Spelling suggestions: "subject:"zellpolarität"" "subject:"cellpolaritet""
1 |
Functional analysis of the Bazooka protein in the establishment of cell polarity in Drosophila melanogaster / Funtionelle Analyse des Bazooka-Proteins während der Etablierung der Zellpolarität in Drosophila melanogasterKrahn, Michael 18 June 2009 (has links)
No description available.
|
2 |
The Role of Lipids in Cellular Architecture and FunctionLopes Sampaio, Julio 15 June 2011 (has links) (PDF)
All cells are delimited by membranes that protect the cell from the surrounding environment. In eukaryotic cells the same principle applies at subcellular level where membranes delimit functional cell organelles. The membrane structure, properties and function are defined in part by their lipid composition. Lipidomics is the large‐scale study of pathways and networks of cellular lipids in biological systems. It involves the identification and quantitation of cellular lipid molecular species and their interactions with other lipids, proteins, and other metabolites. Lipidomics has been greatly facilitated by recent advances in ionization technology and mass spectrometric capabilities which have simplified the sample processing prior to analysis, giving rise to shotgun lipidomics. Shotgun lipidomics is fast, highly sensitive, and can identify hundreds of lipids missed by other methods. However, Glycosphingolipids are an important lipid family that was out of the scope of shotgun lipidomics due to the lack of suitable analytical tools.
The aim of my thesis was two‐fold. The first aim was the establishment of Glycosphingolipid identification and quantification by shotgun approach. This allowed us to perform lipidomic studies with unprecedented comprehensiveness (~300 lipid species from 15 different lipid classes) from low sample amounts and with minimal sample processing. The second was the application of this technology in studies of the role of lipids in several processes like vesicular carrier formation, cell polarization, protein delivery to the plasma membrane and viral budding.
This work resulted in several findings. We found that there is sorting of sphingolipids and sterols into plasma membrane targeted vesicular carriers in budding yeast. When kidney cells change from a mesenchymal to an epithelial morphology there is a profound remodeling of their lipidome, with the synthesis of longer, more saturated, more hydroxylated, and more glycosylated sphingolipids. When these sphingolipids and sterols are depleted in epithelial cells, the apical transport in epithelial cells is impaired. These data strongly support the idea that lipid rafts play an important role in sorting and delivery of lipid and protein cargo to the plasma membrane. Finally, we found that the envelopes of vesicular stomatitis virus and Semliki forest virus assert little specificity in the incorporation of lipids from the plasma membrane. This weak specificity seems to be related to a combination of virus lipid bilayer asymmetry and curvature.
|
3 |
The Role of Lipids in Cellular Architecture and FunctionLopes Sampaio, Julio 09 July 2010 (has links)
All cells are delimited by membranes that protect the cell from the surrounding environment. In eukaryotic cells the same principle applies at subcellular level where membranes delimit functional cell organelles. The membrane structure, properties and function are defined in part by their lipid composition. Lipidomics is the large‐scale study of pathways and networks of cellular lipids in biological systems. It involves the identification and quantitation of cellular lipid molecular species and their interactions with other lipids, proteins, and other metabolites. Lipidomics has been greatly facilitated by recent advances in ionization technology and mass spectrometric capabilities which have simplified the sample processing prior to analysis, giving rise to shotgun lipidomics. Shotgun lipidomics is fast, highly sensitive, and can identify hundreds of lipids missed by other methods. However, Glycosphingolipids are an important lipid family that was out of the scope of shotgun lipidomics due to the lack of suitable analytical tools.
The aim of my thesis was two‐fold. The first aim was the establishment of Glycosphingolipid identification and quantification by shotgun approach. This allowed us to perform lipidomic studies with unprecedented comprehensiveness (~300 lipid species from 15 different lipid classes) from low sample amounts and with minimal sample processing. The second was the application of this technology in studies of the role of lipids in several processes like vesicular carrier formation, cell polarization, protein delivery to the plasma membrane and viral budding.
This work resulted in several findings. We found that there is sorting of sphingolipids and sterols into plasma membrane targeted vesicular carriers in budding yeast. When kidney cells change from a mesenchymal to an epithelial morphology there is a profound remodeling of their lipidome, with the synthesis of longer, more saturated, more hydroxylated, and more glycosylated sphingolipids. When these sphingolipids and sterols are depleted in epithelial cells, the apical transport in epithelial cells is impaired. These data strongly support the idea that lipid rafts play an important role in sorting and delivery of lipid and protein cargo to the plasma membrane. Finally, we found that the envelopes of vesicular stomatitis virus and Semliki forest virus assert little specificity in the incorporation of lipids from the plasma membrane. This weak specificity seems to be related to a combination of virus lipid bilayer asymmetry and curvature.
|
4 |
Identification and functional characterization of PTK7 ligands in Xenopus laevis / Identifizierung und funktionelle Charakterisierung von PTK7-Liganden in Xenopus laevisPeradziryi, Hanna 04 May 2011 (has links)
No description available.
|
5 |
The choreography of yeast matingGiese, Wolfgang 14 December 2016 (has links)
Die Forschung an der Hefe Saccharomyces cerevisiae – auch als Bäckerhefe bekannt – hat sich für die biologische Grundlagenforschung als unentbehrlich erwiesen und führte zu wichtigen Erkenntnissen in der Erforschung von Krankheiten wie Krebs. Am Beispiel der Paarung von Hefezellen werden in dieser Arbeit wesentliche Aspekte der eukaryotischen Zellbiologie untersucht. In der Haplophase des Lebenszyklus der Hefe, treten haploide Zellen als Paarungstyp MATa oder MATα auf. Diese Paarungstypen kommunizieren über Pheromone, die in ein extrazelluläres Medium abgesondert werden und von Zelloberflächenrezeptoren des komplementären Paarungstyps erkannt werden. Hefezellen wachsen in die Richtung eines möglichen Paarungspartners, da sie sich nicht aktiv bewegen können. Die Auswertung von empirischen Daten aus der Fluoreszenzmikroskopie und Rasterkraftmikroskopie (AFM) mit mathematischen Modellen ermöglichte die Rekonstruktion wesentlicher Prozesse der Hefepaarung: (i) Interzelluläre Kommunikation über die Sezernierung und Rezeption von Pheromonen, (ii) Aufbau der Zellpolarität als Reaktion auf die Pheromonantwort, (iii) Induktion und Mechanik der Zellformänderung. Folgende Modelle wurden dazu entwickelt: (i) Die interzelluläre Kommunikation wurde unter Verwendung von zellulären Automaten mit Hilfe von Reaktions-Diffusions (RD) Gleichungen modelliert. Das Modell zeigte, dass die gegenseitige Stimulierung und erhöhte Pheromonabsonderung zu einer verbesserten Abstimmung in der Paarung in der Zellpopulation führt. (ii) Ein Turing- und ein Phasenseparations- Mechanismus wurden als Modelle zum Aufbau der Zellpolarität verwendet. Volumen-Oberflächen gekoppelte RD Gleichungen wurden analytisch und numerisch mit der Finite-Elemente-Methode (FEM) untersucht. (iii) Die Zellwandveränderung wurde mit klassischer Kontinuumsmechanik und der FEM Methode modelliert. Dies ermöglichte eine Beschreibung der reversiblen elastischen und der irreversiblen plastischen Verformungen der Zellwand. / Research on the yeast Saccharomyces cerevisiae – also known as baker’s yeast – has been essential not only for fostering basic biological knowledge but even more so for contributing towards understanding diseases such as cancer. In this thesis, general biological phenomena occurring in eukaryotic cells are investigated, exemplified by the mating process of yeast. In the haploid phase of their life cycle, yeast cells occur as mating type MATa or MATα, both of which communicate via pheromones that are secreted in an extracellular medium and can be sensed by cell-surface receptors of the complementary mating type. In order to mate, yeast cells grow towards a potential mating partner, since they are not able to actively move. Mathematical models on the basis of fluorescence and atomic force microscopy (AFM) data were developed. The key aspects of the yeast mating process that I examined were (i) intercellular communication of cells via pheromones, (ii) the initial symmetry break and implementation of cell polarity, and (iii) subsequent morphogenetic changes. The methods used and findings were as follows: (i) Pheromone secretion and sensing motifs were modelled using cellular automata models based on reaction-diffusion (RD) equations. My models show that mutual stimulation and increased pheromone secretion between cells improves mating efficiency in cell populations. (ii) To explain yeast mating decisions, two possible model types for cell polarity were tested: a Turing-type and a phase-separation mechanism. Bulk-surface RD equations were investigated analytically and numerically using the finite element method (FEM). Typical cell shapes were reconstructed in 2D and 3D. (iii) The cell wall was modelled using classical continuum mechanics that allows for reversible elastic and irreversible plastic cell wall deformation. Mathematical modelling demonstrated that all three processes investigated are precisely orchestrated and interlocked during yeast mating.
|
6 |
The role of the novel endosomal protein Rush hour (CG14782) in endosomal trafficking in Drosophila melanogaster / Die Rolle des endosomalen Proteins Rush hour (CG14782) in der Regulation der Endocytose in Drosophila melanogasterGailite, Ieva 03 May 2010 (has links)
No description available.
|
7 |
From cells to tissuesMerkel, Matthias 02 December 2014 (has links) (PDF)
An essential prerequisite for the existence of multi-cellular life is the organization of cells into tissues. In this thesis, we theoretically study how large-scale tissue properties can emerge from the collective behavior of individual cells. To this end, we focus on the properties of epithelial tissue, which is one of the major tissue types in animals. We study how rheological properties of epithelia emerge from cellular processes, and we develop a physical description for the dynamics of an epithelial cell polarity. We apply our theoretical studies to observations in the developing wing of the fruit fly, Drosophila melanogaster.
In order to study epithelial mechanics, we first develop a geometrical framework that rigorously describes the deformation of two-dimensional cellular networks. Our framework decomposes large-scale deformation into cellular contributions. For instance, we show how large-scale tissue shear decomposes into contributions by cell shape changes and into contributions by different kinds of topological transitions. We apply this framework in order to quantify the time-dependent deformation of the fruit fly wing, and to decompose it into cellular contributions.
We also use this framework as a basis to study large-scale rheological properties of epithelia and their dependence on cellular fluctuations. To this end, we represent epithelial tissues by a vertex model, which describes cells as elastic polygons. We extend the vertex model by introducing fluctuations on the cellular scale, and we develop a method to perform perpetual simple shear simulations. Analyzing the steady state of such simple shear simulations, we find that the rheological behavior of vertex model tissue depends on the fluctuation amplitude. For small fluctuation amplitude, it behaves like a plastic material, and for high fluctuation amplitude, it behaves like a visco-elastic fluid.
In addition to analyzing mechanical properties, we study the reorientation of an epithelial cell polarity. To this end, we develop a simple hydrodynamic description for polarity reorientation. In particular, we account for polarity reorientation by tissue shear, by another polarity field, and by local polarity alignment. Furthermore, we develop methods to quantify polarity patterns based on microscopical images of the fly wing. We find that our hydrodynamic description does not only account for polarity reorientation in wild type fly wings. Moreover, it is for the first time possible to also account for the observed polarity patterns in a number of genetically altered flies. / Eine wesentliche Voraussetzung für die Existenz mehrzelligen Lebens ist, dass sich einzelne Zellen sinnvoll zu Geweben ergänzen können. In dieser Dissertation untersuchen wir, wie großskalige Eigenschaften von Geweben aus dem kollektiven Verhalten einzelner Zellen hervorgehen. Dazu konzentrieren wir uns auf Epitheliengewebe, welches eine der Grundgewebearten in Tieren darstellt. Wir stellen theoretische Untersuchungen zu rheologischen Eigenschaften und zu zellulärer Polarität von Epithelien an. Diese theoretischen Untersuchungen vergleichen wir mit experimentellen Beobachtungen am sich entwickelnden Flügel der schwarzbäuchigen Taufliege (Drosophila melanogaster).
Um die Mechanik von Epithelien zu untersuchen, entwickeln wir zunächst eine geometrische Beschreibung für die Verformung von zweidimensionalen zellulären Netzwerken. Unsere Beschreibung zerlegt die mittlere Verformung des gesamten Netzwerks in zelluläre Beitrage. Zum Beispiel wird eine Scherverformung des gesamten Netzwerks auf der zellulären Ebene exakt repräsentiert: einerseits durch die Verformung einzelner Zellen und andererseits durch topologische Veränderungen des zellulären Netzwerks. Mit Hilfe dieser Beschreibung quantifizieren wir die Verformung des Fliegenflügels während des Puppenstadiums. Des Weiteren führen wir die Verformung des Flügels auf ihre zellulären Beiträge zurück.
Wir nutzen diese Beschreibung auch als Ausgangspunkt, um effektive rheologische Eigenschaften von Epithelien in Abhängigkeit von zellulären Fluktuationen zu untersuchen. Dazu simulieren wir Epithelgewebe mittels eines Vertex Modells, welches einzelne Zellen als elastische Polygone abstrahiert. Wir erweitern dieses Vertex Modell um zelluläre Fluktuationen und um die Möglichkeit, Schersimulationen beliebiger Dauer durchzuführen. Die Analyse des stationären Zustands dieser Simulationen ergibt plastisches Verhalten bei kleiner Fluktuationsamplitude und visko-elastisches Verhalten bei großer Fluktuationsamplitude.
Neben mechanischen Eigenschaften untersuchen wir auch die Umorientierung einer Zellpolarität in Epithelien. Dazu entwickeln wir eine einfache hydrodynamische Beschreibung für die Umorientierung eines Polaritätsfeldes. Wir berücksichtigen dabei insbesondere Effekte durch Scherung, durch ein anderes Polaritätsfeld und durch einen lokalen Gleichrichtungseffekt. Um unsere theoretische Beschreibung mit experimentellen Daten zu vergleichen, entwickeln wir Methoden um Polaritätsmuster im Fliegenflügel zu quantifizieren. Schließlich stellen wir fest, dass unsere hydrodynamische Beschreibung in der Tat beobachtete Polaritätsmuster reproduziert. Das gilt nicht nur im Wildtypen, sondern auch in genetisch veränderten Tieren.
|
8 |
Analysis of the role of the atypical cadherin Fat2 during tissue elongation in the developing ovary of Drosophila melanogaster / Analyse der Rolle des atypischen Cadherins Fat2 bei der Gewebestreckung während der Ovarentwicklung von Drosophila melanogasterAurich, Franziska 29 May 2017 (has links) (PDF)
Tissue elongation is an important requirement for proper tissue morphogenesis during animal development. The Drosophila egg chamber is an excellent model to study the molecular processes underlying tissue elongation. An egg chamber is composed of germline cells that are enveloped by a somatic follicle epithelium. While the egg chamber matures, the drastic increase of the egg chamber’s volume is accompanied by a shape change from round to oval. Egg chamber elongation coincides with a circumferential alignment of F-actin filaments, microtubules, and fibrils of the extracellular matrix (ECM). Additionally, egg chambers rotate around their future long axis. It has been proposed that this rotation aligns F-actin filaments and ECM fibrils. The circumferentially aligned F-actin and ECM fibrils form a molecular corset that promotes egg chamber elongation. The atypical cadherin Fat2 is required for egg chamber rotation, the circumferential alignment of F-actin, microtubules, and ECM fibrils and for egg chamber elongation. However, the molecular mechanisms by which Fat2 influences egg chamber elongation remain unknown.
In my thesis I performed a structure-function analysis of Fat2. I generated a Fat2 version that lacks the intracellular region and a second version, which lacks both intracellular region and the transmembrane domain and tested their ability to compensate for Fat2 functions in fat2-/- mutant egg chambers. My results reveal that the intracellular region is required for the microtubule alignment, and for egg chamber rotation. In contrast, the intracellular region is not required for F-actin and ECM alignment, and for egg chamber elongation.
Hence, my findings for the first time demonstrate that egg chamber rotation is not required for F-actin and ECM fibril alignment and that egg chamber elongation can occur independently from egg chamber rotation. My work uncouples some of the parallel processes that take place during oogenesis and changes the view on the mechanisms that drive tissue elongation in this important model system. / Das Strecken von Geweben ist ein wichtiger Prozess bei der Gestaltbildung während der Entwicklung von Organismen. Die Eikammer von Drosophila ist ein hervorragendes Modellsystem, um die Gewebestreckung zu untersuchen. Eine Eikammer besteht aus Keimbahnzellen und einem einschichtigen Follikelepithel, das die Keimbahn umschließt. Während die Eikammer heranwächst durchläuft sie eine drastische Gestaltveränderung von rund nach oval. Zeitgleich zur Streckung der Eikammer weist das Follikelepithel parallel angeordnete F-actin−Filamente, Mikrotubuli und Fasern der extrazellulären Matrix (ECM) auf, welche die Eikammer ringsum umlaufen. Zudem rotieren die Eikammern um ihre zukünftige Längsachse. Bisher nahm man an, die Rotation würde für die Ausrichtung der F-actin−Filamente, Microtubuli und ECM-Fasern gebraucht werden. Die Anordnung der F-actin−Filamente und ECM-Fasern bilden dann ein molekulares Korsett, das die Gewebestreckung fördert.
Das atypische Cadherin Fat2 wird für die Rotation der Eikammern, die umlaufende Anordnung der F-actin–Filamente, Microtubuli und ECM-Fasern sowie für die Streckung der Eikammern benötigt. Die Mechanismen, mit denen Fat2 die Gewebestreckung beeinflusst, sind allerdings unbekannt. In meinem Projekt führte ich eine Struktur-Funktions-Analyse von Fat2 durch.
Ich generierte eine Version von Fat2 mit einer Deletion der kompletten intrazellulären Region und eine zweite, die weder die intrazelluläre Region noch die Transmembran-Domäne besitzt und testete, ob diese Versionen die Funktionen von Fat2 in fat2-/- mutanten Eikammern kompensieren können. Meine Ergebnisse zeigen, dass die intrazelluläre Region für die Anordnung der Mikrotubuli und für die Rotation der Eikammern gebraucht wird. Die intrazelluläre Region wird jedoch weder für die Anordnung von F-actin–Filamenten und den ECM-Fasern noch für die Streckung der Eikammer benötigt. Meine Erkenntnisse zeigen erstmalig, dass die Streckung der Eikammern ohne Rotation stattfinden kann. Meine Arbeit entkoppelt damit mehrere parallel stattfindende Prozesse während der Entwicklung der Eikammer und eröffnet einen neuen Einblick in die Mechanismen der Gewebestreckung in diesem wichtigen Modellsystem.
|
9 |
From cells to tissuesMerkel, Matthias 21 November 2014 (has links)
An essential prerequisite for the existence of multi-cellular life is the organization of cells into tissues. In this thesis, we theoretically study how large-scale tissue properties can emerge from the collective behavior of individual cells. To this end, we focus on the properties of epithelial tissue, which is one of the major tissue types in animals. We study how rheological properties of epithelia emerge from cellular processes, and we develop a physical description for the dynamics of an epithelial cell polarity. We apply our theoretical studies to observations in the developing wing of the fruit fly, Drosophila melanogaster.
In order to study epithelial mechanics, we first develop a geometrical framework that rigorously describes the deformation of two-dimensional cellular networks. Our framework decomposes large-scale deformation into cellular contributions. For instance, we show how large-scale tissue shear decomposes into contributions by cell shape changes and into contributions by different kinds of topological transitions. We apply this framework in order to quantify the time-dependent deformation of the fruit fly wing, and to decompose it into cellular contributions.
We also use this framework as a basis to study large-scale rheological properties of epithelia and their dependence on cellular fluctuations. To this end, we represent epithelial tissues by a vertex model, which describes cells as elastic polygons. We extend the vertex model by introducing fluctuations on the cellular scale, and we develop a method to perform perpetual simple shear simulations. Analyzing the steady state of such simple shear simulations, we find that the rheological behavior of vertex model tissue depends on the fluctuation amplitude. For small fluctuation amplitude, it behaves like a plastic material, and for high fluctuation amplitude, it behaves like a visco-elastic fluid.
In addition to analyzing mechanical properties, we study the reorientation of an epithelial cell polarity. To this end, we develop a simple hydrodynamic description for polarity reorientation. In particular, we account for polarity reorientation by tissue shear, by another polarity field, and by local polarity alignment. Furthermore, we develop methods to quantify polarity patterns based on microscopical images of the fly wing. We find that our hydrodynamic description does not only account for polarity reorientation in wild type fly wings. Moreover, it is for the first time possible to also account for the observed polarity patterns in a number of genetically altered flies.:1 Introduction
1.1 The development of multi-cellular organisms
1.2 Biology of epithelial tissues
1.3 The model system Drosophila melanogaster
1.4 Planar cell polarity
1.5 Physical description of biological tissues
1.6 Overview over this thesis
2 Tissue shear in cellular networks
2.1 Geometry of tissue deformation on the cellular scale
2.2 Decomposition of the large-scale flow field into cellular contributions
2.3 Cellular contributions to the flow field in the fruit fly wing
2.4 Discussion
3 Rheological behavior of vertex model tissue under external shear
3.1 A vertex model to describe epithelial mechanics
3.2 Fluctuation-induced fluidization of tissue
3.3 Discussion
4 Quantitative study of polarity reorientation in the fruit fly wing
4.1 Experimentally quantified polarity patterns
4.2 Effective hydrodynamic theory for polarity reorientation
4.3 Comparison of theory and experiment
4.4 Discussion
5 Conclusions and outlook
Appendices:
A Algebra of real 2 × 2 matrices
B Deformation of triangle networks
C Simple shear simulations using the vertex model
D Coarse-graining of a cellular Core PCP model
E Quantification of polarity patterns in the fruit fly wing
F Theory for polarity reorientation in the fruit fly wing
G Boundary conditions for the polarity field in the fruit fly wing
Table of symbols
Bibliography / Eine wesentliche Voraussetzung für die Existenz mehrzelligen Lebens ist, dass sich einzelne Zellen sinnvoll zu Geweben ergänzen können. In dieser Dissertation untersuchen wir, wie großskalige Eigenschaften von Geweben aus dem kollektiven Verhalten einzelner Zellen hervorgehen. Dazu konzentrieren wir uns auf Epitheliengewebe, welches eine der Grundgewebearten in Tieren darstellt. Wir stellen theoretische Untersuchungen zu rheologischen Eigenschaften und zu zellulärer Polarität von Epithelien an. Diese theoretischen Untersuchungen vergleichen wir mit experimentellen Beobachtungen am sich entwickelnden Flügel der schwarzbäuchigen Taufliege (Drosophila melanogaster).
Um die Mechanik von Epithelien zu untersuchen, entwickeln wir zunächst eine geometrische Beschreibung für die Verformung von zweidimensionalen zellulären Netzwerken. Unsere Beschreibung zerlegt die mittlere Verformung des gesamten Netzwerks in zelluläre Beitrage. Zum Beispiel wird eine Scherverformung des gesamten Netzwerks auf der zellulären Ebene exakt repräsentiert: einerseits durch die Verformung einzelner Zellen und andererseits durch topologische Veränderungen des zellulären Netzwerks. Mit Hilfe dieser Beschreibung quantifizieren wir die Verformung des Fliegenflügels während des Puppenstadiums. Des Weiteren führen wir die Verformung des Flügels auf ihre zellulären Beiträge zurück.
Wir nutzen diese Beschreibung auch als Ausgangspunkt, um effektive rheologische Eigenschaften von Epithelien in Abhängigkeit von zellulären Fluktuationen zu untersuchen. Dazu simulieren wir Epithelgewebe mittels eines Vertex Modells, welches einzelne Zellen als elastische Polygone abstrahiert. Wir erweitern dieses Vertex Modell um zelluläre Fluktuationen und um die Möglichkeit, Schersimulationen beliebiger Dauer durchzuführen. Die Analyse des stationären Zustands dieser Simulationen ergibt plastisches Verhalten bei kleiner Fluktuationsamplitude und visko-elastisches Verhalten bei großer Fluktuationsamplitude.
Neben mechanischen Eigenschaften untersuchen wir auch die Umorientierung einer Zellpolarität in Epithelien. Dazu entwickeln wir eine einfache hydrodynamische Beschreibung für die Umorientierung eines Polaritätsfeldes. Wir berücksichtigen dabei insbesondere Effekte durch Scherung, durch ein anderes Polaritätsfeld und durch einen lokalen Gleichrichtungseffekt. Um unsere theoretische Beschreibung mit experimentellen Daten zu vergleichen, entwickeln wir Methoden um Polaritätsmuster im Fliegenflügel zu quantifizieren. Schließlich stellen wir fest, dass unsere hydrodynamische Beschreibung in der Tat beobachtete Polaritätsmuster reproduziert. Das gilt nicht nur im Wildtypen, sondern auch in genetisch veränderten Tieren.:1 Introduction
1.1 The development of multi-cellular organisms
1.2 Biology of epithelial tissues
1.3 The model system Drosophila melanogaster
1.4 Planar cell polarity
1.5 Physical description of biological tissues
1.6 Overview over this thesis
2 Tissue shear in cellular networks
2.1 Geometry of tissue deformation on the cellular scale
2.2 Decomposition of the large-scale flow field into cellular contributions
2.3 Cellular contributions to the flow field in the fruit fly wing
2.4 Discussion
3 Rheological behavior of vertex model tissue under external shear
3.1 A vertex model to describe epithelial mechanics
3.2 Fluctuation-induced fluidization of tissue
3.3 Discussion
4 Quantitative study of polarity reorientation in the fruit fly wing
4.1 Experimentally quantified polarity patterns
4.2 Effective hydrodynamic theory for polarity reorientation
4.3 Comparison of theory and experiment
4.4 Discussion
5 Conclusions and outlook
Appendices:
A Algebra of real 2 × 2 matrices
B Deformation of triangle networks
C Simple shear simulations using the vertex model
D Coarse-graining of a cellular Core PCP model
E Quantification of polarity patterns in the fruit fly wing
F Theory for polarity reorientation in the fruit fly wing
G Boundary conditions for the polarity field in the fruit fly wing
Table of symbols
Bibliography
|
10 |
Analysis of the role of the atypical cadherin Fat2 during tissue elongation in the developing ovary of Drosophila melanogasterAurich, Franziska 10 April 2017 (has links)
Tissue elongation is an important requirement for proper tissue morphogenesis during animal development. The Drosophila egg chamber is an excellent model to study the molecular processes underlying tissue elongation. An egg chamber is composed of germline cells that are enveloped by a somatic follicle epithelium. While the egg chamber matures, the drastic increase of the egg chamber’s volume is accompanied by a shape change from round to oval. Egg chamber elongation coincides with a circumferential alignment of F-actin filaments, microtubules, and fibrils of the extracellular matrix (ECM). Additionally, egg chambers rotate around their future long axis. It has been proposed that this rotation aligns F-actin filaments and ECM fibrils. The circumferentially aligned F-actin and ECM fibrils form a molecular corset that promotes egg chamber elongation. The atypical cadherin Fat2 is required for egg chamber rotation, the circumferential alignment of F-actin, microtubules, and ECM fibrils and for egg chamber elongation. However, the molecular mechanisms by which Fat2 influences egg chamber elongation remain unknown.
In my thesis I performed a structure-function analysis of Fat2. I generated a Fat2 version that lacks the intracellular region and a second version, which lacks both intracellular region and the transmembrane domain and tested their ability to compensate for Fat2 functions in fat2-/- mutant egg chambers. My results reveal that the intracellular region is required for the microtubule alignment, and for egg chamber rotation. In contrast, the intracellular region is not required for F-actin and ECM alignment, and for egg chamber elongation.
Hence, my findings for the first time demonstrate that egg chamber rotation is not required for F-actin and ECM fibril alignment and that egg chamber elongation can occur independently from egg chamber rotation. My work uncouples some of the parallel processes that take place during oogenesis and changes the view on the mechanisms that drive tissue elongation in this important model system.:1 ABSTRACT I
2 ZUSAMMENFASSUNG II
3 TABLE OF CONTENTS III
4 LISTS 7
4.1 List of Abbreviations 7
4.2 List of figures 9
5 INTRODUCTION 11
5.1 Tissue morphogenesis during development 11
5.1.1 Tissue organization by differential cell affinity 11
5.1.2 Cell adhesion is mediated by cadherins12
5.1.3 The cytoskeleton drives cell shape changes 13
5.1.4 Planar polarity is required for tissue-level directionality 17
5.2 Models of tissue elongation 19
5.2.1 Germ-band extension in Drosophila melanogaster 19
5.2.2 Primitive streak formation in the chick embryo 21
5.2.3 Neural tube formation in Xenopus 22
5.3 Drosophila egg chamber as a model system to study tissue morphogenesis 24
5.3.1 Oogenesis in Drosophila 24
5.3.2 Egg chamber as a model for tissue elongation 27
5.3.3 Planar polarized organization of the F-actin cytoskeleton in the follicle epithelium 29
5.3.4 Egg chamber elongation requires a link between extracellular matrix and F-actin cytoskeleton 32
5.3.5 Egg chamber rotation is proposed to be a requisite for egg chamber elongation 34
5.3.6 The atypical cadherin Fat2 provides a key role during egg chamber elongation 35
6 AIMS OF THE THESIS 38
7 MATERIALS AND METHODS 39
7.1 Fly husbandry 39
7.2 Used fly stocks 39
7.3 Phenotypic markers 40
7.4 Ovary dissection for fixation 40
7.5 Antibody stainings 41
7.6 Used antibodies 42
7.7 Drug treatment 42
7.8 Microscopy of fixed samples 43
7.9 Live imaging 43
7.9.1 Imaging of the basal F-actin oscillations 43
7.9.2 Imaging of egg chamber rotation 44
7.10 Generation of the transgenic fosmid constructs 44
7.10.1 General materials required for molecular genetics in E.coli 46
7.10.2 Step 1: Amplification of the tagging cassette 47
7.10.3 Step 2: Transformation of the helper plasmid pRedFlp4 49
7.10.4 Step 3: Red-operon driven insertion of the tagging cassette 50
7.10.5 Step 4: Removal of the KanR gene 50
7.10.6 Step 5: DNA isolation and verification of the correct transgenic construct 50
7.10.7 Step 6: Integration of the transgene into the fly genome 52
7.11 Image analysis and quantifications 53
7.11.1 Statistics 53
7.11.2 Aspect ratio measurements 54
7.11.3 Quantification of GFP localization55
7.11.4 Quantification of tissue-wide Collagen IV alignment 55
7.11.5 Quantification of tissue-wide angles of F-actin and microtubules 57
7.11.6 Analysis of periodicity of F-actin oscillations 58
7.11.7 Quantification of the rotation velocity of egg chambers 60
8 RESULTS 61
8.1 Expression of full-length fat2-GFP gene fully rescues all aspects of the fat258D mutant phenotype 61
8.1.1 Expression of the fat2-GFP gene rescues the fat258D mutant egg shape and sterility 61
8.1.2 Using an ‘Alignment parameter’ SAP to quantify the directionality of cytoskeletal structures and extracellular matrix fibrils 63
8.1.3 Expression of the fat2-GFP gene rescues microtubule alignment of fat258D mutant egg chambers65
8.1.4 Expression of the fat2-GFP gene rescues F-actin and Collagen IV alignment of fat258D mutant egg chambers 67
8.2 Generation of different fat2 mutant transgenes by homologous recombineering 70
8.3 The intracellular region of Fat2 is dispensable for some specific aspects of the Fat2 functions 73
8.3.1 The egg chamber elongation is independent of the intracellular region of Fat2 73
8.3.2. Localization of Fat2 protein depends on the intracellular region of Fat2 76
8.3.3 The alignment of microtubules is dependent on the intracellular region of the protein 78
8.3.4 The intracellular region of Fat2 is required for proper early F-actin and Collagen IV fibril alignment 81
8.3.5 The intracellular region of Fat2 is required for late F-actin and Collagen IV fibril alignment 85
8.3.6 F-actin filaments and ECM fibrils co-align in fat258D mutant stage 8 egg chambers 88
8.3.7 F-actin filaments and ECM fibrils do not co-align in fat258D mutant stage 10 egg chambers 90
8.3.8 The stability of basal F-actin fibers and Collagen IV fibrils mutually depend on each other at stage 8 92
8.3.9 The contractile pulses of F-actin in stage 9 egg chambers are independent of the intracellular region of Fat293
8.3.10 The intracellular region of Fat2 is required for proper egg chamber rotation in the early developmental stages96
8.3.11 The intracellular region of Fat2 is required for proper egg chamber rotation in later developmental stages 99
9 DISCUSSION 103
9.1 Egg chamber elongation can be uncoupled from egg chamber rotation 104
9.2 Egg chamber elongation correlates with a functional molecular corset 107
9.3 Fat2 promotes egg chamber elongation by its extracellular region 109
9.4 Alternative mechanisms potentially drive egg chamber elongation 111
9.5 New model of egg chamber elongation 114
9.6 Future perspectives 116
9.7 Impact on tissue morphogenesis in general 119
10 ACKNOWLEDGEMENTS 120
11 REFERENCES 121
12 APPENDIX 134
12.1 Script for “FFTAlignment.m" 134
12.2 Script for “Test1” 143
12.3 Script for “AverageCellAlignment.m" 143 / Das Strecken von Geweben ist ein wichtiger Prozess bei der Gestaltbildung während der Entwicklung von Organismen. Die Eikammer von Drosophila ist ein hervorragendes Modellsystem, um die Gewebestreckung zu untersuchen. Eine Eikammer besteht aus Keimbahnzellen und einem einschichtigen Follikelepithel, das die Keimbahn umschließt. Während die Eikammer heranwächst durchläuft sie eine drastische Gestaltveränderung von rund nach oval. Zeitgleich zur Streckung der Eikammer weist das Follikelepithel parallel angeordnete F-actin−Filamente, Mikrotubuli und Fasern der extrazellulären Matrix (ECM) auf, welche die Eikammer ringsum umlaufen. Zudem rotieren die Eikammern um ihre zukünftige Längsachse. Bisher nahm man an, die Rotation würde für die Ausrichtung der F-actin−Filamente, Microtubuli und ECM-Fasern gebraucht werden. Die Anordnung der F-actin−Filamente und ECM-Fasern bilden dann ein molekulares Korsett, das die Gewebestreckung fördert.
Das atypische Cadherin Fat2 wird für die Rotation der Eikammern, die umlaufende Anordnung der F-actin–Filamente, Microtubuli und ECM-Fasern sowie für die Streckung der Eikammern benötigt. Die Mechanismen, mit denen Fat2 die Gewebestreckung beeinflusst, sind allerdings unbekannt. In meinem Projekt führte ich eine Struktur-Funktions-Analyse von Fat2 durch.
Ich generierte eine Version von Fat2 mit einer Deletion der kompletten intrazellulären Region und eine zweite, die weder die intrazelluläre Region noch die Transmembran-Domäne besitzt und testete, ob diese Versionen die Funktionen von Fat2 in fat2-/- mutanten Eikammern kompensieren können. Meine Ergebnisse zeigen, dass die intrazelluläre Region für die Anordnung der Mikrotubuli und für die Rotation der Eikammern gebraucht wird. Die intrazelluläre Region wird jedoch weder für die Anordnung von F-actin–Filamenten und den ECM-Fasern noch für die Streckung der Eikammer benötigt. Meine Erkenntnisse zeigen erstmalig, dass die Streckung der Eikammern ohne Rotation stattfinden kann. Meine Arbeit entkoppelt damit mehrere parallel stattfindende Prozesse während der Entwicklung der Eikammer und eröffnet einen neuen Einblick in die Mechanismen der Gewebestreckung in diesem wichtigen Modellsystem.:1 ABSTRACT I
2 ZUSAMMENFASSUNG II
3 TABLE OF CONTENTS III
4 LISTS 7
4.1 List of Abbreviations 7
4.2 List of figures 9
5 INTRODUCTION 11
5.1 Tissue morphogenesis during development 11
5.1.1 Tissue organization by differential cell affinity 11
5.1.2 Cell adhesion is mediated by cadherins12
5.1.3 The cytoskeleton drives cell shape changes 13
5.1.4 Planar polarity is required for tissue-level directionality 17
5.2 Models of tissue elongation 19
5.2.1 Germ-band extension in Drosophila melanogaster 19
5.2.2 Primitive streak formation in the chick embryo 21
5.2.3 Neural tube formation in Xenopus 22
5.3 Drosophila egg chamber as a model system to study tissue morphogenesis 24
5.3.1 Oogenesis in Drosophila 24
5.3.2 Egg chamber as a model for tissue elongation 27
5.3.3 Planar polarized organization of the F-actin cytoskeleton in the follicle epithelium 29
5.3.4 Egg chamber elongation requires a link between extracellular matrix and F-actin cytoskeleton 32
5.3.5 Egg chamber rotation is proposed to be a requisite for egg chamber elongation 34
5.3.6 The atypical cadherin Fat2 provides a key role during egg chamber elongation 35
6 AIMS OF THE THESIS 38
7 MATERIALS AND METHODS 39
7.1 Fly husbandry 39
7.2 Used fly stocks 39
7.3 Phenotypic markers 40
7.4 Ovary dissection for fixation 40
7.5 Antibody stainings 41
7.6 Used antibodies 42
7.7 Drug treatment 42
7.8 Microscopy of fixed samples 43
7.9 Live imaging 43
7.9.1 Imaging of the basal F-actin oscillations 43
7.9.2 Imaging of egg chamber rotation 44
7.10 Generation of the transgenic fosmid constructs 44
7.10.1 General materials required for molecular genetics in E.coli 46
7.10.2 Step 1: Amplification of the tagging cassette 47
7.10.3 Step 2: Transformation of the helper plasmid pRedFlp4 49
7.10.4 Step 3: Red-operon driven insertion of the tagging cassette 50
7.10.5 Step 4: Removal of the KanR gene 50
7.10.6 Step 5: DNA isolation and verification of the correct transgenic construct 50
7.10.7 Step 6: Integration of the transgene into the fly genome 52
7.11 Image analysis and quantifications 53
7.11.1 Statistics 53
7.11.2 Aspect ratio measurements 54
7.11.3 Quantification of GFP localization55
7.11.4 Quantification of tissue-wide Collagen IV alignment 55
7.11.5 Quantification of tissue-wide angles of F-actin and microtubules 57
7.11.6 Analysis of periodicity of F-actin oscillations 58
7.11.7 Quantification of the rotation velocity of egg chambers 60
8 RESULTS 61
8.1 Expression of full-length fat2-GFP gene fully rescues all aspects of the fat258D mutant phenotype 61
8.1.1 Expression of the fat2-GFP gene rescues the fat258D mutant egg shape and sterility 61
8.1.2 Using an ‘Alignment parameter’ SAP to quantify the directionality of cytoskeletal structures and extracellular matrix fibrils 63
8.1.3 Expression of the fat2-GFP gene rescues microtubule alignment of fat258D mutant egg chambers65
8.1.4 Expression of the fat2-GFP gene rescues F-actin and Collagen IV alignment of fat258D mutant egg chambers 67
8.2 Generation of different fat2 mutant transgenes by homologous recombineering 70
8.3 The intracellular region of Fat2 is dispensable for some specific aspects of the Fat2 functions 73
8.3.1 The egg chamber elongation is independent of the intracellular region of Fat2 73
8.3.2. Localization of Fat2 protein depends on the intracellular region of Fat2 76
8.3.3 The alignment of microtubules is dependent on the intracellular region of the protein 78
8.3.4 The intracellular region of Fat2 is required for proper early F-actin and Collagen IV fibril alignment 81
8.3.5 The intracellular region of Fat2 is required for late F-actin and Collagen IV fibril alignment 85
8.3.6 F-actin filaments and ECM fibrils co-align in fat258D mutant stage 8 egg chambers 88
8.3.7 F-actin filaments and ECM fibrils do not co-align in fat258D mutant stage 10 egg chambers 90
8.3.8 The stability of basal F-actin fibers and Collagen IV fibrils mutually depend on each other at stage 8 92
8.3.9 The contractile pulses of F-actin in stage 9 egg chambers are independent of the intracellular region of Fat293
8.3.10 The intracellular region of Fat2 is required for proper egg chamber rotation in the early developmental stages96
8.3.11 The intracellular region of Fat2 is required for proper egg chamber rotation in later developmental stages 99
9 DISCUSSION 103
9.1 Egg chamber elongation can be uncoupled from egg chamber rotation 104
9.2 Egg chamber elongation correlates with a functional molecular corset 107
9.3 Fat2 promotes egg chamber elongation by its extracellular region 109
9.4 Alternative mechanisms potentially drive egg chamber elongation 111
9.5 New model of egg chamber elongation 114
9.6 Future perspectives 116
9.7 Impact on tissue morphogenesis in general 119
10 ACKNOWLEDGEMENTS 120
11 REFERENCES 121
12 APPENDIX 134
12.1 Script for “FFTAlignment.m" 134
12.2 Script for “Test1” 143
12.3 Script for “AverageCellAlignment.m" 143
|
Page generated in 0.0604 seconds