Nuclear Architecture explored by live-cell fluorescence microscopy using laser and ion microbeam ...

248 Pages·2011·21.31 MB·English

by  Hilmar Strickfaden

Department Biologie II
Anthropologie und Humangenetik
Ludwig-Maximilians-Universität München
Nuclear architecture explored
by live-cell fuorescence
microscopy using laser and
ion microbeam irradiation
Hilmar Strickfaden
Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München
Eingereicht am 1. Juli 2010
Nuclear architecture explored by live-cell fuorescence microscopy
using laser and ion microbeam irradiation
Dissertation der Fakultät für Biologie
Der Ludwig Maximilians Universität München (LMU)
vorgelegt von:
Dipl. Biol. Hilmar Strickfaden
aus Freiburg im Breisgau
Gutachter:
Prof. Dr. Thomas Cremer
Prof. Dr. Peter Becker
Tag der mündlichen Prüfung: 20.12.2010
II
Das Leben kommt auf alle Fälle
aus einer Zelle.
Doch manchmal endet’s auch bei Strolchen
in einer solchen.
Heinz Erhardt (1909–1979)
(German Comedian)
III
IV
Table of Contents
1 Summary 1
2 Introduction 5
2.1 The Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.2 Mitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2.1 Prophase .......................................................................................... 9
2.2.2 Prometaphase ................................................................................ 10
2.2.3 Metaphase ...................................................................................... 10
2.2.4 Anaphase .........................................................................................11
2.2.5 Telophase ........................................................................................11
2.2.6 Cytokinesis ......................................................................................11
2.3 Theodor Boveri’s Hypotheses . . . . . . . . . . . . . . . 12
2.4 DNA Repair . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.4.1 MDC1 .............................................................................................. 19
2.4.2 53BP1 ............................................................................................. 20
2.4.3 Rad51 .............................................................................................. 20
2.4.4 Rad52 .............................................................................................. 20
2.5 Microscopy – past, present and future . . . . . . . . . . 21
2.5.1 4Pi Microscopy ............................................................................... 23
2.5.2 PALM (Photoactivation Localization Microscopy) ........................ 23
2.5.3 Vertico SMI ..................................................................................... 23
2.5.4 SIM (Structured Illumination) ......................................................... 23
2.5.5 STED (Stimulated Emission Depletion) ......................................... 23
2.6 Live-cell fuorescence microscopy . . . . . . . . . . . . 24
2.6.1 Advantages and disadvantages of live-cell microscopy .............. 24
2.6.2 Live-cell fuorophores .................................................................... 27
2.6.2.1 GFP (Green Fluorescent Protein) and other fuorescent
proteins ............................................................................ 28
2.6.2.2 Photoactivatable GFP and photoswitchable FPs ............... 30
2.6.2.3 Chromobodies .................................................................. 30
V
3 Materials and Methods 33
3.1 Amplifcation, preparation, and storage of expression
plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.1.1 Transformation of bacteria with plasmids ..................................... 33
3.1.2 Glycerol stocks ............................................................................... 34
3.1.3 Preparation of plasmid DNA .......................................................... 34
3.2 Cell cultivation . . . . . . . . . . . . . . . . . . . . . . . . 36
3.2.1 Thawing cells .................................................................................. 36
3.2.2 Sub-culturing .................................................................................. 37
3.2.3 Seeding cells on coverslips ........................................................... 38
3.2.4 Deep-freezing cells ........................................................................ 38
3.3 Manipulation of living cells . . . . . . . . . . . . . . . . . 39
3.3.1 Transfection of cells by lipofection ................................................ 39
3.3.2 Generation of stable transgenic cell lines ..................................... 40
3.3.3 How to determine the right selection conditions? ........................ 41
3.3.4 Fluorescence labeling of replication foci and / or
chromosomes in living cells .......................................................... 41
3.3.5 Electrolabeling ................................................................................ 43
3.3.6 Microinjection ................................................................................. 44
3.3.7 Sensitizing DNA with BrdU ............................................................ 46
3.3.8 DNA Damage induction via NCS ................................................... 46
3.3.9 Induction of reversibly hypercondensed chromatin (HCC) .......... 46
3.4 Immunofuorescence protocol . . . . . . . . . . . . . . . 47
3.4.1 Fixation and permeabilization ....................................................... 47
3.4.2 Immuno-cytochemistry and DNA counterstaining ....................... 48
3.5 Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.5.1 Confocal microscopy of fxed samples ......................................... 49
3.5.2 Live-cell microscopy ...................................................................... 50
3.5.2.1 Fluorescence-Recovery-after-Photobleaching (FRAP): ...... 51
3.5.2.2 Photoactivation of H4-PA-GFP .......................................... 51
3.5.2.3 Laser microirradiation with light from a 405 nm
continuous diode laser ...................................................... 52
3.6 Rotatable 3D reconstructions . . . . . . . . . . . . . . . 52
3.7 Cell lines and constructs . . . . . . . . . . . . . . . . . . 52
3.7.1 Used cell lines ................................................................................ 52
3.7.2 Used expression plasmids: ........................................................... 53
3.7.3 Stable cell lines created in the course of this thesis: ................... 53
VI
3.8 Consumables and technical equipment . . . . . . . . . . 54
3.8.1 Chemicals and Reagents ............................................................... 54
3.8.2 Media and Solutions ...................................................................... 56
3.8.3 Equipment and other Hardware .................................................... 58
3.8.4 Used Microscopes ......................................................................... 62
3.8.5 Image Processing Software .......................................................... 64

4 Results 67
4.1 Revisiting Theodor Boveri‘s Hypotesis . . . . . . . . . . 67
4.1.1 Generation and characterization of the RPE1
H4-PA-GFP H2B-mRFP cell line .................................................... 67
4.1.1.1 Generation of a stable cell line expressing two types of
fuorescent histones .......................................................... 67
4.1.1.2 Description of the phenotype ............................................ 67
4.1.1.3 Analysis of the karyotype .................................................. 69
4.1.1.4 Growth and cell cycle length ............................................. 69
4.1.1.5 Photoactivation of H4-PA-GFP .......................................... 70
4.1.1.6 Checking photoactivated chromatin for DNA damages ..... 71
4.1.1.7 To which extent is photoactivatable GFP activated by
normal image acquisition in long-term observations? ........ 72
4.1.1.8 Hypercondensed Chromatin Condensation (HCC) in
interphase nuclei doesn’t change nuclear architecture ...... 73
4.1.2 Testing Boveri’s hypotheses and exploring the mechanics
of mitosis .........................................................................................74
4.1.2.1 Chromosomes occupy distinct territories within the
interphase cell nucleus ...................................................... 74
4.1.2.2 Arrangements of chromosome territories are stably
maintained during interphase in most of the observed
cells .................................................................................. 75
4.1.2.2.1 During S-phase large scale movements of
chromosomes are not mandatory ...................... 75
4.1.2.2.2 During interphase CT arrangements persist in
the vast majority of nuclei .................................. 75
4.1.2.3 Quantifcation of confned chromatin diffusion using
photoactivatable chromatin ............................................... 77
4.1.2.4 In a few cells 4D observation led to the discovery of
nuclear rotations around an axis parallel to the plane of
the substratum while mostly keeping their fat cell shape ... 81
4.1.2.5 Nuclear rotation can occur directly before the transition
into mitosis but stops immediately at the onset of
chromatin condensation ................................................... 83
4.1.2.6 Chromosomal neighborhoods are not transmitted from
one cell cycle to the next in RPE1, HeLa and Rat NRK
cells .................................................................................. 85
VII
4.1.2.7 Chromosome proximity pattern change especially in
prometaphase when chromosomes attach to the spindle
and move towards the metaphase plate ........................... 86
4.1.2.8 Unmasking the phenomenon “transmission of global
chromosome positions through mitosis” ........................... 89
4.1.2.9 Neighborhood arrangements established in the
metaphase plate are conserved throughout anaphase
and telophase resulting in rather symmetrical daughter
nuclei ................................................................................ 94
4.1.2.10 Photoactivation around the nuclear rim generates
daughter nuclei with a croissant-like distribution of
photoactivated chromatin ................................................. 96
4.1.2.11 The different faces of mitosis are caused by different
orientations of the mitotic spindle with respect to the
nucleus at the onset of prometaphase .............................. 97
4.1.2.12 Mitosis seems to be a robust, fexible and autonomous
process that takes over control once it is initiated ............. 98
4.2 Experiments at the ion microbeam facility SNAKE . . . . 101
4.2.1 The experimental set-up of SNAKE ............................................. 101
4.2.1.1 A tandem accelerator serves as ion source for SNAKE ... 101
4.2.1.2 The ion microbeam SNAKE ............................................ 102
4.2.2 First experiments at the SNAKE using live-cell microscopy ...... 106
4.2.2.1 Targeted cell irradiation ................................................... 106
4.2.2.2 After ion microbeam irradiation patterns of damage foci
remain stable over several hours in U2OS cells ............... 106
4.2.2.3 MDC1-GFP binds to damaged chromatin ca. 20
seconds after DNA damage induction by ion beam
irradiation ........................................................................ 107
4.2.2.4 Sequential irradiation using transgenic cells expressing
fuorescent repair proteins don’t show the competition
effect .............................................................................. 108
4.2.2.5 High doses of irradiation lead to saltatory
phosphorylation of chromatin .......................................... 108
4.2.2.6 Rad 51-GFP generates flaments inside the nucleus
at DNA damage foci ....................................................... 109
4.3 Exploring the dynamics of DNA damage response after
microirradiation using a continuous laser beam . . . . . 111
4.3.1 Microinjection does not induce visible DNA damage
response with respect to single and double strand breaks ........111
4.3.2 Kinetics of the repair-proteins MDC1-GFP, 53-BP1-GFP,
Rad51-GFP, Rad52-GFP in the undamaged nuclei and after
damage induction by a laser microirradiation .............................113
4.3.2.1 MDC1-GFP ..................................................................... 113
4.3.2.2 53BP1-GFP .................................................................... 115
4.3.3 Rad51-GFP polymerizes into complex flamentous networks ....118
4.3.4 Rad52-GFP dissociates from DNA damage foci after highly
dynamic processes in the nucleus ...............................................118
VIII
4.3.5 Increased chromatin mobility after DNA damage isn’t a
general feature in cells ..................................................................121
4.3.6 Fluorescence signals of damaged chromatin and
kinetochores are mutually exclusive ........................................... 123
5 Discussion 127
5.1 Working with cultured cells . . . . . . . . . . . . . . . . . 127
5.2 Evidence for Boveri’s hypotheses (1909) . . . . . . . . . 130
5.3 Probabilistic CT proximity patterns and non-random
radial arrangements . . . . . . . . . . . . . . . . . . . . 133
5.4 The case for long-range chromatin movements . . . . . 135
5.5 Chromatin diffusion . . . . . . . . . . . . . . . . . . . . . 136
5.6 Comments on Chromatin Conformation capturing
methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
5.7 Implications of probabilistic CT proximity patterns for
large-scale, non-random DNA-DNA interactions . . . . 137
5.8 Dynamics of DNA repair . . . . . . . . . . . . . . . . . . 143
5.9 Concluding remarks: mapping and understanding the
dynamic nuclear architecture requires a systems
biological approach . . . . . . . . . . . . . . . . . . . . . 145
6 References 149
Curriculum Vitae 167
IX
Appendix A 173
Abbreviations: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Appendix B 175
Spectral profles, Filters, Lasers and Fluorophores used in this
Thesis: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Appendix C 179
Shift Measurements at the Spinning Disc Microscope: . . . . . . . 179
Measuring the chromatic shift . . . . . . . . . . . . . . . . . . . . 180
Appendix D 183
Fluorophores used in this Thesis: . . . . . . . . . . . . . . . . . . . 183
Appendix E 187
Used cell lines: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Appendix F 193
Maps of the used expression plasmids: . . . . . . . . . . . . . . . 193
Appendix G 197
Box plots of mitotic events and whole cell cycles based on
live-cell observations: . . . . . . . . . . . . . . . . . . . . . . . . . 197
Appendix H
Die Blastomerenkerne von Ascaris megalocephala und die
Theorie der Chromosomenindividualität. . . . . . . . . . . . . . . 199
Appendix I 227
Overview on fuorescent proteins: . . . . . . . . . . . . . . . . . . 227
Properties of selected Optical Highlighters . . . . . . . . . . . . . 229
Ehrenwörtliche Versicherung 231
Acknowledgements 233
X