Development and characterization of lifeact [Elektronische Ressource] : a versatile marker for the visualization of F-actin / Julia Riedl

Julia Riedl

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 39
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Development and Characterization of Lifeact
- a versatile marker for the visualization of F-actin -





Dissertation zur Erlangung des
Doktorgrades der Naturwissenschaften
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München





Julia Riedl
aus
Germering



München 2010



















Eingereicht am: 02.09.2010
Mitglieder der Prüfungskommission:
Erstgutachter: Prof. Dr. Thomas Cremer
Zweitgutachter: Prof. Dr. Charles David

Tag des Promotionskolloqiums: 21.01.2011
Erklärung

Die Versuche zur vorgelegten Dissertation wurden in der Zeit von Februar
2007 bis Februar 2010 in der Arbeitsgruppe zelluläre Dynamik und
Musterbildung von Dr. Roland Wedlich-Söldner und in der Arbeitsgruppe
Leukozyten-Migration von Dr. Michael Sixt am Max-Planck-Institut für
Biochemie in Martinsried bei München durchgeführt.

Hiermit erkläre ich, daß ich diese Arbeit selbstständig und nur unter
Verwendung der angegebenen Quellen und Hilfsmittel angefertigt habe.

München, den 02. September 2010

______________________________
Julia Riedl


























Für Thomas & meine Familie







Table of contents

TABLE OF CONTENTS
1 SUMMARY........................................................................................................... 9
2 INTRODUCTION ...........................................................................................11
2.1 Actin and the cytoskeleton ..................................................................................11
2.2 Regulators of actin polymerization.....................................................................13
2.3 F-actin structures in mammalian cells................................................................18
2.4 F-actin is involved in many cellular processes and diseases............................20
2.5 Visualization of F-actin in fixed and living samples.........................................22
2.6 Abp140 as actin probe in Saccharomyces cerevisiae................................................24
2.7 Transgenic mice ....................................................................................................25
2.8 Aim of the thesis ...................................................................................................28
3 RESULTS............................................................................................................29
3.1 Identification of Lifeact in Saccharomyces cerevisiae..............................................29
3.2 Biochemical properties of the Lifeact peptide..................................................31
3.3 Expression of Lifeact in mammalian cells.........................................................34
3.4 Comparison of Lifeact with other F-actin markers..........................................37
3.5 Cytoskeletal functions are not compromised by expression
of Lifeact in mammalian cells..............................................................................39
3.6 Labelling mammalian cells and tissues using FITC-Lifeact............................43
3.7 Generation of Lifeact-transgenic mice...............................................................48
3.8 Characterization of the transgenic founders .....................................................49
3.9 Lifeact expression during mouse development ................................................60
3.10 Lifeact expression and F-actin staining in single cells .....................................62
3.11 Functionality of cells.............................................................................................65
3.12 Applications of the Lifeact-mice.........................................................................67
4 STATEMENT of contributions ......................................................................71
5 DISCUSSION ....................................................................................................72
5.1 Development of a new actin marker - Lifeact ..................................................72
5.2 Generation and characterization of Lifeact-transgenic mice ..........................80
- 6 - Table of contents

5.3 Possible applications.............................................................................................84
6 MATERIAL and METHODS .........................................................................87
6.1 Material...................................................................................................................87
6.1.1. Chemicals and Reagents ..............................................................................87
6.1.2. Media..............................................................................................................89
6.1.3. Solutions ........................................................................................................91
6.1.4. Kits .................................................................................................................95
6.1.5. Antibodies .....................................................................................................95
6.1.6. Oligonucleotides...........................................................................................96
6.1.7. Plasmids.........................................................................................................96
6.1.8. Organisms .....................................................................................................97
6.2 Methods..................................................................................................................98
6.2.1. Molecular biological methods.....................................................................98
6.2.1.1. Plasmid DNA purification.................................................................................98
6.2.1.2. Polymerase chain reaction (PCR)......................................................................98
6.2.1.3. DNA restriction digestion..................................................................................99
6.2.1.4. Agarose gel electrophoresis .............................................................................100
6.2.1.5. DNA purification from agarose gels..............................................................101
6.2.1.6. Determination of DNA concentration..........................................................101
6.2.1.7. DNA ligation......................................................................................................101
6.2.1.8. DNA sequencing...............................................................................................101
6.2.1.9. DNA isolation from mouse tail biopsies.......................................................102
6.2.1.10. DNA preparation for pronuclear injection ...................................................102
6.2.1.11. Peptide synthesis ...............................................................................................103
6.2.2. Biochemical methods.................................................................................103
6.2.2.1. Actin binding assay ...........................................................................................103
6.2.2.2. Actin polymerization and depolymerisation assay .......................................104
6.2.2.3. Far UV CD Spectroscopy................................................................................104
6.2.2.4. NMR sample preparation and Spectroscopy ................................................104
6.2.3. Cell culture methods ..................................................................................105
6.2.3.1. Cultivation of mammalian cells.......................................................................105
- 7 - Table of contents

6.2.3.2. Freezing and thawing of cells ..........................................................................105
6.2.3.3. Primary cells .......................................................................................................106
6.2.3.4. Cell lines..............................................................................................................108
6.2.3.5. Transfection of cells..........................................................................................109
6.2.3.6. Stable cell lines...................................................................................................109
6.2.3.7. Scrape-loading of human neutrophils ............................................................110
6.2.3.8. Immune-complex (IC) induced neutrophil activation.................................110
6.2.3.9. Preparation of fertilized murine oocytes .......................................................110
6.2.3.10. In vitro generation of E4.5 Embryos.........................................