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………………………………………………………………………………… 45 3.2.1 PDEδ in the retina…………………………………………………………………… 46 3.2.2 Structure of PDEδ…………………………………………………………………… 46 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…………………………………………………………………… 55 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………………………………………. 89 2.6.2 Chemical transformation of E. coli………………………………………………….. 90 2.7.3 Plasmid DNA preparation…………………………………………………………… 90 2.6.4 DNA sequencing…………………………………………………………………….. 91 2.6.5 Agarose gel electrophoresis…………………………………………………………. 92 2.6.6 Polymerase chain reaction (PCR)…………………………………………………… 92 2.6.7 Gateway cloning……………………………………………………………………... 93 2.7 Establishment of an organotypical retinal explant system from murine retina…………………………………………………………………… 97 2.7.1 Culturing of retinal explants…………………………………………………………. 97 2.7.2 Analysis of explant cultures………………………………………………………. 100 2.7.2.1 Tissue analysis…………………………………………………………………... 100 2.7.2.2 Test for apoptosis - TUNEL assay……………………………………………… 100 2.8 Production of rat and mouse monoclonal CRMP2 specific antibodies…………………………………………………………. 102 2.8.1 Peptide selection……………………………………………………………………. 103 2.8.2 Antibody validation by Western blot………………………………………………. 106 2.8.3 Antibody validation by immunohistochemistry (IHC)…………………………….. 107 2.8.4 Antibody validation by immunoprecipitation............................................................ 107 G. RESULTS…………………………………………………………………. 108 1. ANALYSIS OF RHO GTPASES IN ROD OUTER SEGMENTS (ROS)……………………………………………………. 108 1.1 Identification of the Rac1 interactome in ROS…………………………………… 108 1.1.1 Establishment of the Rac1 IP from soluble and