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On the spontaneous motion of cytoskeletally bound markers [Elektronische Ressource] / vorgelegt von Carina Raupach

179 pages
On the spontaneous motionof cytoskeletally bound markersDer Naturwissenschaftlichen Fakult¨atder Friedrich-Alexander-Universit¨at Erlangen-Nurn¨ bergzurErlangung des Doktorgradesvorgelegt vonCarina Raupachaus DormagenAls Dissertation genehmigt von der Naturwissenschaftlichen Fakult¨atder Universit¨at Erlangen-Nurn¨ bergTag der mundlic¨ hen Prufung:¨ 25.07.2008Vorsitzender der Promotionskommission: Prof. Dr. Eberhard B¨anschErstberichterstatter: Prof. Dr. Ben FabryZweitberichterstatter: Prof. Dr. Fran¸cois GalletContents1 Introduction 12 The cell 52.1 General properties of eukaryotic cells . . . . . . . . . . . . . . . . . . . 52.2 The cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62.3 Cell adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62.3.1 Integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72.3.2 Focal adhesions . . . . . . . . . . . . . . . . . . . . . . . . . . . 82.4 Cell motility and migration . . . . . . . . . . . . . . . . . . . . . . . . 82.4.1 Intracellular motility . . . . . . . . . . . . . . . . . . . . . . . . 82.4.2 Cellular motility . . . . . . . . . . . . . . . . . . . . . . . . . . 92.5 Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Nanoscale particle tracking 113.1 The technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113.2 Experimental setup . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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On the spontaneous motion
of cytoskeletally bound markers
Der Naturwissenschaftlichen Fakult¨at
der Friedrich-Alexander-Universit¨at Erlangen-Nurn¨ berg
zur
Erlangung des Doktorgrades
vorgelegt von
Carina Raupach
aus DormagenAls Dissertation genehmigt von der Naturwissenschaftlichen Fakult¨at
der Universit¨at Erlangen-Nurn¨ berg
Tag der mundlic¨ hen Prufung:¨ 25.07.2008
Vorsitzender der Promotionskommission: Prof. Dr. Eberhard B¨ansch
Erstberichterstatter: Prof. Dr. Ben Fabry
Zweitberichterstatter: Prof. Dr. Fran¸cois GalletContents
1 Introduction 1
2 The cell 5
2.1 General properties of eukaryotic cells . . . . . . . . . . . . . . . . . . . 5
2.2 The cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.3 Cell adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.3.1 Integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.3.2 Focal adhesions . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.4 Cell motility and migration . . . . . . . . . . . . . . . . . . . . . . . . 8
2.4.1 Intracellular motility . . . . . . . . . . . . . . . . . . . . . . . . 8
2.4.2 Cellular motility . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.5 Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3 Nanoscale particle tracking 11
3.1 The technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3.2 Experimental setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.3 Bead tracking algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.4 Drift and noise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4 Statistics of the spontaneous bead motion 17
4.1 Mean square displacement . . . . . . . . . . . . . . . . . . . . . . . . . 17
4.2 Power spectral density of velocities . . . . . . . . . . . . . . . . . . . . 21
4.3 Turning angle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
4.4 Fluctuations in velocity and directionality in bead motion over time . . 28
4.5 Step size distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.6 Examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
5 Interpretation of the spontaneous bead motion 39
5.1 Stress fluctuations in the extracellular matrix . . . . . . . . . . . . . . 39
5.1.1 Trajectories and mean square displacement . . . . . . . . . . . . 40
5.1.2 Persistence and superdiffusivity . . . . . . . . . . . . . . . . . . 42
5.2 Bead motion versus stress fiber orientation . . . . . . . . . . . . . . . . 44
5.2.1 Experimental approach . . . . . . . . . . . . . . . . . . . . . . . 44
5.2.2 Analysis methods . . . . . . . . . . . . . . . . . . . . . . . . . . 45
5.2.3 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . 46ii CONTENTS
5.3 Two-point microrheology . . . . . . . . . . . . . . . . . . . . . . . . . . 48
5.3.1 The technique and its background . . . . . . . . . . . . . . . . . 48
5.3.2 Application to cytoskeletally bound beads . . . . . . . . . . . . 50
5.3.3 Discussion and interpretation . . . . . . . . . . . . . . . . . . . 56
5.4 Biophysical model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5.5 Cell motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5.5.1 Motility on short time scales . . . . . . . . . . . . . . . . . . . . 63
5.5.2 Motility on long time scales . . . . . . . . . . . . . . . . . . . . 65
5.5.3 Discussion and conclusions . . . . . . . . . . . . . . . . . . . . . 65
6 Application to biophysical questions 69
6.1 Pharmacological treatment . . . . . . . . . . . . . . . . . . . . . . . . . 69
6.1.1 Cytochalasin D . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
6.1.2 ML-7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
6.1.3 Discussion and interpretation . . . . . . . . . . . . . . . . . . . 81
6.2 The influence of mechanical stress on the bead motion . . . . . . . . . 82
6.2.1 Predictions of the biophysical model . . . . . . . . . . . . . . . 82
6.2.2 Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
6.2.3 Spontaneous bead motion . . . . . . . . . . . . . . . . . . . . . 84
6.2.4 Confocal imaging . . . . . . . . . . . . . . . . . . . . . . . . . . 87
6.2.5 Discussion and conclusions . . . . . . . . . . . . . . . . . . . . . 87
6.3 Bead coating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
6.3.1 Integrin densities in the cell . . . . . . . . . . . . . . . . . . . . 90
6.3.2 Predictions of the biophysical model . . . . . . . . . . . . . . . 91
6.3.3 Experimental results . . . . . . . . . . . . . . . . . . . . . . . . 91
6.3.4 Discussion and conclusions . . . . . . . . . . . . . . . . . . . . . 93
6.4 Characterization of different bead sizes . . . . . . . . . . . . . . . . . . 95
6.4.1 Predictions of the biophysical model . . . . . . . . . . . . . . . 96
6.4.2 Experimental results . . . . . . . . . . . . . . . . . . . . . . . . 97
6.4.3 Discussion and conclusions . . . . . . . . . . . . . . . . . . . . . 100
6.5 Bead binding times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
6.5.1 The beginning of the bead binding process . . . . . . . . . . . . 101
6.5.2 Short bead binding times . . . . . . . . . . . . . . . . . . . . . . 105
6.5.3 Longer bead times . . . . . . . . . . . . . . . . . . . . . 108
6.5.4 Poly-L-lysine- versus fibronectin-coated beads . . . . . . . . . . 115
6.5.5 Long time motion . . . . . . . . . . . . . . . . . . . . . . . . . . 121
6.5.6 Bead internalization . . . . . . . . . . . . . . . . . . . . . . . . 124
6.5.7 Discussion and conclusions . . . . . . . . . . . . . . . . . . . . . 126
6.6 Analysis of different tumor cell lines . . . . . . . . . . . . . . . . . . . . 128
6.6.1 Predictions of the biophysical model . . . . . . . . . . . . . . . 128
6.6.2 Experimental results . . . . . . . . . . . . . . . . . . . . . . . . 130
6.6.3 Discussion and conclusions . . . . . . . . . . . . . . . . . . . . . 134
7 Summary 137CONTENTS iii
8 Zusammenfassung 141
A Transition time in the mean square displacement 147
B Power spectral density of velocities 149
B.1 Relationship to the mean square displacement . . . . . . . . . . . . . . 149
B.1.1 Relationship between the power law exponents . . . . . . . . . . 149
B.1.2 Relat between the power law prefactors . . . . . . . . . . 151
B.2 High-frequency branch . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
C Experimental aspects 153
C.1 Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
C.2 Beads and their coating . . . . . . . . . . . . . . . . . . . . . . . . . . 153
C.3 Preparation of experiments . . . . . . . . . . . . . . . . . . . . . . . . . 154
C.3.1 Experiments with Cytochalasin D . . . . . . . . . . . . . . . . . 154
C.3.2 Experiments with ML-7 . . . . . . . . . . . . . . . . . . . . . . 154
C.3.3 Experiments with varying bead binding times . . . . . . . . . . 155
D Optical microscopy 157
D.1 Phase contrast. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
D.2 Fluorescent microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
D.3 Video microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
D.4 Confocaly . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1591. Introduction
In this thesis, the spontaneous motion of cytoskeletally bound markers is investigated.
The cytoskeleton is built of filamentous scaffolding proteins (such as filamentous actin
andmicrotubules). Actinfilamentscrosslinkedwithmyosinmotorproteinsformstress
fibers which are important for force generation. The cytoskeleton in general is essen-
tial for a number of other processes of living cells such as intracellular transport, cell
crawling, cell spreading, cell division, and mechanochemical signal transduction. A
tensed cytoskeletal network is a highly dynamic structure in a continuous and fluctu-
atingstateofdisassemblyandreassembly. Cytoskeletaldisassemblyandreassembly,in
short, cytoskeletal dynamcis, can be measured by the spontaneous motion of micron-
sized particles.
Spontaneous motion is usually associated with Brownian motion. In 1827, Robert
Brown observed the diffusive motion of pollen in water, but he was unable to ex-
plain this motion. Nearly 80 years later, in 1905, Albert Einstein succeeded in finding
the origin of the spontaneous diffusive motion of particles in a fluid, namely ther-
mally driven impacts of the fluid molecules on the particle [1,2]. In contrast, evidence
presented in this thesis and elsewhere [3] indicates that the spontaneous motion of
cytoskeletally bound particles is generally not driven by thermal forces. It rather de-
pends on adenosine-triphosphate (ATP), the energy source of the cell.
The spontaneous motion of cytoskeletally bound particles is related to cell crawling
and locomotion. Both processes, the spontaneous particle motion as well as cell lo-
comotion, are driven by the same mechanism, namely cytoskeletal dynamics. Cell
crawling and locomotion have attracted much interest, long before the spontaneous
particle motion has been investigated. In 1906, Dellinger studied the locomotion of
amoebae from the side view and concluded that amoebae do not crawl or creep but
“walk” by means of contact points [4]. Since the development of the surface contact
microscope by Ambrose in 1956 [5,6] and the interference reflection microscope by
Curtis in 1964 [7], such contact points (adhesions) between cells and the substrate
were observed and their importance for cell locomotion was analyzed in detail [8–10].
The adhesions between the cell and the substrate form when the substrate is coated
with specific proteins (matrix proteins). Reversely, artificial particles can be coated
with these matrix proteins and thus, can form adhesions with the cell similar to the
cell-substrate adhesions [11,12]. Both, cell-substrate and cell-particle adhesions are
mediated by surface adhesion receptors, so-called integrins which were discovered in
the eighties [13]. Later, it was recognized that integrins also play a role in signal
transduction [14–16]. Additionally, it was discovered that integrins interact with the2 Introduction
Figure 1.1: Concept of the bead-integrin-cytoskeleton link. Focal adhesion complexes,
naturally linking the cytoskeleton to the extra-cellular matrix, can also be induced by
a bead coated with an extra-cellular matrix protein (for instance, fibronectin). Such a
bead binds via integrins to the actomyosin stress fibers. The binding process initializes
the formation of a focal adhesion complex.
actin cytoskeleton at the cell interior [17–19] and that integrin-mediated interactions
between matrix proteins and the cell induce the recruitment of many cytoplasmic pro-
teins to the adhesion site [16,20]. These processes were studied by binding artificial
particles coated with matrix proteins to the cell. Such particles, so-called beads, do
not only cause focal adhesion formation but are also phagocytozed by the cell [12].
The motion of matrix-protein coated beads was first studied by Michael Sheetz and
colleagues [21–23] in the nineties. They found that the bead motion changes as the
bead forms integrin-mediated connections to the cytoskeleton as shown in figure 1.1.
They suggested that the same forces moving a cell forward on a substrate are also
responsible for the motion of beads linked to the cytoskeleton via integrins [24]. The
bead tracking methods in these studies were based on video microscopy techniques
which had been developed in the eighties to analyze the spontaneous motion of mi-
croinjected gold particles within cells [25–29].
Only in the last years, the spontaneous motion of cytoskeletally bound particles was
analyzed with statistical methods [3,30,31]. The following interpretations and mech-
anisms for the spontaneos bead motion have been proposed: First, it was reasoned
that the particle cannot move unless the structure (i.e. the cytoskeleton) to which it
is attached rearranges (figure 1.2). Thus, the particle motion would report the rate
of ongoing internal cytoskeletal rearrangements over time [31]. Second, force fluc-
tuations (force hits onto the particle) caused by the action of motor proteins which
provoke deformations of the viscoelastic medium surrounding the particle, offer an-
other possible source for particle motion [32,33]. A third mechanism is provided by
the structure of the cytoskeleton. The particle is thought to be trapped in a cage3
Figure 1.2: Confocal image which shows beads attached to the cytoskeletal network
consisting of stress fibers. The position of the beads are indicated by white circles. The
actin cytoskeleton was stained in red with phalloidin. Scale bar: 10 μm.
formed by the cytoskeletal network which allows the occasional hopping of the bead
outofitscage[3,32,34]. Suchamotionwouldbecharacterizedbyandstalling
events [3].
Although the mechanical coupling of the particle to the cytoskeleton has been rela-
tively well analyzed using a variety of techniques (optical tweezers, magnetic tweezers,
magnetic twisting cytometry) [35–38], the cellular forces moving the particle in the
absence of external forces are not well characterized or understood. This thesis aims
to elucidate the origin of the spontaneous particle motion.
The purpose of this thesis is, first, to characterize the spontaneous motion of cy-
toskeletally bound particles by statistical means, second, to identify its mechanism,
and third, to apply the developed methods to pertinent biophysical questions.
The thesis is organized as follows: In chapter 2, the cell with its most important prop-
ertiesisintroduced. Thecytoskeletontakescenterstagebecausethespontaneousbead
motion is based on the cytoskeletal reorganization processes. In chapter 3, the tech-
nique of nanoscale particle tracking and its implementation and realization (hardware
as well as software) are described. Limitations due to noise and drift are addressed
and a new method for drift correction is presented. In chapter 4, general statistical
properties of the spontaneous bead motion are developed and applied. Their impor-
tance and what they tell us about cells, especially their cytoskeletal remodeling, are
presented and discussed.
An interpretation of the spontaneous bead motion is developed in chapter 5. It is
demonstrated that the statistical properties of the spontaneous bead motion display
similarities to the statistical properties of the deformations of a flexible matrix (poly-
acrylamid gel) the cells are placed onto. Furthermore, it is shown that the orientation4 Introduction
of the stress fiber the bead is attached to determines its motion. Additionally, the
cross correlated motion of bead pairs is investigated. These results are compared to a
biophysical model based on a network of actomyosin stress fibers the beads are con-
nected to. Interestingly, this model can account for many of the statistical properties
of the spontaneous bead motion. Finally, the extend to which the motion of the entire
cell body contributes to the spontaneous bead motion on short time scales (minutes)
and long time scales (hours) is investigated.
In chapter 6, the developed statistical methods are applied to pertinent biophysical
questions. First, in section 6.1, the stress fibers are blocked directly and indirectly
by pharmacological treatment to study the impact of cytoskeletal stress fibers on the
spontaneous bead motion. Next, it is shown that the bead-cytoskeleton connections
can be strengthened by mechanical stimulation. A resulting tightened bead coupling
as well as reinforced stress fibers acting on the beads affect its motion. These results
are discussed in section 6.2. Specific bead coatings induce the formation of a bead-
cytoskeleton coupling, mediated by integrins or without the participation of integrins.
Whether an integrin mediated bead-cytoskeleton link is important for a directed bead
motion is addressed in section 6.3. Different bead sizes are investigated in section 6.4.
The bead size is assumed to affect the strength of the bead-cytoskeleton link and thus,
the spontaneous motion. The temporal development of such bead-cytoskeleton con-
nections(viaintegrinsandwithoutintegrinsandfortwodifferentbeadsizes)isstudied
in section 6.5. The beginning of the bead binding process as well as long bead binding
times up to seven hours reveal an interesting insight into the maturation and deac-
tivation of the bead-cytoskeleton connections. In section 6.6, differently metastatic
tumor cell lines derived from different tissues are investigated. They are distinguished
fromeachotherbyupregualtionofcertainproteins[39,40]andbydifferentmechanical
properties, for instance stiffness [41,42]. Thus, some differences as well as similarities
in the spontaneous bead motion are expected. Indeed, the spontaneous bead motion
was found to correlate with mechanical measurements of cell stiffness and with the
invasiveness of the cells.
The most important results and their interpretation are summarized and discussed in
chapter 7.