423 research outputs found
Biophysical techniques to study cell and matrix properties in the context of single cell migration
Single cell migration in artificial collagen gels as an in vitro model system in the context of cancer are studied. Cell and matrix mechanical properties are determined using atomic force microscopy and an advanced analysis method. Matrix pore-size is studied using a novel approach and analysis method. A novel, minimally invasive approach to determine the amount of displacement of the cell microenvironment due to force generation of single cells during migration in artificial 3D collagen gels is introduced. An automated analysis and user friendly software to analyze high-throughput cell invasion is introduced. These methods are used to study cell migration and mechanical properties of the breast cancer cell lines MDA-MB-231 and MCF-7 and the influence of cell nuclear elasticity is investigated. Using mouse embryonic fibroblasts, the role of focal adhesion kinase (FAK) during cell migration is studied using FAK deficient knock-out cell lines FAK-/- and control FAK+/+ as well as kinase-dead mutants FAKR454/R454 and control FAKWT/WT.:Abstract i
Acknowledgements iii
1 Introduction 1
2 Background 5
2.1 Cancer — An ever-changing Disease 5
2.1.1 Carcinogenesis and Neoplasm 6
2.1.2 Hallmarks of Cancer 7
2.1.3 Metastasis— The malignant Progression of Cancer 7
2.1.4 Metastatic Cascade 9
2.2 The Cell— Where it begins 10
2.2.1 Actomyosin Complex 12
2.2.1.1 Actin Monomer 12
2.2.1.2 Polymerization 12
2.2.1.3 Structures 14
2.2.1.4 Actin Cortex 15
2.2.1.5 Filopodia 16
2.2.1.6 Lamellipodium 16
2.2.1.7 Invadopodium 17
2.2.1.8 Stress Fibers 17
2.2.1.9 Actin in Cancer and Metastasis 17
2.2.1.10 Myosin and Actin 18
2.2.2 Focal Adhesions 19
2.2.3 Microtubules 20
2.2.4 Intermediate Filaments 21
2.2.5 Cellular Stiffness 22
2.2.6 Nuclear Deformability 23
2.3 The Extracellular Matrix— Where it happens 24
2.3.1 Components and Structure 25
2.3.2 Collagen as a Model System 26
2.3.2.1 Collagen I Fibril Formation 27
2.3.2.2 The Rat/Bovine-Collagen-Mix Model System 28
2.4 Single Cell Migration— Why it spreads 29
3 Materials and Methods 31
3.1 Cell Culture 31
3.1.1 Cancer Cells 31
3.1.2 Mouse fibroblasts 32
3.1.3 Pharmacological treatment 34
3.2 Collagen matrices 34
3.3 Cell Elasticity 36
3.3.1 Atomic Force Microscopy 36
3.3.2 Preparation 37
3.3.3 Data Aquisition 38
3.3.4 Data Analysis 38
3.4 Matrix Stiffness 40
3.4.1 Preparation 40
3.4.2 Data Aquisition 41
3.4.3 Data Analysis 41
3.5 Invasion Assay 42
3.5.1 Preparation 42
3.5.2 Data aquisition 44
3.5.3 Data Analysis 44
3.6 Matrix Topology 48
3.6.1 Preparation 49
3.6.2 Data Acquisition 50
3.6.3 Data Analysis 51
3.6.3.1 Binarization 51
3.6.3.2 Pore-Size 53
3.6.3.3 Fiber Thickness 54
3.7 Fiber Displacement 55
3.7.1 Preparation 56
3.7.2 Data Aquisition 56
3.7.3 Data analysis 57
3.7.3.1 Fiber Displacement 59
3.7.3.2 Cell Segmentation 60
3.7.3.3 Shell Analysis 61
3.8 A toolset to understand Single Cell Migration and what influences it 62
4 Results 65
4.1 Cell Elasticity 65
4.1.1 Example Force-Distance Curves 66
4.1.2 Single Cell Elasticity 67
4.2 Matrix Stiffness 69
4.3 Invasion 71
4.4 Matrix Topology 75
4.5 Influence of Cell Nucleus on Cell Migration 79
4.5.1 Cellular Elasticity 79
4.5.2 Invasion 81
4.6 Fiber Displacement 89
4.7 Effect of FAK on Cell Invasion and Fiber Displacement 93
4.7.1 FAK Knock-Out 93
4.7.2 Kinase-dead FAK Mutant 96
5 Discussion 103
References 107Die Einzelzellmigration in künstlichen Kollagennetzwerken als ein in vitro Modellsystem im Kontext von Krebs wurde studiert. Mechanische Eigenschaften von Zellen und der verwendeten Kollagennetzwerke wurden mithilfe der Atomic Force Microscopy (AFM) und weiterentwickelten Analysemethoden bestimmt. Die Porengröße der verwendeten Kollagennetzwerke wurde mit einer neuentwickelten Auswertemethode analysiert. Eine neuartige, minimal-invasive Methode zur Bestimmung der Verformung der Mikroumgebung von Zellen während der Migration verursacht durch Kräftegenerierung der Zelle wird beschrieben. Die Analyse des Invasions-Assays wurde automatisiert und eine nutzerfreundliche Software entwickelt, mit der große Datenmengen ausgewertet werden können. Diese Methoden wurden verwendet, um mechanische Eigenschaften und Migration der humanen Brustkrebszellinien MDA-MB-231 und MCF-7 zu studieren. Die Rolle der focal adhesion kinase (FAK) wurde mithilfe von embryonalen Maus-Fibroblasten studiert. Sowohl eine FAK knock-out Zellinie FAK-/- und Kontrolle FAK+/+, als auch eine kinase-dead Mutante FAKR454/R454 und Kontrolle FAKWT/WT wurden hinsichtlich ihrer Invasion und Verformung der Mikroumgebung analysiert.:Abstract i
Acknowledgements iii
1 Introduction 1
2 Background 5
2.1 Cancer — An ever-changing Disease 5
2.1.1 Carcinogenesis and Neoplasm 6
2.1.2 Hallmarks of Cancer 7
2.1.3 Metastasis— The malignant Progression of Cancer 7
2.1.4 Metastatic Cascade 9
2.2 The Cell— Where it begins 10
2.2.1 Actomyosin Complex 12
2.2.1.1 Actin Monomer 12
2.2.1.2 Polymerization 12
2.2.1.3 Structures 14
2.2.1.4 Actin Cortex 15
2.2.1.5 Filopodia 16
2.2.1.6 Lamellipodium 16
2.2.1.7 Invadopodium 17
2.2.1.8 Stress Fibers 17
2.2.1.9 Actin in Cancer and Metastasis 17
2.2.1.10 Myosin and Actin 18
2.2.2 Focal Adhesions 19
2.2.3 Microtubules 20
2.2.4 Intermediate Filaments 21
2.2.5 Cellular Stiffness 22
2.2.6 Nuclear Deformability 23
2.3 The Extracellular Matrix— Where it happens 24
2.3.1 Components and Structure 25
2.3.2 Collagen as a Model System 26
2.3.2.1 Collagen I Fibril Formation 27
2.3.2.2 The Rat/Bovine-Collagen-Mix Model System 28
2.4 Single Cell Migration— Why it spreads 29
3 Materials and Methods 31
3.1 Cell Culture 31
3.1.1 Cancer Cells 31
3.1.2 Mouse fibroblasts 32
3.1.3 Pharmacological treatment 34
3.2 Collagen matrices 34
3.3 Cell Elasticity 36
3.3.1 Atomic Force Microscopy 36
3.3.2 Preparation 37
3.3.3 Data Aquisition 38
3.3.4 Data Analysis 38
3.4 Matrix Stiffness 40
3.4.1 Preparation 40
3.4.2 Data Aquisition 41
3.4.3 Data Analysis 41
3.5 Invasion Assay 42
3.5.1 Preparation 42
3.5.2 Data aquisition 44
3.5.3 Data Analysis 44
3.6 Matrix Topology 48
3.6.1 Preparation 49
3.6.2 Data Acquisition 50
3.6.3 Data Analysis 51
3.6.3.1 Binarization 51
3.6.3.2 Pore-Size 53
3.6.3.3 Fiber Thickness 54
3.7 Fiber Displacement 55
3.7.1 Preparation 56
3.7.2 Data Aquisition 56
3.7.3 Data analysis 57
3.7.3.1 Fiber Displacement 59
3.7.3.2 Cell Segmentation 60
3.7.3.3 Shell Analysis 61
3.8 A toolset to understand Single Cell Migration and what influences it 62
4 Results 65
4.1 Cell Elasticity 65
4.1.1 Example Force-Distance Curves 66
4.1.2 Single Cell Elasticity 67
4.2 Matrix Stiffness 69
4.3 Invasion 71
4.4 Matrix Topology 75
4.5 Influence of Cell Nucleus on Cell Migration 79
4.5.1 Cellular Elasticity 79
4.5.2 Invasion 81
4.6 Fiber Displacement 89
4.7 Effect of FAK on Cell Invasion and Fiber Displacement 93
4.7.1 FAK Knock-Out 93
4.7.2 Kinase-dead FAK Mutant 96
5 Discussion 103
References 10
The transmembrane protein fibrocystin/polyductin regulates cell mechanics and cell motility
Polycystic kidney disease is a disorder that leads to fluid filled cysts that replace normal renal tubes.
During the process of cellular development and in the progression of the diseases, fibrocystin
can lead to impaired organ formation and even cause organ defects. Besides cellular polarity,
mechanical properties play major roles in providing the optimal apical-basal or anterior–posterior
symmetry within epithelial cells. A breakdown of the cell symmetry that is usually associated with
mechanical property changes and it is known to be essential in many biological processes such as
cell migration, polarity and pattern formation especially during development and diseases such
as the autosomal recessive cystic kidney disease. Since the breakdown of the cell symmetry can be
evoked by several proteins including fibrocystin, we hypothesized that cell mechanics are altered by
fibrocystin. However, the effect of fibrocystin on cell migration and cellular mechanical properties
is still unclear. In order to explore the function of fibrocystin on cell migration and mechanics,
we analyzed fibrocystin knockdown epithelial cells in comparison to fibrocystin control cells. We
found that invasiveness of fibrocystin knockdown cells into dense 3D matrices was increased and
more efficient compared to control cells. Using optical cell stretching and atomic force microscopy,
fibrocystin knockdown cells were more deformable and exhibited weaker cell–matrix as well as
cell–cell adhesion forces, respectively. In summary, these findings show that fibrocystin knockdown
cells displayed increased 3D matrix invasion through providing increased cellular deformability,
decreased cell–matrix and reduced cell–cell adhesion force
Effect of Nuclear Stiffness on Cell Mechanics and Migration of Human Breast Cancer Cells
The migration and invasion of cancer cells through 3D confined extracellular matrices
is coupled to cell mechanics and the mechanics of the extracellular matrix. Cell
mechanics is mainly determined by both the mechanics of the largest organelle in
the cell, the nucleus, and the cytoskeletal architecture of the cell. Hence, cytoskeletal
and nuclear mechanics are the major contributors to cell mechanics. Among other
factors, steric hindrances of the extracellular matrix confinement are supposed to affect
nuclear mechanics and thus also influence cell mechanics. Therefore, we propose that
the percentage of invasive cells and their invasion depths into loose and dense 3D
extracellular matrices is regulated by both nuclear and cytoskeletal mechanics. In order
to investigate the effect of both nuclear and cytoskeletal mechanics on the overall
cell mechanics, we firstly altered nuclear mechanics by the chromatin de-condensing
reagent Trichostatin A (TSA) and secondly altered cytoskeletal mechanics by addition
of actin polymerization inhibitor Latrunculin A and the myosin inhibitor Blebbistatin. In
fact, we found that TSA-treated MDA-MB-231 human breast cancer cells increased
their invasion depth in dense 3D extracellular matrices, whereas the invasion depths
in loose matrices were decreased. Similarly, the invasion depths of TSA-treated MCF-
7 human breast cancer cells in dense matrices were significantly increased compared
to loose matrices, where the invasion depths were decreased. These results are also
valid in the presence of a matrix-metalloproteinase inhibitor GM6001. Using atomic
force microscopy (AFM), we found that the nuclear stiffnesses of both MDA-MB-
231 and MCF-7 breast cancer cells were pronouncedly higher than their cytoskeletal
stiffness, whereas the stiffness of the nucleus of human mammary epithelial cells was
decreased compared to their cytoskeleton. TSA treatment reduced cytoskeletal and
nuclear stiffness of MCF-7 cells, as expected. However, a softening of the nucleus by
TSA treatment may induce a stiffening of the cytoskeleton of MDA-MB-231 cells and
subsequently an apparent stiffening of the nucleus. Inhibiting actin polymerization using
Latrunculin A revealed a softer nucleus of MDA-MB-231 cells under TSA treatment. This
indicates that the actin-dependent cytoskeletal stiffness seems to be influenced by the
TSA-induced nuclear stiffness changes. Finally, the combined treatment with TSA and
Latrunculin A further justifies the hypothesis of apparent nuclear stiffening, indicating that
cytoskeletal mechanics seem to be regulated by nuclear mechanics
Inhomogeneities in 3D Collagen Matrices Impact Matrix Mechanics and Cancer Cell Migration
Cell motility under physiological and pathological conditions including malignant
progression of cancer and subsequent metastasis are founded on environmental
confinements. During the last two decades, three-dimensional cell migration has been
studied mostly by utilizing biomimetic extracellular matrix models. In the majority of
these studies, the in vitro collagen scaffolds are usually assumed to be homogenous,
as they consist commonly of one specific type of collagen, such as collagen type I,
isolated from one species. These collagen matrices should resemble in vivo extracellular
matrix scaffolds physiologically, however, mechanical phenotype and functional reliability
have been addressed poorly due to certain limitations based on the assumption
of homogeneity. How local variations of extracellular matrix structure impact matrix
mechanics and cell migration is largely unknown. Here, we hypothesize that local
inhomogeneities alter cell movement due to alterations in matrix mechanics, as they
frequently occur in in vivo tissue scaffolds and were even changed in diseased tissues.
To analyze the effect of structural inhomogeneities on cell migration, we used a mixture
of rat tail and bovine dermal collagen type I as well as pure rat and pure bovine collagens
at four different concentrations to assess three-dimensional scaffold inhomogeneities.
Collagen type I from rat self-assembled to elongated fibrils, whereas bovine collagen
tended to build node-shaped inhomogeneous scaffolds. We have shown that the
elastic modulus determined with atomic force microscopy in combination with pore size
analysis using confocal laser scanning microscopy revealed distinct inhomogeneities
within collagen matrices. We hypothesized that elastic modulus and pore size govern
cancer cell invasion in three-dimensional collagen matrices. In fact, invasiveness of three
breast cancer cell types is altered due to matrix-type and concentration indicating that
these two factors are crucial for cellular invasiveness. Our findings revealed that local
matrix scaffold inhomogeneity is another crucial parameter to explain differences in
cell migration, which not solely depended on pore size and stiffness of the collagen
matrices. With these three distinct biophysical parameters, characterizing structure
and mechanics of the studied collagen matrices, we were able to explain differences
in the invasion behavior of the studied cancer cell lines in dependence of the used
collagen model
Gravitational lensing of quasar 0957+561 and the determination of H0
We present results of a deep imaging study of the cluster lensing the double quasar Q0957+561. Using data obtained at KPNO and CFHT we have detected distortions in the lensed blue background galaxy population. From these arclet distortions we have constructed a map of the mass density in the field of the double QSO. We use this map to provide constraints on the mass concentrations responsible for the lensing. Under these constraints in conjunction with mass models and the known time delay between the A and B images we place limits on the value of the Hubble constant. © 1995 American Institute of Physics.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87545/2/343_1.pd
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Structural Breakdown of Collagen Type I Elastin Blend Polymerization
Biopolymer blends are advantageous materials with novel properties that may show performances way beyond their individual constituents. Collagen elastin hybrid gels are a new representative of such materials as they employ elastin’s thermo switching behavior in the physiological temperature regime. Although recent studies highlight the potential applications of such systems, little is known about the interaction of collagen and elastin fibers during polymerization. In fact, the final network structure is predetermined in the early and mostly arbitrary association of the fibers. We investigated type I collagen polymerized with bovine neck ligament elastin with up to 33.3 weight percent elastin and showed, by using a plate reader, zeta potential and laser scanning microscopy (LSM) experiments, that elastin fibers bind in a lateral manner to collagen fibers. Our plate reader experiments revealed an elastin concentration-dependent increase in the polymerization rate, although the rate increase was greatest at intermediate elastin concentrations. As elastin does not significantly change the structural metrics pore size, fiber thickness or 2D anisotropy of the final gel, we are confident to conclude that elastin is incorporated homogeneously into the collagen fibers
Effect of PAK Inhibition on Cell Mechanics Depends on Rac1
Besides biochemical and molecular regulation, the migration and invasion of cells
is controlled by the environmental mechanics and cellular mechanics. Hence, the
mechanical phenotype of cells, such as fibroblasts, seems to be crucial for the
migratory capacity in confined 3D extracellular matrices. Recently, we have shown
that the migratory and invasive capacity of mouse embryonic fibroblasts depends on
the expression of the Rho-GTPase Rac1, similarly it has been demonstrated that the
Rho-GTPase Cdc42 affects cell motility. The p21-activated kinase (PAK) is an effector
down-stream target of both Rho-GTPases Rac1 and Cdc42, and it can activate via the
LIM kinase-1 its down-stream target cofilin and subsequently support the cell migration
and invasion through the polymerization of actin filaments. Since Rac1 deficient cells
become mechanically softer than controls, we investigated the effect of group I PAKs
and PAK1 inhibition on cell mechanics in the presence and absence of Rac1. Therefore,
we determined whether mouse embryonic fibroblasts, in which Rac1 was knockedout,
and control cells, displayed cell mechanical alterations after treatment with group I
PAKs or PAK1 inhibitors using a magnetic tweezer (adhesive cell state) and an optical
cell stretcher (non-adhesive cell state). In fact, we found that group I PAKs and Pak1
inhibition decreased the stiffness and the Young’s modulus of fibroblasts in the presence
of Rac1 independent of their adhesive state. However, in the absence of Rac1 the
effect was abolished in the adhesive cell state for both inhibitors and in their nonadhesive
state, the effect was abolished for the FRAX597 inhibitor, but not for the IPA3
inhibitor. The migration and invasion were additionally reduced by both PAK inhibitors
in the presence of Rac1. In the absence of Rac1, only FRAX597 inhibitor reduced their
invasiveness, whereas IPA3 had no effect. These findings indicate that group I PAKs
and PAK1 inhibition is solely possible in the presence of Rac1 highlighting Rac1/PAK I
(PAK1, 2, and 3) as major players in cell mechanics
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