Dynamics of chromatin structure and nuclear multiprotein complexes investigated by quantitative fluorescence live cell microscopy and computational modeling [Elektronische Ressource] / presented by Norman Constantin Kappel

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• Biologie

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• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2009
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 20 Mo

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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 of Natural Sciences
presented by:
Diplom-Biochemiker Norman Constantin Kappel
born in Worms, Germany
Oral examination: July 2009Dynamics of chromatin structure and nuclear
multiprotein complexes investigated by
quantitative fluorescence live cell microscopy
and computational modeling
Referees: Prof. Dr. Roland Eils
Prof. Dr. Harald Herrman-LerdonAcknowledgements
I wish to thank Prof. Dr. Roland Eils for welcoming me to the unique
and highly dynamic research environment he has created within, both,
German Cancer Research Center and Bioquant. He has given me a per-
spective into quantitative biology at the interface of cell biology, physics
and computer science. I would like to thank him for giving me the time
and guidance I needed to develop my own ideas and for his enthusiastic
support regarding all aspects of this work. Last but not least I have ben-
efited from his numerous collaborations which were essential in creating
this work.
I thank Prof. Dr. Harald Herrmann-Lerdon for refereeing this work and
for numerous suggestions and ideas as well as practical advice for con-
ducting the experimental parts of this work. He also supported me with
his profound overview of cell biology and often pointed me into the direc-
tion of interesting work done in my field. In this way his support helped
me to shape particularly the work done on chromatin dynamics.
I wish to thank Prof. Dr. Peter Lichter for sharing some of his laboratory
facilities and for being a member of my PhD commitee.
I also wish to thank Prof. Dr. David Robinson for being a member of my
PhD commitee.
I owe special thanks to Dr. Joel Beaudouin who has been of invaluable
support during this work. His critical advice, many fruitful discussion
and practical support, both in the lab as well as interpreting data have
been of great importance.
Thanks to Prof. David Spector and Dr. Yi-Chun Chen at Cold Spring Har-
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bour Laboratories for a fruitful collaboration on the dynamics of promyelotic
leukemia bodies.
Thanks to Dr. Markus Ulrich who collaborated with me on the chromatin
dynamics project and with whom I collaborated on the design as well as
implementation of Tropical.
Thanks to Dr. Evgeny Gladilin, Dr. Stefan Wörz for collaborations on nu-
clear mechanics, cell tracking and image registration techniques.
I thank Dr. Michaela Reichenzeller for sharing her experience on confocal
microscopy, cell culture and molecular biology as well as for experimental
collaboration and critical discussions on numerous parts of my projects. I
also thank her for good comradeship and her cheerful character.
Many thanks to Dr. Matthias Weiss and Dr. Gernot Guigas for sharing
their FCS module and giving me a practical introduction into the FCS tech-
nique.
I thank Dr. Bernd Kalbfuss for introducing me to FRAP experiments and
numercial simulations for their quantitative analysis.
Thanks to Dr. Christian Bacher for advice on particle tracking with TIKAL. also belong to Dr. Hauke Busch for many fruitful discussions on
topics related to biophysics and systems biology.
Many thanks also to Karina Drobbe and Johannes Fredebohm, two intern-
ship students who created some of the primary data for the project on
nuclear H1° dynamics under my supervision.
I also thank all members of the Herrman-Lerdon lab as well as all members
of the theoretical bioinformatics department for creating a good working
atmosphere throughout my thesis and for giving me a good time.Zusammenfassung
Die Biologie hat sich schnell in eine datengetriebene, quantitave Wissen-
schaft verwandelt. Die Anforderungen an die biologische Bildgebung be-
wegt sich daher in Richtung quantitativer Annotation von Genen in vivo.
In dieser Dissertation habe ich die räumlich-zeitliche Verteilung, sowie
deren molekulare Interaktionen von Proteinpopulationen wie auch von
Multiproteinkomplexen untersucht. Ich habe Methoden entwickelt, mit
denen man biophysikalische Parameter, wie Diffusionskoeffizienten, ano-
male Diffusion und das Bindungsgleichgewicht von Proteinpopulationen
mit Hilfe von Fluoreszenzphotobleichen, numerischer Simulation und Pa-
rameterschätzung bestimmen kann. Bei der Analyse von Multiproteinkom-
plexen erweiterte ich bestehende Ansätze des Single-particle-tracking, um
den exakten Zeitpunkt von Dynamikänderungen einzelner Partikel in le-
benden Zellen automatisch detektieren zu können. Dabei ist es mir gelun-
gen, quantitative Parameter, wie Diffusionskoeffizienten, anomale Diffu-
sion, Geschwindigkeit und Chromatininteraktion zu bestimmen. Die nuk-
leäre Proteinpopulation, die ich untersuchte, war das Linkerhiston H1°
der Maus in Form von GFP-Konstrukten. Ich konnte zeigen, daß Diffu-
sion und Bindung von H1°-GFP an Chromatin mit Fluoreszenzphotoble-
ichen und numerischer Modellierung untersucht werden kann. Somit er-
hielt ich die Diffusionskoeffizienten von Wildtyp-H1°, sowie von sieben
Punktmutanten mit jeweils unterschiedlicher Bindungsaffinität, die von
2 2D 0.01mm /s (höchste Affinität) bis D 0.1mm /s (niedrigste Affinität)
reichte. Außerdem konnte ich die nicht gebundene Fraktion abschätzen,
die entsprechend von 400 ppm bis 3000 ppm reichte. Als Beispiel für
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große Multiproteinkomplexe wählte ich PML nuclear bodies (PML NBs), die
nach ihrem Hauptbestandteil, dem Promyelotischen-Leukämie-Protein be-
nannt sind. Ich untersuchte exakt deren dynamische Bewegung während
der frühen Mitose, die von der Prophase bis zur Prometaphase reicht. Es
konnte während dieses Zeitraums eine dramatische globale Zunahme in
der Beweglichkeit der PML NBs festgestellt werden, bei der die Diffu-
2sionskonstante von D 0.001mm /s während der Interphase auf D
20.005mm /s während der Prophase ansteigt. In ähnlicher Weise erhöhten
sich die Geschwindigkeiten von v 0.7mm/min auf v 1.4mm/min,
was mit einer Abnahme in subdiffusiver Bewegung (also anomaler Dif-
fusion) einher ging. Ich konnte zeigen, daß eine Loslösung von Chro-
matin die wahrscheinlichste Ursache für die Zunahme der Beweglichkeit
darstellt im Gegensatz zu mechanischem Fluss von Nukleoplasma oder
zu Chromatinverdichtung. Schließlich konnte ich die genaue zeitliche Ab-
folge der Beweglichkeitszunahme mit anderen zellulären Ereignissen in
Verbindung bringen. So trat die Zunahme der Beweglichkeit von PML
NBs hauptsächlich nach dem Eindringen von Zyklin B1 in den Zellkern
auf, welches die Zelle unumkehrbar auf Mitose programmiert, sowie vor
der Auflösung der Zellkernmembran.Abstract
Biology has rapidly been transformed into a mainly data-driven, quanti-
tative science. Demands on biological imaging are moving towards quan-
titative annotations of genes in vivo. In this work I have studied in de-
tail the spatio-temporal distribution and the molecular interaction of pro-
tein ensembles as well as of multiprotein aggregates. I have provided the
methodology to estimate biophysical parameters such as diffusion coef-
ficients, anomalous diffusion and the free fraction in the binding equilib-
rium of protein ensembles using fluorescence photobleaching analysis and
numcerical modeling and parameter estimation. On the side of protein
complexes I have extended existing single particle tracking approaches to
allow to automatically detect the exact timing of mobility changes of single
particles in live cells. Here, I was able to provide quantitative parameters
also on the diffusion coefficient, anomalous diffusion, velocity and chro-
matin interaction. The nuclear protein ensemble I studied was murine
linker histone H1° fused to GFP. I was able to show that diffusion and
binding of H1°-GFP to chromatin can be addressed using photobleaching
analysis and numcerical modeling. I have thus obtained diffusion coeffi-
cients for wild-type H1° and seven point mutants with differential binding
2 2affinity ranging from D 0.01mm /s (strongest binder) to D 0.1mm /s
(weakest binder). Likewise, I was able to estimate the free fraction to
range from 400 ppm to 3000 ppm. Exemplary of large multiprotein
complexes I chose PML nuclear bodies (PML NBs), named after their con-
stituent promyelotic leukemia protein. I studied in detail their dynamic
mobility during early mitosis, ranging from prophase to prometaphase.
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