VISIONS OF THE

CELL NUCLEU S

Stephan Diekman nPeter Hemmeric h,

Foreword

vii

Preface

ix

About the Editors

xxi

List of Contributors

xxiii

Chapter 1 : ARCHITECTURE AND DYNAMICS OF MAMMALIAN GENOMES

1Daniele Zink

1. Mitotic Chromosomes and Interphase Chromosome Territories

1

1 .1 . How Is the Substructure of Chromosome Territories Organized?

32. Mitotic Chromosome Bands, the Organization of Replication, and the Functiona l

Architecture of Mammalian Genomes

52 .1 . Specific Features of Mitotic Chromosome Bands

52 .2 . Organization of Replication within Mammalian Cell Nuclei

52 .3 . The Relationship between Chromosomal Banding Patterns and the Nuclea r

Arrangement of Chromatin with a Specific Replication Timing

52 .4 . Polar Chromosome Territories Build up Higher Order Compartment s

with Specific Functional Characteristics

6

3. Positioning of Chromosome Territories within Cell Nuclei

94. How Is Nuclear Architecture Integrated with Gene Regulation?

94 .1 . Ikaros-Regulated Genes

1 04 .2 . Modifications of the Chromatin Structure and Nuclear Positioning

1 14 .3 . The (3-Globin Locus

1 14,4 . Enhancer Elements and Nuclear Positioning

1 2

5. Mammalian Genome Architecture Shows a Lower Degree of Organizatio nin Noncycling Cells

1 2

6. How Dynamic Is the Nuclear Architecture of Mammalian Genomes?

1 3

6 .1 . Mitosis

1 3

6 .2. Replication

1 36 .3 . Interphase Dynamics in Functionally Unaltered Cells

1 36 .4 . Interphase Dynamics in Cells That Switch Their Functional State

1 37. Anchoring of Chromosome Domains

1 4

7 .1 . The Nuclear Lamina

1 4

7 .2 . The Nuclear Matrix

1 48. Interactions Between the Genome and Other Nuclear Components

1 6Acknowledgments

1 7References

17

Chapter 2 : FUNCTIONAL ORGANIZATION OF CHROMOSOMES IN TH EINTERPHASE CELL NUCLEUS

1 9Astrid E. Visser, Roe/ van Driel, Pernette J . Verschure

1. Chromosome Organization

1 91 .1 . Sausages, Spaghetti, and Meatballs : The Discovery of Chromosome Territories 1 91 .2 . Chromosome Territories Are Discrete Units

201 .3 . Substructure of Chromosome Territories

221 .4 . Ultrastructure of Chromosome Territories

232. Spatial Relationship between Chromosome Territories and Nuclear Processes

242 .1 . Spatial Relationship between Large-Scale Chromatin Folding and Replication

242 .2 . Spatial Relationship between Large-Scale Chromatin Folding an d

Transcriptional Activity

2 52 .3 . Chromatin Unfolds Preceding Transcriptional Activation

272 .4 . Functional Nuclear Positioning of Chromosome Territories an d

Subchromosomal Regions

282 .5 . Nuclear Positioning of Chromosome Territories and Specifi c

Chromosome Regions

292 .6 . Positioning Close to Heterochromatin

3 03. Dynamics and Mobility

3 13 .1 . Chromatin Dynamics

3 13 .2 . Mobility of Nuclear Components Other Than Chromatin

3 24. Conceptual View of Chromosome Organization in Interphase

3 2Acknowledgments

34References

3 4

Chapter 3 : ORGANIZATION AND DYNAMICS OF THE CELL NUCLEU SFOR DNA REPLICATION

37Jean-Marc Lemaltre, Marcel Mechali

1. Introduction

3 72. The Chromosome Is Organized for Its Replication

3 82 .1 . Organization of Replication Origins into Foci

3 82 .2. Chromosome Banding

3 82 .3 . Chromatin Loops

4 03. Maintenance of Chromosome Individuality within the Nucleus

4 13 .1 . Compartments of the Interphase Nucleus

4 13 .2 . Chromosome Movements within the Nucleus

4 24. Replication Initiation Is Spatially and Temporally Regulated

4 44 .1 . Choice of the Origin

444 .2 . Replication Timing

465. Perspectives

4 8Acknowledgments

4 8References

4 8

Chapter 4 : HOW ONE NUCLEUS MAKES TWO NUCLEI :THE NUCLEUS IN MITOSIS

5 2Michael Brandeis

1. Introduction

5 22. The Mitotic Cast: The Main Characters-Mitotic Kinases and the APC/Cyclosome 5 2

2 .1 . Mitotic Kinases

53

2 .2 . Ubiquitin-Mediated Degradation by the Anaphase-Promotin g

Complex/Cyclosome

5 4

3 . The Play

5 6

3 .1 . Act 1 : From G 2 to Metaphase

5 73 .2 . The Metaphase Climax

6 2

3 .3 . Act 2 : From Metaphase to G 1

6 2

4 . Concluding Remarks

6 2

Acknowledgments

6 4References

6 4

Chapter 5 : EPIGENETIC FEATURES THAT INFLUENCE NUCLEAR FUNCTIO N

IN HIGHER EUKARYOTES

67

Dean A . Jackson

1. Introduction

672. Basic Features of Gene Expression

6 82 .1 . Activating Gene Expression in Mammalian Cells

6 82 .2 . Dissecting the Complexity of RNA Synthesis in Mammalian Cells

6 82 .3 . Visualizing Genes in Action

6 82 .4 . The Nuclear Distribution of Specific Transcripts

6 92 .5 . Genetic Elements That Influence Levels of Gene Expression : Activating

Gene Expression

6 92 .6 . Dynamic Features of a Transcription Cycle

7 0

3. The Organization of Gene Domains

7 03 .1 . The Dynamics of Gene Expression

7 03 .2 . Chromatin Domains as Units of Gene Expression

7 13 .3 . Chromatin Loops and Gene Domains

7 23 .4 . Insulators and Chromatin Domain Boundaries

7 23 .5 . Locus Control Regions

7 34, Epigenetic Features That Influence Chromatin Structure

7 34 .1 . Positional Requirements for Gene Expression

7 34 .2 . The Spatial Organization of Heterochromatin

744 .3 . Chromatin Status and Activating Genes in Heterochromatin

765 . Nuclear Structure and Gene Expression

7 75 .1 . Gene Location and Gene Expression

7 75 .2 . Manipulating Gene Structure at the Natural Locus

775 .3 . Chromatin Domains as Units of Gene Expression

785 .4 . DNA Loops : A Simplified View

7 86. Global Nuclear Architecture

7 86 .1 . Specialized Sites of Nuclear Function in Mammalian Cells

7 86 .2 . Sites of Transcription in Vivo : The Concept of Transcription Factories

7 96 .3 . A Transcription Cycle at the Active Centers

8 07 . Genomic Imprinting : A Special Case and Instructive Model?

8 07 .1 . Epigenetic Modification of Imprinted Domains

8 17 .2 . Patterns of Transcription from Imprinted Genes

8 17 .3 . Transcription Cycles during the Establishment and Maintenance

of Imprinting

8 28 . Nuclear Compartmentalization

8 28 .1 . The Dynamics of mRNA Processing and Nuclear Transport

8 28 .2 . Global Nuclear Structure

8 48 .3 . Chromosome Territories

84

9. Controlling DNA Replication

8 69 .1 . Setting up a Replication Program in Higher Eukaryotes

8 69 .2 . The Role of Chromosome Structure in Establishing the S Phase

8 810. Conclusions

8 9

References

9 1

Chapter 6 : THE ORGANIZATION OF TRANSCRIPTION IN THE NUCLEU SOF MAMMALIAN CELLS

9 5Pascale V. Guillot, Sonya Martin, Ana Pomb o

1. Introduction

9 52. The Organization of RNA pols in Transcription Factories

9 62 .1 . RNA pol I

962 .2 . RNA pol II

9 72 .3 . RNA pol III

9 92 .4 . Transcription Factories : General Plan

10 13. Nuclear Subdomains with Roles in Transcription

10 1

3 .1 . Nucleolus

10 1

3 .2 . OPT Domain

10 1

3 .3 . Other Nuclear Domains

10 2

3 .4 . Splicing Speckles

10 34. Concluding Remarks

10 3Acknowledgments

10 3References

10 4

Chapter 7 : PRE-mRNA PROCESSING IN THE NUCLEAR LANDSCAPE

106Karla M. Neugebauer, Kimberly M. Kotovic, Jennifer A . Geiger,David Stanek

1. Introduction

10 62. Cotranscriptional and Nucleoplasmic Pre-mRNA Processing

10 62 .1 . RNA Polymerase II Transcription Units

10 62 .2 . 5' End Capping : Coordinated by pol II

10 72 .3 . Pre-mRNA Splicing: Mixed Messages

10 82 .4 . 3 ' End Formation and mRNP Release

11 02 .5 . Histone 3 ' End Formation

1122 .6 . Fine Structure of the Transcription Unit

11 23. Nuclear Bodies and Pre-mRNA Processing

11 2

3 .1 . Nuclear Bodies

11 2

3 .2 . snRNP Trafficking to Cajal Bodies

11 33 .3 . The Subnuclear Distribution of Capping and Polyadenylation Factors

11 54. What Is Nucleoplasm?

11 55. Concluding Remarks

11 6Acknowledgments

11 6References

11 6

Chapter 8 : THE NUCLEOLUS : AN ESSENTIAL NUCLEAR DOMAIN

120J. Gebrane-Younes, V. Sirri, H. R . Jun6ra, P. Roussel,D . Hernandez-Verdun

1. Introduction

1202. The Nucleolus Is the Prototype of the Functional Compartmentalization

of the Nucleus

121

3. The Nucleolus Reflects the Highly Ordered Nuclear Organization

12 34. Nucleolar Organization Is the Consequence of the Coordination betwee n

Transcription and Processing

1244 .1 . The Canonical Three Nucleolar Components Visible by Electron Microscopy 12 44 .2 . Modifications of the Nucleolar Organization

1265. Nucleolar Assembly Takes Place at Precise Cell Cycle Periods or during a

Precise Cell Cycle

1275 .1 . The Regulation of the rDNA Transcription Machinery

1275 .2 . De nova Assembly of the rDNA Transcription Machinery

12 96. Recruitment of the rRNA Processing Machinery Is Time and Space Regulated

12 9

7. The Nucleolus Is at the Parting of the Ways of Several Nuclear Functions

13 28. Conclusions and Remarks

13 3Acknowledgments

13 3References

13 3

Chapter 9 : PROMYELOCYTIC LEUKEMIA NUCLEAR BODIES : CELLULA RFUNCTION AND DISEASE ASSOCIATION

136Thomas Sternsdorf, Monica Gostissa, Hüseyin Sirma, Giannino Del Sal ,Martin Ruthart, M. Lienhard Schmitz, Hans Will, Thomas G. Hofman n

1. Introduction

13 62. PML Bodies : Discovery and Structure

13 72 .1 . The Promyelocytic Leukemia Protein

13 82 .2 . The Coactivator CBP : Is There Transcription at PML Bodies?

13 92 .3 . The Sp l OO Family of Proteins, Potential Transcriptional Regulatory Factors

14 02 .4 . Daxx, a PML-Associated Transcriptional Repressor and Regulator of Apoptosis 14 22 .5 . SUMO-1 Conjugation and PML Bodies

14 33. p53: A VIP (Very Important Protein) Resident of the Nuclear Bodies

14 33 .1 . p53 Activation and Regulation

14 43 .2 . p53 and PML Bodies

14 44. Homeodomain Interacting Protein Kinase 2 : A Kinase Localizing to Nuclear Bodies 146

4 .1 . Identification and Domain Structure of HIPK2

14 64 .2 . HIPK2 : A Protein Kinase Implicated in Many Physiological Functions

1474 .3 . HIPK2 Predominantly Localizes to Nuclear Bodies

1475. PML Nuclear Bodies and Disease

1485 .1 . Autoimmune Diseases

1495 .2 . Spinocerebellar Ataxia Type 1

1495 .3 . Bloom Syndrome and DNA Repair

1495 .4 . The CBP Protein and Proliferative Disorders

1495 .5 . PML Bodies and Viral Infections

15 06. Leukemia and the Nucleus

15 16 .1 . Leukemia and Translocations

15 16 .2 . Leukemia and Translocation Products

15 16 .3 . Translocation Products Targeting Nuclear Bodies

15 1Acknowledgments

15 3References

15 3

Chapter 10 : THE CAJAL BODY : A NUCLEAR GATHERING PLACE

15 9. Karen E. Tucker, A . Gregory Matera

1. Introduction

15 92. Cajal Bodies

15 92 .1 . Cajal Bodies Are Dynamic and Heterogeneous

160

3. Cajal Body Composition, Localization, and Function

16 03 .1 . Coffin

16 0

3 .2 . Splicing Small Nuclear Ribonucleoproteins

16 4

3 .3 . Subnuclear Localization

16 53 .4 . Transcription

16 63 .5 . The Survival of Motor Neurons Protein Complex

16 64. A Nuclear Gathering Place

16 75. Summary

16 8Acknowledgments

16 9References

16 9

Chapter 11 : THE INTERCHROMOSOMAL DOMAIN COMPARTMENT :AN ACTIVE SPACE WITHIN THE NUCLEUS

172Joanna M. Bridger, Michaela Reichenzeller, Harald Herrmann,Peter Lichter

1. Organization of the Cell Nucleus

17 22. Properties of the Interchromosomal Domain Compartment

17 43. Experiments used to Visualize the Interchromosomal Domain Compartment

1743 .1 . RNA in the Interchromosomal Domain Compartment

1743 .2 . Relationships with the Nuclear Matrix

17 54. Introducing a Topological Marker for the Interchromosomal Domai n

Compartment

17 54 .1 . Live Cell Imaging of Ectopically Expressed Vimentin Confirms th e

Existence of a Reticular Dynamic Nuclear Compartment

17 65. Ectopically Expressed Proteins Are Dynamically Processed within th e

Interchromosomal Domain Compartment

17 96. Conclusions and Perspectives

1.8 1Acknowledgments

18 2References

18 2

Chapter 12 : SUBNUCLEAR PATHOLOGY

184Anna von Mikecz, Peter Hemmeric h

1. Introduction

1842. The Clinician's View of the Cell Nucleus

1862 .1 . Nuclear Shape and Chromatin

1862 .2 . Nuclear Inclusions

1872 .3 . Nucleolar Pathology

18 83. Subnuclear Pathology

18 83 .1 . Nuclear Envelope and Laminopathies

18 83 .2 . PML Bodies and Acute Promyelocytic Leukemia

19 23 .3 . Cajal Bodies, Gems, and Spinal Muscular Atrophy

19 23 .4 . Centromeres and Viral Infection

19 43 .5 . Polyglutamine Diseases

19 43 .6 . Nuclear Proteasomes and Systemic Autoimmune Diseases

19 74. Outlook and Perspectives

19 9Acknowledgments

20 0References

200

Chapter 13 : NUCLEAR COMPONENTS AND TUMOR MARKERS

20 4

Rajesh V. Kamath, Daniel J. . Leary, Sui Huang

1. Introduction

20 42. Classification of Tumor Markers

20 52 .1 . Category I Markers

20 52 .2 . Category II Markers

2092 .3 . Category III Nuclear Tumor Markers

21 23. Future Directions

21 64. Summary

217References

217

Chapter 14 : VISUALIZATION AND QUANTIFICATION OF DYNAMIC PROCESSE S

IN LIVE CELL NUCLEI

225

Daniel Gerlich, Roland Ells

1. Introduction

22 52. Multidimensional Microscopy of Living Specimens

22 62 .1 . Wide-Field Epifluorescence Microscopy

22 72 .2 . Confocal Laser Scanning Microscopy

22 72 .3 . Multiphoton Microscopy

22 72 .4 . Multicolor Fluorescence Microscopy

22 73. Quantitative Analysis and Visualization of Two-Dimensional Image Time Series

22 8

3 .1 . Highly Sensitive Object Detection

22 83 .2 . Two-Dimensional Segmentation

22 93 .3 . Dynamic Object Tracking

23 0

3 .4 . Continuous Time-Space Reconstruction

23 0

3 .5 . Quantitative Measurements

23 04. Multidimensional Image Analysis

23 04 .1 . Segmentation of Three-Dimensional Objects

23 14 .2 . Motion Analysis in Four-Dimensions

23 24 .3 . Visualization of Three-Dimensional Structures

23 24 .4 . Continuous Reconstruction of Temporal Sequences and Displa y

in Virtual Reality

23 44 .5 . Quantitative Analysis of Objects in Four-Dimensions

23 54 .6 . Elimination of Global Movements

23 54 .7 . Investigating Deforming Objects

23 6

5. Conclusions and Outlook

23 7Acknowledgments

23 7References

23 7

Chapter 15 : FLUORESCENCE RESONANCE ENERGY TRANSFER :

ANALYSIS OF PROTEIN-PROTEIN INTERACTION S

IN AND AROUND THE NUCLEUS

23 9

1r-Ma Majoul, Alexander Goroshkov, Rainer Duden

1. Introduction

23 92. FRET Theory

24 03. Experimental Setup

24 13 .1 . Instrumentation

241

3 .2. Molecular Biology

24 2

3 .3 . Transfection

24 3

3 .4 . Autofluorescence

24 3

3 .5 . Spectrofluorimetry

24 4

3 .6. Lifetime Imaging

24 4

4. Measuring FRET on the Golgi Membrane

24 4

5. Measuring FRET in the Nucleus with NFCC

24 56. Conclusions

24 57. Perspectives

24 6Acknowledgments

24 6References

24 7

Chapter 16 : FLUORESCENCE CORRELATION SPECTROSCOPY :PERSPECTIVES FOR IN VIVO CHARACTERIZATIO NOF SINGLE-MOLECULE DYNAMICS

248Petra Schwille

1. Introduction

24 82. FCS Theory

25 12 .1, Autocorrelation Analysis

25 12 .2 . Cross-correlation Analysis

25 33. Experimental Setup, Measurement Performance

25 63 .1 . Conventional (One-Photon) FCS

25 63 .2 . Two-Photon FCS

25 74. FCS Applications

25 84 .1 . Consideration of Autocorrelation Amplitudes : Concentratio n

and Aggregation Measurements

25 84 .2 . Consideration of Residence Times : Determining Mobility an d

Molecular Interactions

25 94 .3 . Consideration of Cross-correlation Amplitudes : A Direct Way to Monito r

Association/Dissociation and Enzyme Kinetics

26 34 .4 . Consideration of Fast Flickering : Intramolecular Dynamics and Probing

of the Microenvironment

26 34 .5 . Limitations of Intracellular FCS

2645. Conclusions and Outlook

26 5Acknowledgments

26 5References

26 5

Chapter 17 : MULTIPHOTON MULTICOLOR FLUORESCENCE IN SITUHYBRIDIZATION AND NANOPROCESSING OF CHROMOSOME SWITH NEAR-INFRARED FEMTOSECOND LASER PULSES

268Karsten König

1. Introduction

26 82. Multicolor FISH

26 93. Multiphoton Fluorescence Excitation

27 04. Principle of Multiphoton Multicolor FISH

27 25. Experimental Setup for 3-D-MM-FISH and Nanoprocessing

27 26. Materials and Preparation Methods

27 3

7. Applications of MM-FISH and DNA Imaging

27 47 .1 . Fluorescence Excitation of DNA Counterstains and FISH Fluorophores

27 48. Nanoprocessing of DNA by Multiphoton Effects

277

9 . Summary and Outlook

27 9

Acknowledgments

27 9

References

27 9

Chapter 18 : DAMAGE IN HUMAN CELL NUCLEI IN TOXICOLOGY ,NUTRITION, AND CANCER : VISUALIZATION BY COMETASSAY AND COMET-FISH

28 1Alexander Rapp, Karl Otto Greulich, Anja Schaeferhenrich,Beatrice Pool-Zobe l

1. Introduction

28 1

1 .1 . Principles of the Comet Assay and Historical Overview

28 1

1 .2 . Current Developments in the Comet Assay Technique

28 3

2. Methodological Aspects

284

2 .1 . Basic Comet Assay Steps

284

2 .2 . The Neutral Comet Assay

286

2 .3 . The Alkaline Comet Assay

28 6

2 .4 . A Comparison of the Different Comet Techniques

28 7

2 .5 . Analyzing Comet Assay Specimens

28 8

3. Selected Experiments and Results

28 8

3 .1 . The Interaction of Light at Different Wavelengths with the Cell Nucleus

28 8

3 .2 . The Comet Assay for the Study of Light-Tissue Interactions in Laser Therapy 290

3 .3 . Ionizing Radiation

29 1

3 .4 . Comet Assay in Basic Studies for Cancer Therapies and in the Search

for New Cancer Drugs

29 1

3 .5 . Comet Assay for the Study of Associations of Cancer and Nutrition

29 1

3 .6 . Apoptosis Does Not Necessarily Lead to Damage Detectable in th e

Comet Assay

2 9 9

3 .7 . Comet-FISH and BrdU Comet Assay in "Nuclear Architecture and

Function " Studies

2 9 94. Conclusions and Summary

302

References

302

Index

305