Mitochondrial copper homeostasis in mammalian cells Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Fakultät Mathematik und Naturwissenschaften der Technischen Universität Dresden von Corina Oswald (Diplom-Biochemikerin) geboren am 10.04.1981 in Dohna, Deutschland Gutachter: Prof. Dr. Gerhard Rödel Prof. Dr. Alexander Storch Eingereicht am 30. April 2010 Verteidigt am 13. August 2010 ACKNOWLEDGEMENTS I sincerely thank my supervisor Prof. Dr. Gerhard Rödel for giving me the opportunity to do my PhD in his group and to join the Dresden International Graduate School for Biomedicine and Bioengineering (DIGS-BB). He introduced me to the world of mitochondria, supported and provided me with all resources and comprehension necessary to conduct my research. I thank Dr. Udo Krause-Buchholz for his scientific advice and for helping writing the paper by giving constructive comments on the manuscript. I honestly thank my TAC members Dr. Frank Buchholz and Prof. Dr. Alexander Storch for their interest in this work, for guiding me scientifically, and for stimulating discussions in the TAC meeting. Especially, Dr. Frank Bucholz for giving insightful suggestions as RNAi specialist, and Prof. Dr. Alexander Storch for acting as reviewer of this thesis. The dSTORM images would not have been possible without the very friendly collaboration with Prof. Dr. Markus Sauer and Sebastian van de Linde, Institute for Applied Laser Physics and Laser Spectroscopy of the University of Bielefeld. Thank you! I am furthermore grateful to all former and present lab members for the friendly working atmosphere, for fruitful discussions, for providing advice and assistance in many situations. In particular, I thank the “girls” in the lab – Anja, Kirsten, Susi2, Simone and Uta for struggling together through the ups and downs. I am grateful to Uta Gey, Dr. Kristof Zarschler and Dr. Kai Ostermann for critical proof reading of the thesis. Special thanks are dedicated to my friends for all our unforgettable moments together. I sincerely thank my parents, who helped, supported and encouraged me all the time. Above all, I want to thank Stefan for always being there, motivating and believing in me. CONTENTS CONTENTS List of Figures and Tables iv Abbreviations v Abstract 1 1 Indroduction 2 1.1 Mitochondria and the respriratory chain 2 1.2 The human mitochondrial genome 4 1.3 Homoplasmy and heteroplasmy 6 1.4 Mitochondrial disorders 6 1.4.1 Mutations in mitochondrial DNA 7 1.4.2 Mutations in nuclear DNA 8 1.5 Cytochrome c oxidase 9 1.6 Cytochrome c oxidase assembly 11 1.7 Copper and its trafficking in the cell 13 1.8 Mitochondrial copper metabolism 15 1.9 Cox17 19 1.10 Aims of the thesis 21 2 Materials and Methods 22 2.1 Materials 22 2.1.1 Chemicals and reagents 22 2.1.2 Antibodies 24 2.1.3 Plasmid 24 2.1.4 Kits 25 2.1.5 Marker 25 2.1.6 Enzymes 25 2.1.7 Primers 26 2.1.8 siRNAs 26 2.2 Methods 28 2.2.1 Cell culture 28 2.2.1.1 Cell culture: HeLa cells 28 2.2.1.2 Cell culture: HeLa cells transfected with pTurboRFP-mito 28 i CONTENTS 2.2.1.3 Subcultivation 29 2.2.1.4 Determination of cell number 29 2.2.1.5 Cell storage and thawing 29 2.2.2 Transient transfection of HeLa cells 30 2.2.3 Transfection of HeLa cells with pTurboRFP-mito 30 2.2.4 Immunocytochemistry 31 2.2.5 RNA extraction and quantitative real-time PCR 31 2.2.6 Isolation of mitochondria 32 2.2.6.1 Isolation of mitochondria for BN-PAGE Analysis 32 2.2.6.2 Isolation of mitochondria for localization studies 33 2.2.6.3 Isolation of bovine heart mitochondria 33 2.2.7 Proteinase K treatment of mitochondria and mitoplasts 34 2.2.8 Photometric activity assay 34 2.2.8.1 Citrate synthase activity 34 2.2.8.2 Cytochrome c oxidase activity 35 2.2.9 Blue native polyacrylamide gel electrophoresis (BN-PAGE) 36 2.2.9.1 In gel activity assay 37 2.2.9.2 2D-BN/SDS-PAGE 37 2.2.10 SDS-PAGE and Western blot analysis 37 2.2.11 Direct stochastic optical reconstruction microscopy (dSTORM) 38 2.2.12 Flow cytometric phenotyping 39 2.2.12.1 Determination of cell cyle phase 39 2.2.12.2 Identification of apoptotic cells 40 2.2.12.3 Detection of ROS 41 2.2.13 Oxygen measurement 42 2.2.14 Cu–His supplementation 43 3 Results 44 3.1 Subcellular localization of Cox17 44 3.2 Transient knockdown of COX17 in HeLa cells 46 3.2.1 Knockdown of COX17 mRNA 47 3.2.2 Knockdown of Cox17 protein 49 3.2.3 Effect of COX17 knockdown on the steady-state levels of OXPHOS subunits 50 3.2.4 Effect of COX17 knockdown on the steady-state levels of copper- bearing COX subunits 51 ii CONTENTS 3.2.5 Subdiffraction-resolution fluorescence imaging 51 3.3 Phenotypical characterization 56 3.3.1 Growth analyis 57 3.3.2 Cell cycle analysis 57 3.3.3 Apoptosis assay 59 3.3.4 Detection of ROS 61 3.3.5 Oxygen measurement 63 3.4 Cytochrome c oxidase activity 64 3.5 Characterization of mt OXPHOS complexes 65 3.5.1 BN-PAGE/in gel activity assays 65 3.5.2 Supramolecular organization of COX 67 3.5.3 Molecular organization of Cox17 68 3.5.4 Molecular organisation of copper-bearing COX subunits Cox1 and Cox2 69 3.5.5 Supramolecular organization of RC complexes 70 3.5.6 dSTORM of supercomplexes 72 3.6 Copper supplementation 74 4 Discussion 75 4.1 Dual localization of human Cox17 75 4.2 COX17 knockdown affects steady-state levels of copper-bearing COX subunits Cox1 and Cox2 77 4.3 Supramolecular organization of RC is affected as an early response to COX17 knockdown 79 4.4 Cox17 is primarily engaged in copper delivery to Sco1/Sco2 82 4.5 Copper supplementation alone cannot rescue the COX17 phenotype 84 4.6 Outlook 85 5 Appendix 88 6 PhD publication record 96 7 References 97 iii LIST OF FIGURES AND TABLES LIST OF FIGURES AND TABLES Figure 1.10. Oxidative phosphorylation. 3 Figure 1.20. Model of mammalian I1III2IV1 supercomplex. 4 Figure 1.30. Molecular organization of COX. 10 Figure 1.40. Illustration of the electron flow through the COX. 11 Figure 1.50. Model of the assembly pathway of human COX. 12 Figure 1.60. Pathways of copper trafficking within a mammalian cell. 14 Figure 1.70. NMR solution structures of apo- and Cu1-Cox172S-S. 20 Figure 2.10. Respective positions of the siRNA sequence on COX17 mRNA. 27 Figure 2.20. Quantification of cell cycle distribution of HeLa cells. 40 Figure 2.30. Sample data using Annexin V-FITC Apoptosis Detection Kit. 41 Figure 2.40. DCF fluorescence in HeLa cells. 42 Figure 3.10. Localization of human Cox17. 45 Figure 3.20. siRNA transfection efficiency in HeLa cells. 47 Figure 3.30. Transient knockdown of COX17 mRNA. 48 Figure 3.40. Effect of transient knockdown of COX17 in HeLa cells. 49 Figure 3.50. The principle of dSTORM image analysis. 52 Figure 3.60. Subdiffraction-resolution imaging of immunolabeled pTurboRFP mito HeLa cells transfected with COX17 siRNAs. 55 Figure 3.70. Growth of COX17 knockdown cells. 57 Figure 3.80. Cell cycle analysis. 58 Figure 3.90. Identification of apoptotic cells by flow cytometry. 60 Figure 3.10. ROS production in HeLa cells. 62 Figure 3.11. Respiration rate in COX17 knockdown cells. 63 Figure 3.12. COX activity of COX17 knockdown cells. 64 Figure 3.13. BN-PAGE/in gel activity of digitonin solubilized mitochondria. 66 Figure 3.14. Supramolecular organization of RC complexes. 68 Figure 3.15. 2D-BN/SDS-PAGE of OXPHOS complexes. 70 Figure 3.16. Molecular organization of RC complexes. 72 Figure 3.17. dSTORM of supercomplexes. 73 Table 2.1. List of gel electrophoresis markers. 25 Table 2.2. List of siRNAs. 26 iv ABBREVIATIONS ABBREVIATIONS AEBSF 4-(2-Aminoethyl)benzenesulfonyl fluoride adPEO Autosomal dominant progressive external ophthalmoplegia APS Ammonium persulphate BHM Bovine heart mitochondria BN Blue native BSA Bovine serum albumine CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1- propanesulfonate COX Cytochrome c oxidase CPEO Chronic progressive external ophthalmoplegia CS Citrate synthase Cu-His Copper-histidine DCFH-DA 2′,7′-Dichlorodihydrofluorescein diacetate Ddp Deafness dystonia protein DTNB 5,5’-dithio-bis(2-nitrobenzoate) ddH2O Double distilled water DMEM Dulbecco’s Modified Eagle Medium DOX Doxycycline dNTP Deoxynucleoside triphosphate dSTORM Direct stochastic optical reconstruction microscopy EDTA Ethylendiamin-tetraacetic acid et al. et alii, and others FBS Fetal bovine serum FMN Flavin mononucleotide FITC Fluorescein isothiocyanate FL Fluorescence FRET Fluorescence resonance energy transfer GAPDH Glyceraldehyde 3-phosphate dehydrogenase HMW High molecular weight Hsp60 Heat shock protein 60 HRP Horse radish peroxidase IEF Isoelectric focusing IgG Immunoglobulin G IMM Inner mitochondrial membrane v ABBREVIATIONS IMS Intermembrane space KSS Kearns-Sayre syndrome LDH Lactate dehydrogenase MELAS Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes MERRF Myoclonic epilepsy associated with ragged-red fibres MIDD Maternally inherited diabetes with deafness MOPS 3-[N-Morpholino]propanesulfonic acid mRNA Messenger RNA mt Mitochondrial mtDNA Mitochondrial DNA MW Molecular weight NAD+ β-nicotinamide adenine dinucleotide NADH Reduced β-nicotinamide adenine dinucleotide NARP Neurogenic muscle weakness, ataxia, and retinitis pigmentosa NDUFB9 NADH dehydrogenase (ubiquinone) subunit 9 nDNA Nuclear DNA NMR Nuclear magnetic resonance nt Nucleotide NTB Nitro tetrazolium blue OMM Outer mitochondrial membrane OXPHOS Oxidative phosphorylation PAA Polyacrylamide PBS Phosphate buffered saline PDH Pyruvate dehydrogenase PS Phosphatidylserine PMS Phenazine methosulfate PI Propidium iodide PIC Protease inhibitor cocktail PVDF Polyvinylidene fluoride RC Respiratory chain RFP Red fluorescent protein RITOLS RNA incorporation throughout the lagging strand RNAi RNA interference ROS Reactive oxygen species rRNA Ribosomal RNA RT Room temperature vi ABBREVIATIONS RT-PCR Reverse transcription – polymerase chain reaction S.