A channel independent function of connexin 43 in cell migration [Elektronische Ressource] / Juliane Behrens. Betreuer: Stefan Zahler

Juliane Behrens

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 36
• Langue: Deutsch
• Poids de l'ouvrage: 10 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München




A channel independent function
of connexin 43 in cell migration






Juliane Viktoria Behrens

aus Wesel





2011

Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29.
Januar 1998 (in der Fassung der sechsten Änderungssatzung vom 16. August 2010) von Herrn
Professor Dr. Ulrich Pohl betreut und von Herrn Professor Dr. Stefan Zahler von der Fakultät
für Chemie und Pharmazie vertreten.




Ehrenwörtliche Versicherung
Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.




München, 07.10.2011




Juliane Behrens




Dissertation eingereicht am: 07.10.2011

1. Gutachter: Prof. Dr. Stefan Zahler
2. Gutachter: Prof. Dr. Ulrich Pohl

Mündliche Prüfung am: 14.11.2011






























Success in science is about combining ambition, imagination and knowledge Table of Contents I
Table of Contents

I Introduction.............................................................................................................................. 1
1 Gap Junctions .................................................................................................................... 1
2 Cx43 .................................................................................................................................. 4
2.1 Cx43: function as a channel and as an adapter............................................................ 4
2.2 Proteins interacting with the C-terminal part of Cx43 ................................................ 6
2.2.1 Cortactin ................................................................................................................ 7
2.2.2 The nephroblastoma-overexpressed protein (NOV; CCN3)................................. 8
3 Cell migration.................................................................................................................... 9
3.1 Cell migration and actin dynamics.............................................................................. 9
3.2 Role of Cx43 in cell migration.................................................................................. 13
4 Aim of the study.............................................................................................................. 15
II Materials and Methods ......................................................................................................... 16
1 Materials.......................................................................................................................... 16
1.1 Chemicals .................................................................................................................. 16
1.2 Cell culture ................................................................................................................ 17
1.3 Fluorescent activated cell sorting.............................................................................. 19
1.4 Migration................................................................................................................... 19
1.5 Cloning...................................................................................................................... 19
1.6 Western blotting ........................................................................................................ 21
1.7 Immunoprecipitation ................................................................................................. 23
1.8 Immunofluorescence ................................................................................................. 23
1.9 Staining of the actin cytoskeleton ............................................................................. 24
1.10 Lab Equipment ........................................................................................................ 24
2 Methods........................................................................................................................... 26
2.1 Cell biological methods............................................................................................. 26
2.1.1 Cell culture .......................................................................................................... 26
2.1.1.1 Cell lines....................................................................................................... 26
2.1.1.2 Cell passaging .............................................................................................. 27
2.1.1.3 Cell freezing ................................................................................................. 27
2.1.1.4 Cell thawing ................................................................................................. 27
2.1.2 Lethality curve..................................................................................................... 28
2.1.3 Transfection of HeLa wt cells ............................................................................. 28
2.1.3.1 Transient transfection of HeLa wt cells ....................................................... 28
2.1.3.2 Stable transfection of HeLa wt cells ............................................................ 29
2.1.4 Analysis of cell coupling using fluorescent activated cell sorting (FACS) ........ 30
2.1.5 Analysis of cell migration ................................................................................... 32
2.2 Cloning of cDNAs encoding truncated Cx43 proteins.............................................. 34
2.2.1 Preparation of plasmid DNA............................................................................... 35
2.2.2 Polymerase chain reaction (PCR)........................................................................ 35
2.2.3 Agarose gel electrophoresis................................................................................. 37
2.2.4 Purification of the PCR product.......................................................................... 38
2.2.5 Digestion of DNA with restriction enzymes ....................................................... 38
2.2.6 Isolation of DNA from agarose gels ................................................................... 39
2.2.7 DNA ligation ....................................................................................................... 39
2.2.8 Transformation of DNA in bacteria .................................................................... 39
2.3 Protein biochemistry ................................................................................................. 41
2.3.1 Western blot analysis .......................................................................................... 41
2.3.1.1 SDS-Polyacrylamid gel electrophoresis (SDS-PAGE) ................................ 41
2.3.1.2 Semi-dry blotting.......................................................................................... 42Table of Contents II
2.3.1.3 Protein detection by western blot analysis ................................................... 42
2.3.1.4 Membrane stripping ..................................................................................... 43
2.3.2 Immunoprecipitation ........................................................................................... 43
2.4 Confocal laser scanning microscopy......................................................................... 45
2.4.1 Immunofluorescence staining.............................................................................. 45
2.4.2 Staining of the actin cytoskeleton ....................................................................... 46
2.4.2.1 Staining of migrating HeLa cells ................................................................. 47
2.4.2.2 Staining of HeLa cells after inhibition of p38 MAPK ................................. 47
3 Statistics .......................................................................................................................... 48
III Results ................................................................................................................................. 49
1 Generation and functional analysis of truncated Cx43 proteins ..................................... 49
1.1 Generation and separate expression of the N-terminal and the C-terminal part of
Cx43 in HeLa cells.................................................................................................... 49
1.2 HeLa 43NT-GFP cells were functionally coupled via gap junctions........................ 52
1.3 The C-terminal part of Cx43 increases cell migration of HeLa cells........................ 53
2 Generation of further truncated Cx43 proteins: .............................................................. 54
Cx43(tr298)-GFP and Cx43(tr336)-GFP ........................................................................... 54
2.1 Expression of Cx43(tr298)-GFP and C