Analysis of multiprotein complexes in the mammalian retina [Elektronische Ressource] / Monika Viktoria Beer

Beer Monika

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Analysis of Multiprotein Complexes in the
Mammalian Retina



Monika Viktoria Beer

Dissertation an der Fakultät für Biologie der
Ludwig-Maximilians-Universität München












Vorgelegt von Monika Viktoria Beer
aus Fürstenfeldbruck

München, den 12. August 2008
Cover:
Immunohistochemistry of porcine
retina sections with anti-Rac1 antibody
Erklärung:
Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig und ohne unerlaubte
Hilfe angefertigt habe.
München, 12. August 2008












Dissertation eingereicht: 12. August 2008
Tag der mündlichen Prüfung: 19. Dezember 2008
Erstgutachter: Prof. Dr. Lutz Eichacker
Zweitgutachter: Prof. Dr. Hugo Scheer
Sondergutachter: Dr. Marius Ueffing Monika Beer, Dissertation Table of Contents

TABLE OF CONTENTS
A. ABBREVIATIONS………………………………………………………… 1
B. SUMMARY………………………………………………………………..... 5
C. ZUSAMMENFASSUNG…………………………………………………… .8
D. INTRODUCTION………………………………………………………….. 12
1. THE MAMMALIAN RETINA…………………………………………………….......... 12
1.1 Structure and function……………………………………………………………….. 12
1.1.1 The retinal pigment epithelium……………………………………………………… 13
1.1.2 The photoreceptors…………………………………………………………………... 14
1.1.3 The other retinal cell types…………………………………………………………... 15
1.1.3.1 Other neurons……………………………………………………………………… 15
1.1.3.1.1 Bipolar cells…………………………………………………………………… 15
1.1.3.1.2 Horizontal cells……………………………………………………………….. 16
1.1.3.1.3 Amacrine cells………………………………………………………………… 16
1.1.3.1.4 Ganglion cells…………………………………………………………………. 17
1.1.3.1.5 The interplexiform cells……………………………………………………….. 17
1.1.3.2 Retinal glial cells…………………………………………………………………... 17
1.1.3.2.1 Müller glial cells (MC)………………………………………………………... 17
1.1.3.2.2 Astrocytes……………………………………………………………………... 18
1.1.4 Murine retina………………………………………………………………………… 18
1.1.5 Porcine retina………………………………………………………………………… 19
1.2 The G-protein-coupled receptor rhodopsin………………………………………… 19
1.2.1 Phototransduction……………………………………………………………………. 21
1.2.2 Light-regulated translocation of photoreceptor proteins…………………………….. 23
1.3 Cilia and ciliopathies…………………………………………………………………. 23
1.3.1 Cilia in vertebrate cells………………………………………………………………. 23
1.3.1.1 The photoreceptor connecting cilium……………………………………………… 24
1.3.2 Cilia-related disorders……………………………………………………………….. 26
1.3.2.1 Leber congenital amaurosis……………………………………………………….. 29
1.3.2.1.1 Lebercilin……………………………………………………………………… 30

Monika Beer, Dissertation Table of Contents
2. GTPASES………………………………………………………………………………… 30
2.1 GTP binding proteins in signal transduction………………………………………. 30
2.2 Rho family GTPases………………………………………………………………….. 31
2.2.1 Cell morphology……………………………………………………………………... 32
2.2.2 Cell movement………………………………………………………………………. 33
2.2.3 Cell behaviour……………………………………………………………………… 35
2.2.4 Post-translational C-terminal modifications of Rho GTPases………………………. 35
2.2.5 Regulation of Rho family GTPases………………………………………………… 36
2.2.6 Rho GTPases in the eye……………………………………………………………... 38
2.2.6.1 Rho GTPases in the retina………………………………………………………… 39
2.3.Collapsin response mediator proteins (CRMP)…………………………………….. 40
2.3.1 CRMP2 switches RhoA and Rac1 morphology……………………………………... 40
3. PHOSPHODIESTERASES AND PDEδ………………………………………………... 43
3.1 Phosphodiesterases…………………………………………………………………… 43
3.1.1 PDE6………………………………………………………………………………… 45
3.2 PDEδ
3.2.1 PDEδ in the retina…………………………………………………………………… 46
3.2.2 Structure of PDEδ
3.2.3 PDEδ homologes…………………………………………………………………….. 48
E. AIM OF THE STUDY……………………………………………………... 51
F. MATERIAL AND METHODS……………………………………………. 54
1. MATERIAL………………………………………………………………………………. 54
1.1 Chemicals………………………………………………………………………………54
1.2 General equipment…………………………………………………………………… 54
1.3 Protein chemistry…………………………………………………………………….. 55
1.3.1 Special equipment…………………………………………………………………… 55
1.3.2 Kits…………………………………………………………………………………... 55
1.4 Molecular biology…………………………………………………………………….. 55
1.4.1 Special equipment
1.4.2 Kits…………………………………………………………………………………... 56
1.4.3 E. coli strains………………………………………………………………………… 56
1.4.4 Oligonucleotides……………………………………………………………………... 56
1.4.5 Plasmids and constructs……………………………………………………………... 57
Monika Beer, Dissertation Table of Contents

1.4.5.1 Plasmids…………………………………………………………………………… 57
1.4.5.2 Constructs………………………………………………………………..……….. 57
1.5 Mammalian cell and tissue culture………………………………………………….. 57
1.5.1 Special equipment…………………………………………………………………… 57
1.5.2 Kits…………………………………………………………………………………... 57
1.5.3 Mammalian cell lines………………………………………………………………... 58
1.5.4 Antibodies…………………………………………………………………………… 58
1.6 Software and databases……………………………………………………………… 60
1.6.1 Software……………………………………………………………………………... 60
1.6.2 Databases…………………………………………………………………………….. 61
2. METHODS……………………………………………………………………………….. 62
2.1 Protein chemistry…………………………………………………………………….. 62
2.1.1 Determination of protein concentration……………………………………………... 62
2.1.2 Protein precipitation…………………………………………………………………. 62
2.1.3 Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE)……………………………………………………. 63
2.1.3.1 SDS-PAGE gradient gels………………………………………………………….. 65
2.1.3.1.1 Casting of SDS gradient mini gels with a gradient maker……………………. 65
2.1.3.2 Sample preparation………………………………………………………………... 65
2.1.4 Blue-native PAGE (BN-PAGE)……………………………………………………. 65
2.1.5 Second-dimensional gel electrophoresis (2-DE)…………………………………….. 68
2.1.5.1 Sample preparation………………………………………………………………... 69
2.1.5.2 Rehydration and sample loading…………………………………………………... 70
2.1.5.3 Isoelectric focusing (IEF)………………………………………………………….. 70
2.1.5.4 Equilibration and transfer of the IPG strips………………………………………. 70
2.1.5.5 Second dimension: SDS-PAGE……………………………………………………. 71
2.1.6 Staining of SDS gels………………………………………………………………… 71
2.1.6.1 Silver staining……………………………………………………………………… 71
2.1.6.2 Coomassie staining……………………………………………………………… 72
2.1.6.3 Digitalizing and drying of SDS gels……………………………………………..… 72
2.1.7 Western blot analysis………………………………………………………………... 73
2.1.7.1 Semi dry blotting…………………………………………………………………... 73
2.1.8 Analysis of protein interactions……………………………………………………… 75
Monika Beer, Dissertation Table of Contents
2.1.8.1 Immunoprecipitations from porcine retina………………………………………... 75
2.1.8.2 Immunoprecipitations from ROS…………………………………………………... 75
2.1.8.3 Rac1-GTP Pull Down……………………………………………………………… 76
2.1.8.4 Tandem affinity purification (TAP)………………………………………………... 76
2.1.8.4.1 Transfection of HEK293 cells transiently expressing
the SF-TAP fusion protein……………………………………………………………… 77
2.1.8.4.2 SF-TAP purification protocol (Gloeckner in press)…………………………... 77
2.1.9 Mass spectrometry…………………………………………………………………… 78
2.1.9.1 In-gel proteolysis…………………………………………………………………... 79
2.1.9.1.1 In-gel proteolysis of silver-stained and
Coomassie-stained gels………………………………………………………... 79
2.1.9.2 Matrix-assisted laser desorption/ionization
mass spectrometry (MALDI-MS)…………………………………………………... 79
2.1.9.3 Peptide mass finger printing (PMF) and
tandem mass spectrometry (MS/MS)………………………………………………. 80
2.1.9.3.1 Mass measurement with the Applied Biosystems
4700 Proteomics Analyzer (AB4700)…………………………………………. 81
2.1.9.4 Electrospray ionization mass spectrometry (ESI-MS)…………………………….. 82
2.1.9.4.1 Mass measurement with the Q-TOF II ESI mass spectrometer………………. 82
2.1.9.4.2 Mass measurement with the LTQ OrbitrapXL mass spectrometer…………….83
2.1.9.5 Data processing and criteria
for protein identification …………………………………………………………………. 84
2.2 Isolation of porcine rod outer segments…………………………………………….. 85
2.3 PDEδ subunit activity assay…………………………………………………………. 86
2.4 Light- dependent methylation of Rac1 in ROS
and autoradiography………………………………...………………………………. 87
2.4.1 Immunoprecipitation of methylated Rac1…………………………………………… 87
2.4.2 Identification of methylated ROS proteins by mass spectrometry…………………... 88
2.5 Immunohistochemistry………………………………………………………………. 88
2.6 Molecular biology…………………………………………………………………….. 89
2.6.1 E. coli cultures……………………………………………………………………….. 89
2.6.1.1 Liquid cultures……………………………………………………………………... 89
2.6.1.2 Plating cultures……………………………………………………………………. 89
2.6.1.3 Cryo cultures………………………………………………………………………. 89
Monika Beer, Dissertation Table of Contents
2.6.1.4 Generation of chemically competent E. coli……………………………………