Molekulare Mechanismen in Zelldifferenzierung und Zellteilung [Elektronische Ressource] = Molecular mechanisms in cell differentiation and cell division / vorgelegt von Arminja Nadine Kettenbach

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2007
• Nombre de lectures: 92
• Langue: Deutsch
• Poids de l'ouvrage: 14 Mo

Extrait

Molekulare Mechanismen in
Zelldifferenzierung und Zellteilung


Molecular Mechanisms in Cell Differentiation and Cell Division






DISSERTATION




der Fakultät für Chemie und Pharmazie
der Eberhard-Karls Universität Tübingen

zur Erlangung des Grades eines Doktors
der Naturwissenschaften




2006


vorgelegt von



Arminja Nadine Kettenbach




























Tag der mündlichen Prüfung: 17. April 2007


Dekan: Prof. Dr. Wesemann

1. Berichterstatter: Prof. Dr. McKeon

2. Berichterstatter: Prof. Dr. Rammensee

3. Berichterstatter: Prof. Dr. Werner























Die vorliegende Arbeit wurde unter der Anleitung von

Prof. Dr. Frank D. McKeon

in der Zeit von März 2003 bis August 2006, am Institut für Zellbiologie an der
Harvard Medical School, Boston, USA, durchgeführt.

Prof. Dr. Hans-Georg Rammensee vom Interfakultären Institut für Zellbiologie,
Abteilung Immunologie, Eberhard-Karls-Universität Tübingen, Deutschland,
übernahm die Vertretung der Arbeit vor der Fakultät fuer Chemie und Pharmazie der
Eberhard-Karls-Universität Tübingen.



























































































WIDMUNG

Meiner Mutter in grosser Dankbarkeit gewidmet.

Table of contents

Table of Contents



Part I – Mice deficient for TA-p63

1. Summary………………………………………………………….……………..1

2. Introduction……………………………………………………………………..2

2.1 The gene structure of p63……………………......……………….……2
2.2 Expression of p63…………………………………………………...…..4
2.3 Skin morphogenesis and organization………………………………..5
2.4 Oocyte development and TA-p63.…………………………………..…7
2.5 Mice deficient for all p63 variants…………………………………..….8
2.6 TA-p63 versus ∆N-p63…………………………………...…...………11
2.7 Germline mutation of p63 in human syndromes……………….......12
2.8 Functional interactions of p53 family members ……………...….…13

3. Results………………………………………………...……………………….15
3.1 Generation of TA-p63 deficient mice.......…………………………...15
3.2 The TA-p63 targeting construct………………………………………16
3.3 Generation of TA-p63 (+/-) ES cells………………………………... 17
3.4 From ES cells to mice: Chimeras and TA-p63 deficient mice....….18
3.5 Characterization of p63 expression in TA-p63(-/-) mice……......…19
3.6 TA-p63(-/-) mice are born at a non-mendelian frequency.………...20
3.7 TA-p63(-/-) mice display normal epidermal development………....23

4. Discussion…………………………………………………......……………..32
4.1 Mice deficient for TA-p63……………………………………………..32
4.2 Normal skin development in TA-p63(-/-) mice……………………...33
4.3 Non-mendelian frequency, newborn death, and lack of pregnancies
of TA-p63(-/-) mice…………………………………….......................35

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Table of contents

Part II – Checkpoint signaling

5. Summary……………………………………………………………………….37

6. Introduction……………………………………………………………………38
6.1 Chromosome segregation…………………………………………….38
6.2 The different phase of mitosis………………………………………..39
6.3 Mitotic progression……………………………………...……………..41
6.4 Meiosis…...……………………………………………………………..42
6.5 Mechanism of error prevention and correction…………………......43
6.6 The Spindle Assembly Checkpoint……………………………..……44
6.7 Function and structure of BubR1………………………………….....46
6.8 Sister chromatid cohesion…………………………………………….47
6.9 The Shugoshin protein family………………………………………...48

7. Results…………………………………………………………………………50
7.1 Characterization of BubR1 protein………………………………......50
7.2 Immunoprecipitation of BubR1 from mitotically-arrested HeLa
cells……………………………………………………………………...52
7.3 Differential quantification of BubR1 phosphorylation sites and
interacting proteins by SILAC………………………………………...55
7.4 Investigation of BubR1 phosphorylation by mutagenesis.…………59
7.5 BubR1 and Bub1 domain swap………………………………………63
7.6 Kinases involved in BubR1 phosphorylation………………………..64
7.7 Confirmation of BubR1-Sgo2 interaction.……………………...........67
7.8 Characterization of Sgo2 protein.…………………………………….68
7.9 Depletion of Sgo2 by siRNA………………………………………….70
7.10 Sgo2 depletion induced mitotic arrest in HeLa cells……………….71
7.11 Chromosome mis-alignment and tension defects in Sgo2
siRNA cells……………………………………………………………..73
7.12 Microtubule attachment defects in Sgo2 siRNA cells……………...75
7.13 Sister chromatid cohesion in Sgo2 siRNA cells…………………....77
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Table of contents
7.14 Distortions in the inter-kinetochore of Sgo2 depleted cells………..78
7.15 Kinetochore-microtubule interactions in a monopolar spindle…....83
7.16 Microtubule dependent re-localization of Sgo2………………….....85
7.17 Interaction of BubR1-Sgo2……………………………………………87

8. Discussion……………………………………………………………………89
8.1 Expression and post-translation modifications of BubR1…………89
8.2 Evolution of protein function by post-translational modifications?..90
8.3 Sgo2 is a novel BubR1 interacting protein………………………….91
8.4 Sgo2’s function in maintenance of a rigid kinetochore structure…92

9. Materials……………………………………………………………………….95
9.1 Reagents………………………………………………………………..95
9.2 Materials…………………………………………………………...…...97
9.3 Kits………………………………………………………………………97
9.4 Instruments……………………………………………………………..97
9.5 Software…………………………………………………………..........98
9.6 Buffers…………………………………………………………….........99

10. Methods…………………………………………………………………...….101
10.1 Molecular biology methods……………………………………….101
10.1.1 Polymerase Chain Reaction (PCR)………………………...101
10.1.2 PCR purification and gel extraction………………..............101
10.1.3 Restriction enzyme digest………………............................101
10.1.4 Ligation of DNA fragment………….……............................101
10.1.5 Cloning……………………………………............................101
10.1.6 Transformation of bacteria………......................................102
10.1.7 Plasmid DNA preparation from bacteria............................102
10.1.8 Mutagenesis………………………………………….............102
10.1.9 DNA sequencing................................................................102
10.1.10 SDS-PAGE……………………………………....................102
10.1.11 Coomassie brilliant blue staining.....................................103
10.1.12 Western Blot…................................................................104
-III-

Table of contents
10.1.13 Immunohistochemistry……………………………………..104
10.1.14 Immunoprecipitation………………………………………..105
10.1.15 Immunofluorescence……………………………………….105
10.1.16 Microscopy…………………………………………………..106
10.1.17 Lambda phosphatase treatment…………………………..107
10.1.18 Antigen preparation………………………………………....107
10.1.19 Antibody purification……………………..………….…..….107
10.1.20 Cross-linking.……………………………………………......108
10.1.21 In vitro Kinase assay………………………...................…108
10.1.22 Generation of TA-p63 targeting construct………………..108
10.1.23 Isolation of genomic DNA from ES cells………………….109
10.1.24 Isolation of genomic DNA from mice tail……….110
10.1.25 Polymerase Chain Reaction (PCR) for genotyping……..110
10.1.26 Southern Blot analysis……………………………………...110
10.1.27 Random Prime labeling of Southern Blot Probe………...111

10.2 Cell biology methods………………………………………...…….112
10.2.1 Cell culture……………………………………………………112
10.2.2 SILAC………………………………………………………….112
10.2.3 Cell synchronization………………………………………….112
10.2.4 Drug treatments……………………………………………....112
10.2.5 siRNA transfection……………………………………………113
10.2.6 Retrovirus packing and transduction…………………….…113
10.2.7 Generation of murine embryonic fibroblasts (MEFs)…..…114
10.2.8 Generation of MEF feeder l