Regulation of SRF via Rac-actin-MAL signalling in epithelial cells [Elektronische Ressource] / Stephan Busche

Mpi

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 26
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN


Institut für Organische Chemie und Biochemie
Lehrstuhl für Biotechnologie



Regulation of SRF via Rac-Actin-MAL
Signalling in Epithelial Cells



Stephan Busche



Vollständiger Abdruck der von der Fakultät für Chemie
der Technischen Universität München zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. St. J. Glaser
Prüfer der Dissertation:
1. Priv.-Doz. Dr. N. Budisa
2. Univ.-Prof. Dr. J. Buchner



Die Dissertation wurde am 29.10.2008 bei der Technischen Universität München
eingereicht und durch die Fakultät für Chemie am 03.12.2008 angenommen.




































for my parents Index i

Index

I.  Summary ....................................................................................................................................... 1 
I.  Zusammenfassung ........................................................................................................................ 2 
II.  Introduction .................................................................................................................................. 3 
1.  The Epithelium ............................................................................................................................ 3 
2.  Molecular composition of the apical junctional complex ........................................................... 4 
2.1.  Tight junctions ..................................................................................................................... 4 
2.2.  Adherens junctions .............................................................................................................. 4 
3.  The actin cytoskeleton ................................................................................................................. 6 
3.1.  Actin filament assambly ...................................................................................................... 6 
3.2. ent disassembly ................................................................................................. 8 
3.3.  Actin contractility ................................................................................................................ 8 
3.4.  Actin modifying compounds ............................................................................................... 9 
4.  Small guanosine triphosphatases ................................................................................................. 9 
4.1.  Clostridial toxins as inhibitors of RhoGTPases ................................................................. 10 
5.  Regulation of epithelial cell-cell adhesion ................................................................................ 10 
5.1.  Epithelial junction formation ............................................................................................. 11 
5.1.1.  Contact formation induced activation of GTPases .................................................... 11 
5.1.2.  Actin binding proteins as effectors of GTPases ........................................................ 12 
5.1.3.  Myosin involvement in junction formation ............................................................... 12 
5.1.4.  Additional mechanisms inducing junction formation and stabilization .................... 13 
5.2.  Epithelial junction dissociation ......................................................................................... 13 
5.2.1.  Epithelial-Mesenchymal Transition .......................................................................... 13 
5.2.2.  Scattering ................................................................................................................... 14 
5.2.3.  Calcium as a tool to manipulate epithelial junction dynamics .................................. 15 
5.3.  Discrimination between adherens and tight junction mediated signalling ........................ 15 
6.  Rho-actin-SRF pathway ............................................................................................................ 16 
6.1.  Serum Response Factor ..................................................................................................... 16 
6.2.  Regulation of SRF via the Rho-actin pathway in fibroblasts ............................................ 17 
6.3.  Endogenous SRF target genes ........................................................................................... 18 
7.  Cellular model systems ............................................................................................................. 19 
III.  Aims of this PhD thesis ............................................................................................................... 20 
IV.  Results .......................................................................................................................................... 21 
1.  Serum stimulation does not activate SRF in confluent epithelial cells ..................................... 21 
2.  Calcium as a tool to manipulate the epithelial integrity of MDCK cells................................... 22 
3.  SRF activity upon epithelial cell-cell contact formation ........................................................... 23 
4.  SRF activation induced by the dissociation of epithelial cell-cell contacts ............................... 24 
5.  E-cadherin deficient cell lines do not activate SRF ................................................................... 27 
6.  Time course of cell-cell contact disintegration and SRF induction ........................................... 28 Index ii

7.  Epithelial cell-cell contact dissociation activates the small GTPases Rac1 and RhoA ............. 30 
8.  Active Rac1, but not Rho, induces SRF .................................................................................... 32 
9.  Dissociating epithelial cell-cell contacts activate SRF via monomeric actin ............................ 34 
10.  Actomyosin contractility is not sufficient to activate SRF .................................................... 37 
11.  SRF activation upon epithelial cell-cell contact dissociation is dependent on MAL ............ 39 
12.  Adherens junctions seem to be essential for SRF activation ................................................. 41 
13.  Induction of endogenous SRF target genes ........................................................................... 43 
14.  SRF activation upon junction dissociation independent of calcium ...................................... 44 
V.  Discussion .................................................................................................................................... 45 
VI.  Materials and Methods............................................................................................................... 53 
1.  Materials .................................................................................................................................... 53 
1.1.  Laboratory hardware ......................................................................................................... 53 
1.2.  Chemicals and reagents ..................................................................................................... 54 
1.3.  Drugs and inhibitors used in cellular assays ...................................................................... 55 
1.4.  Kits and miscellaneous materials ...................................................................................... 56 
1.5.  Media, buffers and solutions ............................................................................................. 56 
1.5.1.  Bacterial media .......................................................................................................... 56 
1.5.2.  Cell culture media...................................................................................................... 57 
1.6.  Buffers and solutions ......................................................................................................... 57 
1.7.  Oligonucleotides ................................................................................................................ 59 
1.7.1.  Sequencing primers ................................................................................................... 59 
1.7.2.  Cloning primers ......................................................................................................... 60 
1.7.3.