Identification and characterization of casein kinase 2 as MuSK binding partner [Elektronische Ressource] / vorgelegt von Tatiana Cheusova

Sujets

• Biologie

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2006
• Nombre de lectures: 58
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

Extrait

Identification and characterization of
Casein Kinase 2 as MuSK binding partner





Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades





vorgelegt von
Tatiana Cheusova
aus Nowosibirsk, Russland

2006





Als Dissertation genehmigt von den Naturwissen-
schaftlichen Fakultäten der Universität Erlangen-Nürnberg










Tag der mündlichen Prüfung: 7. Juni 2006

Vorsitzender der Promotionskommission: Prof. Dr. D.-P. Häder
Erstberichterstatter: PD Dr. F. Titgemeyer
Zweitberichterstatter: Prof. Dr. M. Wegner


























To my mother
Table of contents
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Table of contents
Zusammenfassung.................................................................................................................... 1
Summary................................................................................................................................... 2
1. Introduction.......... 3
1.1. Structure and function of the NMJ................................................................................... 3
1.1.1. The presynaptic part is formed by motoneuron......................................................... 4
1.1.2. The postsynaptic part is generated by myotubes....................................................... 4
1.1.3. The role of Schwann cells at the NMJ....................................................................... 5
1.1.4. The basal lamina at the NMJ ..................................................................................... 6
1.1.5. Physiology of the NMJ.............................................................................................. 6
1.2. Development of the NMJ................................................................................................. 7
1.2.1. Origin of cells ............................................................................................................ 7
1.2.2. Establishment of nerve-muscle contact ..................................................................... 8
1.2.3. Postsynaptic differentiation ....................................................................................... 9
1.3. Molecules and signaling cascades involved in the postsynaptic differentiation............ 10
1.3.1. Agrin-MuSK-rapsyn signaling cascade................................................................... 10
1.3.2. Synapse specific transcription ................................................................................. 17
1.3.3. MuSK binding partners ........................................................................................... 19
2. Aim of the study.................................................................................................................. 23
3. Material and methods ........................................................................................................ 25
3.1. Materials ........................................................................................................................ 25
3.1.1. Reagents................................................................................................................... 25
3.1.2. Devices... 26
3.1.3. Oligonucleotides...................................................................................................... 27
3.1.3. siRNAs..................................................................................................................... 31
3.1.4. Enzymes.. 34
3.1.5. Kits and Columns .................................................................................................... 34
3.1.6. Antibodies................................................................................................................ 35
3.1.7. Frequently used solutions ........................................................................................ 36
3.1.8. Cell culture .............................................................................................................. 37
3.1.9. Animals.................................................................................................................... 37
3.2. Methods.......................................................................................................................... 38
3.2.1. Molecular Biology Methods.................................................................................... 38
I Table of contents
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3.2.1.1. Isolation of plasmid DNA ................................................................................. 38
3.2.1.2. Determination of DNA/RNA concentration ..................................................... 38
3.2.1.3. Electrophoretic separation of DNA fragments in agarose gel........................... 39
3.2.1.4. PCR amplification of DNA............................................................................... 39
3.2.1.5. Cloning techniques............................................................................................ 39
3.2.1.6. Plasmid constructs............................................................................................. 40
3.2.1.7. Transformation of E. coli competent cells. ....................................................... 42
3.2.1.8. Site directed mutagenesis.................................................................................. 43
3.2.1.9. Lightcycler PCR................................................................................................ 44
3.2.1.10. Total RNA isolation ........................................................................................ 45
3.2.1.11. Complementary DNA-synthesis (Reverse Transcription) .............................. 45
3.2.1.12. Yeast two hybrid (Y2H) techniques................................................................ 46
3.2.2. Protein Biochemistry Methods ................................................................................ 49
3.2.2.1. Preparation of protein extract from cells and tissues. ....................................... 49
3.2.2.2. Immunoprecipitation ......................................................................................... 49
3.2.2.3. Protein expression and extraction from bacteria............................................... 50
3.2.2.4. GST-pulldown................................................................................................... 51
3.2.2.5. Determination of protein concentration ............................................................ 51
3.2.2.6. Electrophoresis of proteins................................................................................ 51
3.2.2.7. Staining of protein gels ..................................................................................... 52
3.2.2.8. Western blot ...................................................................................................... 53
3.2.2.9. In vitro kinase assay .......................................................................................... 53
3.2.3. Cell culture methods................................................................................................ 54
3.2.3.1. Cultivation of HEK293, Cos7, C2C12, MuSK-deficient myoblasts................. 54
3.2.3.2. Transient transfection of cells ........................................................................... 55
3.2.3.3. Luciferase reporter test...................................................................................... 56
3.2.3.4. Agrin treatment ................................................................................................. 57
3.2.3.5. Application of CK2 inhibitors 57
3.2.3.6. Immunocytochemistry....................................................................................... 57
3.2.3.7. AChR cluster stability assay ............................................................................. 58
3.2.3.8. Quantification analysis of AChR clusters .................................................... 58
3.2.4.Animal care and immunohistochemistry methods ................................................... 58
3.2.4.1. Generation of muscle specific CK2β knockout animals................................... 58
3.2.4.2. Genotyping........................................................................................................ 59
II Table of contents
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3.2.4.3. Surgical Procedures........................................................................................... 60
3.2.4.4. Immunohistochemistry...................................................................................... 60
3.2.4.5. Mycroscopy, imaging and quantification of endplates. .................................... 61
4. Results .................................................................................................................................