The role of p115RhoGEF/Lsc in G-alpha 12/13 protein-coupled receptor signaling in thymocytes and the generation of LARG knockout mice [Elektronische Ressource] / Anke Harenberg

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• Publié par: universitat_ulm
• Publié le: 01 janvier 2004
• Nombre de lectures: 35
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

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FAKULTÄT FÜR NATURWISSENSCHAFTEN
UNIVERSITÄT ULM
The Role of p115RhoGEF/Lsc in G Protein-coupled Receptor12/13
Signaling in Thymocytes
and the Generation of LARG Knockout Mice
DISSERTATION
Zur Erlangung des Doktorgrades (Dr. rer. nat.)
an der Fakultät für Naturwissenschaften
der Universität Ulm
vorgelegt von
Anke Harenberg
aus
Frankfurt/M, Deutschland
Ulm, 2004
2
Amtierender Dekan:
Prof. Dr. R. J. Behm
Erstgutachter:
Prof. Dr. Klaus-Dieter Spindler, Allgemeine Zoologie und Endokrinologie, Universität
Ulm
Zweitgutachter:
Prof. Dr. Thomas Wirth, Physiologische Chemie, Universität Ulm
Tag der Promotion:
5. November 2004
Die Arbeiten im Rahmen der vorliegenden Dissertation wurden am Institut für
Medizinische Strahlenkunde und Zellforschung der Bayerischen Julius-Maximilians-
Universität Würzburg und in der Abteilung Physiologische Chemie der Universität Ulm
durchgeführt und von Herrn Prof. Dr. Klaus-Dieter Fischer betreut.3
ERKLÄRUNG
Ich versichere hiermit, dass ich die vorliegende Arbeit selbstständig angefertig und
keine anderen als die angegebenen Quellen und Hilfsmittel benutzt sowie wörtlich
oder inhaltlich übernommene Textpassagen als solche gekennzeichnet habe.
Anke Harenberg
Ulm, den.......................................TABLE OF CONTENTS 4
TABLE OF CONTENTS
TABLE OF CONTENTS .................................................................................................. 4
ACKNOWLEDGEMENTS................................................................................................ 7
SUMMARY ...................................................................................................................... 8
ZUSAMMENFASSUNG................................................................................................... 9
ABBREVIATIONS ......................................................................................................... 10
1 INTRODUCTION..................................................................................................... 13
1.1 Rho GTPases.............................................................................................. 13
1.1.1 Introduction............................................................................................... 13
1.1.2 Guanine exchange factors for Rho GTPases ........................................... 15
1.1.2.1 Introduction ........................................................................................... 15
1.1.2.2 RGS domain containing GEFs.............................................................. 16
1.1.3 Functions of RhoGTPases........................................................................ 22
1.1.3.1 Rho GTPases and the actin cytoskeleton ............................................. 22
1.1.3.2 RhoA and gene transcription................................................................. 25
1.2 G protein-coupled receptors..................................................................... 26
1.2.1 Signaling pathways downstream of GPCRs 26
1.3 Thromboxane A and its receptor ............................................................ 282
1.3.1 TXA metabolism ...................................................................................... 282
1.3.2 Thromboxane A and the immune system................................................ 302
1.4 Thymocytes ................................................................................................ 31
1.4.1 Thymocyte development .......................................................................... 31
1.4.1.1 Thymocyte development and RhoGTPases.......................................... 34
1.4.1.2 Apoptosis .............................................................................................. 35
1.5 Aims of the project..................................................................................... 40
2 RESULTS ............................................................................................................... 41
2.1 Lsc RhoGEF mediates signaling from thromboxane A to actin2
polymerization and apoptosis in thymocytes ........................................................ 41TABLE OF CONTENTS 5
-/-2.1.1 Defects in TXA -induced actin polymerization in Lsc thymocytes........... 412
2.1.2 Lsc is required for TXA -mediated RhoA activation ................................. 422
2.1.3 TXA activates multiple signaling intermediates in thymocytes................. 462
2.1.4 Lsc is involved in TXA induction of thymocyte apoptosis......................... 502
-/-2.1.5 Normal expression of FasL on Lsc thymocytes ...................................... 53
2.1.6 U46199 induction of apoptosis is independent of ROCK.......................... 53
-/-2.1.7 Increased number of thymocytes in Lsc mice......................................... 55
2.2 Generation of LARG-deficient mice.......................................................... 59
2.2.1 Overview................................................................................................... 59
2.2.2 Construction of an 129/Ola targeting vector ............................................. 60
2.2.2.1 Library screening .................................................................................. 61
2.2.2.2 Analysis of the cosmids......................................................................... 62
2.2.2.3 Cosmid mapping ................................................................................... 63
2.2.2.4 Construction of an 129/Ola targeting vector.......................................... 66
2.2.2.5 Screening for targeted events............................................................... 69
2.2.3 Construction of an 129/Sv targeting vector............................................... 71
2.2.3.1 Cloning of the 129/vector .................................................. 71
3 DISCUSSION.......................................................................................................... 74
3.1 TXA signals to thymocyte apoptosis and actin polymerization through2
Lsc ..................................................................................................................... 74
3.2 Generation of LARG deficient mice.......................................................... 82
3.3 Future perspective..................................................................................... 83
4 MATERIAL AND METHODS.................................................................................. 85
4.1 Materials ..................................................................................................... 85
4.1.1 Chemicals and reagents........................................................................... 85
4.1.2 Kits ........................................................................................................... 88
4.1.3 Cell lines................................................................................................... 88
4.1.4 Primers 88
4.1.5 Antibodies for Western Blotting ................................................................ 89
4.1.6 Antibodies for flow cytometry.................................................................... 90
4.2 Methods ...................................................................................................... 90TABLE OF CONTENTS 6
4.2.1 Mice.......................................................................................................... 90
4.2.2 Cellular biological Methods....................................................................... 90
4.2.2.1 Thymectomy and thymocyte preparation .............................................. 90
4.2.2.2 Flow cytometry...................................................................................... 91
4.2.2.3 Counting cells by microscope using a hemacytometer ......................... 91
4.2.2.4 Stimulation of thymocytes by crosslinking the TCR .............................. 92
4.2.2.5 Apoptosis assay.................................................................................... 93
4.2.3 Biochemistry............................................................................................. 95
4.2.3.1 Actin polymerization.............................................................................. 95
4.2.3.2 Rho/Rac GTP-“pull down”-Assay.......................................................... 96
4.2.3.3 Western blotting 98
4.2.4 Molecular Biology ................................................................................... 100
4.2.4.1 DNA methods...................................................................................... 100
4.2.4.2 Bacterial manipulation......................................................................... 109
4.2.4.3 Cloning strategies ............................................................................... 111
4.2.4.4 Genomic DNA library screening by PCR............................................. 114
4.2.4.5 Cosmids.............................................................................................. 115
4.2.4.6 Generation of the targeting vector....................................................... 121
4.2.5 ES cell culture......................................................................................... 124
4.2.5.1 Solutions ......................................................................................