Functional characterization of alphaPIX/Cool-2/ARHGEF6 as a novel interaction partner for CALEB/NGC/CSPG5 in the regulation of dendritic process and spine formation:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2014
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XV, 133 Bl. Ill., graph. Darst. 30 cm |
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Datensatz im Suchindex
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|---|---|
| adam_text | ?N30
ULM UNIVERSITY UNIVERSITAT
RR
FY
FUNCTIONAL CHARACTERIZATION OF ALPHAPIX/COOL-2/ARHGEF6
AS A NOVEL INTERACTION PARTNER FOR CALEB/NGC/CSPG5 IN
THE REGULATION OF DENDRITIC PROCESS AND SPINE FORMATION
DISSERTATION
ZUR ERLANGUNG DES DOKTORGRADES DR. RER. NAT.
DER FAKULTAT NATURWISSENSCHAFTEN
DER UNIVERSITAT ULM
VORGELEGT VON
SASCHA JOHANNES
AUS BERLIN
2014
HTTP://D-NB.INFO/1056937971
TABLE OF CONTENTS
TABLE OF CONTENTS ILL
LIST OF ABBREVIATIONS VII
ABSTRACT XIV
1 INTRODUCTION 1
1.1 NEURONAL FUNCTIONS AND DEVELOPMENT 1
1.2 MOLECULAR MECHANISMS OF DENDRITOGENESIS 3
1.3 DENDRITIC SPINES AND SYNAPTOGENESIS 6
1.4 NEURON SUBTYPES AND HIPPOCAMPAL STRUCTURE 11
1.5 RHO GTPASES IN NEURONAL DIFFERENTIATION 13
1.5.1 DIVERGENT FUNCTIONS OF RHO G TPASES 13
1.5.2 DOWNSTREAM TARGETS OF RHO G TPASES 14
1.5.3 REGULATORS OF RHO GTPASE SIGNALING 14
1.5.4 REGULATION OF RHOGEFS LIKE APIX 16
1.6 NEURONAL DIFFERENTIATION FACTORS OF THE EGF SUPERFAMILY 17
1.7 CALEB/NGC/CSPG5 18
1.7.1 BIOCHEMICAL ASPECTS OF CALEB 19
1.7.2 EXPRESSION OF CALEB 20
1.7.3 MOLECULAR INTERACTIONS WITH CALEB 21
1.7.4 THE FUNCTIONAL ROLE OF CALEB IN THE NERVOUS SYSTEM 21
1.8 AIM OF THIS STUDY 22
2 MATERIALS AND
METHODS 24
2.1 MATERIALS 24
2.1.1 REAGENTS 24
2.1.2 STANDARD SOLUTIONS 27
2.1.3 EQUIPMENT 28
2.1.4 BACTERIAL STRAINS 29
2.1.5 CELL LINES 29
2.1.6 LAB ANIMALS 30
2.1.7 PLASMIDS 30
2.1.8 OLIGONUCLEOTIDES 33
2.1.9 ANTIBODIES 35
2.1.10 RESEARCH AND ANALYSIS SOFTWARE 37
2.1.11 ONLINE RESSOURCES 38
III
TABLE OF CONTENTS
2.2 METHODS 38
2.2.1 CULTIVATION OF E. COLI 38
2.2.2 GENERATION OF CHEMICALLY COMPETENT E. COLI 38
2.2.3 TRANSFORMATION OF E. COLI 39
2.2.4 CONSERVATION OF BACTERIAL STRAINS 39
2.2.5 ISOLATION OF MRNA 39
2.2.6 CDNA SYNTHESIS 40
2.2.7 PCR 40
2.2.8 DNA GEL ELECTROPHORESIS 41
2.2.9 NUCLEIC ACID PURIFICATION 41
2.2.10 RESTRICTION DIGEST 42
2.2.11 DEPHOSPHORYLATION OF DNA 5 -ENDS 42
2.2.12 PHOSPHORYLATION AND ANNEALING OF OLIGONUCLEOTIDES 42
2.2.13 DNA LIGATION 43
2.2.14 PLASMID DNA ISOLATION 43
2.2.15 QUANTIFICATION OF NUCLEIC ACIDS 43
2.2.16 SEQUENCING 44
2.2.17 CLONING OF MODIFIED CONSTRUCTS 44
2.2.18 GENOTYPING OF TRANSGENIC MICE 45
2.2.19 RECOMBINANT PROTEIN EXPRESSION IN E. COLI 45
2.2.20 PURIFICATION OF GST FUSION PROTEINS FROM E. COLI 46
2.2.21 QUANTIFICATION OF PROTEINS 46
2.2.22 DIALYSIS AND CONCENTRATION OF PROTEIN SOLUTIONS 47
2.2.23 COUPLING OF GST FUSION PROTEINS TO CNBR-ACTIVATED SEPHAROSE 48
2.2.24 PROTEIN LYSATES 48
2.2.25 A FFINITY CHROMATOGRAPHY 49
2.2.26 CO-IMMUNOPRECIPITATION OF PROTEINS 49
2.2.27 GST PULL-DOWN ASSAY 50
2.2.28 SDS-PAGE 50
2.2.29 COOMASSIE-STAIN 52
2.2.30 TRANSFER OF PROTEINS TO PVDF-MEMBRANES 52
2.2.31 DEVELOPMENT OF IMMUNOBLOTS 53
2.2.32 CULTIVATION OF CELL LINES 54
2.2.33 CONSERVATION OF CELL LINES 54
2.2.34 TRANSIENT TRANSFECTION OF CELL LINES 54
2.2.35 PRIMARY HIPPOCAMPAL NEURONS IN CULTURE 55
2.2.36 TRANSFECTION OF PRIMARY HIPPOCAMPAL NEURONS 56
2.2.37 IMMUNOSTAINING OF PRIMARY HIPPOCAMPAL NEURONS 56
2.2.38 IMMUNOHISTOCHEMISTRY WITH FREE-FLOATING VIBRATOME SECTIONS 57
IV
TABLE OF CONTENTS
2.2.39 IMMUNOHISTOCHEMISTRY ON CRYOSECTIONS 58
2.2.40 MICROSCOPY 58
2.3 ANALYSIS 59
2.3.1 STATISTICS 59
2.3.2 RELATIVE IMMUNOFLUORESCENCE INTENSITY 59
2.3.3 TNDET 60
2.3.4 SPINE PARAMETERS 60
3 RESULTS 61
3.1 PROTEIN-PROTEIN INTERACTION SCREEN FOR THE CYTOPLASMIC TAIL OF CALEB
61
3.1.1 AFFINITY CHROMATOGRAPHY IDENTIFIES NEUROFASCIN AS A PUTATIVE
BINDING PARTNER.. 62
3.1.2 IMMUNOPRECIPITATE OF NEUROFASCIN ISOFORM 3 LACKS CALEB 63
3.2 BIOCHEMICAL ANALYSIS OF A NOVEL CALEB-APIX COMPLEX 64
3.2.1 BINDING OF CALEB AND APIX MANIFESTS IN POSITIVE
CO-IMMUNOPRECIPITATIONS
...64
3.2.2 MAPPING OF THE INTERACTION DOMAINS WITH DELETION CONSTRUCTS OF
APIX 66
3.2.3 THE AFFINITY OF CALEB FOR APIX EXTENTS TO OTHER RELATED RHOGEFS 70
3.2.4 MAPPING OF THE INTERACTION DOMAINS WITH DELETION CONSTRUCTS OF
CALEB 71
3.3 ENDOGENOUS EXPRESSION OF CALEB AND APIX 73
3.3.1 CALEB AND APIX INHABIT THE POSTNATAL MOUSE BRAIN 73
3.3.2 CALEB AND APIX ARE PRESENT IN HIPPOCAMPAL TISSUE OF MICE AND RATS
74
3.3.3 CALEB AND APIX COLOCALIZE IN PRIMARY HIPPOCAMPAL NEURONS 75
3.4 FUNCTIONAL ANALYSIS OF CALEB AND APIX IN CULTURED HIPPOCAMPAL
NEURONS 76
3.4.1 APIX INFLUENCES CALEB-MEDIATED DENDRITIC TREE COMPLEXITY 78
3.4.2 APIX AFFECTS CALEB-DRIVEN SPINE MORPHOGENESIS 82
3.5 FUNCTIONAL ANALYSIS OF CALEB-DEFICIENT NEURONS 86
3.5.1 GENERATION OF A CALEB NULL MUTANT 86
3.5.2 THE REDUCTION OF DENDRITIC BRANCHING BY KDCALEB IS CAUSED BY THE
LOSS OF
CALEB 89
3.5.3 SILENCING OF APIX HAS NO EFFECT ON DENDRITIC ARBORIZATION IN
CALEB-LACKING
NEURONS 90
3.5.4 MODULATION OF SPINE DENSITY BY APIX DEPENDS ON CALEB PROTEIN
EXPRESSION. 91
4 DISCUSSION 93
4.1 IDENTIFICATION OF NEUROFASCIN AS A NOVEL INTERACTION PARTNER FOR
CALEB 93
4.2 BIOCHEMICAL ASPECTS ABOUT CALEB, APIX AND THEIR ASSEMBLY 94
4.3 IMPLICATIONS RELATED TO THE EXPRESSION OF CALEB AND APIX PROTEIN
COMPONENTS.... 98
4.4 FUNCTIONAL QUALITIES OF CALEB AND APIX FOR NEURONAL IN VITRO
DIFFERENTIATION 100
4.4.1 RECIPROCAL SHAPING OF DENDRITIC TREE COMPLEXITY BY CALEB AND APIX
100
4.4.2 MODULATION OF SPINE DENSITY BY THE INTERPLAY OF CALEB AND APIX.
101
4.4.3 A PUTATIVE ROLE FOR CALEB AND APIX IN NEURONAL CONNECTIVITY 102
V
TABLE OF CONTENTS
4.5 DEDUCTION OF A MUTUAL SIGNALING PATHWAY FOR CALEB AND APIX 104
4.6 CALEB DEFICIENCY BY TARGETED GENE KNOCKOUT 105
4.7 REFERENCES TO THE LITERATURE 106
4.8 SUGGESTIONS FOR A REGULATORY MECHANISM OF CALEB-INDUCED SIGNALING BY
APIX... 109
4.9 OUTLOOK 112
REFERENCES 114
ACKNOWLEDGEMENTS 130
CURRICULUM VITAE 132
LEGAL STATEMENT 133
VI
|
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| spellingShingle | Johannes, Sascha Functional characterization of alphaPIX/Cool-2/ARHGEF6 as a novel interaction partner for CALEB/NGC/CSPG5 in the regulation of dendritic process and spine formation Epidermaler Wachstumsfaktor (DE-588)4271160-5 gnd Dendritische Zelle (DE-588)4313846-9 gnd |
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| title | Functional characterization of alphaPIX/Cool-2/ARHGEF6 as a novel interaction partner for CALEB/NGC/CSPG5 in the regulation of dendritic process and spine formation |
| title_auth | Functional characterization of alphaPIX/Cool-2/ARHGEF6 as a novel interaction partner for CALEB/NGC/CSPG5 in the regulation of dendritic process and spine formation |
| title_exact_search | Functional characterization of alphaPIX/Cool-2/ARHGEF6 as a novel interaction partner for CALEB/NGC/CSPG5 in the regulation of dendritic process and spine formation |
| title_full | Functional characterization of alphaPIX/Cool-2/ARHGEF6 as a novel interaction partner for CALEB/NGC/CSPG5 in the regulation of dendritic process and spine formation vorgelegt von Sascha Johannes |
| title_fullStr | Functional characterization of alphaPIX/Cool-2/ARHGEF6 as a novel interaction partner for CALEB/NGC/CSPG5 in the regulation of dendritic process and spine formation vorgelegt von Sascha Johannes |
| title_full_unstemmed | Functional characterization of alphaPIX/Cool-2/ARHGEF6 as a novel interaction partner for CALEB/NGC/CSPG5 in the regulation of dendritic process and spine formation vorgelegt von Sascha Johannes |
| title_short | Functional characterization of alphaPIX/Cool-2/ARHGEF6 as a novel interaction partner for CALEB/NGC/CSPG5 in the regulation of dendritic process and spine formation |
| title_sort | functional characterization of alphapix cool 2 arhgef6 as a novel interaction partner for caleb ngc cspg5 in the regulation of dendritic process and spine formation |
| topic | Epidermaler Wachstumsfaktor (DE-588)4271160-5 gnd Dendritische Zelle (DE-588)4313846-9 gnd |
| topic_facet | Epidermaler Wachstumsfaktor Dendritische Zelle Hochschulschrift |
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