The Effect of Thymosin Beta 4 on Cell
Mechanics and Motility
Die Wirkung von Thymosin Beta 4 auf Zellmechanik
und Motilität
Der Naturwissenschaftlichen Fakultät/dem Fachbereich Physik
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur Erlangung des Doktorgrades Dr. rer. nat.
vorgelegt von
Leila Minori Jaafar
aus Kuala Lumpur, Malaysia
Als Dissertation genehmigt von der Naturwissenschaftlichen
Fakultät/vom Fachbereich Physik der Friedrich-Alexander-Universität
Erlangen-Nürnberg
Tag der mündlichen Prüfung: 6. Juni, 2014
Vorsitzender des Promotionsorgans:
Gutachter:
ii
Prof. Dr. Johannes Barth
Prof. Dr. Ben Fabry
Prof. Dr. Ewald Hannappel
Table of Contents
Zusammenfassung ........................................................................................................................................... 1
Summary ............................................................................................................................................................. 3
1
1.1
Introduction................................................................................................................................................ 5
Cell motility: a biochemical and biophysical process ............................................................ 6
1.1.1
The biochemical regulation of cell migration
6
1.1.2 Cellular mechanics and dynamics: biophysical processes regulating cell
migration
9
1.2
Thymosin β4: structure and function ......................................................................................... 12
1.2.1
1.2.2
1.3
2
2.1
The molecular structure of Tβ4
Tβ4: a multi-functional protein in health and pathogenesis
12
13
Open questions ................................................................................................................................... 18
Material and Methods ........................................................................................................................... 20
Biological and biochemical methods .......................................................................................... 21
2.1.1
Cells and cell culture
21
2.1.3
Ligation
22
2.1.2
2.1.4
2.1.5
2.1.6
2.1.7
2.1.8
2.1.9
Cloning cassettes of EGFP-Actin for generating the GSP
22
Cloning of EGFP-Actin into the pIRESpuro3 selection vector
24
Stable transfection into mammalian cell lines
24
Transfection of the EGFP-Actin vector construct
Immunofluorescence and fluorescence microscopy
Synthesis of lipid vesicles
24
25
27
Synthesis of Tβ4-Alexa488 labeled peptide
27
2.2.1
Three-dimensional collagen cell invasion assay
29
2.2.3
Cell-spreading assay
30
2.2
Biophysical methods......................................................................................................................... 29
2.2.2
2.2.4
2.2.5
2.2.6
2.2.7
2.2.8
Two-dimensional cell motility assay
29
Two-dimensional cellular traction microscopy
30
Nano-scale particle tracking
34
Magnetic tweezer microrheology
Differential scanning calorimetry (DSC)
Fluorescence correlation spectroscopy
32
37
39
iii
3
3.1
Results ........................................................................................................................................................ 43
The effect of extracellular Thymosin β4 on the motility of SW480 cells ...................... 44
3.1.1
3.1.2
3.2
Two-dimensional cell motility
44
46
The effect of extracellular Tβ4 on cell morphology ............................................................... 49
3.2.1
3.2.2
3.3
Three-dimensional cell migration
Analyses of SW480 cell morphology in 3D
Analyses of SW480 cell morphology in 2D
49
51
Effect of extracellular Tβ4 on cell compliance, dynamics and contractility................. 54
3.3.1
3.3.2
Creep response and cell mechanics
Two-dimensional cellular tractions
54
57
3.3.3 Nanoscale particle tracking of cytoskeletal-bound beads: studying the
time- dependant activity of Tβ4 on cytoskeletal dynamics
59
3.4
Tβ4 and ILK: The Biphasic Switch in 3D Cell Invasion Assays .......................................... 64
3.4.1
Role of ILK as a molecular switch involved in the Tβ4 biphasic response .......... 64
3.4.2 Bypassing RhoA inhibition: the effect of calyculin-A on Tβ4-induced cell
invasion
66
3.5
Tβ4 – membrane interaction .......................................................................................................... 69
3.5.1 Determining the binding potential of Tβ4 to lipid membranes using
differential scanning calorimetry (DSC)
69
4
3.5.2 Fluorescence correlation spectroscopy (FCS) of membrane-bound
fluorescent Tβ4
71
4.1
Discussion ................................................................................................................................................. 75
The effects of Tβ4 on Cellular Mechanics and Motility ........................................................ 76
4.1.1
Tβ4 regulates the stiffness and contractility of SW480 cells
76
4.1.2 Tβ4 regulation of acto-myosin contractility is involved in the biphasic
response of SW480 cell morphology and motility
78
4.2
4.3
Tβ4 and the cell lipid membrane: a novel mechanism for outside-in signaling......... 82
The Tβ4-ILK Molecular Switch ...................................................................................................... 84
Bibliography..................................................................................................................................................... 88
iv
List of Figures
Figure 1.1 Two-dimensional cell migration. ............................................................................................................ 7
Figure 1.2 The dynamic turnover of the actin cytoskeleton at the leading edge of a cell. .................... 8
Figure 1.3 Molecular structure of Tβ4 ..................................................................................................................... 13
Figure 1.4 Extracellular application and genetic overexpression: the multi-faceted role of
Tβ4 in health and disease......................................................................................................................... 14
Figure 2.1 Cell lines. ........................................................................................................................................................ 21
Figure 2.2 Stable transfection of NIH 3T3 cells with an EGFP-Actin plasmid vector. ......................... 23
Figure 2.3 Close-up of an NIH 3T3 mouse fibroblast stably transfected with the pIRESEGFP-Actin construct. ............................................................................................................................... 25
Figure 2.4 Actin fluorescent staining with Alexa Fluor 546-phalloidin. ................................................... 26
Figure 2.5 Fluorescent staining of vinculin. .......................................................................................................... 27
Figure 2.6 Linking the Alexa Fluor 488 fluorophore to Tβ4 molecules. .................................................... 28
Figure 2.7 Two-dimensional traction microscopy. ............................................................................................ 31
Figure 2.8 2D traction microscopy images............................................................................................................ 31
Figure 2.9 Magnetic tweezer measurements. ...................................................................................................... 33
Figure 2.10 Nano-scale particle tracking of fibronectin coated superparamagnetic beads ............. 36
Figure 2.11 Analysis of nano-scale particle tracking. ....................................................................................... 36
Figure 2.12 Phase transition of synthetic lipid vesicles................................................................................... 38
Figure 2.13 Differential Scanning Calorimetry (DSC): experimental setup............................................. 39
Figure 2.14 Schematic diagram of the Fluorescence Correlation Spectroscopy (FCS)
detection volume. ....................................................................................................................................... 40
Figure 2.15 Principle of Fluorescence Correlation Spectroscopy (FCS). .................................................. 42
Figure 3.1 Analysis of SW480 cell migration in three-dimensional collagen gels. ............................... 45
Figure 3.2 Analysis of two dimensional SW480 cell motility. ....................................................................... 47
Figure 3.3 Biphasic response of SW480 cell morphology to Tβ4 concentration in 3dimensional collagen gels. ...................................................................................................................... 50
Figure 3.4 Biphasic response of SW480 cell morphology to Tβ4 concentration on twodimensional surfaces................................................................................................................................. 53
Figure 3.5 Stiffness of Tβ4 induced SW480 cells. ................................................................................................ 55
Figure 3.6 Viscoelasticity of Tβ4 induced SW480 cells. .................................................................................... 56
Figure 3.7 Traction force maps of SW480 cells: contractility of Tβ4 stimulated cells on 2D
substrates....................................................................................................................................................... 58
v
Figure 3.8 The effect of exogenous Tβ4 on the contractility of SW480 cells on 2D
substrates....................................................................................................................................................... 59
Figure 3.9 Short term effects of extracellular Tβ4 on cytoskeletal remodeling. .................................... 61
Figure 3.10 Long term effects of extracellular Tβ4 on cytoskeletal remodeling. ................................... 62
Figure 3.11 Tβ4-ILK molecular switch. ................................................................................................................... 65
Figure 3.12 The combined effect of calyculin-A and Tβ4 on the 3D invasion of SW480 cells
into collagen gels......................................................................................................................................... 67
Figure 3.13 Lipid-binding qualities of Tβ4 to DMPG/DMPC lipids analyzed using
differential scanning calorimetry (DSC)............................................................................................ 70
Figure 3.14 Lipid membrane binding of Tβ4 measured through fluorescence correlation
spectroscopy. ................................................................................................................................................ 72
Figure 3.15 The effect of acidic lipid head groups on the binding of Tβ4 -Alexa488 on
membranes.................................................................................................................................................... 74
Figure 4.1 3D stucture of Tβ4. ..................................................................................................................................... 83
Figure 4.2 The regulation of SW480 cellular mechanics in response to extracellular Tβ4
concentration – a biphasic molecular switch mechanism. ........................................................ 86
vi
Abbreviations
2D
3D
ADF
ADP
AKT
Arp2/3
ATP
BSA
CCD
Cdc42
cDNA
two dimensional
three dimensional
actin depolymerizing factors
adenosine diphosphate
protein kinase b
actin-related protein 2/3
adenosine triphosphate
bovine serum albumin
charged-coupled device
cell division control protein 42
complementary deoxyribonucleic acid
Kd
kDa
MAPK
MARCKS
MCS
MIP-2
MLC
MLCK
MLVs
binding constant
kilo Dalton
Mitogen-activated protein kinase
myristoylated alanine-rich C kinase substrate
multiple cloning site
macrophage inflammatory protein 2
myosin light chain
myosin light chain kinase
Multilamellar lipid vesicles
dH20
DMEM
DMPC
DMPG
DMPS
DNA
dNTPs
DSC
DTT
EDTA
EGFP
EMT
FAK
FCS
FCS
FITC
GSP
GTP
HEPES
HIV-1
HRP
ILK
IRES
KC
distilled water
Dulbecccco’s modified Eagle’s medium
1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine
Dimyristoyl-L-α-phosphatidylglycerol
1,2-Dimyristoyl-sn-glycero-3-phospho-L-serine
deoxyribonucleic acid
deoxynucleotide triphosphate
differential scanning calorimetry
Dithiothreitol
ethylenediaminetetraacetic acid
enhanced green fluorescent protein
epithelial-mesenchymal transition
focal adhesion kinase
fluorescence correlation spectroscopy
fetal calf serum
fluorescein isothiocyanate
green stoic puppies (EGFP-actin stably transfected NIH 3T3 cells)
guanosine triphosphate
(Hydroxyethyl-Piperazine Ethanesulafonic Acid)-Saline-Albumin-Gelatin
human immunodeficiency virus type 1
horseradish peroxidase
integrin-linked kinase
viral internal ribosome entry site
keratinocyte-derived chemokine
vii
MMP
MSD
MT6-MMP
NF-kB
PAA
PBS
PCR
pCum
PINCH
PMN
Rac
Rho
RhoA
ROCK
SD
SE
SEM
Shc
Src
SUVs
matrix metalloproteinase
mean square displacement
membrane-type 6 metalloproteinases
Nuclear factor κ B
polyacrylamide
phosphate buffer saline
polymerase chain reaction
cumulative probability
particularly interesting new cysteine-histidine rich protein
polymorphonuclear macrophage
related to A and C protein kinase
ras homolog
ras homolog A
Rho-kinase
standard deviation
standard error
scanning electron microscope
SH2-containing collagen-related proteins
Rous sarcoma oncogene cellular homolog
small unilamellar vesicles
Tb5
TGA
Thymosin beta-5
transglutaminase
Tb10
Tb4
Tm
TM
Tris HCl
VEGF
WASP
viii
Thymosin beta-10
Thymosin beta-4
annealing temperature
melting temperature
tris(hydroxymethyl)aminomethane hydrochloric acid buffer
vascular endothelial growth factor
Wiskott-Aldrich syndrome protein
Zusammenfassung
Die Zellmotilität oder die -migration sind fundamentale Prozesse in der Entwicklung
und Funktionsweise von multizellulären Organismen im gesunden wie pathologischen
Zustand, beispielsweise bei der embryonalen Entwicklung und bei der Metastase von
Krebszellen. Zellen bewegen sich dadurch, dass deren Zellgerüste stetig neu aufgebaut
werden und sich dadurch verformen. Die koordinierte Polymerisation von G-ActinMonomeren zu F-Actin-Filamenten und Stressfasern findet dabei am vorangehenden
Rand der Zelle statt. Aufgrund der Polymerisation wirkt dort eine Kraft, die eine
Nettobewegung der Zelle verursacht. Der Zusammen- und Abbau dieser F-ActinFasern werden durch eine Vielzahl Actin-bindender Proteine gesteuert. Um diese
Actin-Filamente bei Bedarf sehr schnell zu produzieren, benötigen Zellen einen
großen Pool an nicht-polymerisiertem G-Actin, das für die Polymerisation zur
Verfügung steht.
Thymosin beta-4 (Tβ4) ist ein kleines, ca. 5 kDa großes Peptid, das einen 1:1 Komplex
mit G-Actin formt und die spontane Polymerisation von G-Actin zu F-Actin hemmt.
Größere Konzentrationen von Tβ4, etwa bis 500 µM, sind in den Zellen aller
Säugetiere vorhanden, Spuren des Peptids können auch im Blutserum nachgewiesen
werden. Kürzlich wurde beobachtet, dass eine zelluläre Überexpression bzw. eine
extrazelluläre Behandlung mit Tβ4 mit dem bösartigen Verlauf und der Migration von
Krebszellen mehrerer Linien korreliert. Darunter auch mit den sonst nichtinvasiven
kolorektalen Krebszellen SW480. Da der Prozess der Zellmigration und die
Verformung des Zellgerüsts essentiell zusammenhängen, untersucht diese Arbeit die
Auswirkung von extrazellulärem Tβ4 auf die Mechanik des Zellgerüsts, die
Kontraktilität und die Motalität von Zellen im Experiment.
Die Untersuchungen der Steifheit und Kontraktilität von Zellen werden mit magnetic
tweezer und 2D-tractions microscopy durchgeführt. Die Ergebnisse zeigen eine
deutliche biphasische Reaktion mit steigenden Konzentrationen an extrazellulären
Tβ4. Der höchste Wert an Steifheit und Kontraktilität wird mit SW480-Zellen und
einer Konzentration an Tβ4 von ca. 0,2 µM erreicht. Es wird nachgewiesen, dass die
biphasische Reaktion in der Steifheit und Kontraktilität der Zellen mit Änderungen in
der Morphologie und Migrationsverhalten derselben einhergeht. Zellen die mit 0,2 µM
Tβ4 behandelt wurden, zeigen eine betont längliche, mesenchymale Morphologie. Ihr
Migrationsverhalten auf einer flachen Oberfläche ist bei dieser Konzentration
ausgeprägt räumlich gerichtet. In Experimenten mit 3D-Kollagengelen verhalten sich
die Zellen bei dieser Konzentration stark invasiv. Eine längliche Zellmorphologie
zusammen mit einer erhöhten Zellkontraktilität bei Behandlung mit 0,2 µM Tβ4 sind
zwei starke Indikatoren für eine verstärkte Zellmigration in einer 3D Kollagenmatrix.
Die Messungen zeigen jedoch keine Auffälligkeiten bei der Behandlung mit
Konzentrationen von Tβ4 größer als 1 µM.
1
Es bleibt zunächst unerklärt, warum durch eine spezifische Konzentration an
extrazellulären Tβ4 eine so starke Reaktion hervorgerufen wird, zumal eine 100mal
höhere Konzentration des Peptids bereits in der Zelle vorhanden ist. Außerdem ist für
Tβ4 derzeit auch kein Zelloberflächenrezeptor oder eine andere Möglichkeit für
Signalübertragung von außerhalb der Zelle nach innen bekannt. Die
Aminosäureanalyse von Tβ4 zeigt jedoch, dass ca. 60% des Peptids mit Clustern von
basischen und hydrophoben Aminosäuren besetzt ist. Es ist bekannt, dass basische
Aminosäuren mit sauren Bereichen auf einer Lipidmembran wechselwirken und dass
hydrophobe Aminosäuren als Anker wirken können. Diese beiden Eigenschaften
würden es dem Peptid erlauben an Zellmembrane zu binden und in einer
Konfiguration darin einzudringen, die es dem Peptid erlaubt mit weiteren
membranassoziierten Partnern zu interagieren, um weitere intrazelluläre Signale zu
veranlassen. Die Bindung von Tβ4 an synthetische Lipidmembranen wird in dieser
Studie mittels differential scanning calorimetry (DSC) und fluorescence correlation
spectroscopy (FCS) nachgewiesen.
Mit der Einbettung und Bindung von extrazellulärem Tβ4 an die Zellplasmamembran
wird somit ein möglicher Mechanismus aufgezeigt, wie die lokale Konzentration des
Peptids stark erhöht werden kann. Gleichzeitig wird beobachtet, dass die
Diffusiongeschwindigkeit des Peptids stark abnimmt und somit die
Reaktionswahrscheinlichkeit mit anderen membranassoziierten Signalproteinen wie
beispielsweise integrin-linked kinase (ILK) und particularly interesting new cysteinehistidine rich protein (PINCH) erhöht wird.
Die Messergebnisse aus dieser Studie und die oben beschriebenen Beobachtungen
zeigen, dass membrangebundenes Tβ4 mit Membran- und Zellgerüsten-assoziierter
Proteine (ILK und PINCH) wechselwirken und dabei ein funktionales Komplex
ausbilden kann. Bei geringer Konzentration fördert dieses Komplex die
Zellkontraktilität. Hohe Konzentrationen des Komplexes hemmen die Kontraktilität
hingegen, was zu einer biphasischen Reaktion bei ansteigender Konzentration an
extrazellulären Tβ4 führt. Diese Arbeit zeigt somit auf, dass eine Stimulierung mit
extrazellulären Tβ4 als biphasischer molekularer Schalter wirken kann, um
beispielsweise die Motilität von SW480 Zellen zu steuern.
2
Summary
Cell motility or migration is a process crucial for the development and function of a
multi-cellular organism in a normal or pathological state, for example, during
embryonal development and cancer metastasis. In a moving cell, the cytoskeleton is
remodeled dynamically. Coordinated polymerization of G-actin monomers into F-actin
filaments and stress fibers at the leading edge of the cell provides force for the net
movement of the cell. The assembly and disassembly of these F-actin fibers are
regulated by a myriad of actin-binding proteins. To assemble actin filaments rapidly
when needed, cells keep a large pool of non-polymerized G-actin ready for
polymerization.
Thymosin beta-4 (Tβ4), a small 5 kDa peptide, forms a 1:1 complex with G-actin and
inhibits the spontaneous polymerization of G-actin to F-actin. Tβ4 is found in trace
amounts in serum and in abundant concentrations of up to 500 µM in virtually every
mammalian cell. Recently, the cellular overexpression and extracellular
administration of Tβ4 has been correlated with the malignant progression and
migration of several tumor cell lines including the otherwise non-invasive SW480
colorectal cancer cells. As the process of cell migration involves substantial changes in
the cytoskeleton, the effect of extracellular Tβ4 on cytoskeletal mechanics,
contractility, and motility was measured in this study.
Cell stiffness and contractility measurements using the magnetic tweezer and 2D
tractions microscopy showed a distinct biphasic dose response with increasing
concentrations of extracellular Tβ4; the highest level of stiffness and contractility was
recorded in SW480 cells stimulated with 0.2 µM Tβ4. The biphasic response in cell
stiffness and contractility were accompanied accordingly with changes in cell
morphology and migration. Cells treated with 0.2 µM Tβ4 displayed a pronounced
elongated, mesenchymal morphology, migrated with higher directional persistence on
flat 2D surfaces, and became highly invasive in a 3D collagen invasion assay. An
elongated cell morphology, together with an increased contractility, are two potent
factors for enhanced cell migration in a 3D collagen matrix in response to stimulation
with 0.2 µM Tβ4. All parameters return, however, to baseline levels at concentrations
larger than 1 µM.
It is puzzling how minute amounts of extracellular Tβ4 can elicit such a massive
response, considering that a more than 100 fold higher concentration of the peptide is
already present in the cell. Furthermore, a cell surface receptor or another way of
outside-in signaling for Tβ4 has yet to be found. Amino acid analysis of Tβ4 showed
that clusters of basic and hydrophobic residues make up about 60% of the entire
3
peptide. Basic amino acids are known to interact with acidic domains on a lipid
membrane and hydrophobic residues may function as an anchor. Both factors would
enable the peptide to bind and insert into the cell membrane in a configuration
allowing it to interact with membrane-associated partners to trigger downstream
signaling events. Differential scanning calorimetry (DSC) and fluorescence correlation
spectroscopy (FCS) measurements using fluorescently labeled Tβ4 and synthetic lipid
membranes were used to demonstrate the binding of Tβ4 to the synthetic lipid
membranes. The insertion and binding of extracellular Tβ4 to the cell plasma
membrane offers a mechanism of how the local concentration of the peptide can be
greatly increased. At the same time its diffusion speed is greatly decreased so as to
increase the reaction probability with other membrane-associated signaling proteins
such as integrin-linked kinase (ILK) and particularly interesting new cysteinehistidine rich protein (PINCH).
Data from this study together with previous observations suggest that membranebound Tβ4 interacts with membrane and cytoskeleton associated proteins, ILK and
PINCH, to form a functional complex that promotes cell contractility at lower
concentrations, and suppresses contractility at higher concentrations resulting in a
biphasic response to increasing concentrations of applied Tβ4. As a conclusion, this
study postulates that extracellularly applied Tβ4 may act as a biphasic molecular
switch to regulate cell motility in SW480 cells.
4
1
Introduction
1
Introduction
1.1
1.1.1
Cell motility: a biochemical and biophysical process
Cell motility or migration is a process crucial for the development and function of a multicellular organism in a normal or pathological state, for example, during embryonal
development and cancer metastasis. One of the main processes involved in cell motility
functions is the regulation of the actin filament assembly and disassembly in the
cytoskeleton. Although three types of cytoskeletal filaments are involved in cell motility,
namely actin filaments, microtubules and intermediate filaments, actin filaments play the
leading role [1-2]. In this chapter, the biochemical and biophysical aspects of the
cytoskeleton and its associated constituents in cell motility are described.
The biochemical regulation of cell migration
The movement of a cell is controlled by coordination of several complex biochemical
and biomechanical mechanisms. At the start of the migration process on two
dimensional surfaces, a cell develops protrusions at its leading edge (see Figure 1.1).
These structures, which can be sheet-like (lamellipodia) or finger-like (filapodia), are
driven to protrude forth by the polymerization of actin monomers (G-actin) into actin
filaments (F-actin). New focal adhesions, multi-protein assemblies through which the
mechanical force and regulatory signals of a cell are transmitted, are formed at the
front of the cell. As the cell contracts, focal adhesions are detached at the rear and the
cell body is translocated forward. Cycles of cell contraction and relaxation occur
through the interaction of head domains of myosin, an ATP dependent motor protein,
and actin filaments. This acto-myosin contraction is regulated by the phosphorylation
of the light chain domain of myosin (MLC: myosin light chain) [1].
Each phase during cell movement, as described above, is modulated by biochemical
signaling mechanisms controlling actin polymerization and acto-myosin contraction.
Responsible for this regulation are mainly the Rho family of GTPases (signal
transducers), which include Cdc42, Rac and Rho [1, 3-4]. Cdc42 is involved with the
formation of filapodia, whereas Rac with that of lamellipodia. The activation of Rho
increases actin polymerization, the development of stress fibers and acto-myosin
contraction. Cdc42 and Rac can both activate the Wiskott-Aldrich syndrome protein
(WASP), which leads to the recruitment of Arp2/3 and actin polymerization. The
activation of LIM-kinase 1 by Cdc42 and LIM-Kinase 2 by Rac leads to the increase in
cofilin phosphorylation which inhibits the disassembly of actin filaments. Rho,
however, stimulates the formation of stress fibers and focal adhesions through the
activation of Rho-kinase (ROCK). Not only does ROCK phosphorylate MLC, which is
responsible for acto-myosin contraction, it can inhibit the dephosphorylation and
deactivation of MLC by inhibiting MLC phosphatase.
6
Introduction
1
Figure 1.1 Two-dimensional cell migration. Four phases can be distinguished during the
process of a cell moving on a two-dimensional surface (extracellular substrate seen in blue).
(1) Protrusion: at the leading edge of a cell, actin polymerization stimulated through Rac and
Cdc42 leads to the formation membrane protrusions in the shape of the finger-like filapodia
or sheet-like lamellipodia. (2) Adhesion: new focal adhesions (light yellow) are formed.
These connect the actin cytoskeleton to the extracellular substrate via cell surface integrins.
(3) Translocation: Forces generated by acto-myosin (green rods) contraction modulated by
Rho cause the forward translocation of the cell body and nucleus. (4) Retraction: Lastly,
disassembly of focal adhesions at the rear end of the cell causes the retraction of the trailing
edge.
7
1
Introduction
The dynamic turnover of the cytoskeleton from filamentous F-actin to G-actin
monomers and back can be examined largely in the cell lamellopodia, the major organ
in responsible for moving the cell forward. Proteins taking part in this dynamic
process are localized in the leading edge of the lamellipodia. The spatial orientation of
key proteins allows for the highly specific compartmentalization of actin-filament
assembly at the front and disassembly at the rear of the lamellopodia, which together
with myosin motors generates a directional force for forward movement of the cell
[2].
Figure 1.2 The dynamic turnover of the actin cytoskeleton at the leading edge of a cell.
Shown above is a schematic diagram of the dynamic process of the assembly of G-actin
monomers into F-actin and disassembly again into G-actin monomers. This cycle occurs
typically at the leading edge of a moving cell. (Diagram taken from [2])
8
Introduction
1
As seen in Figure 1.2, starting at the leading edge of the lamellipodia of a moving cell,
the actin fibers form a dense branched network with barbed ends of F-actin filaments
facing the edge of the cell and pointed end of the filaments forming Y-shaped, ~70°
angle junctions with other filaments. These filaments are joined together by the
Arp2/s complex. Upon the activation by the WASP (1), the Arp 2/3 complex can
nucleate G-actin assembly into F-actin [5] (2) and caps the free pointed ends of the
filaments, starts filament growth at the side of another filament and/or catch the
pointed end of an existing filament (3) [6]. Actin-filaments at the leading edge are
made of mostly ATP-actin and ADP-actin and do not bind ADF/cofilin. Filament
growth at the leading edge of a lamellipodia is a rapid process when compared to
depolymerization of filaments. Further elongation of the actin fibers is prevented by
capping proteins (4). The ADF/cofilin complex promotes the depolymerization of
ADP-actin at the distal end of the lamellipodia (5). ADF-cofilin and ADP-Actin
complexes that break away from the F-actin filament ends are in equilibrium with the
ADF-cofilin and ADP-actin monomers (6). The slow process of nucleotide exchange on
the G-actin monomer is inhibited by ADF-cofilin but is increased by profilin (7) [7-8].
Dissociated ADP-actin then undergoes nucleotide exchange with ATP to form ATPactin. At this end, Thymosin beta 4 (Tβ4) binds to the free G-actin monomers and
sequesters them to make a ready pool of intracellular G-actin to be polymerized into
F-actin when needed (8) [9-10]. The interaction of G-actin and Tβ4 is, however,
dependent on the binding of nucleotides ADP and ATP to G-actin. The binding affinity
of Tβ4 to ATP-G-actin is about 50 times higher than to ADP-G-actin [11]. The G-actin
binding protein profilin promotes the nucleotide exchange of ADP-G-actin to ATP-Gactin, whereas Tβ4 inhibits this process.
During cell migration, mechano-chemical signaling occurs in an outside-in and insideout manner through the interaction of the cytoskeleton, the focal adhesion complex
proteins and integrins, cell surface extracellular matrix receptors. Upon binding
ligands in the extracellular matrix, integrins transmit these extracellular signals into
the cell by modulating the activity of Rho GTPases and focal adhesion proteins such as
focal adhesion kinase (FAK), Src, integrin-linked kinase (ILK) and Shc [12-15]. These
proteins in turn transmit the signals to the cytoskeleton.
1.1.2
Cellular mechanics and dynamics: biophysical processes regulating cell
migration
In the past, the phenomenon of a moving cell has been explained through the various
molecular and biochemical signaling pathways. However, the process of cell motility
has distinct physical aspects which have remained undiscovered until recently. For
example, cell traction and adhesion forces are involved cell morphology and
anchoring of the cell onto extracellular substrates during migration. The cell also
9
1
Introduction
generates contractile forces to propel itself forward on two-dimensional surfaces as
well as to overcome physical barriers within a three-dimensional matrix. In a moving
cell, the cytoskeleton and focal adhesions are force-transmitting structures which
undergo constant dynamic reorganization.
Recently, efforts have been made to elucidate the purely physical and mechanical
processes governing cell motility. The mechanics and dynamics of the cytoskeleton
and its constituents, parameters both of which influence the rate of cell migration,
have been studied extensively using high-force magnetic tweezers. With this method,
the application of forces can be precisely applied to the cytoskeleton. This method can
be used to apply up to 100 nN force onto Ø 4.5 fibronectin-coated beads which bind to
the cytoskeleton via cell surface integrins [16-17]. Experiments with the magnetic
tweezer have been used to uncover the non-linear material properties and viscoelastic
characteristics of the cytoskeleton of various cell types. Through these experiments, it
was discovered that cells control their mechanical properties by generating an
internal cytoskeletal prestress powered actively by motor proteins. Further studies
were carried out using the magnetic tweezer to examine the force transducing
function of focal adhesion proteins including vinculin [18-19] and FAK [20] Recent
studies also using this method have shown that the loss of filamin A leads to a more
viscous-like cytoskeleton in M2 human melanoma cells [21].
Increased cell stiffness has been correlated with increased cell contractility,
characteristic for motile cells [19]. Using two-dimensional traction microscopy, a
method developed initially by Pelham and Wang et al. [22] , contractile forces
produced by individual cells can be measured. In this method, cells are cultivated on
collagen-coated polyacrylamide (PAA) gel with a known Young’s modulus. The
contractility of each cell can be characterized as the elastic strain energy stored in the
extracellular matrix because of cell tractions. For example, studies with vinculin wildtype and vinculin deficient mouse embryonal fibroblasts have shown that vinculinexpressing cells generated higher traction forces when compared with vinculin
knockout cells. As a result, vinculin expressing fibroblasts are able to invade through a
three-dimensional collagen matrix by utilizing traction forces to actively overcome
steric barriers in the extra-cellular matrix [23]. Recent advances in three-dimensional
traction microscopy can be used to extend studies of cellular forces in cell invasion
into a three dimensional environment [24-25].
As mentioned previously, the dynamic reorganization of the cytoskeleton is one of the
major processes occurring in a migrating cell. Cytoskeletal dynamics can be examined
by tracking the spontaneous motion of cytoskeletal-bound markers such as
fibronectin coated beads [26]. This technique enables the measurement of stress
fluctuations within the cytoskeleton, which is constantly transmitting stresses to the
10
Introduction
extra-cellular matrix, especially during the process of migration. The fibronectin
coated beads bound to the cytoskeletal network of acto-myosin stress fibers and were
observed to move, powered by ATP, in a directionally persistent super-diffusive
manner in virtually all cell types. Through these biophysical measurements, a physical
model of a tensed and continuously reorganizing system has complemented the
widely established knowledge on the biochemical process of the constant assembly
and disassembly of actin stress fibers in the cytoskeleton.
In the past, simple methods have been used to qualitatively examine cell migration
such as the Boyden-Chamber assay and in vitro scratch/wound assays. However, these
methods do not precisely quantify the speed and persistence of cell movement. Recent
advances in microscopy techniques have overcome these barriers. By using nanoscale particle tracking methods similar to studying stress fluctuations in the
cytoskeleton, bright cell-internal structures such as the nucleus can be used as an
indicator for the entire cell and can be tracked. With this, the mean square
displacement of a moving cell can be obtained, which gives information on the speed
and persistence of its movement. The latter is particularly interesting for studies using
chemoattractants. Great improvement in three dimensional microscopy and
automated methods of tracking cells in collagen gel has enabled the accurate
quantification of cell invasion [23, 27].
11
1
1
Introduction
1.2
1.2.1
Thymosin β4: structure and function
Among other cytoskeleton-associated proteins, Tβ4 has been determined as a key peptide
in the regulation of the cytoskeleton. In this section, a detailed description of the
structure and function of this peptide is given. Furthermore, the role of Tβ4, as described
in past literature, in health and pathological development is discussed.
The molecular structure of Tβ4
Thymosin β4 (Tβ4) is a small 5 kDa peptide belonging to the family of thymosins
originally isolated from the calf thymus. The 43-amino acid polypeptide has its
isoelectric point at a pH of 5.1 (slightly acidic) and is water soluble [28]. Although
largely unstructured in water, NMR spectroscopic analysis shows that Tβ4 tends to
form α-helices in aqueous solutions containing fluorinated alcohols. The two helices
formed involve amino acid residues 4 – 16 and 30 – 40 [29-30]. This biologically
active peptide is present in high concentrations (of up to 500 µM) in virtually every
mammalian cell line [31-33], being highly conserved across all species. Up to 1% of
the whole Tβ4 blood level has been found in serum largely due to its release from
damaged cells. Erythrocytes remain the exception; no Tβ4 has been found in these
cells [28, 34].
Tβ4 is known as the main intracellular G-actin sequestering protein, as established by
Safer et al. initially in human platelets [35-36]. Tβ4 was found to form a 1:1 complex
with G-actin and, thereby, inhibit the spontaneous polymerization of G-actin through
steric hindrance [28, 37]. On the molecular level, the sequence motif 17LKKTETQEK25
in Tβ4 has been established by previous studies to be directly involved with G-actin
binding. Further NMR analysis has come to the conclusion that for the binding and
sequestration of G-actin, the N-terminus of Tβ4 must form an α-helix [29-30].
Additionally, the three hydrophobic residues (6M, 9I and 12F) and the first three
residues of the actin-binding motif (17L, 18K and 19K) are essential for the interaction.
12
Introduction
Figure 1.3 Molecular structure of Tβ 4 Adapted from Safer et al [37], Kabsch et al. [38] and
Huff et al [28]). Tβ4 is a 5 kDa peptide with 43 amino acid residues. Shown above is a
schematic list of the three main domains, the two α-helices spanning from residues 4 – 16
and 30 – 40 and the G-actin binding ’LKKTETQEK’ motif. Tβ4 binds to the G-actin subdomains
3, 1 and 2 through the interaction of G-actin residues E167, D1-E4 and Q41 with the Tβ4
residues K3, K18 and K38. Through steric hindrance, Tβ4 inhibits the spontaneous
polymerization of G-actin monomers.
1.2.2
Tβ4: a multi-functional protein in health and pathogenesis
T β 4 in dermal wound closure
The course of wound healing involves stages of regulated process such as
inflammation, cell proliferation, the formation of blood vessels, migration of
fibroblasts and keratinocytes, and deposition of tissue collagen. Tβ4 has been shown in
different in vitro and in vivo animal studies to play a major role in wound healing. The
concentration of Tβ4 is highest in platelets and polymorphonuclear leukocytes. These
cells are responsible for releasing factors which attract other cells involved in closing
the wound. Wound serum is known to have a Tβ4 concentration of up to 13 µg/ml,
largely due to release from damaged platelets[39].
13
1
1
Introduction
Figure 1.4 Extracellular application and genetic overexpression: the multi-faceted role
of Tβ 4 in health and disease. (adapted from [34]) Aside from its classical role as an actin
sequestering protein, Tβ4 has been documented to induce other cellular processes. Here, a
distinction between the extracellular induction and the genetic overexpression of Tβ4 has
been made. In situ animal studies have shown that extracellular application of Tβ4 has lead to
cardio-protective effects following a myocardial infarct [40] and enhanced wound healing in
the dermis and cornea [41-46]. In these studies, it has been reported that Tβ4 interacts with
PINCH which results in the activation of integrin-linked kinase (ILK). In addition to its
enhancing effects on cell motility, ILK modulates the activity of AKT (protein kinase B) in cell
survival processes. Furthermore, the release of inflammatory cytokines, chemokines and
proteases are regulated. Genetic overexpression of Tβ4 in SW480 cells, however, has led to
the epithelial-mesenchymal transition (EMT) of SW480 cells. Through the simultaneous
down-regulation of E-cadherin, up-regulation of b-catenin and MMP-7, the increase in the
invasiveness of SW480 cells and the subsequent distant metastases of human colorectal cells
in mouse models have been documented [47-48].
In the wound, Tβ4 has been documented to promote several processes of wound
healing. Firstly, it increases the formation of new blood vessels from the pre-existing
vasculature, a process known as angiogenesis, which is essential for tissue growth
14
Introduction
[41-42]. Both keratinocyte and endothelial cell migration into the wound are then
increased by Tβ4 stimulation as shown in in vitro and in vivo studies [49]. The actinbinding domain of the peptide, 17LKKTETQEK25, was found to be responsible for the
increase in cell migration activity and vessel sprouting [50]. Tβ4 has been shown to
increase matrix metalloproteinase (MMP) production in vitro, which is essential for
migrating endothelial cells, fibroblasts, keratinocytes, chondrocytes and monocytes
[51-52].
Usual symptoms of a chronic wound are prolonged inflammation, dysfunctional cell
proliferation and collagen deposition. Thus, the process of wound healing is also
enhanced by the anti-inflammatory effects of Tβ4. Here, the N-terminus of the
molecule is believed to be responsible for the inhibition of inflammatory cell activity
in wounds [34].
T β 4 in cardiac repair
Heart disease including myocardial infarction, stroke and peripheral vascular
disorders has been shown to be one of the leading causes of death. Myocardial
infarction is the acute loss of myocardial function due to vascular occlusion of a
coronary artery. Bock-Marquette et al.[40] have shown that with the administration of
Tβ4 immediately following an induced myocardial infarct in mice, Tβ4 can prevent cell
death of heart tissue. It also increases the motility and survival of myocytes after
hypoxia [53-54].
The postulated mechanism of action is that exogenously administered Tβ4 upregulates
the expression of PINCH, a LIM domain protein. Tβ4 binds to PINCH and forms a
functional complex with integrin-linked kinase (ILK), a scaffolding protein [40]. It has
also been shown that Tβ4 interacts with the kinase region of ILK [55]. Through the
kinase activity of ILK, Akt2 is phosphorylated, which increases the production of
MMP-2, a metalloproteinase responsible for matrix degradation. The process of
breaking down extracellular matrix, a hindrance to movement, is necessary during the
migration of myocardial cells. In addition to myocardial cell migration, the effect of
exogenous Tβ4 on the increased migration of endothelial cells in the embryonic mouse
heart was also recorded. All these effects, taken together, contribute to an enhanced
myocyte survival after hypoxia and an improved cardiac function.
T β 4 in corneal repair and anti-inflammatory processes
Tβ4 has been implicated with corneal repair in several animal models [45-46] through
several mechanisms. Upon external application in the eye, Tβ4 promotes cell-cell and
cell-matrix contacts and clarity of the cornea after injury. It also increases epithelial
cell migration of the conjunctiva. Unlike in the heart and dermis, Tβ4 speeds up the
healing of the cornea by down regulating the expression of metalloproteinases, chiefly
15
1
1
Introduction
those which break down gelatin (MMP-2 and MMP-9), as well as membrane-type
metalloproteinases (MT6-MMP) [56]. Tβ4 is also found to inhibit injury-induced
apoptosis.
Prolonged and massive inflammation usually exacerbates wounds in the eye caused
by chemical burns and has an influence on healing. Uncontrolled inflammation is
mainly caused by the infiltration of polymorphonuclear macrophages (PMNs) in the
eye wound and surrounding stroma. Studies have shown that topical administration
of Tβ4 inhibits the gross migration of PMNs into damaged tissue by decreasing the
expression of chemokines, keratinocyte-derived chemokine (KC) and macrophage
inflammatory protein 2 (MIP-2), which act as main chemotactic agents for PMNs [45].
T β 4 in tumor metastases
The process of tumor metastasis involves several stages: tumor cell migration and
invasion, angiogenesis and, finally, tumor cell growth and proliferation [57-58]. The
overexpression of thymosins (Tβ4, Tβ10 and Tβ15) has been recorded in tumors
including thyroid and breast tumors and in melanoma and fibrosarcoma [59-60]. Cha
et al. [61] have previously undertaken in vitro and in vivo studies to elucidate the
effect of Tβ4 on the highly malignant B16-F10 cell line cultured from lung tumors. In
these studies, Tβ4 cDNA was introduced into B16-F10 cells through a genetically
engineered adenoviral infection. The resulting overexpression of Tβ4 was correlated
with the increase in levels of secreted vascular endothelial growth factor (VEGF), a
factor essential for tumor growth and angiogenesis. Further in vitro studies of B16F10 cells showed an increase in cell invasion with Tβ4 overexpression although no
change in levels of MMPs (MMP-2 and MMP-9).
SW480 colon carcinoma: epithelial-mesenchymal transition
The cellular overexpression of Tβ4 has been implicated with the malignant
progression of SW480 colon carcinoma cells. Wang et al. have shown that SW480 cells
overexpressing Tβ4 demonstrate increased levels of growth and invasivity in in vitro
and in vivo mouse models. Here, invasivity of cells is directly connected with the
elevated levels and activity of MMP-7, b-catenin and c-myc, all well recognized agents
in the process of cell invasion. Furthermore, SW480 cells overexpressing Tβ4
exhibited lower levels of E-cadherin, a protein responsible for the integrity of cell-cell
junctions [62].
The overexpression Tβ4 has been linked with the epithelial-mesenchymal transition of
SW480 cells [47, 62]. In this process, cells exhibit a loss of cell adhesion through the
loss of E-cadherin activity. These cells undergo a change of morphology into a more
mesenchymal phenotype typical for migratory cells in normal embryonic
development and the malignant progression of non-invasive cells. In these studies,
16
Introduction
increased levels of Tβ4 expression in SW480 cells correlate with the upregulation of
ILK. Together with the loss of E-cadherin, the increase in activity of ILK and its
downstream effectors, including Akt, is postulated to induce EMT and enhanced
invasivity of SW480 cells.
17
1
1
Introduction
1.3
Open questions
What are the effects of T β 4 on cytoskeletal mechanics and dynamics?
Extensive studies have been undertaken to elucidate the molecular and biochemical
role of Tβ4 in the cellular signaling pathway. These studies have shown that not only
does Tβ4 play a major role in the regulation of the cytoskeleton as a G-actin
sequestration protein; it also interacts with other cytoskeletal associated proteins
such as ILK and PINCH. Through these interactions, Tβ4 can elicit various cellular
reactions involved in healing and repair processes as well as in cancer metastases. The
underlying mechanism behind these processes is the increase in the motility of the
cells. The molecular pathways governing these processes have only been partially
understood and the results of previous studies show only a correlation between the
extracellular application of Tβ4 and the endogenous intracellular overexpression of
the protein with cell motility and invasion. No quantitative studies have yet been
made to assess how Tβ4 affects cell motility. The effects of Tβ4 on cell motility imply a
distinct change in the mechanics and dynamics of the cytoskeleton. Until now, these
aspects have not been examined in any of the cellular models.
Exogenous application vs. intracellular over-expression?
In the past, studies concerning Tβ4 have been made with the external addition of the
peptide as well as with the genetic overexpression in cells and often the observed
effects of both types of studies, such as increased cell motility, have been interpreted
as originating from the same mechanism. In investigating how Tβ4 can affect
cytoskeletal mechanics and dynamics, these two aspects; exogenous application and
endogenous genetic overexpression, must be examined separately. This is especially
true when considering the simple fact that the cytoskeleton and its constituents which
interact with Tβ4 cannot be accessed in the same manner from inside and from
outside the cell. The following studies are concerned mainly with the effect of
exogenous Tβ4 on cellular mechanics and dynamics.
Exogenous T β 4 : How can so little do so much?
Tβ4 is present at a high concentration of up to 500 µM in virtually every cell in
humans and mammals [63-64]. Up to 1% of the total Tβ4 concentration is circulating
in blood serum found in wound fluid [28, 65] and is believed to originate from
damaged platelets. Although this may be the case, studies have shown that even nanomolar concentrations of the peptide can induce a major cellular response. The
molecular mechanism of Tβ4 entry into the cell is currently unknown. Until now, no
known cell surface receptor has been found for Tβ4 cellular outside-in signaling.
18
Introduction
Clinical studies: a biphasic dose response – a biophysical phenomenon?
As recorded in previous studies, the role of Tβ4 as an agent in wound healing, cardiac
repair, anti-inflammatory pathways and in cancer metastases has made it a target for
clinical application. Clinical trials of using Tβ4 have been carried out for the treatment
of a number of diseases including venous stasis ulcers [66], myocardial infarction [67],
chronic non-healing corneal ulcers [68], epidermolysis bullosa and for dermal wounds
[69]. In these clinical trials, the efficacy of the drug, synthetic Tβ4, was examined in a
dose dependant manner. In the clinical application for the treatment of venous stasis
ulcers and in the healing of dermal wound, separate and independent clinical trials
were carried on patients with increasing concentrations of Tβ4 (0.01%, 0.03% and
0.1% topical peptide concentration) with a placebo used as a control [66, 70].
Remarkably, the most rapid healing in both cases was observed at the mid-dose level
of applied Tβ4; a clear biphasic response to increasing Tβ4 doses. Here the question
arises of why the mid-dose elicited the highest efficacy and how this phenomenon is
correlated to cell mechanical responses.
19
1
2 Material and Methods
Material and Methods
2.1
2.1.1
Biological and biochemical methods
Cells and cell culture
The SW480 cell line was established from a primary human colorectal
adenocarcinoma and can be acquired commercially at America Type Culture
Collection (cat. No. CCL-228 ATCC, Manassas, VA). NIH 3T3 cells (cat. No. CRL-1658
ATCC, Manassas, VA) originate from primary mouse embryonic fibroblast cells.
Immortalized cells with a stable growth rate were established from the original
culture after 20–30 generations. Both established cell lines were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1 mg/mL DGlucose, 100 U/ml Penicillin/Streptomycin and 10% fetal calf serum (FCS) (v/v)
(Biochrom) at a constant temperature of 37°C and 5% CO2 on cell culture surface
treated flasks (Nunclon Surface, Nunc). The GSP-cell line is a product of an NIH 3T3
cell stably transfected with a plasmid vector (pIRESpuro3) carrying a gene for EGFPActin. This cell line was maintained in DMEM supplemented with 1 mg/mL D-Glucose,
100 U/ml Penicillin/Streptomycin, 10% FCS (v/v) (Biochrom) with 1 µg/ml
Puromycin (Sigma) at a constant temperature of 37°C and 5% CO2. Cells were
passaged at 80% confluency.
Figure 2.1 Cell lines. (A) SW480 human colorectal carcinoma cells, Hoffmann modulation
contrast image with 20x objective (B) NIH 3T3 mouse fibroblasts, field contrast image with
10x objective
21
2
2
Material and Methods
For routine passaging, 5 ml Trypsin (Biochrom AG) was used to detach cells from Tflasks. Prior to measurements, cells were suspended in 10 ml Accutase (PAA
Laboratories GmbH) to ensure gentle detachment. Cells were incubated at 37°C
during detachment. When using non-treated culture surfaces (glass-bottom wells),
10 g/ml fibronectin in PBS (phosphate buffered saline) was applied to the surfaces
and incubated overnight at 4°C prior to seeding.
2.1.2
Cloning cassettes of EGFP-Actin for generating the GSP
The cloning cassette with the EGFP-Actin (Enhanced GFP) target DNA was produced
through PCR (polymerase chain reaction). The PCR reaction mixture contained 100 ng
of template DNA 0.5 µM of each primer, 200 µM dNTPs (Nucleotide Solution Mix, New
England Biolabs) and 1.0 U polymerase (Phusion ® Hot Start DNA-Polymerase,
Finnzymes). The mixture was added to a final volume of 50 µL with dH2O. The PCR
program cycle was set in four steps. Initial denaturation of DNA was done at 95°C for
30 s. This was followed by 34 cycles of denaturation at 98°C, annealing at gradually
rising temperatures of between 67°C and 71.1°C and extension at 72°C. A final
extension step at 72°C was then done for 10 minutes. The Tm (annealing temperature)
was varied to account for uncertainties resulting in differences between the Tm of the
2 primers used (see 2.1.4). After amplification, the PCR mixtures were purified using a
PCR purification kit (Qiagen). The DNA products were checked by gel-electrophoresis
on 1% Agarose gel before purification and used subsequently in digestion reactions
with the appropriate restriction enzymes (New England Biolabs) according to the
manufacturer’s protocol. DNA fragments were purified again after digestion.
2.1.3
Ligation
Ligation of the DNA target insert and the vector backbone was done by incubating the
insert with the vector (in this case both digested with NheI and BamHI restriction
enzymes) at a vector:insert mol ratio of 3:1 and 5:1. 20 mM ATP, 5x ligase buffer and
2 U ligase (T4 DNA-Ligase, Invitrogen) was added to the reaction mixture and topped
off with dH20 to a final volume of 10 µL. Reactions were incubated at either 14°C
overnight or at room temperature for 3 h before heat-inactivation at 65°C for 15 min.
A third of the mixture was used to be transformed into DH5α E. coli strain by heat
shock according to standard protocols [71].
22
Material and Methods
Figure 2.2 Stable transfection of NIH 3T3 cells with an EGFP-Actin plasmid vector. (1)
The EGFP-Actin cassette was cloned with 5’-NheI and BamHI-3’ restriction sites through
polymerase chain reaction. Template DNA for the EGFP-Actin cassette was obtained from a
pEGFP-Actin vector (Clontech). The EGFP-Actin DNA cassette was ligated into the
pIRESpuro3 backbone at the multiple cloning site (MCS) resulting in a pIRES-EGFP-Actin
vector, (2). The pIRES-EGFP-Actin plasmid vector was transformed into the DH5-α E. coli
strain (3) to multiply the plasmid. The plasmid vector was purified using a conventional
purifying kit, EndoFree Maxi Kit (Qiagen) (4). NIH 3T3 mouse fibroblasts were transfected
with the resulting plasmid vector using the Effectane (Qiagen) transfection method (5).
Puromycin (1 µg/ml) was used as a selective agent for further cultivation. Limited dilution of
transfected cells (1 cell in every 4 of 96x wells) enables the growth of positive clones from a
single cell. Clones with EGFP-actin are maintained in DMEM with 100 U/ml
Penicillin/Streptomycin, 10% FCS and 1 µg/ml Puromycin.
23
2
2
Material and Methods
2.1.4
Cloning of EGFP-Actin into the pIRESpuro3 selection vector
The pIRESpuro3 vector (Clontech) contains a viral internal ribosome entry site (IRES)
which allows for the bicistronic expression of the resistance gene and the target gene
of interest. This enhances the translation of both the Puromycin resistance gene in
combination with the target DNA. A cloning cassette of EGFP-Actin with restriction
sites for NheI (5’) and BamHI was created through PCR using the primers (NheI-EGFPActin forward) 5’-GTC AGC GCT ACC GGT CGC C-3’and EGFP-Actin-BamHI reverse) 5’ATC CCT AGA AGC ATT TGC GGT GG-3’ using the EGFP-Actin vector (Clontech) as a
template (see 2.1.2). Digestion and ligation were performed as described previously.
The cloned DNA product was transformed into DH5α E. coli by heat-shock according
to standard protocols and subsequently purified using the EndoFree Plasmid Maxi Kit
(Qiagen).
2.1.5
Transfection of the EGFP-Actin vector construct
Transfection of the NIH 3T3 cells was done by incubating 1 µg of the pIRES-puro3EGFP-Actin vector construct with the 10 µl Effectane Transfection Reagent (Qiagen)
and 8 µl Enhancer according to the manufacturer’s protocol. The DNA-Effectane
complexes were added drop-wise with constant gentle mixing onto 80% confluent
cells grown on Ø 35 mm cell-culture treated wells in 2 ml normal growth medium
(DMEM with 100 U/ml Penicillin/Streptomycin and 10% FCS). Transfected cells were
cultivated further for 24 h before further observation.
2.1.6
Stable transfection into mammalian cell lines
Prior to stable transfection, Puromycin resistance of NIH 3T3 and SW480 cells were
examined. 3 x 105 cells in Ø 35 mm wells were incubated with concentrations of
Puromycin ranging from 1 to 10 µg/ml over a period of three weeks. As a control, no
Puromycin was added to the cells. Cell medium (DMEM with 100 U/ml
Penicillin/Strepromycin and 10% FCS) containing the antibiotic was replaced every 2
days. Throughout this period, cell growth was observed. An optimum Puromycin
concentration of 1 µg/ml was chosen for both cells lines.
24
Material and Methods
Figure 2.3 Close-up of an NIH 3T3 mouse fibroblast stably transfected with the pIRESEGFP-Actin construct. Cells were seeded on glass bottom wells. The live image was made
using a 63x objective.
24 h after transfection (see 2.1.5), growth medium was replaced. 48 h after
transfection, 1.0 µg/ml Puromycin was added to the growth medium for selection.
Puromycin-containing medium was replaced every 2 days regularly until small
colonies of resistant cells were visible. Limited dilution of the cells was performed in
96-well plates at a calculated concentration of 1 cell in every 4 wells to ensure singlecell populations. These cells were grown with enriched DMEM (4.5 mg/ml D-glucose,
20% FCS) containing 1.0 µg/ml Puromycin.
2.1.7
Immunofluorescence and fluorescence microscopy
Up to 5 x 104 cells were seeded on 10 µg/ml fibronectin-coated glass cover slips
(Menzel, Braunschweig, Germany) and incubated overnight at 37°C and 5% CO2.
Adherent cells were fixed with 3% paraformaldehyde for 20 min prior to permeation
with 0.2% Triton-X-100 for 5 min. Cells were blocked using 0.5% BSA (bovine serum
albumin) in PBS for 1 h at room temperature. After washing, primary antibodies of
anti-actin and anti-vinculin (Sigma) were diluted 1:8,000 and 1:1,000,000,
respectively, with 0.5% BSA in PBS and incubated with the fixed cells at room
temperature for 1 h. Secondary HRP (Amersham) or FITC (Jackson ImmunoReaserch)
labeled anti-mouse antibodies were then diluted at 1:1,000 and 1:200, respectively,
and incubated for another hour with the fixed cells. For actin staining, 1 µg/ml Alexa
25
2
2
Material and Methods
Fluor 546-phalloidin (Molecular Probes, Eugene, Oregon) was added during
incubation with the secondary antibody. Between each step, cells were washed with
0.5% BSA in PBS. The cell samples were mounted on microscope slides using Mowiol
(Sigma) mounting solution. All fluorescent microscopy was carried out on a Leica
microscope DM16000 with both phase contrast and fluorescence modi. The objectives
for magnification used in the imaging and other experiments were 20x/0.4NA-,
40x/0.6NA- and 63x/1.3NA. Data acquisition was performed by a CCD (chargedcoupled device) camera (ORCA ER, Hamamatsu).
Figure 2.4 Actin fluorescent staining with Alexa Fluor 546-phalloidin. (A) The diffuse
actin fibers in SW480 cells are made visible using a 10x objective. (B) The cortical actin
network of an NIH 3T3 cell seen through a 63x objective.
26
Material and Methods
Figure 2.5 Fluorescent staining of vinculin. Focal adhesions of an NIH 3T3 cell are made
visible with vinculin fluorescent staining. Images were taken with a 63x objective.
2.1.8
Synthesis of lipid vesicles
Multilamellar lipid vesicles (MLVs) were synthesized by dissolving Dimyristoyl-L-αphosphatidylcholin (DMPC), Dimyristoyl-L-α-phosphatidylglycerol (DMPG), or 1,2dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DMPS) (Avanti Polar
Lipids, Birmingham, Alabama) in a 2:1 (v/v) mixture of chloroform/methanol in an
Erlenmeyer flask. Through nitrogen evaporation and subsequent vacuum desiccation
for 2 h, a dry lipid film was produced on the glass wall of the flask. The lipid film was
then resuspended in a buffer solution containing 20 mM HEPES (pH 7.4), 2 mM EDTA,
5 mM NaCl and 0.2 mM DTT. The resulting MLVs were left to equilibrate overnight at
35°C. Fluorescence correlation spectroscopy measurements require the use of small
unilamellar vesicles (SUVs) produced by dissolving the lipid film in 10 mM potassium
phosphate buffer (pH 7.4) followed by equilibration and finally sonication for 10 min
before use.
2.1.9
Synthesis of Tβ4-Alexa488 labeled peptide
Tβ4 and scrambled Tβ4 (used as a negative control) were obtained courtesy of
RegenerRex Biopharmaceuticals (J.J. Finkelstein, 15245 Shady Grove Rd, 470
Rockville, MD 20850-6243). The amino acid sequence of scrambled Tβ4 synthetic
peptide is: acetyl-EIPKETEKFDKETMEQKSIDKSQLEKPEAQNKELPTSKSGDKA. The
27
2
2
Material and Methods
labeling of Tβ4 with Alexa-488 was done according to methods as described in [72].
Using this procedure, 500 µg Alexa Fluor488 cadaverine sodium salt (Invitrogen) and
250 µg Tβ4 was incubated with 0.2 U guinea pig transglutaminase in a reaction
mixture containing 50 mM Tris HCl, pH 7.4; 15 mM CaCl2; 3 mM DTT while adding
0.3 mM TGA. This transamidation reaction labels the Tβ4 peptide with Alexa Fluor488
at glutamine residues. The labeled product was characterized by using reverse phaseHPLC and MALDI-TOF analysis.
Figure 2.6 Linking the Alexa Fluor 488 fluorophore to Tβ 4 molecules. Using the
transglutaminase enzyme as a transamidation agent, Alexa Fluor 488 cadaverine sodium salt
reacts with the Tβ4 peptide in which the fluorophore is linked to glutamine residues of the
peptide.
28
Material and Methods
2.2
2.2.1
Biophysical methods
Three-dimensional collagen cell invasion assay
Collagen gel were prepared by mixing 775 µl collagen R (2 mg/ml rat collagen type I;
Serva, Heidelberg, Germany) and 775 µl collagen G (4 mg/ml bovine collagen type I;
Biochrom). The mixture was then neutralized by adding 135 µl 26.5 mM NaHCO3
buffer, 43 µl 1 mM NaOH and 135 µl 10x DMEM. 1.2 ml of the preparation was
pipetted into Ø 35 mm cell culture dishes left to polymerize for 2 h at 37°C, 5% CO2
and 95% humidity. 2 ml DMEM was layered on top of the resulting gel to prevent
dehydration of the matrices before overnight incubation at 37°C, 5% CO2 and 95%
humidity.
After equilibration, the DMEM layer was replaced with a 2 ml suspension of 1 x 105
cells in DMEM (with 1 mg/ml D-Glucose, 2 mM L glutamine, 100 U/ml
Penicillin/Streptomycin). As Tβ4 can be found in serum, cells were cultured without
FCS. Tβ4 was added to the medium upon cell seeding. Cells were left to migrate within
the gel for 72 h. In experiments using calyculin-A (Calbiochem), cells were left to
adhere to the gel for 24 h before 10 nM or 50 nM of calyculin-A was added for 2 h.
Afterwards, the culture dish was washed with pre-warmed (37°C) DMEM, and cells
were allowed to migrate for another 48 h. Gels were then fixed with 2.5%
glutaraldehyde. Nuclei of individual cells were stained with Hoechst 33342. The
number of invaded cells and their invasion depths were determined in 25 randomly
selected fields of view.
2.2.2
Two-dimensional cell motility assay
1 x 104 cells were seeded in DMEM medium in a 35 mm dish and stimulated with
various concentrations of Tβ4 (0.05 – 1.0 µM) 24 h before measurements. Phase
contrast images of cells (x10 magnification) in 10 random fields of view were taken
every 60 s for a period of 3.5 h. Cells were kept in a stable environment throughout
the entire measurement in a heated chamber mounted onto the microscope set at
37°C with a constant supply of CO2. Cell movement was calculated as a measure of
mean square displacement (MSD) through a Fourier-based difference-withinterpolation image analysis [73]. The MSD followed the power law as below:
𝑀𝑆𝐷 = 𝐷 ∙ (∆𝑡/𝑡0 )𝛽 + 𝑐
where t0 is the time interval between recorded images (60 s), the pre-factor D
represents the apparent diffusivity of movement, and the exponent β is a measure of
movement persistence [74]. Values of β ~1 are seen with cells migrating randomly,
and β ~2 for cells moving ballistically. The additive constant c
represents
29
2
2
Material and Methods
measurement noise. In our case, because of the large time interval between successive
images, this constant is negligible.
2.2.3
Cell-spreading assay
5 x 104 cells were seeded on 10 µg/ml fibronectin-coated glass cover slips in a 35 mm
dish. After overnight incubation at 37°C and 5% CO2, adherent cells were fixed with
3% paraformaldehyde for 15 min at room temperature. After washing with 0.5 % BSA
in PBS solution, cells were stained for 30 min with 1 µg/ml Alexa Fluor546-phalloidin
(Molecular Probes, Eugene, Oregon) at room temperature. Cell nuclei were stained
with 1 mg/ml Hoechst 33342 dye (Pierce) for 5 min at room temperature. Fluorescent
and bright field images were taken from 10 – 20 random fields of view. Each cell
nucleus was counted to represent a single cell. The projected area of a cell was
calculated as the area bounded by the stained actin cytoskeleton measured with a
custom image processing program written in MATLAB.
2.2.4
Two-dimensional cellular traction microscopy
Polyacrylamide (PAA) gels (6.1% acrylamide/0.31% bis-acrylaminde) used for
traction microscopy measurements were cast on 75 mm x 25 mm silane-coated
microscope glass slides (Menzel) as described in [22]. For silanization the glass slides
were immersed first in 0.1 N NaOH for 5 min and, after drying, in 2.0% (3Aminopropyl)-trimethoxysilane for another 5 min. The slides were then washed in
dH2O twice before further immersion in 2.5% glutaraldehyde for 30 min. The slides
were ready for use after washing twice again in dH2O and drying at room
temperature. Green fluorescent carboxylated beads were suspended in the PAA gel
before casting into 10 x 10 mm frames (Abgene, Thermo) on the silanized glass slides.
The gel suspension was centrifuged upside down at 288 g and 4°C for 3 min to ensure
a uniform distribution of the fluorescent beads in a thin layer on the upper surface of
the gel. After polymerization, the gels were cross linked with Sulfo-SANPAH (Pierce
Biotechnology) and activated under UV-light for 5 min at room temperature.
Following two wash intervals with 50 mM HEPES buffer, the PAA gel were coated with
50 µg bovine Collagen G (Biochrom AG) in 5 mM HEPES buffer overnight at 4°C. The
Young’s modulus of the gels was 12.8 kPa ± 0.8 kPa as measured by a magnetically
driven plate rheometer.
30
Material and Methods
Figure 2.7 Two-dimensional traction microscopy. Serum starved SW480 cells are left to
adhere overnight onto collagen-coated PAA gel with a Young’s modulus of 12.8± 0.8 kPa.
Upon seeding, concentrations of Tβ4 were added to the cells. Fluorescence and bright field
images at 20x are taken before and after detaching the cells with a mixture of Cytochalasin
D/Trypsin. The images were analyzed to determine the displacement of the fluorescent
markers.
Figure 2.8 2D traction microscopy images. (A) Bright field image of SW480 cells on the
surface of PAA gel. (B) Fluorescence image of the underlying fluorescent beads in the same
field of interest as in (A).
1.5 ml DMEM suspension (without FCS) containing 2 x 104 cells was pipetted onto the
gel. The cell suspension was contained within a silicone ring (In Vitro, Göttingen,
Germany) attached to the glass slide. For measurements with Tβ4, concentrations of
31
2
2
Material and Methods
the peptide were added to the cell suspension while seeding. Cells were left to adhere
on the gel overnight at 37°C, 5% CO2 and 95% humidity. During tractions
measurements, cells were detached from the gel using a mixture of 80 µM
cytochalasin D and 0.25% trypsin. The displacement of fluorescent markers below the
surface of the gel was determined using a Fourier-based difference-with-interpolation
image analysis [74]. Cell tractions were then calculated from an unconstrained
deconvolution of the gel surface displacement field measured before and after
detachment of the cells [75].
2.2.5
Magnetic tweezer microrheology
The magnetic tweezer device is described in [17]. Fibronectin beads used in this
method were produced by incubating 4x108 superparamagnetic epoxylated beads
(Ø 4.5 µm, Dynabeads, Invitrogen) with fibronectin (5 µg per 1x107 beads; Roche) for
24 h at 2°C. After incubation, beads were washed with 0.1% BSA in PBS. Fibronectin
coated beads were kept in this buffer at 4°C. Beads were sonicated directly each time
before adding to cells to avoid clumping. 1.5 x 105 cells were seeded in DMEM medium
without FCS on Ø 35 mm cell-culture treated dishes (Nunclon) 24 h before tweezing
measurements. Cells were stimulated with different concentrations of Tβ4 upon
seeding. On the day of the measurements, 2 x 105 fibronectin coated beads were
added to each dish with adherent cells and incubated for 30 min at 37°C with 5% CO2.
Directly before measurements, culture dishes were washed with fresh, pre-warmed
medium to remove unbound beads.
32
Material and Methods
Figure 2.9 Magnetic tweezer measurements. (A) Fibronectin-coated beads are bound to
the cytoskeleton through cell surface integrins. Force directed toward the tip of the needle
pulls at the bead resulting in its displacement. Inset: bright-filed image of the needle tip
pulling at a fibronectin-coated bead. (B) Staircase-like protocol: forces exerted onto the bead
are raised sequentially (inset). Bead displacement over time is then recorded over forces
from 0.5 nN to 10 nN. Images are modified from [76].
33
2
2
Material and Methods
A fibronectin coated bead is bound to the cytoskeleton via integrins spanning the cell
membrane. The magnetic tweezer needle (HyMu80 alloy, Carpenter) was aimed by a
micromanipulator (Injectiman NI-2, Eppendorf) 20-30 µm away from a bound bead
on a cell. A solenoid coil around the needle generated a magnetic field with a high field
gradient at the tip of the needle. The resulting force pulled at the fibronectin-coated
bead attached to the cell. Bright-field images of the cell, bead and needle were taken
continuously during the measurements with a charged-coupled device (CCD) camera
(ORCA ER, Hamamatsu) at a rate of 40 frames/s. Bead positions were tracked using an
intensity-weighted centre of mass algorithm. A preset force was kept constant by
updating the current flowing through the solenoid or by moving the solenoid to keep
the distance between the needle-tip and the bead constant. Acquisition of images was
triggered and synchronized with the solenoid current generator. All measurements
were performed on a heated microscope stage.
With each force step with an amplitude of ∆F applied onto a fibronectin-coated bead,
the bead moves with the displacement d(t) towards the tip of the needle. The creep
response J(t) , which is the ratio of d(t)/ ∆F, followed a power law:
𝐽(𝑡) = 𝑎 (∆𝑡/𝑡0 )𝑏
for all amplitudes of force where t0 is a reference time arbitrarily set to 1 s. The
inverse of the prefactor a (in units of nN/µm) is a measure of cell stiffness and the
exponent b describes the visco-elastic properties of the cytoskeleton. b ~ 1 indicates a
Newtonian fluid-like behavior, whereas b ~ 0 an elastic solid-like behaviour. A
stepwise increasing force protocol was performed with steps of 0.5 nN, 1.0 nN, 1.5 nN,
2 nN, 3 nN, 4 nN, 5 nN, 6 nN, 8 nN and 10 nN, with each force-step lasting for 1 s.
Values of 1/a and b were determined at each force step.
2.2.6
Nano-scale particle tracking
Nano-scale tracking of cytoskeleton-bound beads was described by [73]. 4 x 104 cells
were seeded in DMEM medium without FCS on flat bottomed Ø 6.4 mm cell culture
wells (Corning® Sigma) and left to adhere overnight at 37°C and 5% CO2. Prior to
measurements approx. 2 x 104 pre-sonicated fibronectin-coated beads were added to
the culture wells. The same Ø 4.5 µm fibronectin-coated beads were used as described
previously in the magnetic tweezers method (see 2.2.5). The beads were incubated
with the cells for 30 min at 37°C and 5% CO2. For overnight measurements,
concentrations of Tβ4 were added during seeding. Short term stimulation was done by
adding Tβ4 during measurements. Bright field images were recorded at a rate of
8.3 images/s with a CCD camera (ORCA ER, Hamamatsu) on an inverted microscope
with a 10x, 0.3 NA objective (Leica) placed on a vibration isolated table (Newport,
Irvine CA). 100-200 bead positions were tracked continuously for 5 min using an
34
Material and Methods
intensity-weighted center of mass algorithm with an accuracy of 10 nm (rms). All
measurements were performed in a cell chamber mounted onto the microscope stage
maintained at 37°C with a constant supply of CO2. The stage drift, which was
estimated from the mean motion of all beads in the field of view, was accounted for in
order to calculate the accurate bead positions.
The mean-squared displacement of the beads was calculated as:
2
⟨∆𝑟 2 (∆𝑡)⟩ = ⟨�→ (𝑡 + ∆𝑡 )– → (𝑡) � ⟩
𝑟
𝑟
where → is the position of the bead and ∆𝑡 is the time lag. The brackets ⟨… ⟩ is the time
𝑟
average. The MSD of bead movement is fitted to the power law using a least-squares
fit and, thus, the equation can be rewritten as:
𝑀𝑆𝐷 = 𝐷 ∙ (∆𝑡/𝑡0 )𝛽 + 𝑐
The power-law cofactor D (in the unit of nm2) is a measure of diffusivity of the bead
motion influenced by the stability of the cytoskeleton and the binding quality of the
bead to the cytoskeleton via cell-surface integrins. The persistence of bead movement
is reflected in the β exponent. Values of β = 1 indicates Brownian/diffusive, β < 1
subdiffusive, β > 1 superdiffusive and β = 1 ballistic motion of the bound bead.
35
2
2
Material and Methods
Figure 2.10 Nano-scale particle tracking of fibronectin coated superparamagnetic
beads Beads are attached to the cytoskeletal network through integrins. Bead displacement
due to movement of the cytoskeleton is recorded. Inset: an SEM image of a cell with attached
beads (taken by C. Mierke, bar indicates 10 µm).
Figure 2.11 Analysis of nano-scale particle tracking. A) The trajectory of cytoskeletal
bound beads is shown as a series of dots. Bead positions are recorded every 120 ms.
Measurements are taken for 5 minutes.(B) The mean square displacement (MSD) of a bead in
correlation with ∆t following the power law. This particular analysis is from a recording of
trajectories of a bead bound to an NIH 3T3 cell.
36
Material and Methods
2.2.7
Differential scanning calorimetry (DSC)
Preliminary measurements of Tβ4 – lipid interaction were made using this method.
Basically, DSC was used to measure the melting phase transition temperature and the
phase transition enthalpy of lipid vesicles in the presence of Tβ4. In membranes,
phospholipids exist in a gel-like (ordered) or fluid-like phase (disordered) depending
on the temperature. In the ordered arrangement, acyl residues of the fatty acids are in
the energetically favorable trans-conformation. A transition into the more disorderly,
fluid phase requires energy, leading to the change into the more energetically
unfavorable gauche fatty acid conformation. The energy needed for this
conformational change can be determined by the specific heat capacity of the lipids
(Cp). Some lipids go from the gel-phase through intermediate phase changes, the
lamellar (Lβ) then the ripple phase (Pβ) before reaching the fluid phase at the melting
temperature (TM) (see Figure 2.12). With the non-covalent interaction of a peptide
with the acyl-residues, a disruption of the lipid arrangement occurs. As a result, less
energy in the form of transition enthalpy is needed to cause a phase transition, thus, a
reduction in the TM.
In DSC measurements, the sample and reference solution is heated at a preset heating
rate of:
𝛽 = ∆𝑇⁄∆𝑡
of 0.5°C/min to +30°C, and cooled at a rate of 1°C/min to +7°C until the phase
transition enthalpy reached equilibrium (Q100, TA Instruments). The difference in the
heat capacity ∆C(T) of the sample and reference is:
∆𝐶(𝑇) = 𝐶𝑃 − 𝐶𝑅 = ∆𝑃(𝑇)⁄𝛽
where 𝐶𝑃 and 𝐶𝑅 are the specific heat capacities of the probe and reference,
respectively.
The integration of the heat capacity, ∆C, over the change in temperature gives the
phase transitions enthalpy, ∆H. A phase transition was observed at around 23°C,
depending on the molar ratio of DMPG and DMPC lipids used. Analysis of the raw data
recorded during DSC measurements was performed using custom-programmed
software in MATLAB. Small unilamellar vesicles (SUVs) used in these experiments
were synthesized as described in 2.1.
37
2
2
Material and Methods
Figure 2.12 Phase transition of synthetic lipid vesicles. Shown above is a thermogram of
the transition phases of DMPG/DMPC lipid vesicles with increasing temperature. A peak
temperature of TV is reached between the lamellar (Lβ) and the ripple phase (Pβ). The melting
point, TM, marks the maximum specific heat which is reached before the lipid bilayer shifts
from the ripple phase into the fluid phase (Lα). The onset of the rise in specific heat is marked
by Ts .TL marks the end of the main phase transition. Diagram taken from [77].
38
Material and Methods
Figure 2.13 Differential Scanning Calorimetry (DSC): experimental setup. The
temperatures of the sample (1) and reference (2) are increased with the heater (3). Loss of
heat is prevented by the insulation coat (4). Temperatures are recorded by the temperature
observation point (5). PP and PR are heat outputs of the sample and reference, respectively.
2.2.8
Fluorescence correlation spectroscopy
The binding of Tβ4 to lipid membranes was examined further with the use of
fluorescence correlation spectroscopy (FCS). This method was used to measure the
diffusion of Tβ4-Alexa488 labeled peptide in the presence of various concentrations of
lipid vesicles. A custom FCS system was built as described in [78]. The setup consisted
of an excitation laser (20 mW, 488 nm; Changchun Dragon Lasers) with a beam that
was spatially filtered and expanded by a lens (f1 = 30 mm), a 50 µm pinhole, and a
second lens (f2 = 75 mm) before being passed through an aperture. The laser power
was reduced to ~100 µW with a neutral density filter. The excitation beam was then
passed through a beam-splitter and reflected by a dichroic mirror into an oil
immersion objective (NA 1.25, 100x). Low-fluorescence immersion oil was used to
decrease background fluorescence. Fluorescence emitted from the samples was
passed through the same objective and reflected by the dichroic mirror into an
avalanche photodiode (APD) with an active area of 625 µm². Scattered laser light at
488 nm was eliminated by a blocking filter. Fluctuations in the excitation laser
intensity were monitored by a photodiode. Longer correlation times of fluorescence
39
2
2
Material and Methods
intensity fluctuations were expected for fluorescent peptides bound to larger lipid
vesicles compared to free fluorescent peptides.
Figure 2.14 Schematic diagram of the Fluorescence Correlation Spectroscopy (FCS)
detection volume. Free fluorescent peptide (Tβ4-A488) and fluorescent peptide bound to
much large lipid vesicles diffuse in and out of the illuminated volume, causing characteristic
fluctuations in the detected fluorescence intensity. A detection volume of ca. 1 µm3 was used
in the setup.
DMPC and DMPS SUVs were synthesized as described in 2.1.8 and sonicated again
directly before measurements. The focal volume was determined to be 1 µm3 through
calibration with Alexa-488 (D = 435 µm2 s-1 and Rhodamine 6G solution (D = 414 µm2
s-1) [79]. Measurements were performed at 25 kHz for 10 s. Data analysis of
fluorescence auto-correlation was done using a custom program developed in
MATLAB.
The principle of fluorescence correlation spectroscopy has its basis in the analyses of
correlations in fluorescence fluctuation intensities. According to this principle, longer
correlation times are expected of fluorescent peptides bound to larger lipid vesicles
than free fluorescent peptides travelling in and out of the detection volume. The
normalized autocorrelation function of the fluorescence fluctuations, 𝐺(𝜏), calculated
from the fluorescence intensity, 𝐹(𝑡), detected in the sample volume is as follows:
40
𝐺(𝜏) =
⟨𝛿𝐹(𝑡) . 𝛿𝐹(𝑡 + 𝜏)⟩
⟨𝐹(𝑡)⟩2
Material and Methods
The autocorrelation signal was fitted to
𝐺(𝜏) =
𝑉𝑒𝑓𝑓
1
1
∙
∙
< 𝐶 > (1 + 𝜏𝜏𝐷)
1
𝑟
𝜏
�1 + (𝑧00 )² ∙ 𝜏𝐷
where Veff is the effective observation volume; <C> is the average concentration of
particles; τD is the correlation time for translational diffusion; r0 and z0 are the radial
and axial dimensions of the observation volume.
Dissociation constants for the binding for Tβ4 to SUVs, Kd, were obtained by fitting τD
versus lipid concentration [L] to the Michaelis-Menten equation
𝜏𝐷 =
𝜏𝐷,𝑚𝑎𝑥 [𝐿]
𝐾𝑑 + [𝐿]
where τD,max is the maximum correlation time when all fluorophores are bound to
SUVs. τD,max was obtained from 𝜏𝐷 =
(𝑟0 )2
4𝐷
[78], and D is the diffusion constant of SUVs
(8.08x10-12 m2/s) calculated from the Stokes-Einstein equation, assuming an average
radius of 30 nm [80].
41
2
2
Material and Methods
Figure 2.15 Principle of Fluorescence Correlation Spectroscopy (FCS). (A) A fluorescent
peptide moving in Brownian motion in and out of the detection volume has a much shorter
correlation time (τfree) than a fluorescent peptide bound to a slower-moving lipid vesicle. This
produces characteristic fluorescence intensity patterns, with longer bursts of intensities for
bound peptide recorded over time. Autocorrelation analysis of intensity fluctuations results
in an autocorrelation function against correlation time. (B) In an ideal two-component
system, the autocorrelation function for τfree is much smaller that of τbound. When the fraction
of free and bound fluorescent peptide are the same, however, the molar partition coefficient
can be calculated as 𝐾 = 1⁄[𝑙𝑖𝑝𝑖𝑑].
42
3 Results
3
Results
3.1
3.1.1
The effect of extracellular Thymosin β4 on the motility of SW480 cells
Previous studies have shown that Tβ4 is directly involved in regulating cell motility. In the
metastases of human colorectal carcinoma, the over-expression of Tβ4 is believed to be
responsible for the increased invasion of malignant cells [48]. In vivo and in vitro studies
on embryonal cardiomyocytes and endothelial cells have shown that induction with
extracellular Tβ4 has resulted in increased cell migration [40]. Increased migration of
endothelial progenitor cells (EPCs) in response to stimulation with extracellular Tβ4 has
also been recorded [81]. Although this phenomenon has been qualitatively discussed, the
biophysical mechanisms underlying the dose response of extracellular Tβ4 induction on
cell migration is still not known. In this chapter, the dose-dependent correlation of
increasing Tβ4 concentrations to the migration of SW480 colon carcinoma cells is
quantitatively examined.
Three-dimensional cell migration
The migration of SW480 cells in response to increasing extracellular doses of Tβ4 was
characterized using a three-dimensional collagen gel assay. The gels used in this assay
had an average shear modulus of 58 Pa and a thickness of 500 ± 100 µm (mean ± SD).
The lattice of collagen fibers within each gel had an average pore size of 1.3 ± 0.2 µm
(mean ± SE)[27]. The SW480 cells were treated with Tβ4 directly upon seeding onto
the gels. The cells were induced with a range of Tβ4 concentrations (0.05 µM – 1 µM).
0.2 µM scrambled Tβ4 peptide was used as a negative control. The cells were cultured
for 3 days before labeling their nuclei with Hoechst 33342 vital stain for cell counting
and measuring the invasion depth.
The invasivity of the cells was quantified by obtaining the invasion depths of the cells
in multiple randomly chosen fields of view. The cumulative probability of finding a cell
(pCum) at or below a given invasion depth is expressed in the invasion profiles (see
Figure 3.1 (A)). As a robust measure of cell invasiveness, a characteristic invasion
depth was defined as the depth that a given percentile of cells have reached or
exceeded. A threshold was chosen at pCum = 0.1; the characteristic invasion depth
used in this study is the depth that 10% of all counted cells had reached. This
approach was selected to accurately demonstrate the differences in migration at
different Tβ4 concentrations while at the same time remaining conservative, ensuring
a high number of cell counts for sampling.
The SW480 colon cancer cell line is known to be non-invasive, and these cells
normally do not invade collagen gels. However, cells induced with Tβ4 at
concentrations between 0.05 µM and 0.5 µM were able to migrate into the collagen
gel. The cells showed a biphasic migratory response to increasing concentrations of
Tβ4; SW480 cells were most invasive at an applied Tβ4 concentration of 0.2 µM. Cells
induced with 1 µM Tβ4 exhibited a marked decrease in invasiveness, with a migration
profile almost equivalent to that of the control (0.2 µM scrambled Tβ4).
44
Results
Figure 3.1 Analysis of SW480 cell migration in three-dimensional collagen gels. (A) A
Tβ4 dose dependant cellular invasion profile; pCum is the cumulative probability of finding a
cell at a given depth and lower. (B) Invasion depth of cells at pCum of 10; 10% of cells are
seen at this depth or lower.
45
3
3
Results
Closer analysis of the invasion profile shows that 10% of SW480 cells treated with the
control have migrated into the gel to a depth of 99 µm or below. Compared to the
control, cells induced with Tβ4 at concentrations of 0.1 µM and 0.2 µM have migrated
over threefold deeper into the collagen gels. Tβ4 concentrations used above and below
these levels resulted in a reduced cell migration. At an induction with 1 µM Tβ4,
however, cells migrate in the same manner as the control; 10% of cells are seen at a
depth of only 110 µm and below. These results demonstrate a clear biphasic response
in cell migration to the dose of exogenous Tβ4 applied to SW480 cells (see Figure
3.1 (B)).
3.1.2
Two-dimensional cell motility
Two-dimensional cell motility of SW480 cell was analyzed to acquire more
information on the effect of Tβ4 on the dynamics of cell movement. Sub-confluent
SW480 cells were induced with 0.2 µM Tβ4, the peak working concentration as seen in
the 3D-invasion assays, and at the maximum dose of 1 µM where cell motility had
receded. Scrambled Tβ4 was used as a negative control. 24 h after induction, bright
field time lapse images from multiple fields of view were taken every 60 seconds over
three and a half hours. Using the images, the movement of each single cell was
recorded by tracking bright nuclear structures with an intensity-weighted center-ofmass algorithm [26, 82]. From the recorded trajectory of these nuclear structures, the
mean square displacement (MSD) of an entire cell was obtained.
The MSD is a measure of the Euclidean distance the cell has moved within a given time
span. This was calculated for each cell using the following equation
∆𝑟 2 (∆𝑡) = ⟨[(𝑟(𝑡 + ∆𝑡) − 𝑟(𝑡)]2 ⟩t
where 𝑟 is the two-dimensional position of the cell and ∆𝑡 the time lag. The brackets
indicate an average over absolute times 𝑡. The MSD follows the power-law
relationship as a function of the time lag. Thus, the MSD can be fitted according to the
power-law as below
𝑀𝑆𝐷 = 𝐷 ∙ (∆𝑡/𝑡0 )𝛽
where the 𝐷 coefficient, bearing a unit of µm2, is a measure of the apparent diffusivity.
The 𝛽 exponent describes the movement as diffusive (Brownian motion) when 𝛽 = 1,
sub-diffusive when 𝛽 < 1, super-diffusive when 𝛽 > 1 and ballistic when 𝛽 = 2. The
persistence of cell movement can be obtained by assessing the turning angles between
two successive trajectories of the cell within a time lag segment. Since this is strongly
coupled to the 𝛽 exponent, the 𝛽 exponent is used as an indicator for the persistence
of movement [26].
46
Results
Figure 3.2 Analysis of two dimensional SW480 cell motility. Cells were seeded subconfluently on Ø 35 mm plastic dishes and were stimulated with Tβ4 directly upon seeding.
Time lapse measurements were taken after 24 h of induction. (A) MSD plots of cells after
induction with Tβ4 concentrations of 0.2 µM (seen in red), 1 µM (blue) and the control using
0.2 µM scrambled Tβ4 peptide (green). Black lines indicate the power-law fit according to
𝐷 (∆𝑡/𝑡0 )𝛽 + 𝑐 . The values of the coefficient 𝐷 are given in (C) and 𝛽 in (D). (B) Examples of
cell trajectories tracked in a measurement lasting 3.5 h. Dots show successive cell positions
tracked every 60 seconds. Seen in red and blue are trajectories of cells treated with 0.2 and
1.0 µM Tβ4. The control is shown in green. Cells treated with 0.2 µM Tβ4 moved faster on
short time scales (higher apparent diffusivity values) and showed highly persistent
movement (MSD exponent β = 1.40). In contrast, control cells and cells treated with 1.0 µM
Tβ4 displayed sub-diffusive to diffusive movement (β = 0.90 and β= 0.98, respectively) and
3-fold lower apparent diffusivity values.
47
3
3
Results
As shown in Figure 3.2 (A), the log average of the MSDs of all SW480 cells from each
concentration of Tβ4 used and the control was plotted as a function of the time lag. In
all cases, the MSDs increased with the time lag, ∆𝑡, in accordance with a power law.
The solid lines represent the least squares fit to the data. From this line, the 𝐷
coefficient and the 𝛽 exponent were obtained. A marked difference can be seen in the
MSDs slopes of cells treated with 0.2 µM Tβ4 and cells treated with the control or
1.0 µM Tβ4. This is reflected in the 𝐷 coefficients and the 𝛽 exponents as shown in
Figure 3.2 (C) and (D). The 𝐷 coefficient of cells treated with 0.2 µM Tβ4 was more
than three-fold higher than in cells treated with 1 µM Tβ4 and the control. Analysis of
the 𝛽 exponents show a highly persistent (super-diffusive; 𝛽 = 1.40) movement of
SW480 cells treated with 0.2 µM Tβ4. Cells induced with 1.0 Tβ4 and the control
demonstrate sub-diffusive to diffusive movement (𝛽 = 0.90 and 𝛽 = 0.98,
respectively). Taken together, these results show clearly that the persistent
directional movement and average velocity of an SW480 cell stimulated with 0.2 µM
Tβ4 allows it to travel much larger distances. As seen with the results obtained from
the 3D invasion assays, these results also demonstrate a clear biphasic dose response
of Tβ4 concentration to 2D cell motility.
0
48
Results
3.2
3.2.1
The effect of extracellular Tβ4 on cell morphology
Cells employ a myriad of strategies to overcome tissue barriers in the process of invasion.
One of these is a distinct change in cell morphology called the epithelial-mesenchymal
transition (EMT) which occurs during malignant progression of metastatic carcinomas.
As the name suggests, EMT is marked by a change from an epithelial to a mesenchymal
phenotype following primarily the loss of cell-cell adhesion mediated by the downregulation of E-cadherin expression. This results in solitary cells bearing a more
elongated, fibroblastic morphology [83]. Overexpression of the Tβ4 gene has been shown
to trigger EMT in SW480 colorectal carcinoma cells [62]. Until now, the effects of
extracellular Tβ4 stimulation of SW480 on cell morphology have not been investigated.
Analyses of SW480 cell morphology in 3D
The morphology of SW480 cells was observed using three dimensional collagen gel. 5
x 104 cells were seeded onto the gels in Ø 35 mm dishes and were incubated for 3 days
before image stacks through the entire gel were taken. As in the previous
experiments, the cells were treated with 0.2, 1.0 µM Tβ4 and 0.2 µM scrambled Tβ4
peptide (control) upon seeding.
Wild-type SW480 cells exhibit a round, pancake-like phenotype and, when seeded
sub-confluently (5 – 10 x 104 cells), tend to group together into large monolayer
clusters of between 5 – 10 cells. This is seen clearly with wild-type SW480 cells lying
on the surface of 3D collagen gels (Figure 3.3; 0 µM Tβ4 at a depth of 0 µm). After
incubation with 0.2 µM Tβ4, SW480 cells undergo a drastic change in their
morphology and are found to have invaded into the collagen gel (Figure 3.3; 0.2 µM
Tβ4 at a depth of 100 µm). On the surface of the gel, induced cells assume an
elongated, spindle-like morphology, forming long filapodia stretching throughout the
gel (see white arrows in Figure 3.3) and are found largely as single cells within the
collagen gel. These fibroblastic cells which have invaded as far as 100 µm into the gel
and beyond can be identified with the same outer characteristics. SW480 cells treated
overnight with 1 µM Tβ4 were observed to be in mixed states on the surface of
collagen gels, with populations of induced cells with a spindle-shaped, fibroblastic
morphology (see red arrows in Figure 3.3) as well as round cells similar to the wildtype gathered in clusters. Although some cells are elongated, they do not invade into
the gel and remain largely on the surface. The phenomenon of mixed phenotypes was
not seen in either the control group or with cells induced with 0.2 µM Tβ4.
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Figure 3.3 Biphasic response of SW480 cell morphology to Tβ 4 concentration in 3dimensional collagen gels. SW480 cells were seeded onto collagen gel in Ø 35 mm dishes.
0.2 µM, 1 µM Tβ4 and 0.2 µM scrambled Tβ4 (control) were added upon seeding. Cells were
incubated for a total of 3 days before images of 25 random fields of view through the entire
depth of the gels. Here, sample images are shown of the surface of the gels (0 µm depth) and
at a depth of 100 µm. On the surface of the gel, large clusters of round cells are seen after
induction with the control. Cells induced with 0.2 µM Tβ4 have undergone EMT and exhibit
long filapodia (white arrows). At this Tβ4 concentration, cells have migrated up 100 µm into
the gel and beyond. Cells incubated with 1 µM Tβ4 are seen to be in mixed phenotypes; round
cells in clusters as well as spindle-like, fibroblastic cells (red arrows) are found on the surface
of the gel. Cells treated with the control and 1 µM Tβ4 are not found at depths beyond 100 µm.
Bars measure 20 µm.
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3.2.2
Analyses of SW480 cell morphology in 2D
Cell morphology and spreading area on 2D surfaces were examined to accurately
quantify the changes in phenotype as a response to exogenously applied Tβ4. Cells
cultured on two-dimensional glass slides were incubated for 24 h with PBS (control)
and 0.2 µM and 1 µM doses of Tβ4. Fixed cells were then stained with phalloidin to
visualize actin fibers. From 25 random fields of view, fluorescent images were taken
to calculate the cell area with the actin staining serving as a cell area boundary.
As seen in Figure 3.4 (A), wild-type SW480 cells are typically round and tend to form
clusters in a monolayer as seen previously on 3D gel surfaces. Cells treated overnight
with 0.2 µM Tβ4 undergo distinct morphological changes, being much larger and
better spread. These cells have a polarized morphology, with a clear leading (white
arrow) and trailing edge (red arrow), while forming long filapodia. Cells induced with
1 µM Tβ4 have also formed filapodia but tend to cluster and revert to a shape similar
to that of the control, with no clear cellular polarization.
An analysis of the calculated cell area in Figure 3.4 (B) shows that SW480 cells
treated with 0.2 µM Tβ4 are 40% larger than the control (PBS induced; average
spreading area= 905 µm2). The spreading area of cells induced with 1 µM Tβ4 is 26%
larger when compared with the control. These results show a clear biphasic Tβ4 dose
response in cell spreading and are consistent with all measurements discussed
previously. A cell count of each concentration group as seen in Figure 3.4 (C) revealed
that SW480 cells treated with 0.2 µM showed more than a 3-fold higher incidence of
single cells (13%) within a field of view when compared with the PBS control (4.5%).
Consistent with the biphasic dose response, a reduction to 7% single cells were found
in the population treated with 1.0 µM Tβ4.
Results from 3D and 2D analyses of cell morphology have shown that stimulation with
extracellular Tβ4 has induced EMT similar to the overexpression of Tβ4 in SW480
cells. A major factor involved in this transition may be the cell-cell disassociation
caused by the loss of E-cadherin and a massive cytoskeletal reorganization [84]. The
transition from cell-cluster to single cells is seen with SW480 cells induced with
extracellular 0.2 µM Tβ4 and intracellular genetic overexpression of the peptide [48,
62]. The event of cell polarization into a leading and a trailing edge indirectly causes
the reorientation of traction-generation of the cell and the formation of protrusions on
one side of the cell and, thus, an increase in directional motility [85]. The increase in
cell area as seen with 0.2 µM Tβ4 treated cells indicates higher cellular traction forces
generated for this directional movement. However, unlike the effects of genetic
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intracellular overexpression, the effect of extracellular Tβ4 is distinctly biphasic with
respect to increasing peptide concentration. This implies that the molecular
mechanism with which extracellular Tβ4 regulates cell morphology and motility in
SW480 cells may involve other mechanisms to that of intracellular Tβ4. This point will
be examined further in the proceeding sections.
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Figure 3.4 Biphasic response of SW480 cell morphology to Tβ 4 concentration on twodimensional surfaces. (A) SW480 cells were stained with fluorescently labeled phalloidin
to visualize actin fibers. Cells were treated overnight with either PBS (control), 0.2 µM Tβ4 or
1 µM Tβ4. Twenty-five random fields of views were taken to quantitatively measure the cell
spreading area marked by the actin boundary. The cells induced with 0.2 µM Tβ4 showed a
marked polarization into a leading (white arrow) and trailing edge (red arrow). Cells of the
control group showed the round wild-type morphology, forming clusters. Cells treated with
1 µM Tβ4 have formed filapodia but do not exhibit any signs of polarization. Scale bars are
20 µm. (B) Analysis of cell spreading area. Cell of the control (PBS) have an average cell
spreading of 905 µm2 ± 10 µm2 (mean ± SE). 0.2 µM Tβ4 induced cells are 40% larger than the
control (1257 ± 17 µm2) and 1 µM Tβ4 induced cells are 26% larger (1135 ± 12 µm2). Bars
indicate standard errors. (C) Percentage of single cells found on 2D culture surfaces. 4.6% ±
0.5% of cells treated with PBS are single cells. 12.9 % ± 0.8% of cells treated with 0.2 µM Tβ4
and 7.1 % ± 0.4% of cells treated with 1 µM Tβ4 are single cells. Bars indicate standard errors.
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3.3
3.3.1
Effect of extracellular Tβ4 on cell compliance, dynamics and
contractility
The process of directional migration involves the polarization of a cell into a leading and
trailing edge. At the leading edge of the cell, protrusions are generated by the
polymerization of actin filaments which are stabilized by adhesions to the substrate lying
under the cell [86-87]. Generation of traction forces by actin-myosin contraction enables
the formation of adhesions at the leading edge and the disassembly of adhesions at the
rear to allow for the forward locomotion of the cell [88]. This is a dynamic process which
requires constant remodeling of the cytoskeleton and turnover of adhesions. In this
chapter, the effects of extracellular Tβ4 on the cytoskeletal mechanics, dynamics and
cellular tractions of SW480 cells are examined.
Creep response and cell mechanics
The magnetic tweezer method was employed to examine the effect of exogenously
applied Tβ4 on cytoskeletal stiffness and viscoelasticity. Measurements were
performed on SW480 cells incubated with fibronectin-coated Ø 4.5 µm superparamagnetic beads. The fibronectin coating links the beads to the cytoskeleton and
acto-myosin fibers through cell-surface integrins [89]. The magnetic tweezer exerts an
external force onto the bead, thereby actively deforming the cytoskeleton in the
process. This method allows for the quantification of several physical properties of the
cytoskeleton. Firstly, the stiffness of the cell, which is a measure of the ability of the
cytoskeleton to withstand the forces, and secondly its hysteresivity or loss tangent,
which is a measure the turnover rate of cytoskeletal bonds in response to the active
external force [17, 90]. A staircase-like sequence of increasing force steps from 0.5 nN
to 10 nN was applied on fibronectin coated super-paramagnetic beads bound to the
cytoskeleton. The displacements of the beads were tracked online followed by
statistical analysis. Separate measurements on SW480 cells were made to observe the
response of 24 h induction with either the control (PBS) or Tβ4 concentrations of
0.2 µM and 1 µM as done in previous measurements.
The displacement 𝐽(𝑡) of the fibronectin coated bead in response to a force step of
magnitude Fstep follows the power law [90] as below:
𝐽(𝑡) = 𝐹𝑠𝑡𝑒𝑝 𝑎(𝑡/𝑡0 )𝑏
The 𝑎 coefficient is the compliance of the cytoskeleton equal to the inverse stiffness.
This coefficient indicates the extent of cytoskeletal deformation caused by a unit force.
The power-law exponent 𝑏 describes the viscoelasticity of the cytoskeleton. A value of
𝛽 = 0 is obtained from a purely elastic solid whereas a value of 𝑏 = 1 is obtained from a
purely viscous fluid. A 𝑏 exponent value of between 0 and 1 is expected for
viscoelastic systems such as the cytoskeleton. b is related to the loss tangent, or
hysteresivity η of the system by η=tan(b π/2). Within each group of measurements, a
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log normal distribution of stiffness values (1/𝑎) and a normal distribution of the
power-law exponent were recorded. Due to this, the geometric mean of stiffness and
the arithmetic mean of the power-law exponent were calculated [17]. The average
response of the cells was taken as the value averaged over all cells within each
concentration group.
Figure 3.5 Stiffness of Tβ4 induced SW480 cells. Cells stimulated overnight with the PBS
control, 0.2 µM and 1.0 µM Tβ4 were incubated with fibronectin-coated super-paramagnetic
beads. External force applied to the bead were increased step-wise from 0.5 – 10 nN. (A)
Displacement of the bead following the direction of the force followed the power law,
𝐽(𝑡) = 𝑎(𝑡/𝑡0 )𝑏 . The stiffness of the cell is given by the inverse of the power-law coefficient ,
a, in units of nN/µm at all force steps. (B) A biphasic response in stiffness was recorded at
external forces of 0.5 nN; cells stimulated with 0.2 µM Tβ4 were more than twofold stiffer
(13.3 nN/µm) than the control (5.8 nN/µm) and cells stimulated with 1.0 µM Tβ4
(6.2 nN/µm). Bars indicate the standard error.
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A non-linear response of bead displacement 𝐽(𝑡) to increasing force was seen with
cells in all groups as shown in Figure 3.5 (A). Bead displacement decreased with the
increase of force less than proportional as a result of cellular stress stiffening. Nonlinear stress stiffening is a behavior typical of cross-linked biomaterial such as
extracellular matrix and the actin cytoskeleton [91-92]. Stress stiffening of cells
treated with 1 µM Tβ4 and the control were similar; both populations showed a
twofold increase in stiffness at 10 nN compared to at 0.5 nN. However, cells induced
with 0.2 µM Tβ4 displayed less stress-stiffening; only a one and a half fold increase in
stiffness was recorded over the force range. These results suggest that cells treated
with 0.2 µM Tβ4 have an inherently more pre-stressed cytoskeleton and as a result
have a smaller increase in stress-stiffening in response to higher external force
exerted onto the cytoskeleton. Closer analysis of cell stiffness at an external force of
0.5 nN revealed that cells treated with 0.2 µM Tβ4 showed a slightly more than twofold increase in stiffness compared to the PBS control. No statistically significant
difference was found between the stiffness values of cells induced with 1 µM and that
of the control.
Figure 3.6 Viscoelasticity of Tβ 4 induced SW480 cells. (A) Power law exponent b
represents the viscoelastic properties of the cells as a function of increasing force. (B) Power
law exponent b at an external force of 0.5 nN; no changes in viscoelasticity was seen at all
concentrations. Bars indicate the standard error.
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The dynamic turnover rate of cytoskeletal bonds was examined by analyses of the
viscoelastic 𝑏 exponent. The exponent of this power-law creep response tended to
decrease at a Tβ4 concentration of 0.2 µM especially at higher force values, as
expected for stiffer cells, although the differences compared to control conditions or
Tβ4 concentrations of 1 µM were not statistically significant (see Figure 3.6).
3.3.2
Two-dimensional cellular tractions
As previously recorded in literature, cell stiffness correlates directly with prestress
and internal contractility [93]. Cell contractility is caused by the ATP-dependent
action of myosin, which cross-link actin filaments to form stress fibers. This is a
crucial step required in cell adhesion, cell movement and cell division [94]. The
contractile force a cell exerts on a two dimensional collagen substrate was examined
through 2D traction microscopy. Polyacrylamide (PAA) gels with a known stiffness of
Young’s modulus = 12.5 kPa were coated with collagen to enable cell adhesion. Due to
cell tractions, elastic strain energy is stored in the gel [75]. The total strain energy of
each cell, 𝑈, is obtained as the sum of local tractions causing gel deformation following
the equation given below:
1
𝑈 = �2� �→ �→� ∙ →�→� 𝑑𝑥𝑑𝑦
𝑇
𝑟
𝑢
𝑟
Here, → �→� is the traction vector and →�→� the displacement vector. The resulting
𝑇
𝑟
𝑢
𝑟
value of elastic strain energy is given in units of pico-Joules per cell. As cellular
tractions are dependent on the spreading area of the cells, the calculated strain energy
was normalized to the cell area giving a value in strain energy density with a unit of
pJ/µm2.
Marked differences in cell contractility were measured between Tβ4 induced cells and
the controls. Compared to the PBS control (0.108 pJ/cell ± SE 0.028), cells treated
overnight with 0.2 µM Tβ4 exhibited a threefold increase (0.430 pJ/cell ± SE 0.063)
and cells induced with 1 µM Tβ4 showed an 85% increase (0.235 pJ/cell ± SE 0.03) in
elastic strain energy. The differences became more pronounced when the cell
spreading area is taken into consideration as shown in Figure 3.8 (B). SW480 cells
show an apparent biphasic response to the increasing dose of Tβ4 in contractility, with
the highest contractility at stimulation with 0.2 µM Tβ4. These results are consistent
with those recorded with cell stiffness measurements as described previously in
section 3.3.1.
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Figure 3.7 Traction force maps of SW480 cells: contractility of Tβ 4 stimulated cells on
2D substrates. Cells were seeded on collagen coated PAA gels (Young’s modulus = 12.5 kPa)
and immediately treated with exogenous Tβ4. 2D- traction microscopy was performed after
24 h incubation with the peptide. On the left are bright field images of the cells, scale bars
indicate 20 µm. Traction field maps of cells are given on the right. Color bars indicate
differences in local tractions in units of Pa. Cells were sampled from random fields of view.
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Figure 3.8 The effect of exogenous Tβ 4 on the contractility of SW480 cells on 2D
substrates. (A) Strain energy of SW480 cells was calculated from cellular tractions
integrated over the spreading area of each cell. The highest level of strain energy per cell
(0.430 pJ/cell ± SE 0.063) was recorded on cells treated with 0.2 µM Tβ4 compared with that
of cells induced with the PBS control (0.108 pJ/cell ± SE 0.028) and 1.0 µM Tβ4 (0.235 pJ/cell
± SE 0.028). (B) The cell spreading area was taken into consideration when calculating the
strain energy density. Cells treated overnight with 0.2 µM Tβ4 have a strain energy density
(343.8 µN/m ± SE 34.3) significantly more than that of the control (144.4 µN/m ± SE 14.4)
and cells treated with 1.0 µM Tβ4 (207.3 µN/m ± SE 20.7). Bars indicate the standard error.
3.3.3
Nanoscale particle tracking of cytoskeletal-bound beads: studying the
time- dependant activity of Tβ4 on cytoskeletal dynamics
Cytoskeletal dynamics can be quantified by directly tracking the spontaneous
movement of fibronectin coated beads (Ø 4.5 µm), which are directly connected to the
cytoskeleton through cell-surface integrins. In comparison with methods that actively
employ external force such as the magnetic tweezer, this method detects the internal
cellular forces that cause bead movement, mainly the formation of stress fibers and
the reorganization of the cytoskeleton, both of which involve actin in filamentous and
monomeric form as well as motor proteins such as myosins. To analyze the
differences between the short term and long term effects of Tβ4 on the cytoskeletal
remodeling of SW480 cells, this method was used on cells stimulated for 30 min
(short term) and 24 h (long term) with exogenous Tβ4.
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Cytoskeletal dynamics was measured by tracking the position of bound beads at 10
times per second over a period of 5 min. The mean square displacement (MSD) of the
moving
bead
is
fitted
according
to
the
power
law
equation,
𝛽
𝑀𝑆𝐷 = 𝐷 ∙ (∆𝑡/𝑡0 ) + 𝑐 as seen previously in section 3.1.2 (2D cell motility
measurements). Here, the fit parameter, D, carries a unit of µm2 and measures the
diffusivity of bead motion, correlating directly to the amplitude of its movement. D
values in each of the scenarios (PBS control and induction with either 0.2 µM or 1 µM
Tβ4) were found to be distributed over a wide range in a log-normal manner. Thus, an
accurate method of obtaining the average MSD for a given concentration would be to
use geometric mean of all MSD values [26]. The power-law exponent, β, describes the
persistence of bead motion.
Bead diffusion measurements of SW480 cells after 30 min incubation (short term)
revealed a sharp decrease in diffusivity (pre-factor D) values with cells stimulated
with both 0.2 µM and 1 µM compared with the control (see Figure 3.9 (A)). D values of
cells incubated with 0.2 µM Tβ4 (1.94 x 10-4 µm2 ± 0.13 x 10-4) (mean ± SE) were 42%
smaller and with 1 µM (1.62 x 10-4 µm2 ± 0.15 x 10-4) 51% lower than that of the
control (3.33 x 10-4 µm2 ± 0.27 x 10-4). β coefficient values of cells treated with 0.2 µM
Tβ4 (1.47 ± 0.015) and 1 µM Tβ4 (1.44 ± 0.017) were significantly higher than that of
the PBS control (1.39 ± 0.013). No statistically significant differences could be seen
between β coefficient values of cells treated with 0.2 µM or 1 µM Tβ4.
Diffusivity values are influenced by a network of tension-carrying stress fibers
spanning the entire cell, which are constantly being restructured. Following short
term stimulation, lower bead diffusivity recorded in cells induced with both 0.2 µM
and 1 µM Tβ4 is a result of the restricted movement of beads bound tightly to the
cytoskeleton. This phenomenon is indicative of the higher amounts of polymerized
actin fibers compared to the total amount of actin in these cells, causing rigidity of the
cytoskeleton. On the other hand, we assume the persistence (β coefficient) values to
reflect the directional persistence of cytoskeletal remodeling events. The higher bead
persistence values of cells stimulated with 0.2 µM and 1 µM Tβ4 indicate that the actin
fibers of these cells are more aligned than those of the control. Taken together, these
results show that short term induction of cells with Tβ4 causes a pronounced
remodeling of the cytoskeleton due to the formation of stress fibers.
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Figure 3.9 Short term effects of extracellular Tβ 4 on cytoskeletal remodeling. The
spontaneous motion of fibronectin coated Ø 4.5 µm beads followed the power-law,
𝑴𝑺𝑫 = 𝑫 ∙ (∆𝒕/𝒕𝟎 )𝜷 + 𝒄. (A) After 30 min of induction, power-law cofactor, D, has
decreased with increasing concentrations of Tβ4. This indicates the immediate increase in
actin polymerization into stress fibers causing less diffusivity in bead movement due to the
effect of Tβ4 application. (B) The increase in the power-law exponent, β, values indicate an
alignment of cytoskeletal components. No statistically significant differences were recorded
with β values between cells induced with 0.2 µM and 1.0 µM Tβ4. Bars indicate one standard
error.
Long-term stimulation (>24 h) of SW480 cells reveal a biphasic response to increasing
concentrations of Tβ4. A decrease in bead diffusivity values of cells incubated with
0.2 µM Tβ4 (2.45 x 10-4 µm2 ± 0.16 x 10-4) (mean ± SE) compared with the PBS control
(3.22 x 10-4 µm2 ± 0.24 x 10-4) was recorded. Diffusivity values of cells treated
overnight with 1 µM Tβ4 (2.96 x 10-4 µm2 ± 0.30 x 10-4) are not significantly lower
than that of the control. Analyses of bead persistence, power-law factor β, show
distinct changes in the turnover rate of the cytoskeleton. Cells treated overnight with
0.2 µM Tβ4 have significantly lower β values Tβ4 (1.33 ± 0.012) compared with the
PBS control (1.39 ± 0.012). However, compared to the control, cells induced overnight
with 1 µM Tβ4 show a significant increase in bead persistence (1.47 ± 0.015) (see
Figure 3.10). Thus, long term stimulation with Tβ4 shows an apparent biphasic
response in cytoskeletal dynamics; cells treated overnight with 0.2 µM Tβ4 still show
increased formation of stress fibers resulting in lower diffusivity in the motion of
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bound fibronectin-coated beads compared to the control and 1.0 µM Tβ4. These
results are consistent with increase in stiffness and contractility seen in preceding
measurements in magnetic tweezer and 2D traction microscopy. The biphasic
response in the power-law β exponent show cells treated with 0.2 µM Tβ4 have a more
dynamic turnover rate of the actin cytoskeleton than that of cells treated with the
control or 1 µM Tβ4.
Figure 3.10 Long term effects of extracellular Tβ 4 on cytoskeletal remodeling.
(A) Significantly lower bead diffusivity, power-law cofactor, D, values were seen with 0.2 µM
Tβ4 treated cells after 24 h when compared with cells treated with 1.0 µM Tβ4 and the
control. No significant differences were seen between the control and 1.0 µM induced cells.
Here, only cells treated with 0.2 µM Tβ4 have retained increased levels of actin
polymerization. (B) The lowest β values were recorded on cells treated with 0.2 µM Tβ4 is
indicative of the significantly higher turnover rate of the cytoskeleton at this concentration.
Cells induced with 1.0 µM Tβ4 show a higher β value compared with the control. After 24 h of
induction, a biphasic response to increasing Tβ4 concentrations can be seen in SW480
cytoskeletal dynamics and production of actin stress fibers. Bars indicate one standard error.
When comparing the effects of long term and short term Tβ4 stimulation of SW480
cells, it becomes evident that the initial increase in the polymerization of actin fibers,
reflected by the drop in bead diffusivity (power law D coefficient values) after 30 min
stimulation with Tβ4, is transient. After 24 h, less stress fibers are formed in
stimulated cells, as seen with the slight rise in D values after 0.2 µM Tβ4 stimulation
and a rise to the same as the control with 1.0 µM shown in Figure 3.10 (A).
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Considering the dynamic reorganization of the cytoskeleton, Tβ4 induces a short term
decrease in the turnover rate of actin polymerization, seen with the increase in the
power-law exponent β. However, after 24 h, only cells treated with 0.2 µM exhibit a
more dynamic cytoskeleton. Taken together, the biphasic cellular response to Tβ4,
with a combination of higher formation of actin stress fibers and a more dynamic
turnover can be seen only on longer timescales with a peak at 0.2 µM stimulation.
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3.4
3.4.1
Tβ4 and ILK: The Biphasic Switch in 3D Cell Invasion Assays
Biphasic cellular regulation is by no means an isolated phenomenon. This type of
regulation has been found in the MAPK [95-97] and NF-kB signaling pathways [98]. In
both examples, two components in the signaling cascade are required to induce a
biphasic response. The first is involved in triggering the system’s activation whereas the
second regulates the concentrations of the members in the system to elicit the biphasic
response. Thus, an over-expression or reduced expression of a protein within the system
will lead to inhibition of the signaling cascade. This mechanism, also known as
combinatorial inhibition, may be involved in the biphasic response seen in cellular
mechanics and dynamics to Tβ4. Combinatorial inhibition usually occurs in the presence
of a molecule capable of binding and interacting with multiple molecules such as in
scaffolding and adapter proteins. In this chapter, we will examine this hypothesis closely
and present a model to explain the biphasic phenomenon involving Tβ4 and ILK.
Role of ILK as a molecular switch involved in the Tβ4 biphasic response
Several processes are involved in cell migration. As discussed previously, a major
factor in this event is the generation of contractile forces within the cell and the
transmission of these forces to the substrate [99]. Cell contractility is caused by actinmyosin contraction regulated by the phosphorylation of the myosin light chain (MLC)
domain. With its role as a Ca2+ independent myosin light chain kinase, studies have
linked ILK with cell contractility through its ability of phosphorylating MLC [100].
Recently, however, a contradictory role of ILK in the regulation of MLC has been
documented. Elevated activity of ILK has been known to negatively regulate RhoA.
RhoA plays an indirect role in the MLC pathway [101-102]. Through its effector, Rho
Kinase (ROCK), MLC phosphatase is phosphorylated and remains inactive, thus,
unable to dephosphorylate and inactivate myosin light chain. However, through the
inhibition of RhoA by ILK, MLC phosphatase is maintained in the active state and leads
to the inhibition of actin-myosin contraction.
In the cellular biphasic response seen throughout all our experiments, it is assumed
that exogenous Tβ4 activates ILK which in turn regulates actin-myosin contractility via
the direct phosphorylation of MLC or the negative regulation of RhoA. At this point,
these opposing pathways are not necessarily mutually exclusive events. Initially,
increasing concentrations Tβ4 triggers higher levels of active ILK which function as a
Ca2+ independent MLC kinase. This in turn leads to increasing levels of actin-myosin
contractility directly related to increased cell motility. At levels of Tβ4 exceeding
0.2 µM, increasing levels of RhoA inhibition overrides the ability of ILK to
phosphorylate MLC leading to a decrease in cell motility (see Figure 3.11).
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Figure 3.11 Tβ 4-ILK molecular switch. Upon initial activation by Tβ4, ILK assumes the role
of a Ca2+ independent [103] MLC kinase and phophorylates MLC at Thr18/Ser-19 which leads
to increased actin-myosin mediated contraction and increased cell motility. At higher levels
of exogenously applied Tβ4, the activity of ILK as a regulator of RhoA increases which leads to
increasing inhibition of ROCK. As a result, higher levels of active MLC phosphatase
dephosphorylate MLC, causing less actin-myosin contraction and a decrease in cell motility.
In this model, ILK acts as a molecular switch in the biphasic cellular response to increasing
levels of extracellular Tβ4.
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3.4.2
Bypassing RhoA inhibition: the effect of calyculin-A on Tβ4-induced cell
invasion
Tβ4 induced SW480 cells were treated with calyculin-A, an MLC phosphatase inhibitor
[104-105] to study the effect of bypassing ILK-induced RhoA inhibition. Through MLC
phosphatase inhibition, phosphorylated MLCs are kept at higher levels, thereby,
causing further stimulation of actin-myosin contraction [106]. Invasion assays were
carried out on SW480 cells incubated with Tβ4 concentrations of 0.2 µM and 1.0 µM
and 0.2 µM scrambled Tβ4 as a control. At each Tβ4 concentration level, invasion
assays were carried out with increasing levels of calyculin-A (0 nM, 10 nM and 50 nM).
Cells were allowed to invade into the collagen gel overnight before stimulation with
calyculin-A.
The general effect of MLC phosphatase inhibition is demonstrated clearly in the
control measurements with SW480 cells incubated with 0.2 µM scrambled Tβ4 and
10 nM and 50 nM calyculin-A stimulation. At pCum 10%, cells stimulated with
calyculin-A have invaded almost twofold deeper into the gel than the control. No
significant difference was seen between cells stimulated with 10 nM or 50 nM
calyculin-A. This trend is also observed in further invasion assays involving
stimulation with Tβ4. Cells stimulated with calyculin-A and 0.2 µM Tβ4 show a 20%
increase in invasion depth when compared with non-stimulated cells. However, when
compared with cells stimulated with only 0.2 µM Tβ4, cells induced with both
calyculin-A and 0.2 µM Tβ4 show up to 30% lesser invasion depth. A 28% increase in
cell invasivity is seen further with cells stimulated with both 1.0 µM Tβ4 and calyculinA when compared with non-stimulated cells. A dramatic increase of more than
twofold in invasivity of cells stimulated with both calyculin-A and 1.0 µM Tβ4 is seen,
however, when compared to cells stimulated with only 1.0 µM Tβ4.
Taken together, these results show that the motility of SW480 cells can be enhanced
through maintaining cell contractility by sustaining MLC phosphorylation. A steady
increase in three-dimensional cell motility is seen as a result of both the effects of
calyculin-A and Tβ4 stimulation. Experiments with both 1.0 µM Tβ4 and calyculin-A
show clearly that the role of ILK as a regulator of RhoA, which is assumed to be the
cause of the decrease in the motility of cells stimulated with only 1.0 µM Tβ4, is
compensated by enhancing MLC phosphorylation through calyculin-A. On the other
hand, the finite number of MLC available for phosphorylation is a limiting factor
leading possibly to a plateau in the combined effect of the two stimuli which can be
expected when applying higher concentrations of Tβ4 and calyculin-A.
The peak level of SW480 invasion seen with cells stimulated with 0.2 µM Tβ4 was not
observed with cells treated with both calyculin-A and 0.2 µM Tβ4. At this point, we can
66
Results
assume that the overall synergistic effect of Tβ4 and calyculin-A on MLC
phosphorylation is limited. MLC phosphorylation is not the only pathway determining
the increase in cell invasion in connection with Tβ4 stimulation of SW480 cells.
Figure 3.12 The combined effect of calyculin-A and Tβ 4 on the 3D invasion of SW480
cells into collagen gels. 3D invasion profiles of SW480 cells were analyzed after incubation
with both calyculin-A (at concentrations of 10 and 50 nM) and Tβ4. (A) Influence of only
calyculin-A on 3D cell invasion. Due to sustained cell contractility as a result of continued
phosphorylation of MLC, cells treated with 10 and 50 nM Calyculin invade twofold deeper
67
3
3
Results
into collagen gels. (B) Influence of calyculin-A combined with 0.2 µM Tβ4 on 3D cell invasion.
Cells treated calyculin-A and 0.2 µM Tβ4 invade 30% less than cells treated with only 0.2 µM
Tβ4. (C) Influence of calyculin-A combined with 1.0 µM Tβ4 on 3D cell invasion. Cells treated
with both calyculin-A and 1.0 µM Tβ4 were able to invade 28% deeper into gels than when
treated with only 1.0 µM Tβ4. (D) Summary: A steady linear increase in cell invasion depth
was recorded with the addition of calyculin-A to increasing concentrations of Tβ4. At a Tβ4
concentration of 0.2 µM Tβ4, however, cells treated with only Tβ4 were able invade 30%
further into collagen gel than with the addition of calyculin-A. This shows that prolonged cell
contractility due to the phosphorylation of MLC alone is not the only factor determining the
invasivity of SW480 cells after stimulation with Tβ4.
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Results
3.5
3.5.1
Tβ4 – membrane interaction
Until now, the effect and pathway of exogenously applied Tβ4 on cells is not known. The
mechanisms of entry or a cell surface receptor for this peptide have not yet been
discovered. So far, previous studies have given no explanation as to why minute
concentrations of Tβ4, as low as 0.1% of intracellular concentrations, are able to elicit
pronounced changes in cellular mechanics resulting in altered morphology and motility.
Due to its molecular properties, which will be examined further in this chapter, Tβ4
presents itself as a plausible candidate for membrane binding.
Determining the binding potential of Tβ4 to lipid membranes using
differential scanning calorimetry (DSC)
Amino acid analysis of Tβ4 showed that clusters of basic and hydrophobic residues
make up about 60% of the entire peptide. Basic amino acids are known to interact
with acidic domains on a lipid membrane. In addition, hydrophobic residues in the
peptide may enable its insertion into a lipid membrane and function as an anchor
[107]. NMR spectroscopic analysis of Tβ4 in solution has shown that the peptide is
largely unstructured in water and consists of two α helices in fluorinated alcohols
[30], the first helix extending from residues 4-16, and a second helix from residues 3040. Among the 43 amino-acids of the entire peptide, 9 residues are basic and 1 residue
is aromatic [108]. These characteristics combined make Tβ4 a good candidate for
membrane binding. Similar modes of membrane association have been reported for
other proteins such as myristoylated alanine-rich C kinase substrate, MARCKS [107]
and Human Immunodeficiency Virus Type 1, HIV-1 [109].
Using differential scanning calorimetry, studies were carried out to explore the lipidbinding ability of Tβ4. Theoretically, the insertion of the peptide into the lipid
membrane and interaction with the hydrophobic acyl chains causes the expansion,
realignment and destabilization of the phospholipid membrane. This effect lowers the
transition temperature of the lipid membrane from the gel to the fluid phase. Evidence
of this phenomenon is given as the decrease in the heat capacity and phase transition
enthalpy.
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3
Results
Figure 3.13 Lipid-binding qualities of Tβ 4 to DMPG/DMPC lipids analyzed using
differential scanning calorimetry (DSC). (A) DSC thermogram of 175 µM Tβ4 and the
control in a lipid mixture with a molar ration of 70:30 DMPC to DMPG. The distinct reduction
in the Tm of the Tβ4 lipid vesicle mixture indicates the insertion of the peptide into the lipid
bilayers. (B) Relative transition enthalpy (∆H/∆H0) as a function of increasing peptide
concentration. Raising the negative charge of the lipids caused only a slight decrease in
peptide insertion into the membrane. As expected, no changes in relative transition enthalpy
were recorded with increasing concentrations of insulin used as the control.
In this experiment, small unilamellar vesicles (SUVs) with different ratios of
DMPC/DMPG were incubated with increasing concentrations of Tβ4 (from 60 µM to
175 µM peptide). In these experiments, insulin, a molecule confirmed to having no
membrane-binding properties, was used as a control. An example of a thermogram
comparing the specific heat of 175 µM Tβ4 in lipid with a molar ratio of 70:30 DMPC to
DMPG and lipid only is shown in Figure 3.13 (A). A distinctive drop in the Tm of Tβ4 in
lipid indicates the insertion of the peptide into lipid bilayers.
In Figure 3.13 (B), the relative transition enthalpy (∆H/∆H0) as a function of
increasing peptide concentration is plotted for DMPC/DMPG lipid vesicles with molar
ratios of 70:30 and 30:70 to vary the strength of the negative charge of the lipid
vesicles. For both lipid types, a drop in the relative transition enthalpy was measured
with increasing peptide concentration. Raising the negative charge of the lipids caused
a slight decrease in peptide insertion into the membrane. As expected, no changes in
relative transition enthalpy were recorded with increasing concentrations of insulin.
These results are a clear indication of the membrane binding properties of Tβ4.
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Results
3.5.2
Fluorescence correlation
fluorescent Tβ4
spectroscopy
(FCS)
of
membrane-bound
Although DSC is a well established method for examining peptide-membrane
insertion, it cannot accurately measure binding kinetics. Moreover, this method is not
suitable for binding measurements for nanomolar concentrations, as loss of peptide
and lipid are bound to occur as they adsorb onto the walls of measurement containers
and pipettes. The fluorescence correlation spectroscopy (FCS) method, however,
allows for a direct measurement of single-molecules in solution.
Originally, FCS had been utilized to study molecular diffusion and reaction dynamics
[79, 110-111] . This method involves the measurement of fluorescence intensity
fluctuations caused by the movement of fluorescently labeled Tβ4 moving in and out
of a detection volume (1 µm3) defined by the focus of an excitation laser beam.
Unbound fluorescent peptide diffusing rapidly through the volume causes a highly
fluctuating fluorescence signal. Peptides bound to the much larger SUVs move at much
slower rates and result in a longer correlated pattern of bursts in the fluorescence
signal. Auto-correlation analysis of the fluorescence fluctuations yields information on
the diffusion kinetics of bound and unbound fluorescent Tβ4. Using this method, we
are able to resolve the single-molecule diffusion of the fluorescently-labeled Tβ4 on
lipid membranes.
The binding of Tβ4 to synthetic lipid membranes was examined with fluorescence
correlation spectroscopy (FCS) using Alexa488-labeled Tβ4 and SUVs. The SUVs had
PS/PC ratios of 1:5 and 1:6 to mimic cellular membranes, which have concentrations
between 10-30% of monovalent acidic lipids (mainly DMPS) [107]. While
measurements were made with increasing concentrations of lipid, the concentration
of Tβ4 was kept constant at a minimum of 50 ng Tβ4-Alexa488 /300 µl lipid buffer to
ensure that a minimum number of fluorophores on average (1..10) will be in the
detection volume at a time during the measurement. Control measurements were
done on Tβ4- Alexa488 in lipid buffer only. Single photon counts were registered at a
rate of 250 kHz for 10 s to maximize the resolution of yielded information.
Autocorrelation plots 𝐺(𝜏) obtained from each measurement were fitted according to
the normalized autocorrelation function (see Materials and Methods 2.2.8).
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Results
Figure 3.14 Lipid membrane binding of Tβ 4 measured through fluorescence
correlation spectroscopy. (A) Autocorrelation curves of Tβ4-Alexa488 in increasing
concentrations of 1:6 PS/PC lipids. There is no significant difference in lag times of the
measured fluorescent signal. (B) An increase in the amount of acidic lipids (1:5 PS /PC)
results in a significant right shift of autocorrelation curves with increasing lipid
concentrations. These results suggest longer lag times (τ) of the fluorescent peptide in the
detection volume, consistent with the binding of fluorescently labeled Tβ4 on much larger
SUVs. The concentration of Tβ4-Alexa488 was kept to a minimum (50 ng Tβ4/300 µl lipid
buffer) to limit the particles in the detection volume for measurement efficiency.
Autocorrelation curves are taken as an average of 10 measurements. (C) A typical pattern of
photon count rate as a function of time for Tβ4-Alexa488 in lipid buffer only. (D) Fluorescent
signal pattern of Alexa488- Tβ4 in 5 µM 1:5 PS/PC. Labeled SUVs result in a photon count rate
bursts and higher signals.
72
Results
Figure 3.14 (A) shows the autocorrelation plot of Tβ4-Alexa488 in increasing lipid
concentrations (PS/PC of 1:6) and the control (no lipids). Each curve is obtained from
an average of 10 measurements. As seen in Figure 3.14 (A) The addition of a 1:6
DMPS/DMPC lipid mixture (concentration ranging from 1 – 5 µM) to an Alexa488-
labeled Tβ4 solution caused only a marginal change in the correlation time (τD) when
compared to control (no lipids added), indicating that the peptide did not bind to or
insert into the SUV lipid membrane. At a DMPS/DMPC ratio of 1:5, however, the
correlation time increased dramatically with increasing lipid concentrations,
indicating that Tβ4 bound to the lipid membrane as seen in Figure 3.14 (B) and Figure
3.15, confirming that Tβ4 and not the fluorophore bound to the SUVs. Taken together,
these results show that Alexa488-Tβ4 is able to bind to lipid membranes, and
furthermore indicate that basic residues in the Tβ4 peptide are mainly responsible for
lipid binding through their interaction with acidic lipid head groups. A similar
mechanism of binding was reported previously for the effector domain of MARCKS
(residues 151-175) [107].
The insertion and binding of extracellular Tβ4 to the cell plasma membrane offers a
mechanism of how the local concentration of the peptide can be greatly increased
while at the same time its diffusion is greatly decreased so as to increase the reaction
probability with other membrane-associated signaling proteins such as ILK and
PINCH [112-113]. Previous studies have shown the co-localization of actin-free Tβ4
and ILK around cell edges in migratory cells [55]. In addition, the binding affinity of
Tβ4 with G-actin is much higher (dissociation constant, Kd = 0.5 – 2.5 µM, [114])
compared to binding with the lipid membrane (Kd = 72 µM as measured in this study
using FCS and lipid vesicles with a DMPS/DMPC ratio of 1:5). Therefore, intracellular
Tβ4 is not expected to display a pronounced association with the cell membrane in the
presence of G-actin. This explains our finding that the binding of small concentrations
of extracellular Tβ4 to the lipid membrane can induce dramatic cellular responses
even in the presence of large intracellular concentrations of Tβ4.
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3
Results
Figure 3.15 The effect of acidic lipid head groups on the binding of Tβ4 -Alexa488 on
membranes. Correlation times of Tβ4-Alexa488 in the presence of SUVs for varying lipid
concentrations with a PS/PC ratio of 1:5 (squares) or 1:6 (triangles). As control, Alexa488
fluorophore without Tβ4 was added to lipids with a PS/PC ratio of 1:5 (circles). Error bars
(mean ± SE, n=10) are not shown when smaller than data symbols.
74
4 Discussion
4
Discussion
4.1
4.1.1
The effects of Tβ4 on Cellular Mechanics and Motility
The results obtained from the course of this work have shown that Tβ4, when applied
exogenously to SW480 cells, does not act classically as an actin sequestering protein. Past
studies give evidence of Tβ4 having multiple functions [34]. The following section
discusses in depth how Tβ4 can affect the mechanics of the cytoskeleton and the
regulation of SW480 cell motility.
Tβ4 regulates the stiffness and contractility of SW480 cells
The stiffness of a cell is a measure of the number of the interactions and the elasticity
of bonds in the cytoskeletal network which transfers mechanical forces from the cell
to the fibronectin-coated bead. The degree of internal cellular prestress, cell geometry,
the thickness of the cell below the fibronectin-coated bead, the number of cell surface
integrins bound to the bead, and the number of focal adhesions and associated
proteins involved in binding the bead are all factors that influence the stiffness of a
cell. It has been shown so far that up to a concentration of 0.2 µM, that increasing
amounts of exogenously applied Tβ4 results in the increase in stiffness of SW480 cells.
The forces during the tweezing experiments that are needed to detach the fibronectin
coated bead from the cell characterize the bead binding strength. As with cell stiffness,
the bead binding strength is related directly to the number of molecular interactions
that relay mechanical forces between the integrin-bound bead and the cytoskeleton
[18-19]. These include the connections in the cell involved in the out-in force
transmission, such as the fibronectin-coated bead with the integrins, the integrins
with the focal adhesion proteins, and the focal adhesion proteins with the
cytoskeleton. Central in this network are mechano-transducers in the focal adhesion
complex. Bead binding strength also depends on how these structures yield to the
forces. In experiments involving externally induced forces of up to 10 nN, no
difference in bead detachment was seen across all concentrations of Tβ4 used and the
control. This leads to the conclusion that Tβ4 does not compromise the effect of force
transmitting elements between integrins, focal adhesions and the cytoskeleton.
The yielding of the cell to pulling forces exerted onto the fibronectin-coated bead can
also be measured by recording the differential cell stiffness at increasing forces.
Irreversible yielding can occur through induced stress and will decrease the stiffness
of the cell. The stretching of the cytoskeleton under stress results in the non-linear
response of cellular mechanics. According to most models, this phenomenon has its
origins from the prestress or contractile tension of the filamentous cytoskeletal
network. Cellular prestress is a mechanism responsible for the stability of cell shape
[93, 115] and is caused by acto-myosin contraction. The prestress can be estimated
from the tractions generated through the adhesion of cells onto the extracellular
matrix. In tweezing experiments, stepwise increasing external force from 0.5 nN to
76
Discussion
10 nN in 0.2 µM Tβ4 treated cells produced less increase in stiffness (one and a half
fold increase) when compared with the control and 1 µM Tβ4 treated cells (both over
twofold increase in stiffness). Taken the observations in bead detachment and cellular
prestress together, it can be concluded that although Tβ4 may not alter passive force
transmission through the focal-adhesion complex, up to a maximum working dose of
0.2 µM, it does, however, influence the active acto-myosin contraction regulating
cytoskeletal pre-stress in SW480 cells.
In each individual creep measurement across all concentrations of Tβ4, the recorded
response of bead displacement over time followed a power law, in agreement with
past literature [89-90, 116-119]. A higher power-law exponent value, b, would
indicate a more viscous system and, in sum, a higher turnover of interactions that
transmit the externally exerted stress intracellularly. When comparing the power-law
exponent, b, of SW480 cells, no statistically significant difference was recorded at all
force steps throughout all Tβ4 concentrations and the control. Even at forces of 10 nN,
no change of the power-law exponent was observed for all concentrations. Thus, Tβ4
does not result in a change of yielding of the cell to increasing induced stress, nor does
it contribute to the fluidization of the force transmitting structures within the cell. As
in the observations in stress stiffening, these results confirm that Tβ4 does not affect
the transmission of externally induced forces from the extracellular environment,
through the integrins and focal adhesion to the cytoskeleton. Taken together, all the
results so far have shown that Tβ4 does not cause fluidization of the cytoskeleton as
would be the case if the peptide would act predominantly as a classical actinsequestering protein.
While magnetic tweezing assays record the active cellular forces resisting the
externally induced deformation of the cytoskeleton, cellular traction microscopy
measures the contractile prestress, or the intrinsic strain energy stored within the
matrix. Although SW480 cells do not exhibit a pronounced development of stress
fibers, they generate substantial traction forces when compared to other cell types
[75, 120]. SW480 cells treated with 0.2 µM Tβ4 showed more than a fourfold increase
in strain energy generated per cell when compared with the negative control. A tight
correlation between cell stiffness and contractility has been shown in previous studies
[19, 121]. Extensive experimental evidence have shown that cell traction forces are
generated through acto-myosin contraction induced by the phosphorylation of the
myosin light chain (MLC) by myosin light chain kinase (MLCK) and Rho-kinase [3, 88,
122]. In line with the effect of Tβ4 on cellular stiffness and prestress discussed
previously, these results show that up to a concentration of 0.2 µM, Tβ4 plays a role in
actively influencing the acto-myosin contraction in SW480 cells.
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4
4
Discussion
4.1.2
Tβ4 regulation of acto-myosin contractility is involved in the biphasic
response of SW480 cell morphology and motility
Maximum levels of cellular stiffness and contractility of SW480 cells were reached
using a Tβ4 concentration of 0.2 µM. The use of Tβ4 concentrations higher than 0.2 µM
causes a reduction in cell stiffness; SW480 cells treated overnight with 1 µM Tβ4 did
not show a significant difference in stiffness or cytoskeletal prestress to those treated
with the control. The same phenomenon was observed in the traction microscopy
assays using increasing amounts of Tβ4 for the stimulation of SW480 cells. Cells
treated with 1.0 µM Tβ4 showed a reduction in strain energy per cell when compared
with cells treated with 0.2 µM Tβ4. As discussed previously, no difference in bead
detachment and cellular yielding to externally induced distortion was recorded across
all force steps at all concentrations of Tβ4. These results indicate that the biphasic
response to Tβ4 seen in SW480 cell stiffness and tractions is due to its active biphasic
regulation of acto-myosin contractility. At this point so far, the assays indicate that the
biphasic cellular response of Tβ4 stimulation does not affect the mechano-coupling
processes of force transmission mediated by the focal adhesions.
The cell interacts with its environment by applying tractions through the integrins to
adhere to the extracellular substrate or matrix [123]. In a feedback loop, the tension
from the cell is resisted by the extracellular matrix and results in an out-in signaling,
which triggers cellular responses that include cell motility, differentiation or growth.
The cycle of acto-myosin contractility is one of the major processes governing the
tension within the cell. As discussed previously, acto-myosin interaction is promoted
through the phosphorylation of the regulatory myosin light chain (MLC) [124-125] by
MLCK (myosin light chain kinase). The activity of MLCK is controlled by the activity of
ROCK and RhoA. Through the action of ROCK, MLC is not dephosphorylated and,
thereby, remains in an active state.
The extracellularly applied Tβ4 forms a complex with PINCH to activate ILK, a focal
adhesion scaffolding protein that interacts directly with cell surface integrins and that
is known to be activated by Tβ4 [40]. Here, ILK plays a dual role; it acts as a Ca2+
independent myosin light chain kinase [103] as well as a RhoA regulator [102].
Initially, at lower levels of Tβ4, ILK promotes the phosphorylation of MLC, resulting in
increased acto-myosin contractility as confirmed in the magnetic tweezer and
tractions microscopy experiments; up to a working concentration of 0.2 µM Tβ4 cells
become stiffer and more contractile. At concentrations of Tβ4 exceeding 0.2 µM,
however, the action of ILK as a RhoA regulator may inhibit the action of ROCK.
Through the dephosphorylation and inactivation of MLC, acto-myosin contraction
decreases, as seen in assays with 1.0 µM Tβ4 induction of SW480 cells. In this effect,
78
Discussion
the interaction of Tβ4 and ILK becomes essentially a cellular sensor for outside-in
signaling governing cell contractility.
The consequences of the biphasic regulation of acto-myosin contractility are
manifested directly in the morphology and motility of SW480 cells. On 2D surfaces,
cells treated with 0.2 µM Tβ4 were observed to adopt a more polarized form with a
distinct formation of a leading edge and trailing edge. Past literature gives evidence
that point to a pivotal role for myosin II activity in maintaining cell polarity [87]. The
ability of cells to move on 2D surfaces [27, 99] is influenced by the adhesion strength,
adhesion dynamics and cytoskeletal remodeling. In this effect, a cycle of detachment
and adhesion to the extracellular substrate occurs in a migrating cell [87]. The
migration speed is inversely correlated with the adhesion strength of the cell to the
extracellular substrate. Cells which are highly adherent have typically large integrin
clusters and low migration speeds. This phenomenon was observed in 2D cell motility
assays of SW480 cells. Although cells treated with 0.2 µM Tβ4 were seen to move
slower (lower apparent diffusivity values, D, in µm2 per second), they showed a high
persistence in movement (power-law coefficient value). With an extracellular
induction of 1.0 µM Tβ4, lower levels of tractions and cell migration persistence were
recorded in SW480 cells. Thus, the effect of Tβ4 at a maximum working concentration
of 0.2 µM on 2D cell migration is the combination of increased acto-myosin induced
contractility and the persistence of cell movement.
Compared to movement on 2D substrates, a three dimensional extracellular matrix
provides a physical obstruction to cell movement. According to Zaman et al. [126127], four biophysical processes influence the speed of cell invasion in 3D
environments: 1) contractile forces are needed to overcome the steric hindrance of
the extracellular matrix, 2) these forces are transmitted to the surrounding matrix
through cell surface adhesions (integrins) and must be in a balance with detachment
from the matrix to allow the cell to move itself forward. 3) As the cell moves through
the matrix, it undergoes massive changes in cell morphology; the cell’s intrinsic force
resisting the change in shape must be small otherwise a dynamic remodeling of the
cytoskeleton must accommodate for this change, 4) The physical obstruction in form
of elastic forces provided by the matrix must be low enough for the cell to squeeze
through otherwise the cell must degrade the ECM through the production of matrix
proteinases [27].
Again, a distinctive biphasic response in three dimensional cell morphology and
migration of SW480 cells in collagen gels was also observed with increasing
concentrations of Tβ4 induction. Previous studies have shown than cells
overexpressing Tβ4 have increased levels of matrix metalloproteinase-7 (MMP-7) [48]
coupled with lower levels of E-cadherin causing the disruption of cell-cell contacts.
79
4
4
Discussion
This phenomenon has been correlated with the increase in ILK activity [62]. The
combination of all these biochemical responses leads to the degradation of the ECM
and the epithelial-mesenchymal transition of the originally non-malignant SW480
cells, enabling a single cell to break away from a non-motile cell colony and invade
through the ECM.
Although levels of MMP-7 and E-cadherin were not measured in SW480 cells induced
with extracellular Tβ4 in the assays performed, changes in cell shape as seen in the
overexpression of Tβ4 in SW480 cells was observed in 3D collagen gels; cells treated
with 0.2 µM Tβ4 developed a mesenchymal morphology with a fibroblastic
appearance and long extended filapodia. Here, it can also be assumed that up to a
concentration of 0.2 µM, the effect of acto-myosin contractility increases, as does the
decrease in E-cadherin associated with the altered shape of the cells to a more
fibroblastic morphology. These effects combined with the production matrix
proteinases have enabled cells to move furthest within a 3D collagen matrix as shown
in the invasion assays with an induction of 0.2 µM.
With a treatment of 1.0 µM Tβ4, however, cells show a mixed morphology of round
cells in clusters and differentiated fibroblastic-like cells. The reduction in invasivity of
the SW480 cells is in part due to the lower levels contractility of these cells as a result
of RhoA inhibition. At this point, it is not known whether the levels of MMP-7 or Ecadherin expression due to Tβ4 induction have contributed to this effect. For more
conclusive evidence, this must be looked into further.
The inhibition of cell contractility at higher levels of Tβ4 induction (at 1.0 µM working
concentration) was bypassed using calyculin-A in 3D cell invasion assays. As a MLC
phosphatase inhibitor, calyculin-A sustained phosphorylation of MLC and, thereby,
maintained the contractility of SW480 cells. Use of calyculin-A (10 nM and 50 nM) and
Tβ4 of up to 1 µM together produced a progressive increase in 3D cell invasivity. No
biphasic response with the increase in Tβ4 working concentration in cell treatment
was recorded. In 3D invasion assays with calyculin-A and Tβ4 induction of 0.2 µM, the
recorded SW480 cell invasivity was lower than that of with using 0.2 µM Tβ4 alone.
Taking these results together, it is shown that sustaining overall cell contractility
alone will not account for the increase in cell invasion. It can be concluded here that
the balance in cell adhesion, contractility and dynamic remodeling of the cytoskeleton
is essential for the motility and, finally, the invasivity of a cell. Shifts in this systematic
balance will have a dramatic effect on cell migration. In sum, in addition to the
regulation of MLC phosphorylation through a putative Tβ4-PINCH-ILK pathway, it may
be also the influence of that pathway on RhoA activity and the associated effects on
80
Discussion
cell polarity, adhesion behaviour and cytoskeletal dynamics that contribute to the
pronounced biphasic response in cell invasiveness.
81
4
4
Discussion
4.2
Tβ4 and the cell lipid membrane: a novel mechanism for outside-in
signaling
The cellular plasma membrane is not only a biological barrier between the cell interior
and the extracellular environment; it is involved in a myriad of cellular process including
cell adhesion, biochemical signal transduction [128] and molecular transport. The
cellular lipid membrane provides an anchor to the cytoskeleton, a scaffold for
cytoskeletal and membrane associated proteins involved in outside-in and inside-out
signal transduction. This section discusses the novel mechanism of Tβ4 lipid binding and
its consequences in the signaling cascade and cellular responses.
Firstly, the binding of Tβ4 to synthetic lipid membranes in the form of small
unilamellar lipid vesicles (SUVs) was recorded in DSC measurements using a mixture
of DMPC/DMPG lipid vesicles. Increasing the overall negative charge of the lipids by
increasing the DMPG lipid portion resulted in a slight increase in binding capacity of
the peptide only at very high concentrations (175 µM). As a slightly negatively
charged protein with an isoelectric point of 5.1, Tβ4 will only bind weakly to neutral
lipids. Consequently, by increasing the negative charge of the lipids, electrostatic
binding of Tβ4 to the membrane was not enhanced significantly. In DSC
measurements, the change in specific heat is recorded over a large time span and
gives only a general indication of the binding capacity of Tβ4 to SUVs. Due to this, the
kinetics of peptide-lipid binding cannot be resolved with this method. Furthermore,
measurement inaccuracy due to the loss of peptide and lipids, which usually adsorb to
the walls of assay containers and pipettes during the course of the experiment, cannot
be avoided.
The binding of Tβ4 to synthetic lipid membranes was examined further and confirmed
with the FCS method using Alexa488 labeled Tβ4 and a PS/PC lipid vesicle mixture. As
expected, increasing the mole fraction of acidic lipids (PS) and, thereby, allowing more
electrostatic interaction between the basic residues on the Tβ4 peptide and the lipid
vesicles resulted in the dramatic increase in Tβ4-lipid binding. The FCS technique has
been used extensively to study intermolecular interactions of complimentary DNA
oiligomers [78], protein-protein interactions between membranes, and the binding of
myristoylated alanine-rich C kinase substrate (MARCKS) [107] to synthetic lipid
vesicles. Not only does FCS allow for the use of only minute quantities of Tβ4 peptide,
single-molecule events of protein-lipid binding can be recorded. The main advantage
of using this method is that it allows for the measurement at very short time scales,
which enables the quantification of the protein-lipid binding kinetics.
While the basic structure of the cellular membrane is given by the lipid bilayers, its
functions are carried out by membrane proteins. The number and types of proteins
can differ vastly according to the specific function of the membrane and type of cell
[85]. Membrane proteins are known to span or insert into a lipid bilayers through a
82
Discussion
single or multiple α-helices. Many of these proteins are amphipathic; meaning some
regions of the protein are hydrophobic and some hydrophilic. Hydrophobic residues
of the peptide can react with the hydrophobic tails of the phospholipid molecules
located inside the lipid bilayers, whereas the hydrophilic residues lie in water either
on the cytoplasmic side or the extracellular side of the membrane [85].
Figure 4.1 3D stucture of Tβ 4. Shown is the structure of the Tβ4 peptide as generated by the
molecular visualization software, PyMOL. In this conformation, the peptide is made up of two
α-helices, the first from residues 4 – 16 and the second from 30 – 40. 60 % of the peptide is
made up of basic and hydrophobic residues; basic residues seen in blue, hydrophobic in red.
NMR spectroscopic analysis of Tβ4 in solution have shown that the wild-type Tβ4
peptide is largely unstructured in water and are made up of two α-helices in
fluorinated alcohols [30]; the first helix extending from residues 4 – 16 and a second
helix from residues 30-40. Amino acid analysis of Tβ4 showed that clusters of basic
and hydrophobic residues make up about 60% of the entire peptide (Figure 4.1).
Among the 43 amino-acids making up the entire peptide, 9 residues are basic and 1 is
aromatic [108]. These characteristics combined, make Tβ4 a good candidate for
membrane binding. Similar modes of membrane association have been recorded for
other proteins such as MARCKS [107] and HIV-1 [109]. The insertion or translocation
of proteins, in this case Tβ4, either from the extracellular environment to the cell
plasma membrane will not only increase the concentration of the peptide at this
location in the membrane up to a thousand fold [107], it will reduce significantly the
diffusion time so as to increase the reaction probability with other membraneassociated signalling proteins, such as ILK and PINCH. Thus, the mechanism of Tβ4
binding to the lipid membrane would explain the phenomenon of how induction with
minute concentrations of Tβ4 can elicit a dramatic cellular response.
83
4
4
Discussion
4.3
The Tβ4-ILK Molecular Switch
In this section, a putative model for the biphasic response of Tβ4 on cellular response on a
global scale is elucidated. A direct correlation will be made from the biophysical results
measured and observed in the course of this work with past biochemical experiments
done in vitro and in vivo. Finally, future implications of this work in clinical therapeutic
application are discussed.
Previous studies have associated Tβ4 with increased cellular motility and invasivity.
The genetic over-expression of Tβ4 caused the epithelial-mesenchymal transition and
the subsequent increase in invasivity of SW480 colorectal carcinoma cells, an
otherwise non-malignant cell line [47-48, 62]. In embryonal cardiomyocytes,
extracellular treatment with Tβ4 induced increased migration in cardiac outflow tract
explants [40, 129]. Both phenomena have been associated with the activity of ILK. In
previous work, however, no distinction has been made between the effects of
extracellular induction and genetic overexpression of Tβ4 cellular responses. The
dose-dependent effect of Tβ4 application and overexpression on cell motility has yet
to be quantified.
The results of this work have demonstrated the direct biphasic cellular response of
SW480 extracellular induction in cell stiffness, contractility and invasivity. A novel
mechanism of the Tβ4 binding to the cell membrane has also been shown. Close
analysis of the recorded cellular biophysical responses and Tβ4 –membrane binding in
this work combined with extensive knowledge of the biochemical pathways involved
with Tβ4 expression, extracellular stimulation and its interaction with ILK retrieved
from past literature have contributed to the following model.
84
Discussion
85
4
4
Discussion
Figure 4.2 The regulation of SW480 cellular mechanics in response to extracellular Tβ 4
concentration – a biphasic molecular switch mechanism. A schematic diagram of the
biphasic cellular responses in stiffness, spreading, tractions and motility due to increasing
levels of exogenous Tβ4 stimulation; at low extracellular concentrations, Tβ4 activation of ILK
results in the phosphorylation of MLC, acto-myosin contractility and consequent increase in
cell stiffness and contractility leading to increased motility. At concentrations above 0.2 µM,
Tβ4 and ILK activity leads to the inhibition of RhoA and a decrease in MLC phosphorylation.
It is known that Tβ4 circulates in serum and wound fluid; its accumulation is assumed
to be a product of cellular secretion or its release from damaged cells [34]. Near the
wound, a concentration gradient is present with the highest concentrations of Tβ4
directly at the wound site (see Figure 4.2 (B)). As passively circulating cells approach
the wound, the cells responsive to Tβ4 stimulation such as SW480 cells, ‘sense’ the Tβ4
concentration gradient. Tβ4 starts to bind to the cell membrane and then interacts
with ILK, a membrane associated protein. Local Tβ4 concentrations at the membrane
are amplified, thus, even minute amounts of extracellular Tβ4 can trigger a response.
At lower Tβ4 concentrations, Tβ4 activates ILK which acts as a Ca2+ independent MLC
kinase, increasing MLC phosphorylation and, consequently, causing acto-myosin
mediated cell contractility. As a result, the cell moves persistently in the direction of
the concentration gradient. Above a threshold level of Tβ4 in the extracellular
environment, the further activation of ILK results in the negative regulation of RhoA
(see Figure 4.2 (C)), leading gradually to the ablation of cell contractility and
invasivity as it reaches the site of the wound. In the wound environment, the cell
generates enough tractions to adhere at the site [87] without further movement.
The role of Tβ4 as a molecular switch in cell motility lies, firstly, in its role as a
membrane-binding peptide and, secondly, in its interaction with ILK as a molecular
switch. The extent of the cellular response to Tβ4 concentrations in the extracellular
environment may depend on the individual characteristics of the membranes in
different cell lines such as the fraction of acidic phospholipids and the overall
electrostatic charge of the membrane. Thus, it may be assumed that not all cells will
respond in the concentration gradient as shown in SW480 colorectal carcinoma cells.
The complicated dual role of ILK in regulating cell contractility by, on one hand,
negatively regulating RhoA [102, 130] and, on the other hand, phosphorylating MLC as
a Ca2+ independent MLC kinase has already been documented extensively in the past
[131]. However, by elucidating the biphasic cellular response of Tβ4, the two opposing
pathways are reconciled in the function of Tβ4 as a molecular switch in regulating
SW480 cell motility.
86
Discussion
As demonstrated in this work, it is clear, that several aspects must still be considered
in developing Tβ4 as a potential therapeutic agent. Firstly, studies involving the topical
or extracellular application of Tβ4 should be separated from those involving the
overexpression of the peptide. Secondly, the overall consequences of Tβ4 application
must be considered in the mitigation of each pathological condition. For example, in
the course of accelerating wound healing, concerns must be raised with regard to the
role Tβ4 plays in tumor metastases and invasivity. Until now, no in vitro or in situ
experimental data has been recorded on the effect of Tβ4 application on existing
tumors. Finally, the sensitivity of individual cell types to Tβ4 concentrations may vary
greatly due to the unique membrane characteristics distinguishing one cell type from
the other.
87
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Acknowledgements
First and foremost, I would like to thank my supervisor, Prof. Ben Fabry, for his
guidance and support. Through Prof. Fabry, I was able to build a network of contacts
and participate in numerous international conferences to pursue further my scientific
endeavors. Many thanks also to Prof. Wolfgang Goldmann for the insightful comments
and constructive criticism.
I would like to express my gratitude to Prof. Ewald Hannappel, who introduced me to
the world of Thymosins and, in particular, Thymosin beta 4. His profound knowledge
in this field had singularly inspired me to begin my work with Tβ4. The motivation and
feedback he provided as well as the constant supply of the potent peptide were all key
ingredients to the success of this study. A special acknowledgement here to Christine
App, Prof. Hannappel’s PhD student, for producing the much coveted fluorescently
labeled Tβ4 and all her help throughout my dissertation.
For funding my research and making this study possible, I would like to thank the
International Max-Planck Research School Physics of Light and their staff.
Thanks to all my colleagues during my stay at the institute: Carina, Phillip, Daniel,
Claudia, Martina, Navid, Nadine, Johannes, Anna and Thorsten not only for motivating
me but also for supporting my strengths and enduring my weaknesses. I would like to
give special credit to Nadine for her direct contribution to the FCS setup and
measurements. Very special thanks to my roommates, Anna and Thorsten, for their
constant and everlasting friendship and good conversation, scientific and otherwise,
and to Johannes, our auxiliary member, for showing me the lighter side of physics.
Thank you to Prof. Ana Smith for her inexhaustible enthusiasm, wisdom and
optimism.
Last but never least; I would like to express my deepest gratitude to all my family
members for their unwavering support, encouragement and patience throughout the
years leading to the completion of this dissertation. This work is dedicated to my
daughters, Sanna and Ellen, my constant source of strength and inspiration.
95
Publications/Conferences
Lorenz C, Brunner JG, Kollmannsberger P, Jaafar L, Fabry B, Virtanen S.
Effect of surface pre-treatments on biocompatibility of magnesium.
Acta Biomater. 2009 Sep;5(7):2783-9. doi: 10.1016/j.actbio.2009.04.018. Epub 2009
May 4
Jaafar L, App C, Lang N, Hannappel E, and Fabry B
The Effect of Extracellular Thymosin β4 on Cell Mechanics and Motility
PLoS One, 2014 Jan, submitted
Cell Biomechanics Meeting, Paris (Sept. 2005)
Invited talk
Title : The Role of Vinculin in Cellular Mechanotransduction
ASCB Annual Meeting, San Francisco (Dec. 2005)
Poster Session
Title : Role of Vinculin in Cytoskeletal Dynamics and Regulation
Cell Biomechanics Meeting, Barcelona (Sept. 2007)
Invited talk
Title : Effects of Thymosin β4 on Cytoskeletal Organization
Biophysical Society 50th Annual Meeting, Salt Lake City (Feb. 2006)
Poster Session
Title : Vinculin Regulates Cytoskeletal Dynamics and Prestress in the Cell
Thymosins in Health and Disease (Oct.. 2009)
Poster Session
Title : Effects of Thymosin β4 on Cytoskeletal Organization
96
Curriculum Vitae
Personal information
Birth place and date:
Kuala Lumpur, Malaysia, 09.07.1975
Address:
Max-Busch-Str. 22
91054 Erlangen
leilaatwork@yahoo.com; leila.jaafar@areva.com
Citizenship:
Malaysisch
Tertiary Education
2006- present
2005 – 2006
2000 –2005
1999 –2000
1994 –1997
Doctoral studies in Biophysics
Biophysics Group (Prof. B. Fabry)
FAU Erlangen-Nuremberg, Germany
Diploma thesis in Molecular Biology
Department of Microbiology (Prof. M. Niederweiß)
FAU Erlangen- Nuremberg, Germany
Undergraduate studies in Biology (Diploma)
FAU Erlangen- Nuremberg, Germany
German language course for the
Deutschen Sprachprüfung für den Hochschulzugang
(DSH)
Undergraduate studies in Medicine (MBBS)
University of New South Wales, Sydney, Australia
Primary/Secondary Education
1993 –1994
1987 –1992
UNSW Foundation Certificate
University of New South Wales Foundation Course
Kuala Lumpur, Malaysia
Malaysian High School Certificate
Sri Aman Secondary School
Kuala Lumpur, Malaysia (1987-1992)
97
1982 –1986
Awards
2006 – 2010
Elementary school
Louise Archer Elementary School
Vienna, Virginia, USA
Stipend for doctoral studies
International Max-Planck Research School for
Physics of Light
Job Experience
2013 - present
2010 - 2013
1999 - 2000
98
Project manager
R&D/Product Development, Containment Systems
AREVA GmbH
Computational Engineer
Radiation Protection & Neutron Fluence
AREVA GmbH
Software User-Interface and Testing for Syngo
Siemens Medical Solutions
Siemens AG