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Development of Nanobodies to Image Synaptic Proteins in Super-Resolution Microscopy’ Independently and with No Other Aid Or Sources Than Quoted

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Development of Nanobodies to Image Synaptic Proteins in Super-Resolution Microscopy’ Independently and with No Other Aid Or Sources Than Quoted Development of Nanobodies to Image Synaptic Proteins in Super-Resolution Microscopy Dissertation in partial fulfilment of the requirements for the degree Doctor rerum naturalium at the Georg-August University Göttingen within in the International Max Planck School for Molecular Biology of the Göttingen Graduate School for Neurosciences, Biophysics and Molecular Biosciences submitted by Manuel Maidorn from Gronau (Leine), Germany Göttingen, September 2017 Examination Board Thesis Committee and Examination Board: Prof. Dr. Silvio Rizzoli (first referee) Department of Neuro- and Sensory Physiology University Medical Center Göttingen Göttingen, Germany Prof. Dr. Peter Rehling (second referee) Department of Cellular Biochemistry University Medical Center Göttingen Göttingen, Germany Prof. Dr. Mikael Simons Institute of Neuronal Cell Biology German Center for Neurodegenerative Diseases / Technical University Munich Munich, Germany Extended Examination Board: Prof. Dr. Henning Urlaub Bioanalytical Mass Spectrometry Max Planck Institute for Biophysical Chemistry Göttingen, Germany Prof. Dr. Jörg Enderlein Institute for Biophysics Georg-August-University Göttingen Göttingen, Germany Prof. Dr. Markus Bohnsack Institute for Molecular Biology University Medical Center Göttingen Göttingen, Germany III Every day for us something new Open mind for a different view And nothing else matters. Affidavit I hereby declare that I prepared this dissertation entitled ‘Development of Nanobodies to Image Synaptic Proteins in Super-Resolution Microscopy’ independently and with no other aid or sources than quoted. Göttingen, September 2017 (Manuel Maidorn) VII Für Margarete und Manfred Table of Contents Examination Board .......................................................................................................... III Affidavit ......................................................................................................................... VII Acknowledgement ......................................................................................................... XIV List of Publications ......................................................................................................... XVI List of Figures ................................................................................................................ XVII List of Tables .................................................................................................................. XIX List of Equations ............................................................................................................. XX List of Abbreviations ...................................................................................................... XXI Abstract ............................................................................................................................ 1 Zusammenfassung (German Abstract) ............................................................................... 2 1. Introduction ............................................................................................................. 3 1.1 Basic Concepts of Light Microscopy ......................................................................................... 3 1.2 Super-Resolution Microscopy Techniques ............................................................................... 5 1.2.1 STED-Microscopy ................................................................................................................ 5 1.2.2 STORM / PALM .................................................................................................................... 7 1.2.3 Other Approaches for Sub-Diffraction Imaging .................................................................. 7 1.3 Labelling Tools used in Fluorescence Microscopy ................................................................... 9 1.3.1 Detection of Proteins via Recombinant Tags ...................................................................... 9 1.3.2 Affinity-Based Detection ................................................................................................... 10 1.4 Affinity Probes in Microscopy ................................................................................................ 12 1.4.1 Antibodies ......................................................................................................................... 12 1.4.2 Small Probes...................................................................................................................... 13 1.4.3 In vitro Selection Techniques ............................................................................................ 17 1.4.4 Nanobodies ....................................................................................................................... 18 1.5 Comparison of Nanobodies to Conventional Probes ............................................................. 19 1.6 Imaging Approaches to Study the Synaptic Architecture ...................................................... 23 1.7 Aims of the Project................................................................................................................. 27 XI 2. Material and Methods ........................................................................................... 28 2.1 Materials and Reagents ......................................................................................................... 28 2.1.1 General Chemicals ............................................................................................................ 28 2.1.2 Buffers, Solutions and Media ............................................................................................ 30 2.1.3 Molecular Kits ................................................................................................................... 34 2.1.4 Antibodies ......................................................................................................................... 34 2.1.5 Purified Proteins ............................................................................................................... 35 2.1.6 Consumables ..................................................................................................................... 36 2.1.7 Instrumentation / Equipment ........................................................................................... 37 2.1.8 Software ............................................................................................................................ 39 2.2 Methods and Protocols .......................................................................................................... 40 2.2.1 Molecular Cloning / Gene Constructs ............................................................................... 40 2.2.2 Glycerol Stocks .................................................................................................................. 44 2.2.3 Bacterial Protein Expression and Purification ................................................................... 45 2.2.4 SDS PAGE........................................................................................................................... 47 2.2.5 Preparation of Competent Bacteria .................................................................................. 47 2.2.6 Cell-Line Culture and Transfection.................................................................................... 49 2.2.7 Immunostainings .............................................................................................................. 50 2.2.8 Fluorescence Imaging ....................................................................................................... 50 2.2.9 Protein Purification from Mammalian Cell Lines .............................................................. 52 2.2.10 Primary Neuronal Culture ................................................................................................. 52 2.2.11 Brain Slice Preparation ...................................................................................................... 53 2.2.12 Tissue Isolation ................................................................................................................. 53 2.2.13 Western Blotting ............................................................................................................... 54 2.2.14 Animal Immunization ........................................................................................................ 54 2.2.15 Nanobody Library Construction ........................................................................................ 55 2.2.16 Phage Display .................................................................................................................... 60 2.2.17 Validation and Characterization of Nanobody Candidates............................................... 64 2.2.18 Labelling of Nanobodies ................................................................................................... 65 2.2.19 Affinity Measurements ..................................................................................................... 69 2.2.20 Image Analysis and Evaluation ......................................................................................... 69 2.2.21 Molecular Modelling ......................................................................................................... 69 XII 3. Results ..................................................................................................................
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