Induction and regulation of plasma membrane blebbing by SH4 domains and the diaphanous related formin FHOD1 [Elektronische Ressource] / presented by Sebastian Hannemann

Sebastian Brant

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 36
• Langue: English
• Poids de l'ouvrage: 40 Mo

Extrait

Dissertation

submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of

Doctor of Natural Sciences


Induction and Regulation
of Plasma Membrane Blebbing
by SH4-Domains and the
Diaphanous Related Formin FHOD1










presented by
Diplom-Biologe Sebastian Hannemann
born in: Hildesheim, Germany
Oral-examination:


































Referees: Prof. Dr. Hans-Georg Kräusslich
Prof. Dr. Oliver Till Fackler









Meiner Familie


















Goethe, 1808

"Grau, teurer Freund, ist alle Theorie,
Und grün des Lebens goldner Baum."

Johann Wolfgang von Goethe
Faust, der Tragödie erster Teil, Studierzimmer




INDEX
1 PUBLICATIONS.........................................................................................................I
2 ABBREVIATIONS......................................................................................................II
3 SUMMARY...............................................................................................................1
4 ZUSAMMENFASSUNG ...............................................................................................3
5 INTRODUCTION........................................................................................................5
5.1 The Microfilament System............................................................................................................. 5
5.1.1 The Actin Protein and Microfilaments ........................................................................................... 6
5.1.2 The Dynamics of the Microfilament System.................................................................................. 8
5.1.3 Diaphanous Related Formins ..................................................................................................... 17
5.1.4 The Regulation of the Microfilament System During Cell Motility............................................... 21
5.2 Plasma Membrane Blebbing....................................................................................................... 23
5.2.1 Formation of PM Blebs and the Contribution of the Cortical Actin.............................................. 25
5.3 Src Kinases and SH4-Domain Containing Proteins.................................................................... 28
6 OBJECTIVES OF THE STUDY ...................................................................................34
7 MATERIAL AND METHODS ......................................................................................36
7.1 Materials...................................................................................................................................... 36
7.1.1 Reagents..................................................................................................................................... 36
7.1.2 Consumables .............................................................................................................................. 36
7.1.3 Sterilization.................................................................................................................................. 36
7.1.4 Cell Culture Media and Solutions................................................................................................36
7.1.5 Bacterial Growth Media............................................................................................................... 37
7.1.6 Eukaryotic Cell Lines................................................................................................................... 37
7.1.7 Bacterial Strains .......................................................................................................................... 39
7.1.8 siRNA Oligonucleotides..... 39
7.1.9 Plasmids............... 39
7.1.10 Antibodies ................................................................................................................................... 42
7.1.11 Standard Buffers ......................................................................................................................... 43
7.1.12 Software............... 47
7.2 Molecular Biology.......... 48
7.2.1 Transformation of Bacteria.......................................................................................................... 48
7.2.2 Preparation of Plasmid DNA.. 48
7.2.3 Determination of DNA Concentration.......................................................................................... 48
7.2.4 Digestion of DNA with Restriction Endonucleases ..................................................................... 48
7.2.5 Electrophoretic Separation of DNA............................................................................................. 49
7.3 Biochemistry................................................................................................................................ 49
7.3.1 Production of Cell Lysates..... 49
7.3.2 Determination of Overall Protein Concentration ......................................................................... 49
7.3.3 Discontinuous SDS-PAGE..........................................................................................................49
7.3.4 Western Blotting.......................................................................................................................... 50
7.3.5 Immunoprecipitation (IP) and in vitro Kinase Assay (IVKA)........................................................ 50
7.3.6 Purification of rabbit-α-FHOD1 Antibody .................................................................................... 51
7.4 Cell Biology.............. 52
7.4.1 Cultivation of Adherent Cell Lines............................................................................................... 52
7.4.2 Transient Transfection of Animal Cells ....................................................................................... 52
7.4.3 RNAi Based Knockdown of Endogenous FHOD1 ...................................................................... 53
7.4.4 Induction of Gene Expression..................................................................................................... 54
7.4.5 Microscopy .................................................................................................................................. 54
7.4.6 Inhibition of PM Blebbing by Drug-Treatment............................................................................. 56
7.4.7 Induction, Detection and Inhibition of Apoptosis......................................................................... 57
7.4.8 VSV-G Transport Assay.............................................................................................................. 57
7.4.9 Adhesion and Proliferation Assay............................................................................................... 57



7.4.10 Measurement of Cell Size........................................................................................................... 58
7.4.11 SRE Transcription Assay ............................................................................................................ 58
7.4.12 Wound Healing Assay for 2D-Motility ......................................................................................... 58
7.4.13 Chemotaxis Assay ...................................................................................................................... 59
7.4.14 Invasion Assay for 3D-Motility..................................................................................................... 59
7.4.15 Golgi Disruption Assay................................................................................................................ 60
7.4.16 Measurement of F-actin Content by Flow Cytometry ................................................................. 60
7.4.17 Endocytosis Assays .................................................................................................................... 60
8 RESULTS..............................................................................................................62
8.1 SH4-Domain Mediated PM Blebbing .......................................................................................... 62
8.1.1 Generation of Stable SH4-Domain Cell Lines............................................................................. 62
8.1.2 Expression of SH4-Domains Induces PM Blebbing.................................................................... 64
8.1.3 Localization of the SH4-Domain to the PM is Required for Production of PM Blebs.................. 65
8.1.4 N18-HASPB Induced PM Blebs are Highly Dynamic ................................................................. 68
8.1.5 N18-HASPB Induced PM Blebs are Non-Apoptotic 70
8.1.6 SH4-Domain Mediated PM Blebbing is a Rho Regulated Process .