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A DNA-microarray screening: {_d63-catenin [delta-catenin], a new mediator of Eph-ephrin signaling [Elektronische Ressource] / eingereicht von Luca Dolce

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For Takeshi B. AcknowledgementsI wish to thank my thesis supervisor Dr. Rüdiger Klein who gave me the opportunity to do my PhD work in his lab and directed my scientific work all these years.Special thanks to Dr. Tomoko Iwata for offering me a fruitful collaboration and for supervising the first part of my PhD studies.I thank all my collaborators, especially Guido Panté and Lu Qun for providing tools to do my studies and interesting discussion. Special thanks also to lab members Joaquim Egeaand Jenny Köhler who very kindly provided DNA constructs and antibodies.I thank Angel Nebreda at EMBL as member of my thesis committee for his precious suggestions.Special Thanks to Sidney Cambridge for critically reading and proofreading this thesisand for his friendship. I acknowledge all members of the Klein lab for providing a great environment both in the lab and in the Biergarten. Especially I am grateful to Svetla Dimitrova, Joaquim Egea, Jenny Köhler, Archana Mishra, Laura Knott, Elsa Martinez, Taija Mäkkinen, Stefan Weinges and Manuel Zimmer, for their help, critical discussions and friendship. I would like to thank all the people at the Max-Planck Institute of Neurobiology who have supported and encouraged my work, especially the members of the labs of Amparo Acker-Palmer and Gaia Tavosanis for their discussions during our joint group meetings.

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Published 01 January 2005
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For Takeshi B. Acknowledgements
I wish to thank my thesis supervisor Dr. Rüdiger Klein who gave me the opportunity to
do my PhD work in his lab and directed my scientific work all these years.
Special thanks to Dr. Tomoko Iwata for offering me a fruitful collaboration and for
supervising the first part of my PhD studies.
I thank all my collaborators, especially Guido Panté and Lu Qun for providing tools to do
my studies and interesting discussion. Special thanks also to lab members Joaquim Egea
and Jenny Köhler who very kindly provided DNA constructs and antibodies.
I thank Angel Nebreda at EMBL as member of my thesis committee for his precious
suggestions.
Special Thanks to Sidney Cambridge for critically reading and proofreading this thesis
and for his friendship.
I acknowledge all members of the Klein lab for providing a great environment both in the
lab and in the Biergarten. Especially I am grateful to Svetla Dimitrova, Joaquim Egea,
Jenny Köhler, Archana Mishra, Laura Knott, Elsa Martinez, Taija Mäkkinen, Stefan
Weinges and Manuel Zimmer, for their help, critical discussions and friendship.
I would like to thank all the people at the Max-Planck Institute of Neurobiology who
have supported and encouraged my work, especially the members of the labs of Amparo
Acker-Palmer and Gaia Tavosanis for their discussions during our joint group meetings.
2A DNA-Microarray screening: -Catenin, a new
mediator of Eph-ephrin signaling
Dissertation
Der Fakultät für Biologie der
Ludwig-Maximilians Universität München
Eingereicht am 20.5.2005 von
Luca Dolce
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GFirst “Gutachter“: Dr. Rüdiger Klein
Second “Gutachter“: Dr. Angelika Böttger
The work presented in this thesis was performed from March 2001 to August 2001 in the
laboratory of Dr. Rüdiger Klein at the European Molecular Biology Laboratories (EMBL)
Heidelberg, Germany; and from September 2001 to April 2005 at the Max-Planck
Institute of Neurobiology Munich, Germany.
4Erklärung
Ich versichere, da ich meine Dissertation selbstständig, ohne unerlaubte Hilfe
angefertigt, und mich dabei keiner anderen als der von mir ausdrücklich bezeichneten
Hilfen und Quellen bedient habe.
Die Dissertation wurde in der jetzigen oder ähnlichen Form bei keiner anderen
Hochschule eingereicht und hat noch keinen sonstigen Prüfungszwecken gedient.
______________________ ______________________
(Ort, Datum) (Luca Dolce)
5
1. TABLE OF CONTENTS
1. TABLE OF CONTENTS…………………………………………………………….6
2. ABBREVIATIONS……………………………..…………………………………….9
3. PREFACE……...…………………………...…………...…………………………....14
4. SUMMARY…………………………………………...…………………...…………16
5. INTRODUCTION………………………………..………………………………….17
5.1 Introduction outline………………………………………………………………….18
5.2 The generation of the dendritic tree………………………………………………….19
5.2.1 Dendrite outgrowth………………………………………………………………...20
5.2.2 Dendrite guidance………………………………………………………………….21
5.2.3 Dendrite branching…………………………………………………………………21
5.2.3.1 Role of Rho family GTPases in dendrite branching……………………………..23
5.2.3.2 Downstream cellular effectors of Rho family GTPases ………………………...25
5.2.3.3 Extracellular cues that trigger Rho family GTPases signalling cascade…………28
5.2.3.4 Spine formation…………………………………………………………………..29
5.3 Eph receptors and their ephrin ligands……………………………………………….33
5.3.1 The Eph class of receptor tyrosine kinases………………………………………...33
5.3.2 Ephrin ligands……………………………………………………………………...34
5.3.3 Signaling mechanisms by Eph receptors and ephrin ligands………………………36
5.3.3.1 Mechanisms of Eph receptor forward signaling…………………………………46
5.3.3.2s of ephrin ligand reverse signalling………………………………...40
5.3.4 Effects of Eph-ephrin signaling……………………………………………………43
5.3.4.1 Forward and reverse signaling during axon guidance…………………………...43
5.3.4.2 Eph/ephrins and synaptic plasticity……………………………………………...45
5.4 cDNA Microarrays…………………………………………………………………..47
5.5 New candidate molecules for dendritic development: Catenin………..………….50
5.5.1 Molecular structure………………………………………………………………...50
5.5.2 Expression pattern………………………………………………………………….52
5.5.3 The biological functions of Catenin…………………………………………….56
5.5.3.1 Catenin in cell junctions………………………………………………………56
5.5.3.2 Catenin in dendrites and synapses…………………………………………….59
6
G
G
G
G5.5.3.3 Catenin and actin cytoskeleton dynamics……………………………………..60
6. RESULTS…………………………………………...………………………………..64
6.1 Identification of novel Eph-ephrin downstream effectors using a cDNA microarray
screening: Catenin…………………………………………………………………….65
6.2 Catenin and Eph-ephrin signaling………………………………………………...76
6.2.1 Catenin is phosphorylated by Eph-forward signaling but not by ephrin-reverse.76
6.2.2 Catenin and EphA4 interact physically…………………………………………79
6.3 Eph receptor forward signaling, but not ephrin reverse signaling, leads to the
formation of -Catenin aggregates ………………………………………………………87
6.4 Catenin and Eph-ephrin signaling are required for the establishment of proper
dendritic morphology…………………………………………………………………….92
6.4.1 -Catenin and the dynamics of filopodia formation………………...…………....109
6 -Catenin and HeLa cell migration………………………………………………...116
7. DISCUSSION…………………...…………………………………………………..120
7.1 Outlook……………………………………………………………………………..134
8. MATERIALS AND METHODS……………………………………………….….135
8. 1 Buffers and solutions……………………………………………………………....135
8.2 Media and antibiotics for bacterial culture ………………………………………...136
8.3 Bacterial strains……………………………………………………………………..137
8.4 Media and supplements for tissue culture…………………………………………..137
8.5 Media and supplements for primary culture of neurons……………………………138
8.6 Cell lines……………………………………………………………………………138
8.7 Solutions for cell Transfection……………………………………………………...139
8.8 Solutions for Biochemistry…………………………………………………………139
8.9 Plasmids…………………………………………………………………………….140
8.9.1 Expression constructs…………………………………………………………….140
8.10 Antibodies…………………………………………………………………………141
8.11 Chemicals and commercial kits…………………………………………………...143
8.12 Molecular biology…………………………………………………………………144
8.12.2 Enzymatic treatment of DNA…………………………………………………...144
8.12.3 Separation of DNA on agarose gels……………………………………………..145
8.12.4 Purification of DNA……………………………………………………………..145
8.12.5 Transformation of competent E. coli by electroporation………………………..145
8.13 Semi-quantitative real-time PCR………………………………………………….146
8.13.1 cDNA preparation……………………………………………………………….146
8.13.2 PCR primers and templates……………………………………………………...146
8.13.3 LightCycler RT-PCR…………………………………………………………....146
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GGG
G
G
G
G
G
G8.14 Primary culture of cortical neurons for biochemistry …………………………….147
8.14.1 Stimulation of cells……………………………………………………………...147
8.15 Primary culture of neurons for immunocytochemistry, cell imaging and time lapse
imaging…………………………………………………………………………………148
8.15.1 Transfection of cell lines and primary neurons………………………………….148
8.15.2 Time-lapse imaging……………………………………………………………..149
8.15.3 Immunocytochemistry and cell imaging………………………………………...149
8.16 Microarray…………………………………………………………………………150
8.16.1 Microarray production…………………………………………………………..150
8.16.2 RNA extraction and preparation of fluorescent probes…………………………151
8.16.3 Microarray hybridization………………………………………………………..151
8.16.4 Microarray analysis……………………………………………………………...151
8.16.5 Insert cloning and generation of probes for various uses……………………….152
8.17 Boyden chamber assay…………………………………………………………….152
8.18 Sholl analysis……………………………………………………………………...153
8.19 Biochemistry………………………………………………………………………153
8.19.1 Cell lysis………………………………………………………………………...153
8.19.2 Immunoblotting and immunoprecipitation……………………………………...154
8.20 Histology…………………………………………………………………………..154
8.20.1 Vibratome sections for in situ hybridization…………………………………….154
8.20.2 In situ hybridization……………………………………………………………..155
9. BIBLIOGRAPHY……………………………………………………………...….157
10. CURRICULUM VITAE………………………………………………..………..174
82. ABBREVIATIONS
Abi-1 Abl interacting protein-1
Abl Abelson kinase
ACp Anterior Commissure, posterior
AMP Adenosin-5’-monophosphate
AMPA D-2-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid
AP Adaptor protein
APS Ammonium-persulfate
Arg Abelson related gene
ARP2/3 Actin related protein 2/3
ATP Adenosin-5’-triphosphate
BBS BES buffered saline
BDNF Brain-derived neurotrophic factor
BES N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid
Borax Sodium tetraborate
bFGF Basic fibroblast growth factor
BSA Bovine serum albumine
CAM Cell adhesion molecule
2+
CamKII Ca /Calmodulin-dependent kinase II
cAMP Cyclic AMP
CAP Cbl associated protein
Cbp Csk binding protein
9Cdc42 Cell division cycle 42
cDNA Complementary DNA
CNS Central nervous system
2+CREB Ca /cAMP-response element binding protein
Csk C-terminal Src kinase
CXCR CXC motif receptor
DIV Days in vitro
DMEM Dulbecco’s modified Eagles medium
DNA Deoxyribonucleic acid
D-PBS Dulbecco’s phosphate buffered saline
E Embryonic day
EDTA Ethylenediamine-tetra acetic acid
EGFP Enhanced green fluorescent protein
Eph Erythropoietin-producing hepatocellular
Ephexin Eph-interacting exchange protein
Ephrin Eph family receptor interacting protein
ERK Extracellular regulated kinase
EYFP Enhanced yellow fluorescent protein
FAK Focal adhesion kinase
FBS Fetal bovine serum
FNIII Fibronectin type III
Fyn Fgr/Yes-related Novel gene
G Protein GTP binding protein
GAP GTPase activating protein
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