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Quantitative analysis of the structural dynamics of mitotic chromosomes in live mammalian cells [Elektronische Ressource] / Felipe Mora-Bermúdez

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140 pages
Ruprecht-Karls-Universität Heidelberg Naturwissenschaftlich-Matematische Gesamtfakultät Quantitative Analysis of the Structural Dynamics of Mitotic Chromosomes in Live Mammalian Cells Dissertation Felipe Mora-Bermúdez Heidelberg, April 2006 2 Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of doctor rerum naturalium Presented by M.Sc. Biologist Felipe Mora-Bermúdez Born in San José, Costa Rica Oral examination:___________________ 3 Quantitative Analysis of the Structural Dynamics of Mitotic Chromosomes in Live Mammalian Cells Referees: Dr. Damian Brunner European Molecular Biology Laboratory (EMBL) Meyerhofstrasse 1, D-69117, Heidelberg, Germany. Prof. Dr. Renato Paro, Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH). Im Neuenheimer Feld 282, 69120 Heidelberg, Germany Examiners: Dr. Iain Mattaj European Molecular Biology Laboratory (EMBL) Meyerhofstrasse 1, D-69117, Heidelberg, Germany. Prof. Dr. Peter Lichter Deutsches Krebsforschungszentrum (DKFZ) Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
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Ruprecht-Karls-Universität Heidelberg
Naturwissenschaftlich-Matematische Gesamtfakultät






Quantitative Analysis of the Structural
Dynamics of Mitotic Chromosomes in Live
Mammalian Cells

















Dissertation

Felipe Mora-Bermúdez

Heidelberg, April 2006






2

Dissertation

submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany

for the degree of

doctor rerum naturalium




















Presented by
M.Sc. Biologist Felipe Mora-Bermúdez
Born in San José, Costa Rica





Oral examination:___________________


3



Quantitative Analysis of the Structural
Dynamics of Mitotic Chromosomes in Live
Mammalian Cells













Referees:
Dr. Damian Brunner
European Molecular Biology Laboratory (EMBL)
Meyerhofstrasse 1, D-69117, Heidelberg, Germany.

Prof. Dr. Renato Paro,
Zentrum für Molekulare Biologie der Universität Heidelberg
(ZMBH). Im Neuenheimer Feld 282, 69120 Heidelberg, Germany


Examiners:
Dr. Iain Mattaj
European Molecular Biology Laboratory (EMBL)
Meyerhofstrasse 1, D-69117, Heidelberg, Germany.

Prof. Dr. Peter Lichter
Deutsches Krebsforschungszentrum (DKFZ)
Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

4




The following work was performed at the European Molecular Biology
laboratory (EMBL), Heidelberg, from October 2002 to April 2006, in the
laboratory and under the supervision of Dr. Jan Ellenberg.












I herewith declare that, under supervision, I independently wrote the following
doctoral dissertation, using none other than the sources and aids listed. Also,
during this work, the principles and recommendations in “Verantwortung in
der Wissenschaft” (Responsibility in Science), by the Ruprecht-Karls-
Universität Heidelberg, were observed.







th20 ofApril, 206
_______________ _________________________
Date Signature





5









Bruscamente la tarde se ha aclarado
porque ya cae la lluvia minuciosa.

Jorge Luis Borges
































(Suddenly the afternoon has cleared
as now the thorough rain is falling.)

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I Table of Contents


I Table of Contents _____________________________________________ 7
II Acknowledgments ___________________________________________ 10
III Abbreviations ______________________________________________ 11
IV Zusammenfassung __________________________________________ 13
V Summary __________________________________________________ 14
VI Publications________________________________________________ 15
VII Introduction 16
VII. 1 Chromatin _______________________________________________________ 16
VII. 1.1 Chromatin as information carrier ___________________________________ 16
VII. 1.2 Functional states of chromatin _____________________________________ 17
VII. 1.3 Basal chromatin composition ______________________________________ 18
VII. 2 Chromosome Properties ____________________________________________ 20
VII. 2.1 Nucleosome arrays ______________________________________________ 20
VII. 2.2 Higher orders of chromosome structure ______________________________ 20
VII. 2.3 Chromosome mechanics__________________________________________ 22
VII. 3 The Chromatin Cycle 23
VII. 3.1 Cohesins link genome replication and segregation _____________________ 24
VII. 3.2 Dual chromosomes must compact, resolve and attach before segregation____ 24
VII. 3.3 How is mitotic chromosome compaction orchestrated?__________________ 26
VII. 4 The study of Chromosome Compaction _______________________________ 27
VII. 4.1 Live microscopy to study chromosomes _____________________________ 28
VII. 4.2 How far can live microscopy go? ___________________________________ 29
VII. 4.3 Measures of chromosome compaction 31
VII. 5 Motivation and Aims of this Project __________________________________ 33

VII. 6 Specific Goals of this Project ________________________________________ 33



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VIII Material and Methods _____________________________________ 36
VIII. 1 Material ________________________________________________________ 36
VIII. 1.1 Laboratory equipment and reagents ________________________________ 36
VIII. 1.2 Stock chemicals and solutions 37
VIII. 1.3 Enzymes, markers, antibodies and nucleic acids_______________________ 38
VIII. 1.4 Bacteria ______________________________________________________ 40
VIII. 1.5 Mammalian cells _______________________________________________ 41
VIII. 2 Methods 43
VIII. 2.1 Molecular biology methods_______________________________________ 43
VIII. 2.2 Bioinformatic sequence analysis ___________________________________ 44
VIII. 2.3 Cell biology methods____________________________________________ 44
VIII. 2.4 Confocal microscopy and image processing methods __________________ 47
VI Results ____________________________________________________ 53
VI. 1 Maximal chromosome compaction occurs by axial shortening in anaphase and
depends on dynamic microtubules _________________________________________ 53
VI. 1.1 A volumetric assay for large-scale chromatin compaction ________________ 53
VI. 1.2 Chromatin occupies minimal volume in anaphase, not metaphase __________ 55
VI. 1.3 Assays to measure chromosome length during anaphase _________________ 57
VI. 1.4 Single chromosome arms shorten along their telomere-centromere axis after
segregation ___________________________________________________________ 60
VI. 1.5 An assay for the action kinetics of microtubule-perturbing drugs in anaphase _ 62
VI. 1.6 Requirements for anaphase chromosome shortening: a new role for
microtubules in mitosis _________________________________________________ 64
VI. 1.7 Acute perturbation of chromatid shortening impairs rescue of segregation
defects in condensin-depleted cells ________________________________________ 68
VI. 1.8 The reduction of global chromatin volume in anaphase depends also on
dynamic microtubules __________________________________________________ 70
VI. 2 Additional Assays for Chromosome Compaction at Different Scales:
Development and applications ____________________________________________ 72
VI. 2.1 A fluorescence distribution assay to measure compaction at medium scale ___ 72
VI. 2.2 Role of PNUTS in mitotic compaction probed with the fluorescence
distribution assay ______________________________________________________ 73
VI. 2.3 Further development of the fluorescence distribution analysis _____________ 76
VI. 2.4 A FRET assay for chromosome compaction at the molecular scale _________ 78
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VI. 3 Additional Results on Chromatin Organization _________________________ 84
VI. 3.1 Automated recognition, tracking and analysis of mitotic cells _____________ 84
VI. 3.2 Dynamics of Chromatin Proteins____________________________________ 84
VII Discussion ________________________________________________ 85
VII. 1 Applying Chromosome Compaction Assays____________________________ 85
VII. 1.1 Limitations of measuring chromosome volumes in live cells by confocal
microscopy ___________________________________________________________ 85
VII. 1.2 Comparison of the volumetric and intensity distribution assays ___________ 86
VII. 1.3 The quantitative study of the PNUTS-PP1 system may reveal key aspects of
chromatin organization__________________________________________________ 88
VII. 1.4 Comparison of the volumetric and time-lapse FRET assays ______________ 89
VII. 1.5 Limitations and perspectives of the EGFP-ReAsh FRET reporter__________ 90
VII. 1.6 Comparison of the two assays for the anaphase chromosome lengths_______ 91
VII. 2 Biological Aspects of Anaphase Chromatin Supercompaction_____________ 93
VII. 2.1 Novel anaphase chromosome dynamics______________________________ 93
VII. 2.2 Mechanism of microtubule dependence for chromosome compaction ______ 95
VII. 2.3 The function(s) of axial shortening of chromosomes ____________________ 96
VIII References _______________________________________________ 99
IX Appendix _________________________________________________ 110
IX. 1 Macro Codes for the Intensity Distribution Assay ______________________ 110
IX. 1.1 Masker-Quantifier ______________________________________________ 110
IX. 1.2 Focalizer______________________________________________________ 112
IX. 2 Publications 114

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II Acknowledgments

I would like to sincerely thank Jan for welcoming me in the group and mentoring me through this
project. From the personal and professional points of view, it has been a pleasure to work here.
Jan has been a personally kind but scientifically and technically challenging supervisor, a
combination that I have cherished. I am thankful for the opportunity he has given me to express
and follow my ideas, while at the same time directing, improving and correcting them.

Simply all of this work has been catalyzed by the constant training and help from each member of
the group. Not a single day goes by in which I don’t realize how the supportive atmosphere in the
lab makes the work more enjoyable. Together with Jan, the fabulous team that was Jo ёl, Daniel,
Gwén and Péter during the first part of this project is to thank for what I know about imaging
and image processing. I have had great collaborations with Daniel, Jo ёl and more recently with
Aurélien, and the results are an important part of the work I here present. Gwén’s input and his
macros have also greatly facilitated this work, and I had loads of fun with Péter in establishing
imaging assays with the Leica. Nathalie’s molecular biology and lab-organizing skills have made
even tedious protocols more enjoyable, and Katharina made the biochemistry-trainned rookie feel
more at ease in the early times. Lucia has also brightened-up the lab, and our spinning and
resurging, expanding and diffusing conversations have been a lot of fun. I need two more
minutes... before I can thank Antje for her witty way of easing-up the day and, together with
Melina and Elisa, for the help with the Zusammenfasung and making sure I have lunch every day.
Esther and Claudia were fun colleagues and the MitoCheck cousins have always been helpful with
reagents and discussions.

I have also enjoyed fruitful collaborations with groups outside of embl, for example with Helga
and Philippe at the University of Oslo. Also with Nathalie, Karl and Roland at the DKFZ, within
the Mitocheck frame. Promising results from these ongoing projects are also included here.

The Gene Expression and Cell Biology/Biophysics crowds have been a fun bunch to have
around, go for a beer, etc. I thank Melpi, our honorary group member, for nice discussions and
help with the DeltaVision, Mikko for help with biochemistry experiments and for his friendship,
Julien for our ongoing assay development, Veronika for help with parts of this manuscript,
people in the 2002 predoc generation for their friendship. Christina and Sylvia, who also
organized Matthias’s indispensable Kaffee, were also always helpful.

My friends have been supportive and always fun to be with, to relax, to think about something
else when weeks and months of lablife would yield only background, but also to share the
brighter moments. Bea, Roland, Silvia, Stoilka, die wunderbare “Pinte Truppe”... thank you.

I would like to deeply thank mi familia for always caring and supporting me, through the
difficulties faced before and during these times, and through the easy moments too. Every letter
and number, every nm and µl, every bit and byte and neural pulse offered here carries an inherent
gratitude to all of you.

My appreciation goes also to Damian, Iain and Renato for being part of my thesis committee and
supervising this project, and to Peter Lichter for being part of my defense committee.

This Project would obviously not have been possible without the EMBL environment and the
funding provided by the DFG (grant No. EL 246/1-1 and grant No. EL 246/2-1/2)

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