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Investigation of age related alterations occurring on the protein and the lipid level by using STED microscopy [Elektronische Ressource] / presented by Rebecca Medda

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174 pages
Dissertationsubmitted to theCombined Faculties for the Natural Sciences and for Mathematicsof the Ruperto-Carola University of Heidelberg, Germanyfor the degree ofDoctor of Natural Sciencesconducted atMax Planck Institute for Biophysical Chemistryin Göttingenpresented byDiplom-Biologin Rebecca MeddaBorn in Neuwied am RheinOral-examination: 20.11.2009Investigation of age-related alterationsoccurring on the protein and the lipid levelby using STED MicroscopyReferees: Prof. Dr. Klaus-Armin NaveProf. Dr. Dr. h.c. Stefan W. HellTo my familyContents1 Introduction 151.1 Fluorescence Microscopy in Cell Biology . . . . . . . . . . . . . . . . . 151.1.1 Fundamentals of Light Microscopy in Cell Biology . . . . . . . . 151.1.2 Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . 161.1.3 Specific Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . 181.1.4 Fluorescence Correlation Spectroscopy . . . . . . . . . . . . . . 191.2 Fluorescence Nanoscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 251.2.1 STED microscopy . . . . . . . . . . . . . . . . . . . . . . . . . 261.3 The plasma membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . 281.3.1 The Phospholipids and Cholesterol . . . . . . . . . . . . . . . . 291.3.2 The ’Raft’ Controversy . . . . . . . . . . . . . . . . . . . . . . . 321.4 Proteins Interacting with Constituents of the Plasma Membrane . . . . . . 331.5 The human neuroblastoma cell line SH-SY5Y . . . .
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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
conducted at
Max Planck Institute for Biophysical Chemistry
in Göttingen
presented by
Diplom-Biologin Rebecca Medda
Born in Neuwied am Rhein
Oral-examination: 20.11.2009Investigation of age-related alterations
occurring on the protein and the lipid level
by using STED Microscopy
Referees: Prof. Dr. Klaus-Armin Nave
Prof. Dr. Dr. h.c. Stefan W. HellTo my familyContents
1 Introduction 15
1.1 Fluorescence Microscopy in Cell Biology . . . . . . . . . . . . . . . . . 15
1.1.1 Fundamentals of Light Microscopy in Cell Biology . . . . . . . . 15
1.1.2 Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . 16
1.1.3 Specific Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.1.4 Fluorescence Correlation Spectroscopy . . . . . . . . . . . . . . 19
1.2 Fluorescence Nanoscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 25
1.2.1 STED microscopy . . . . . . . . . . . . . . . . . . . . . . . . . 26
1.3 The plasma membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
1.3.1 The Phospholipids and Cholesterol . . . . . . . . . . . . . . . . 29
1.3.2 The ’Raft’ Controversy . . . . . . . . . . . . . . . . . . . . . . . 32
1.4 Proteins Interacting with Constituents of the Plasma Membrane . . . . . . 33
1.5 The human neuroblastoma cell line SH-SY5Y . . . . . . . . . . . . . . . 34
1.5.1 Redifferentiation of SH-SY5Y . . . . . . . . . . . . . . . . . . . 34
1.6 The Cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
1.6.1 The Neuronal Cytoskeleton . . . . . . . . . . . . . . . . . . . . 36
1.6.2 Organization of neurofilaments . . . . . . . . . . . . . . . . . . . 37
1.6.3 Post-translational Modifications of Neurofilaments . . . . . . . . 39
1.6.4 Role in Neurodegeneration . . . . . . . . . . . . . . . . . . . . . 41
2 Motivation 43
3 Results 45
3.1 Post-translational modifications of neurofilaments visualized by STED
Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
7Contents
3.1.1 Appearance of Intermediate Filament Proteins during Redifferen-
tiation of SH-SY5Y . . . . . . . . . . . . . . . . . . . . . . . . 46
3.1.2 Reciprocal Expression of Vimentin and Doublecortin . . . . . . . 49
3.1.3 Visualization of Post-Translational Modifications of Neurofila-
ments using STED Microscopy . . . . . . . . . . . . . . . . . . 50
3.1.4 Hyperphosphorylation upon Glucose Deprivation . . . . . . . . . 51
3.1.5 The Role of p38 Mitogen Activated Protein Kinase in Glucose
Deprived cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.1.6 The Role of c-Jun N-terminal Kinase in Glucose Deprived cells . 57
3.2 Lipid Diffusion in the Plasma Membrane . . . . . . . . . . . . . . . . . . 59
3.2.1 Reduction of the Focal Volume by Combining STED with Fluo-
rescence Correlation Spectroscopy . . . . . . . . . . . . . . . . . 60
3.2.2 Incorporation of Lipids into the Plasma Membrane . . . . . . . . 60
3.2.3 The Influence of the Fluorescent Label on the Lipid Behavior . . 66
3.2.4 Measurements of Lipid Dynamics in the Plasma Membrane . . . 72
3.2.5 Influence of Cholesterol on the Diffusion of SM, GM1 and Cer . . 74
3.2.6 Effectivity of Depletion . . . . . . . . . . . . . . . . 77
3.2.7 Approximation of the Spatial Dimensions of Microdomains . . . 78
4 Discussion and Outlook 81
4.1 Post-translational Modifications of Neurofilaments . . . . . . . . . . . . 81
4.1.1 The Effects of Retinoids on Cancer Cells . . . . . . . . . . . . . 81
4.1.2 Phosphorylation of Neurofilaments . . . . . . . . . . . . . . . . 83
4.1.3 Phosphatases in Neurodegeneration . . . . . . . . . . . . . . . . 83
4.1.4 Responses to Metabolic Stress . . . . . . . . . . . . . . . . . . . 85
4.1.5 Viability of Deprived Cells . . . . . . . . . . . . . . . . . . . . . 87
4.1.6 Doublestaining of phosphorylated and O-GlcNAcylated epitopes . 87
4.1.7 Investigation of the Post-Translational Modifications using STED
Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
4.2 Incorporation of Labeled Lipids in the Plasma Membrane . . . . . . . . . 90
4.2.1 Fusion of Liposomes with the Plasma . . . . . . . . . 90
4.2.2 Plasma Membrane Sheet Generation . . . . . . . . . . . . . . . . 91
4.2.3 Serum Albumins - Lipid Carriers in the Blood . . . . . . . . . . . 91
8Contents
4.2.4 Proper Incorporation of the Labeled Lipids in the Plasma Membrane 92
4.2.5 Diffusion of Phospholipids with Saturated and Unsaturated Fatty
Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
4.2.6 Outer or Inner Leaflet? . . . . . . . . . . . . . . . . . . . . . . . 94
4.2.7 Cholesterol Depletion . . . . . . . . . . . . . . . . . . . . . . . 95
4.2.8 The Interactions of the Cytoskeleton with Constituents of the Plasma
Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
4.2.9 Temperature Dependence on Lipid Diffusion . . . . . . . . . . . 97
4.2.10 Potential Artifacts due to the High Intensities of the STED and
Excitation Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . 98
4.3 Specific Labeling in High Resolution Microscopy . . . . . . . . . . . . . 100
4.3.1 Alternative Affinity Markers . . . . . . . . . . . . . . . . . . . . 100
4.3.2 in vitro Labeling of Recombinant Proteins . . . . . . . . . . . . . 103
4.3.3 N-V Centers: Extremely Stable Labels in High Resolution Mi-
croscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
5 Materials and Methods 109
5.1 Buffers and Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
5.2 Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
5.2.1 Liposome generation . . . . . . . . . . . . . . . . . . . . . . . . 110
5.2.2 Generation of Plasma Membrane Sheets . . . . . . . . . . . . . . 111
5.2.3 Formation of the lipid-BSA complex . . . . . . . . . . . . . . . . 112
5.2.4 Cholesterol depletion . . . . . . . . . . . . . . . . . . . . . . . . 115
5.3 Organisms and organism specific methods . . . . . . . . . . . . . . . . . 116
5.3.1 Cultivation of Escherichia coli . . . . . . . . . . . . . . . . . . . 116
5.3.2 Transformation of E. coli . . . . . . . . . . . . . . . . . . . . . . 117
5.3.3 Isolation and purification of plasmid DNA from E. coli . . . . . . 117
5.3.4 Mammalian cell lines . . . . . . . . . . . . . . . . . . . . . . . . 118
5.3.5 Media and cultivation . . . . . . . . . . . . . . . . . . . . . . . . 119
5.3.6 Re-differentiation of SH-SY5Y . . . . . . . . . . . . . . . . . . 120
5.3.7 Test on mycoplasma . . . . . . . . . . . . . . . . . . . . . . . . 120
5.3.8 Transfection of mammalian cells . . . . . . . . . . . . . . . . . . 121
5.4 Protein biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
9Contents
5.4.1 Western analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 122
5.4.2 Kinase assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
5.5 Specific labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
5.5.1 DNA dyes, TMA-DPH and mito trackers . . . . . . . . . . . . . 127
5.5.2 Dye Conjugation of proteins . . . . . . . . . . . . . . . . . . . . 129
5.5.3 Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . 131
5.6 Light microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
5.6.1 Wide field Fluorescence microscopy . . . . . . . . . . . . . . . . 134
5.6.2 Confocal microscopy . . . . . . . . . . . . . . . . . . . . . . . . 134
5.6.3 STED-FCS setup . . . . . . . . . . . . . . . . . . . . . . . . . . 135
5.6.4 Supercontinuum STED . . . . . . . . . . . . . . . . . . . . . . . 136
6 Abbreviations 139
Bibliography 141
7 Supplementary 165
8 List of Publications 169
8.1 Publications Related to Thesis . . . . . . . . . . . . . . . . . . . . . . . 169
8.2 in Cooperation (Selection) . . . . . . . . . . . . . . . . . . 170
9 Contribution to Conferences 171
10 Acknowledgment 173
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