Table Of ContentQuantitative Fluorescence Microscopy
of Protein Dynamics in Living Cells
Shehu Mustapha Ibrahim
The work described in this thesis was performed at the Department of Pathology of
the Josephine Nefkens Institute, Erasmus MC, Rotterdam and Department of
Biophysics and Cell Biology, University of Debrecen, Medical and Health Science
Centre, Hungary.
Quantitative Fluorescence Microscopy of Protein
Dynamics in Living Cells
Kwantitatieve Fluorescentiemicroscopie van Eiwitdynamiek
in Levende Cellen
Proefschrift
ter verkrijging van de graad van doctor
aan de Erasmus Universiteit Rotterdam
op gezag van de rector magnificus
Prof. dr. S.W.J. Lamberts
en volgens besluit van het College voor Promoties.
De openbare verdediging zal plaatsvinden op
donderdag 21 december 2006 om 09:00 uur
door
Shehu Mustapha Ibrahim
Geboren te Ilorin, Nigeria
Promotiecommissie
Promotor: Prof.dr. J.H.J. Hoeijmakers
Overige leden: Prof.dr.ir. J. Trapman
Prof.dr. J. Szöllősi
Dr. R. Krams
Copromotores: Dr. A.B. Houtsmuller
Dr. G. Vereb
Contents
Abbreviations 7
Chapter 1 General Introduction 9
Chapter 2 Light Driven Dynamics of GFP Fluorescence 49
Emission in Living Cells
Chapter 3 Dynamics of Nuclear Proteins: 73
Complementarities of Fluorescence Correlation
Microscopy and Photobleaching in Intra-Cellular
Mobility Measurements
Chapter 4 In the Absence of DNA Damage the Nuclear Mobility 109
of Most Nucleotide Excision Factors is Mainly
Determined by their Molecular Size
Chapter 5 Recruitment of the Nucleotide Excision 131
Endonuclease XPG to sites of UV-induced Damage
Depends on Functional TFIIH
Chapter 6 Translational Mobility of EGF Receptor Fusion 169
Proteins over Short and Long Distances –
Complementarity of Fluorescence Correlation
Microscopy and FRAP in Cellular Diffusion
Measurements
Chapter 7 Cholesterol-dependent Clustering of IL-2Rα 223
and its Colocalization with HLA and CD48 on T
Lymphoma Cells suggests their Functional
Association with Lipid
Summary 249
Samenvating 253
Acknowledgements 257
List of publications 259
Curriculum Vitae 261
Abbreviations
AOM Acousto-optical modulator
AR Androgen receptor
ARE Androgen response element
BCIP Bromo-4-chloro-3-indolyl phosphate
CAF I Chromatin assembly factor I
CHO Chinese hamster ovary
CLSM Confocal laser scanning microscopes
CPD Cyclobutane pyrimidine dimers
CS Cockayne syndrome
DBD DNA binding domain
DBD DNA-binding domain
DM Dichroic mirror
EBFP Enhanced blue fluorescent protein
ECFP Enhanced cyan fluorescent protein
EGF Epidermal growth factor
EGFP Enhanced green fluorescent protein
EGFR Epidermal growth factor receptor
ERCC Excision repair cross complementing protein
ESPT Excited state proton transfer
EYFP Enhanced yellow fluorescent protein
FCM Fluorescence correlation microscopy
FCS Fluorescence correlation spectroscopy
FP Fusion protein
FRAP Fluorescence recovery after photobleaching
FRET Fluorescence resonance energy transfer
FWHM Full width half-maximum
GFP Green fluorescence protein
GG-NER Global genome NER
IC Internal conversion
IL-2 Interleukin-2
IL-2R Interleukin-2 receptor
ISC Intersystem crossing
LBD ligand-binding domain
NA Numerical aperture
NBT Nitro blue tetrazolium
NER Nucleotide excision repair
NLS Nuclear localisation signal
NTD N-terminal transactivation domain
PBS Phosphate-buffered saline
PMT Photomultiplier tube
RCFPs Reef coral fluorescent proteins
ROI Region of interest
RPA Replication Protein A
RT Rom temperature
S0 Electronic ground state
S1, S2 and so on Singlet excited energy states
SNOM Scanning near-field optical microscopy
SR Steroid receptor
T1 Excited triplet state
TC-NER Transcription-coupled NER
TIR Total internal reflection
TrfR Transferrin receptor
TTD Trichothiodystrophy
UV Ultraviolet
wt-GFP Wild-type green fluorescent protein
XP Xeroderma pigmentosum
XPA Xeroderma pigmentosum A
XPB Xeroderma pigmentosum B
XPC Xeroderma pigmentosum C
XPD Xeroderma pigmentosum D
XPE Xeroderma pigmentosum E
XPF Xeroderma pigmentosum F
XPG Xeroderma pigmentosum G
XPV Xeroderma pigmentosum V
Chapter
1
General Introduction
In fluorescence microscopy, the ability of fluorescent molecules to emit light of specific
wavelengths following the absorption of light of shorter wavelengths is utilized to
observe the molecules directly or to indicate the position of fluorescently tagged target
molecules in a cell. Recent advances in laser and computer technologies have lead to the
availability of commercial confocal laser scanning microscopes (CLSM) thereby making
fluorescence microscopy widely accessible. Furthermore, the development of genetically
encoded fluorescent proteins have lead to advances in the analytical techniques used for
the evaluation of protein localization, dynamics and interactions in the cellular
environment.
A perfect fluorescence assay is one, which could monitor every protein directly
and map its behaviour in time. However, at present no single imaging modality can assess
all aspects of the complicated processes a protein undergoes in carrying out its functions
in the living cell. Proteins are mostly observed indirectly with the use of fluorescent tags,
which are assumed not to interfere with the behaviour of the target protein being studied.
High-resolution digital imaging of fusion proteins (FP) enables protein localization and
colocalization to be monitored in time. More sophisticated quantitative techniques, such
as fluorescence recovery after photobleaching (FRAP) and fluorescence correlation
spectroscopy (FCS) are used to study protein dynamics and protein-protein interactions.
Although FRAP is a bulk assay method and FCS is more effective when only a few
molecules are monitored at a time, the complementary use of both methods provides a
more accurate insight into the movement of proteins and their interactions with cellular
components in living cells.
Most fluorescence assay methods rely on fluorescence changes to quantify the
dynamics leading to these changes. Therefore an accurate understanding of protein
behaviour does not only require the use of more than one assay technique where
Chapter 1 10
