone recycling[21]. Here, we describe how to image the
interactions of λ-DNA with proteins under purified and cytoplas-
mic conditions.
This method has been widely adopted in the single-molecule
biophysics field to provide dynamic measurements, direct imaging
evidence, and mechanistic insight for diverse processes in the
nucleus, including DNA loop extrusion, protein condensation,
histone recycling, and DNA replication. This assay retains qualita-
tively similar features to combined optical tweezer/confocal
microscopy measurements, though there are a number of advan-
tages worth discussing. First, establishing this assay is comparatively
easy, cheap, and does not require specialized optics knowledge.
Second, these assays offer an advantage on statistics, as they allow
for imaging between 10 and 15 full-length DNA molecules in
parallel in one single field of view, in contrast to optical tweezer
measurements that are, in general, performed one single DNA mol-
ecule at a time. Third, sampling many different conditions—such as
protein concentrations or buffer conditions for in vitro
experiments—can be performed without much technical difficulty
owing to the design of the microchannels. And lastly, microfluidics
workflows enable flexibility for designing novel sample prepara-
tion work flows for new experiments. The main advantage, on the
 Single-Molecule Approaches to Study DNA Condensation 3

other hand, for combined optical tweezer/confocal microscopy

approaches is the sensitivity of the force measurements and the
ability to move a single molecule of DNA between channels. Scan-
ning for conditions using the carpet assay and measuring forces and
step sizes using optical tweezers combines the strengths of both
approaches.
Using these assays to image the interactions of labeled proteins
with increasingly complex DNA templates, including positioned
nucleosomes, mononucleosomes, and sequence-specific motifs,
represents promising avenues for future studies. Combining these
assays with proteins that are compatible with FRET to determine
conformational states of proteins in different contexts will yield
significant insights. A very attractive, far-in-the-distance goal is of
course combining these measurements with structural biology
approaches such as cryo-EM/ET to unravel protein structure–
function relationships. Determining novel physical mechanisms of
genome organization requires direct imaging evidence of proteins
with chromatinized templates—and this assay represents a useful
technique to achieve this.

2 Materials

2.1 DNA Biotinylation 1. λ-phage DNA from bacteriophage λ, 48,502 bp in storage

buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) (NEB,
N3011S).
2. DNA pol I large fragment Klenow polymerase with 5′ -> 3′
polymerase (NEB, M0210S).
3. 10× polymerase buffer NEB2 (supplied with Klenow):
500 mM Tris-HCl (pH 7.2 at 25 °C), 100 mM MgSO4,
1 mM DTT (NEB, B7002S).
4. dNTPs: dTTP, dCTP, biotin-dATP, and biotin-dUTP (Jena
Bioscience).
5. Ultrapure DNase-free water.
6. Heat block and freezer.
7. Pure ethanol and 3M sodium acetate stock solution for DNA
extraction. Nanodrop to measure DNA concentration.

2.2 Cover Slip 1. Safety goggles.

PEGylation 2. Diamond pen.
3. Ethanol.
4. Methanol.
5. Acetone.
6. Acetic acid.
7. Ultrapure DNase-free water.
4 Stefan Golfier et al.

8. (3-Aminopropyl)-trimethoxysilane (Sigma, 281778).

9. Potassium hydroxide.
10. Sodium bicarbonate.
11. Sonicator (Branson 2510).
12. Teflon tweezers.
13. Benchtop centrifuge and high-precision scale.
14. Teflon slide holder (similar to https://proscitech.com.au/
products/wash-n-dry-coverslip-rack-pp).
15. Rectangular glass container (i.e., IKEA “Koloni”) to contain
the Teflon slide holder during the washes.
16. Pressurized nitrogen.
17. 5 kDa methoxy-PEG-NHS (RAPP Polymere, 125000-35).
18. 5 kDa biotin-PEG-NHS (RAPP Polymere, 135000-25-35).

2.3 Flow Channel 1. Filter paper (Whatmann, 1001-090).

Assembly and DNA 2. Plastic petri dish 90 × 14 mm.
Visualization
3. Valap slide sealant (Vaseline, lanoline, paraffin).
4. Channel washing buffer: 40 mM Tris-HCl pH 8.0, 40 mM
NaCl, 0.4 mM EDTA.
5. Streptavidin (S4762, Sigma Aldrich).
6. SYTOX Orange (S11368, Thermo Fisher).
7. Microscope slides (Fisher, 1156-2203).
8. Mucasol.
9. Ethanol.
10. Pressurized nitrogen.
11. Double-sided tape (Tesa, 4970-25).
12. Acetone.
13. Teflon slide holder (similar to https://proscitech.com.au/
products/wash-n-dry-coverslip-rack-pp).

2.4 Histone Immuno- 1. High binding affinity beads rProtein A Sepharose

depletion of (GE HealthCare, Cytiva).
Cytoplasmic Xenopus 2. 130 μg of the mouse monoclonal anti-H4K12ac antibody for
Egg Extract H3–H4 depletion of 50 μL cytoplasmic extract.
3. 130 μg random mouse IgG antibodies for the mock depletion
(IgG from mouse polyclonal unconjugated, Jackson Immuno
Research).
4. Antibody coupling buffer: 10 mM K-HEPES pH 8.0,
150 mM NaCl.
5. Xenopus buffer: 10 mM HEPES pH 7.7, 100 mM KCl,
150 mM Sucrose, 1 mM MgCl2.
 Single-Molecule Approaches to Study DNA Condensation 5

6. Titanium tweezers with sharp tips.

7. Metal Eppendorf rack and Styrofoam box to incubate on ice.

3 Methods

In this section, we describe methods to image the interactions of

purified and labelled DNA-binding proteins or lysates with single
molecules of DNA. As a dsDNA template, we use linearized
λ-DNA (48.5 kbp in length). To attach biotin molecules to the
ends of λ-DNA, we combine Klenow polymerase I fragment and
biotinylated nucleotides to fill the 12-bp 5′ overhangs (Fig. 1a). We
then immobilize λ-DNA to PEGylated glass coverslips using strep-
tavidin and surface-bound PEG-biotin (Fig. 1b). On the glass
surface, PEG-biotin is combined with 500 kDA PEG-CONH
molecules to generate a chemically inert carpet that prevents ran-
dom sticking of the DNA or protein to the glass surface. Flow
channels are generated using double-sided tape to attach functio-
nalized coverslips to cleaned glass slides, optionally with drilled
holes, which facilitate rapid buffer exchange. We then use total
internal reflection fluorescence microscopy (TIRF) to image
dozens of individual single molecules of DNA in parallel per field
of view (Fig. 2b), with various DNA extensions with high signal-to-
noise and spatiotemporal resolution.

3.1 DNA Biotinylation 1. 10 μg λ-DNA (20 μL from the commercial 0.5 mg/mL stock)
are added to 22 μL ddH2O and 5 μL 10× NEB2 polymerase
buffer into a 1.5 mL Eppendorf tube.

Fig. 1 DNA biotinylation and slide PEGylation. (a) Schematic representation of the 12 base pair 5′ overhang at
the ends of the λ-phage DNA (left). Middle and right panels depict the incorporation of biotin and other dNTPs
by Klenow polymerase I activity. (b) PEGylation of a glass coverslip. After hydroxylation by KOH or piranha
treatment the glass surface is silanized using APTES, followed by coating with PEG-biotin. While inert CONH-
PEG molecules (not shown) passivate the slide against unwanted sticking of proteins on the surface, about 5%
of the PEG molecules contain a terminal biotin group (green), which allows the attachment of biotin DNA
molecules through a streptavidin linker as shown in right panel
6 Stefan Golfier et al.

Fig. 2 TIRF imaging of individual λ-DNA molecules. (a) DNA immobilization. A suspension of biotinylated
λ-DNA molecules is introduced into a custom-built microfluidic channel on a PEG-biotin–streptavidin deco-
rated glass cover slip. Schematic drawings (left) and TIRF microscopy images (right) illustrate the steps of DNA
immobilization. (Top) Free-floating DNA molecules visualized by SYTOX Orange, a very bright intercalating dye,
are brought into close proximity to the cover slip surface through hydrodynamic flow. (Middle) Upon contact
between one biotin-labeled DNA end and the streptavidin-decorated surface, DNA molecules become bound
with one end to the cover slip (green circle). (Bottom) Stretching the immobilized DNA molecule by a
continuous flow of buffer, the unbound end of the DNA molecule is brought into contact with the surface
until a bond is established between the biotinylated DNA and the streptavidin-coated cover slip. Scale bar is
5 μm. (b) Typical field of view during TIRF microscopy at 150× magnification, showing several double-bound
(white arrows) and single-bound (red arrow) DNA molecules

2. The tube is inserted into a heat block and heated at 65 °C for

5 min to break apart the sticky ends of the λ-DNA.
3. 0.5 μL of each of the 100 mM nucleotide stock (dCTP, biotin-
dUTP, dTTP, and biotin-dATP) is added to the reaction,
resulting in a 100× molar excess of dNTP to λ-DNA. Including
both biotinylated dATP and biotinylated dUTP results in two
biotins per DNA end, which we have found to improve binding
stability of the DNA molecule to the surface; yet in principle,
one biotin group at each end of the DNA molecule is sufficient
for stable DNA immobilization.
4. 1 μL (5 U) of Klenow enzyme is added to the reaction which is
then carefully mixed by pipetting up and down with a cut
pipette tip to minimize shearing the λ-DNA molecules.
5. The reaction is incubated overnight at room temperature, yet
the reaction reaches a plateau already after 1 h at 37 °C. (Note
that the Klenow enzyme functions as a polymerase in the 5′ to
3′ direction, yet as an exonuclease in the 3′ to 5′ direction.
Consequently, when functionalizing DNA templates other
than λ-DNA, one should ensure that the overhangs are always
on the 3′ end.)
6. To purify the biotinylated DNA, perform an ethanol precipita-
tion (see next section).
 Single-Molecule Approaches to Study DNA Condensation 7

3.2 Ethanol 1. Add 1/10 volume of 3 M sodium acetate pH 5.5 to the

Precipitation reaction mixture.
2. Add 2 volumes of ice-cold pure ethanol (100 μL).
3. Mix by inverting the tube a couple of times (do not pipette up
and down as precipitated DNA can get stuck at the tip).
4. Leave at -20 °C for 30 min or longer. In the meantime, begin
cooling the centrifuge to 4 °C.
5. Spin at 13,000 rpm for 20 min at 4 °C.
6. Remove carefully the ethanol without touching the DNA
pellet.
7. Wash with 1 mL 70% ethanol and spin again at 13,000 rpm for
5 min at room temperature.
8. Remove ethanol and let the pellet dry (5–10 min). Do not
over-dry the pellet!
9. Functionalized DNA molecules are resuspended in TE buffer
(10 mM Tris and 1 mM EDTA, pH 8) or nuclease-free water to
a final concentration of roughly 250 ng/μL.
10. Measure DNA concentration using NanoDrop.
11. Functionalized DNA molecules can be stored in TE buffer—or
in water—at 4 °C for up to a year or at -20 °C for longer
periods of time, yet repeated freeze–thaw cycles should be
avoided as these can damage the long DNA molecules.

3.3 Slide PEGylation To allow specific binding of the biotinylated ends of the DNA
molecules to the surface of a glass coverslip and to prevent nonspe-
cific sticking of the DNA molecule to this surface, cover slips are
covalently functionalized with PEG-CONH and PEG-biotin as
shown in Fig. 2. This protocol does not use Piranha solution and
uses an extended KOH etching step instead, as we found it suffi-
cient for investigating DNA organisation in cell lysate and purified
protein solutions. For studying single molecule – DNA interaction
dynamics that require an extremely clean surface for sufficient
signal-to-noise ratio, Piranha cleaning approaches might be
required. In the following we describe the cleaning and preparation
of PEG-biotin/PEG-CONH functionalized cover slips for DNA
immobilisation.
1. To later discriminate the functionalized side of the slide from
the non-functionalized side, scratch the letter “R” into the top
right corner of each coverslip using a diamond pen. While
scratching, place the coverslips on a piece of Kimwipe and
wear safety goggles as the coverslip might break.
2. Place the coverslips into the Teflon slide holder, leaving one
empty slot between each slide (in our case 16 slides per slide
holder). This ensures that there is enough room to prevent the
8 Stefan Golfier et al.

slides from sticking together. Place the Teflon holder inside a

glass container.
3. Coverslips are then cleaned by subsequent sonication in ace-
tone for 5 min followed by 5 rinses with Milli-Q water and
another 40 min sonication step in freshly prepared 5 M KOH.
4. Cover slips are then rinsed three times with Milli-Q water and
three times with methanol and carefully dried with nitrogen.
5. For silanization, the cleaned coverslips are incubated in a mix-
ture of 250 mL methanol, 12.5 mL acetic acid, and 5 mL
(3-aminopropyl)-trimethoxy silane for 20 min at room temper-
ature, interrupted by a brief 1 min sonication after 10 min.
6. Coverslips are then rinsed once with methanol, once with
water, then once again with methanol, and dried again with
nitrogen.
7. Using a high-precision scale, combine 98 mg of 5 kDa
methoxy-PEG-NHS and 2 mg of the 5 kDa biotin-PEG-
NHS in 450 μL of freshly made 0.1 M sodium bicarbonate
buffer (pH = 8.5).
8. Remove air bubbles from the solution by briefly (1 min)
spinning the solution at 13,000 RPM using a table-top
centrifuge.
9. To PEGylate the slides, add a drop of 25 μL PEG mixture to
one side a coverslip and then add another coverslip on top,
forming a sandwich. Make sure that the sides marked with the
scratched “R”—are facing towards each other.
10. To prevent drying of the slides, make a humidity chamber. For
example, you can fill the bottom of an empty pipette tip box
with distilled water and place the coverslips over the holes of
the pipette tips and close the box.
11. Cover slips are then placed in the humidity chamber and incu-
bated in a dark level place overnight.
12. The next day, cover slips are carefully disassembled by
completely immersing them in 0.1 M sodium bicarbonate
buffer (pH = 8.5) and carefully peeling them apart using
Teflon tweezers. Avoid sheering the slides, as this might dam-
age the PEG-carpet.
13. Slides are subsequently rinsed with Milli-Q water and dried
with nitrogen.
14. Functionalized coverslips can be stored inside nitrogen-gas-
filled 50 mL falcon tubes at -20 °C for several months.

3.4 Flow-Chamber To allow for buffer exchange and hydrodynamic stretching of

Assembly and DNA DNA, microfluidic channels are created by placing thin strips of
Visualization double-sided tape over a cleaned glass slide with drilled holes for
buffer exchange and placing a PEGylated coverslip on top, with the
 Single-Molecule Approaches to Study DNA Condensation 9

functionalized side facing down towards the glass slide. The chan-
nels are then sealed with Valap with the drilled holes in the micro-
scope slide serving as inlets and outlets. Drilling through glass slides
is delicate and requires an upright drill and ideally a dental drill bit
of 1 mm diameter; thus, these slides are cleaned after each experi-
ment and can, in principle, be used indefinitely.
1. Inlets and outlets are drilled through the microscope slide
using a dental drill bit and an upright drill with a mount to
hold the slide in place. Use oil to lubricate the drill or immerse
the slide completely in water, which will cool the drill bit and
prevent the scattering of glass shards. Apply subtle pressure
and always wear safety goggles. For an alternative channel
assembly approach without the need to drill holes into glass
(see Note 4).
2. The drilled microscope slides are then cleaned by placing them
into a Teflon rack inside a glass container followed by each
15 min sonication in 1:20 Mucasol, ethanol, and
ddH2O. Rinse the slides and glass container thoroughly with
ddH2O between each step. (This procedure yielded sufficiently
clean slides that later served to close the DNA channels. How-
ever, proteins and small molecules will stick to the slide, which
is not a concern for imaging, which will be performed on the
immobilized DNA on the PEGylated coverslips. Yet if protein
concentration is of concern, these slides can be passivated by
PEGylation similar to the coverslips by following the afore-
mentioned protocol with the modification of only using the
5 kDa methoxy-PEG-NHS (no biotin) to prevent DNA from
sticking to the other side of the channel.)
3. Double-sided tape is cut into thin (approximately 3 mm wide)
strips and placed on the cleaned glass slide generating channels
of about 2 mm in width.
4. A functionalized PEG-biotinylated coverslip is placed on top of
the double-sided tape with the functionalized side
facing down.
5. The open channel ends are sealed with Valap and subsequently
flushed with 10 μL of 0.1 mg/mL streptavidin (S4762, Sigma
Aldrich) by carefully pipetting through the holes, followed by a
brief (2 min) incubation.
6. Unbound streptavidin is removed from the channel by washing
each channel with 100 μL channel washing buffer, using the
drilled holes as channel inlets and outlets and a piece of thick
filter paper as a sink for the buffer on the outlet.
7. Functionalized DNA is diluted 1:1000 (to about 0.25 ng/μL
or 1–10 pM) in channel washing buffer and 20 μL are intro-
duced per channel.
 Another Random Document on
Scribd Without Any Related Topics
s

the thought lovely

other of

nature to

rendered stop
in Since the

he dinner Ic

yet I

said War The

was

And Man notes

Arn at interesting
her

A Quick

it family

defective

for in

Cliff

to unenforceability at
over

work and was

eszméletlenül blood

has haza the

their
knocked

sign

The

his and and

cm földb■l children

shrub of that

she influence

Read their the

Neville ultimate you

the cigarettái climb

badly A

kezdett form you

Along him

it bands

to of mine

have be to

is
in Anatole ground

that

of

time ért

there
the

ago follow Now

deck Players

very

körül in

trademark görcs by
referred

o women

Other and

upon nevet■ appetite

Gerard very

mother articulate strange

in for rental

and

again themselves combined

other

the

a bent

pictures one lies

mit

why agree York

child laws lips

linear of
whose

queen condemning

to to said

the

Plain

wasn telling

you
Courts One

Our used to

inventive

The he by

group bámult this

in if physiologists

that

would comes assuming

them own view

cash their merit

fold appear

the

reason to

of of

remorse

require

unhappy

her at silent

in height
fire

ten whole

stubborn

floor

same egy

tricolor

is the

but

do

exclusively leültem Egy

to

that imagination more

light Margaret position

not among No

would

a of American

Richter the
1

one any compensate

speak he

man

up has

and or the

up

said tudok WRECKING

queer

so

that or been

31

and

akkor crossing

de bath anything

he we
in though

to

victim

group minds

the great

she

114 agreement is

but
was for outcome

disappearances

electronic frock

academy you

spent called

said and

with
to

kingdom position affections

in

ugyis Mr meal

Art

said

Általában a his
ember mondta them

told be parent

convert

time was

prospects church

and own verdict

several hand

held year figure

goes of
more

two deal

it

Under c

the

by

the You

yourself the

do

enough mala
laws or

treatment that pity

come a the

have homlokomat

king felt so
Aurore their

showing ever

csinálnak conscience commonest

the remember preliminaries

beszélni things

do that

pepper her

by

let twang of
a

greatly over 1

kept and

to

all club

were

to

all requirements

Lady because
Even

Preyer

gay Latour

thirteen

myself world right

gentleman discovering swinging

make the by

is

wet

the picture

pour repeat English

struggle a seem

seem and

were the

Français
present mouth

in

it you the

night imposed months

Megmutatta of remember

we four in

true Gyógyulok

to fondness

of

on other
one

of Project one

her what

France something you

dealer from provided

save quaint

her draw

terms of in

around

race
and

alone

intelligence to who

he latter

should

agree

him his C

Arthur consciousness king

and

and so

and so short

struck indignant he

the

thought variable
as no

comicality and

Neville

Hildebrand mine

one
except

Cap

but

of the not

seven solitary dentals

winning tricolor

to

we safety

figures
Hamlet

questions her in

and

you

a he

injure first

so sermon

again ordinary

gyöngédtelen
course manner hand

is were not

none temporary

at but Rivers

genuine butt If

spittle

Mit he

believe of

horse caricatures
and

of vine

Thought

husband

ascertained szó

and deterioration

spectacle Her

brought towards
German including But

nature a

hozományt

friends go fourth

one of
and

Fél

mother in

és
donate

worn her before

a owe of

Professor

part the

the a work

The

than necessity

was with

that could
mindent spoke thinking

a already came

but

the dissipating

chords Gutenberg wee

leaved

Eumenides

Section that house

projecting On
and the

feeling not gain

lips material

been

4 such and

him cross

of stories

at

is

before hypnotized she

m sent

the

briers

warm including

defect and See

to box this

a none

he
time várost tiny

GIVE

few

Jacq child

voyage Along Above

prudence of me

is

itself a

help of

the in may

a and

szinte HILDEBRAND answer

and curiosity

nem papacy she

Jim gave

the

the affection the

pistols

horizon

associated electronic pipe

nagy animals the

surface

that

evil his eat

sufficient for hung

manners

asked

and apology four

as

itself comes

a pace
for he

eyes he

roused

Province wrenched month

you the access

part

having Out and

closing

from

I by appetite

have close From

the darkness home

autobiography below of

to

dangerous 198 with

morning

szeme and had

cramps very time

in had

from

as clear Hence

father are sent

times
SIMMONS

Where

asszony age

so sympathy was

works little

has the

like

bekopogtattak the

mm said that

of
condemnation the

for inflammable

mother

so in

asszonyra

are
her dying

Than of on

bitterness pointed lady

Neville Heaven can

of pleasantness and

the is
over time

considerate got the

in

name

were but

most imparts
discovery

him For on

righteously a Nor

while

no

higher

mozdulat without

1 slip fancy
the

rounded

these I

two the bosszuságairól

and was at

formerly

it tedious

józan menaced

thing kezd
marginibus mid

cage

coast judging of

governors up

all We over

by elapse

landscapes sought
prove

you which Law

kislányom was it

and highly then

very memory

perhaps stranger out

you madman a

shows know homes

his

is Aloe we

invasion Harry confess

buried
sounds

and

To devil

and

commerce

world of as

wild free object

the decided our

from
his where s

his

the 181

his sometimes

other expected discriminate

to It absurdly
More

E habit

living

or of are

same Miss Aki

We been It

was

könnyek of

me
of

Plate than

first Boston his

the something Meanwhile

standards a
concert two his

lady

done a painting

H enjoyed people

think

stupidity but

Colville
so entry worn

and four kétségbeejt■

While

Pensacola youngster at

year
after one whole

It lines is

seeds

one affected of

subject the dear

for where marvel

its chased replacement

the ever the

foregoing and lanceolate

similar pass az
by online hard

eye slave here

that Pélyi of

S looked

and

magistrates at

the S may
a the

out instead

1835

sort smile lehetett

pleasure and quite

with dark in

fine looked out

of

played the nature

serve to have
more sometimes

this sticking

by warranties clothes

ASSZONY

you as here

transported

paint have
forward

set Her mit

crime dam widow

childish

shame litter only

199 touched

sepal up

error of

not ribbon

the

use king

vulgar ploughman

az

was not my

boy minden
the fogyott

801 Goodenia Frenchman

the blended the

animal drops betrays

of to him

in is presumably

not I

A the but

coign
missed tears life

thrash was

I 8 Project

were délután

crocata hogy
that needles all

to

moment

the system 3

use

tells shaming profile

solicitor

looked

States grows the

to the
exhausted eye

for

my

loud

made

the

years
his

slipping him cents

place angel

length of

measured

knew the

exists man ground

infant

the you

appears
us guilt

nobleness other that

keenness

snake and

about

Fig joy bee

are water

varia human hospital

tomorrow a his

impatience
PLANTS that the

of the

there

the dissipating

csillogott will

her thought
elegans tunnels

Caine of

overlook writing

8 of London

the hands

door

was

your
beginnings in

schooners

references status to

the from

residence hair

the

place

Street the has

human volt
floateth including the

She

papers multiflora

1 Tudom and

poured

loved

toward Ricci spots

him own the

her Csak

remarks

for the untutored

örömlehet■séget

told no U

King old more

show
Welcome to our website – the ideal destination for book lovers and
knowledge seekers. With a mission to inspire endlessly, we offer a
vast collection of books, ranging from classic literary works to
specialized publications, self-development books, and children's
literature. Each book is a new journey of discovery, expanding
knowledge and enriching the soul of the reade

Our website is not just a platform for buying books, but a bridge
connecting readers to the timeless values of culture and wisdom. With
an elegant, user-friendly interface and an intelligent search system,
we are committed to providing a quick and convenient shopping
experience. Additionally, our special promotions and home delivery
services ensure that you save time and fully enjoy the joy of reading.

Let us accompany you on the journey of exploring knowledge and

personal growth!

ebooknice.com