Biogenesis of peroxisomes in mammalian cells [Elektronische Ressource] : Characterization of the Pex11 proteins and their role in peroxisomal growth and division / Hannah Katharina Delille. Betreuer: Ralf Jacob

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AUS DEM INSTITUT FÜR KLINISCHE ZYTOBIOLOGIE UND ZYTOPATHOLOGIE
GESCHÄFTSFÜHRENDER DIREKTOR: PROF. DR. ROLAND LILL
DES FACHBEREICHS MEDIZIN DER PHILIPPS-UNIVERSITÄT MARBURG







BIOGENESIS OF PEROXISOMES IN MAMMALIAN CELLS:
CHARACTERIZATION OF THE PEX11 PROTEINS AND THEIR ROLE
IN PEROXISOMAL GROWTH AND DIVISION





INAUGURAL-DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER HUMANBIOLOGIE
(DR. RER. PHYSIOL.)

DEM FACHBEREICH MEDIZIN DER PHILIPPS-UNIVERSITÄT MARBURG
VORGELEGT VON

HANNAH KATHARINA DELILLE
AUS KÖLN

MARBURG, 2010 Angenommen vom Fachbereich Medizin der Philipps-Universität Marburg am:
11.11.2010
Gedruckt mit Genehmigung des Fachbereichs.

Dekan: Prof. Dr. Matthias Rothmund
Referent: Prof. Dr. Ralf Jacob
Korreferent: Prof. Dr. Uta-Maria Bauer TABLE OF CONTENTS | i
TABLE OF CONTENTS
1 INTRODUCTION 1
1.1 Peroxisomes – an overview 1
1.1.1 General features of the organelle 1
1.1.2 Metabolic functions of peroxisomes 2
1.1.3 Peroxisomal disorders 6
1.1.3.1 Peroxisomal Biogenesis Disorders 6
1.1.3.2 Single Peroxisomal Enzyme Deficiencies 8
1.2 Peroxisome biogenesis 10
1.2.1 Import of matrix proteins 11
1.2.2 Import of membrane proteins 13
1.2.3 “Growth and division” vs.d “e novo synthesis” 15
1.2.4 Peroxisomal dynamics 18
1.2.4.1 Proliferation 18
1.2.4.2 Degradation 19
1.2.4.3 Inheritance and motility 20
1.3 The division machinery 23
1.3.1 Peroxisomal fission by dynamin-like proteins 24
1.3.2 Fis1 – an adapter protein 26
1.3.3 Pex11 proteins in peroxisome proliferation 29
1.4 Objectives 35
2 MATERIAL AND METHODS 37
2.1 Equipment 37
2.2 Consumables 39
2.3 Chemicals and reagents 40
2.3.1 Chemicals 40
2.3.2 Loading dyes and markers 42
2.3.3 Kits 42
2.3.4 Cell culture reagents 42
2.4 Immunological reagents 43
2.4.1 Primary antibodies 43
2.4.2 Secondary antibodies 44
2.5 Molecular biology reagents 44
2.5.1 Enzymes and other reagents 44
2.5.2 Plasmids 45 ii | TABLE OF CONTENTS
2.5.3 Constructs 46
2.5.4 Primer 47
2.6 Frequently used buffers and solutions 48
2.7 Cell lines 52
2.8 Cell culture 52
2.8.1 Cell passage 53
2.8.2 Cell freezing 53
2.8.3 Mycoplasma detection 54
2.8.3.1 Hoechst staining test 54
2.8.3.2 Mycoplasma PCR test 54
2.8.4 Transfection of mammalian cells 55
2.8.4.1 PEI transfection 56
2.8.4.2 DEAE-Dextran transfection 56
2.8.4.3 Electroporation 57
2.8.4.4 Microinjection 57
2.8.4.5 Lipofection 58
2.8.5 RNA interference 59
2.9 Microscopic techniques 60
2.9.1 Immunofluorescence 60
2.9.2 Fluorescence microscopy 61
2.9.2.1 Image deconvolution 62
2.9.3 Confocal microscopy 62
2.9.4 Quantitative examination 63
2.9.5 Live cell imaging 63
2.9.6 FRAP 64
2.9.7 HaloTag technology 65
2.9.8 Electron microscopy 65
2.9.8.1 Buffer and solutions 66
2.9.8.2 Embedding in EPON 67
2.9.8.3 Immunoelectron microscopy 67
2.9.8.4 Alkaline DAB staining 68
2.10 Biochemical techniques 68
2.10.1 Preparation of cell lysates 68
2.10.2 Preparation of peroxisome-enriched fractions 69
2.10.3 Protein precipitation 70
2.10.4 Measurement of protein concentration 70
2.10.5 SDS-PAGE 71
2.10.6 Immunoblotting 72
2.10.7 Immunoprecipitation 73
2.10.7.1 Co-immunoprecipitation 73
2.10.7.2 Cross-linking 75
2.10.7.3 Endogenous co-immunoprecipitation 75 TABLE OF CONTENTS | iii
2.10.7.4 Peroxisome immunoprecipitation 76
2.11 Molecular biology techniques 76
2.11.1 RNA isolation 76
2.11.2 cDNA synthesis 77
2.11.3 PCR 78
2.11.4 Semi-quantitative RT-PCR 80
2.11.5 Agarose gel electrophoresis 81
2.11.6 Gel extraction 82
2.11.7 Digestion with restriction enzymes 82
2.11.7.1 Preparative RE digestion 82
2.11.7.2 Analytical RE digestion 83
2.11.8 DNA precipitation 83
2.11.9 Dephosphorylation 84
2.11.10 Ligation 84
2.11.11 Bacterial culture 85
2.11.11.1 Preparation of competent bacterial cells 85
2.11.11.2 Chemical transformation 86
2.11.12 Plasmid isolation 86
2.11.13 Measurement of DNA and RNA concentrations 87
3 RESULTS 89
3.1 Pex19-dependent targeting of hFis1 to peroxisomes 89
3.1.1 Interaction of Pex19p and hFis1 90
3.1.2 Targeting of hFis1 to peroxisomes but not to mitochondria depends on
Pex19p 94
3.1.3 Summary 98
3.2 Comparative characterization of Pex11pα, Pex11pβ, and Pex11pγ 99
3.2.1 The mammalian Pex11 isoforms differ in their membrane elongation-
inducing properties 99
3.2.2 Pex11pα, Pex11pβ, and Pex11pγ differ in their Trit-1on X00 sensitivity 103
3.2.3 Pex11pγ-induced peroxisomal tubules are highly motile 105
3.2.4 Summary 106
3.3 Pex11pβ-mediated growth and division of mammalian peroxisomes follows a
maturation pathway 108
3.3.1 Pex11pβ-YFP induces tubular peroxisomal accumulations (TPAs) and
inhibits the formation of spherical peroxisomes 108
3.3.2 TPA formation appears to be specific for Pex11p -YFP 110
3.3.3 C-terminal truncations of Pex11p  inhibit peroxisome elongation 110
3.3.4 TPAs represent a pre-peroxisomal membrane compartment composed
out of tubular membrane extensions and mature globular peroxisomes 113
3.3.5 TPAs show a distinct distribution of matrix proteins 115 iv | TABLE OF CONTENTS
3.3.6 TPAs show different distribution of PMPs 116
3.3.7 Tubular membrane extensions are formed by pre-existing peroxisomes 120
3.3.8 TPA formation is also induced by manipulation of hFis1 125
3.3.9 Pex11pβ but not Pex11pα and Pex11pγ induces TPA formation when
co-expressed with YFP-Pex11pβ 127
3.3.10 Pex11pγ has higher membrane mobility than Pex1 1pβ 130
3.3.11 Summary 132
3.4 Hypertubulation of peroxisomes 133
3.4.1 Cumulative effects of Pex11pβ expression, DLP1 silencing, and
microtubule depolymerisation on peroxisomal elongation 133
3.4.2 Induction of reticular peroxisomal structures 136
3.4.3 Silencing of DLP1 induces long and branched TPAs, which are stable
without microtubules 137
3.4.4 Formation of TPAs does not require the microtubule cytoskeleton 138
3.4.5 Peroxisome hypertubulation is not caused by Pex11 upregulation 139
3.4.6 Summary 140
4 DISCUSSION 141
4.1 Pex19p-mediated peroxisomal import of hFis1 142
4.2 Characterization of the mammalian Pex11 proteins 147
4.2.1 Insights into the functions of Pex11 protein – elongation and
constriction of peroxisomes 147
4.2.2 Lipid binding and the different Pex11p C-termini 148
4.2.3 Membrane mobility and tubule motility 150
4.3 Pex11pβ-mediated growth and division of mammalian peroxisomes follows a
maturation pathway 152
4.3.1 Pex11pβ-YFP – a novel tool to study peroxisome growth and division 152
4.3.2 New insights in Pex11p-medβ iated growth and division 154
4.3.3 Pex11pβ – a “morphogenic” peroxin? 158
4.4 Is there Pex11p-independent elongation and proliferation of peroxisomes? 161
4.5 Future perspectives 166
5 SUMMARY 168
6 ZUSAMMENFASSUNG 170
7 REFERENCES 173
8 APPENDIX 202 TABLE OF CONTENTS | v
8.1 Abbreviations and definitions 202
8.1.1 Abbreviations 202
8.1.2 Unit definitions 206
8.2 Table of figures 207
8.3 Curriculum vitae 210
8.4 Verzeichnis der akademischen Lehrer 211
8.5 Acknowledgements 212
8.6 Ehrenwörtliche Erklärung (mit Publikationsliste) 213