Quantifying adhesive interactions between cells and extracellular matrix by single-cell force spectroscopy [Elektronische Ressource] / vorgelegt von Anna Verena Taubenberger

Sujets

• Biologie

Informations

• Publié par: technische_universitat_dresden
• Publié le: 01 janvier 2009
• Nombre de lectures: 49
• Langue: English
• Poids de l'ouvrage: 43 Mo

Extrait

TECHNISCHE UNIVERSITÄT DRESDEN

Fakultät Maschinenwesen




Quantifying adhesive interactions between cells and
extracellular matrix by single-cell force spectroscopy



DISSERTATION

Zur Erlangung des akademischen Grades
Doktoringenieur
(Dr.-Ing)



vorgelegt von

Anna Verena Taubenberger

geboren am 27.03.1980
in Kulmbach



Dresden, 04.05.2009



Gutachter/ thesis reviewers: Prof. Daniel J. Müller
Prof. Anthony Hyman
Prof. Dietmar W. Hutmacher


Tag der Verteidigung/
Day of thesis defense: 07.10.2009
















Die Projekte, die Inhalt der Dissertation sind, wurden in der Zeit von 06/2005 bis
04/2009 in der Arbeitsgruppe für zelluläre Maschinen am Biotechnologischen
Zentrum der TU Dresden bearbeitet.
Betreuender Hochschullehrer war Prof. Daniel J. Müller.

The projects that are part of this thesis were conducted between 06/2005 and 04/2009
in the group of cellular machines at the Center of Biotechnology of the TU Dresden.
The thesis supervisor was Prof. Daniel J. Müller.
Contents
Page
Abbreviations, symbols and units 1
Summary (english and german) 4
Chapter 1. Background- Interactions between cells and extracellular matrix 8
1.1 The extracellular matrix (ECM) 8
1.1.1 Composition 8
1.1.2 Effects of ECM on cellular functions 12
1.1.3 ECM receptors 13
1.2 Integrins 14
1.2.1 General aspects 14
1.2.2 Integrin structure 15
1.2.3 Integrin regulation 17
1.2.4 Biological functions of integrins 23
1.3 Conclusions 24
Chapter 2. Cell adhesion assays- overview 25
2.1 Bulk assays 25
2.1.1 Techniques using hydrodynamic shear flow 25
2.1.2 Centrifugation assay 28
2.1.3 Further methods 28
2.2 Single-cell force spectroscopy (SCFS) techniques 29
2.2.1 Micropipettes 30
2.2.2 Optical tweezers 31
2.2.3 Magnetic tweezers 31
2.2.4 Conclusions 32
2.3 AFM force microscopy-based SCFS 34
2.3.1 Principle- force spectroscopy mode 34
2.3.2 AFM-SCFS- experimental setup 36
2.3.3 Interpretation of SCFS F-D curves 39
2.3.4 AFM-based SCFS- state of the art 44
2.4 Conclusions 47
Chapter 3. The Bell-Evans model 48
3.1 Basic reaction equations & bond kinetics for receptor-ligand interactions 48
3.2 Dissociation kinetics near and far from equilibrium 50
3.3 Binding strength 54
3.4 Conclusions 56Contents
Page
Chapter 4. Quantifying early steps of a b -integrin mediated cell adhesion to2 1
collagen type I 57
4.1 Abstract 57
4.2 Introduction 58
4.3 Results & Discussion 61
4.3.1 Background 61
4.3.2 Investigating single a b -integrin mediated adhesion events 692 1
4.3.3 Dependence of a b -mediated adhesion on contact time 752 1
4.3.4 Role of actomyosin contractility in cooperative integrin binding 81
4.3.5 Visualizing paxillin redistribution during SCFS 83
4.4 Conclusions & Outlook 86
Chapter 5. Effects of cryptic integrin binding site exposure in collagen type I on
osteoblast adhesion and matrix mineralisation 88
5.1 Abstract 88
5.2 Introduction 89
5.3 Results & discussion 92
5.3.1 Characterization of Col and pdCol matrices 92
5.3.2 Analysing cellular interactions with Col and pdCol 95
5.3.3 Analysing effects of Col and pdCol on cell growth and differentiation 100
6.4 Conclusions & Outlook 104
Chapter 6. Quantifying adhesion of myeloid progenitors to bone marrow
derived stromal cells 107
6.1 Abstract 107
6.2 Introduction 108
6.3 Results & discussion 113
6.3.1 Quantifying overall cell adhesion between 32D and BMSC 113
6.3.2 Analyzing single rupture events in F-D curves 115
6.3.3 32D cell adhesion to FN and collagen type I coated surfaces 116
6.3.4 Role of b -integrins in mediating adhesion of 32D cells to BMSC 1181
6.3.5 b -integrin protein and mRNA levels 1201
6.3.6 32D cell attachment to FN secreted by BMSC 122
6.4 Conclusions & Outlook 124
Final remarks 126
References 127
Index of figures and tables 145
Appendix (inclusively experimental procedures) 147
-1Abbreviations, symbols and units
Abbreviations
AFM atomic force microscopy
ABL Ableson leukemia virus
BCR break point cluster region
BCR/ABL fusion protein due to gene fusion of BCR and ABL
BMSC bone marrow stromal cells
BDM butandione-2-monoxime
BSA bovine serum albumin
CAM cell adhesion molecule
CAM-DR cell adhesion-mediated drug resistance
CHO chinese hamster ovary
CML chronic myeloid leukemia
Col collagen type I matrix used in this work
DAB 3.3´-diaminobenzidine
DDR discoidin domain receptor
DFS dynamic force spectroscopy
DMEM Dulbeccos modified eagle medium
DNA deoxyribonucleic acid
ECM extracellular matrix
EDTA ethylenediaminetetraacetic acid
EGTA ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid
EM electron microscopy
FAK focal adhesion kinase
FCS fetal calf serum
F-D force-distance
FITC fluorescein isothiocyanate
FN fibronectin
GFOGER glycine-phenylalanine-hydroxyproline- glycine-glutamate-arginine
HRP horse radish peroxidase
ICAM intercellular adhesion moleculeAbbreviations, symbols and units
-2IL-3 interleukin-3
IM imatinib mesylate
Itgb1 gene encoding b -integrin1
JAM junctional adhesion molecule
LFA lymphocyte function-associated molecule
mAB monoclonal antibody
MEM minimal essential medium
MIDAS metal ion dependent adhesion site
MMP matrix metallo-protease
MSC mesenchymal stem cell
pAB polyclonal antibody
PBS phosphate buffered solution
pdCol partially denatured collagen type I matrix used in this work
PFA paraformaldehyde
RGD arginine-glycine-aspartic acid
RNA ribonucleic acid
RPMI Roswell Park Memorial Institute
RT room temperature
SCFS single-cell force spectroscopy
SD standard deviation
SMFS single-molecule force spectroscopy
TRITC tetramethylrhodamine isothiocyanate
VCAM vascular cell adhesion molecule
VLA very late antigen
Symbols
2a effective cantilever area [mm ]eff
2 2A area [mm ][m ]
E elasticity [Pa]
f, F force [N][pN][nN]
*f mean or most probable rupture force [pN]
F detachment force [pN][nN]D
h effective cantilever height [mm]effAbbreviations, symbols and units
-3j single rupture event
-23k Boltzman constant (1.3806504*10 J/K)B
k effective spring constant [pN/nm]eff
-1k rate of bond dissociation [sec ]off
-1 -1k rate of bond formation [M *sec )]on
L ligand
h viscosity [Pa*s]
N number of ligands, receptorsL,R
p pressure [Pa]
P probability
r effective loading rate [pN/sec]eff
-1rpm rotations per minute [min ]
R receptor
t lifetime [sec]
t membrane nanotube unbinding event
v velocity [mm/sec]
W detachment work [J]D
Units
-10 Å Angstrom (10 m)
°C degree Celsius
K Kelvin
M Molar (mol/l)
m meter
-6!mm mikrometer (10 m)
2N Newton (kg*m/s )
-9!!nm nanometer (10 m)
-9 nN nanonewton (10 N)
-12 pN piconewton (10 N)
2Pa Pascal [N/m ]
sec seconds
-4Summary
Interactions of cells with their environment regulate important cellular functions
and are required for the organization of cells into tissues and complex organisms. These
interactions involve different types of adhesion receptors. Interactions with extracellular
matrix (ECM) proteins are mainly mediated by the integrin family of adhesion molecules.
Situations in which integrin-ECM interactions are deregulated cause diseases and play a
crucial role in cancer cell invasion. Thus, the mechanisms underlying integrin-binding
and regulation are of high interest, particularly at the molecular level.
How can cell-ECM interactions be studied? While there are several methods to
analyze cell adhesion, few provide quantitative data on adhesion forces. One group,
single-cell force spectroscopy (SCFS), quantifies adhesion at the single-cell level and can
therefore differentiate the adhesive properties of individual cells. One implementation of
SCFS is based on atomic force microscopy (AFM); this technique has been employed in
the presented work. Advantageously AFM-SCFS combines high temporal and spatial cell
manipulation, the ability to measure a large range of adhesion forces and sufficiently
high-force resolution to allow the study of single-molecule binding events in the context
of a living cell. Since individual adhesion receptors can be analyzed within their
physiological environment, AFM-SCFS is a powerful tool to study the mechanisms
underlying integrin-regulation.
The presented work is split into six chapters. Chapter one gives background information
about cell-ECM interactions. In chapter two, different adhesion assays are compared and
contrasted. The theoretical Bell-Evans model which is used to interpret integrin-mediated
cell adhesion is discussed in chapter three. Thereafter, the three projects that form the
core of the thesis are detailed in chapters four through six.
In the first project (chapter 4), a b -integrin mediated cell adhesion to collagen type I,