Biogenesis and Maintenance of Cytoplasmic Domains in Myelin of the Central Nervous System Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen within the doctoral program Molecular Biology of Cells of the Georg-August University School of Science (GAUSS) submitted by Caroline Julia Velte from Usingen, Germany Göttingen 2016 Members of the Thesis Committee: Prof. Dr. Mikael Simons, Reviewer Max Planck Institute of Experimental Medicine, Göttingen Prof. Dr. Andreas Janshoff, Reviewer Georg-August University, Göttingen Prof. Dr. Dirk Görlich Max Planck Institute of Biophysical Chemistry, Göttingen Date of the thesis defense: 27th of June 2016 Don't part with your illusions. When they are gone, you may still exist, but you have ceased to live. (Mark Twain) III Affidavit I hereby declare that my PhD thesis “Biogenesis and Maintenance of Cytoplasmic Domains in Myelin of the Central Nervous System” has been written independently with no other aids or sources than quoted. Furthermore, I confirm that this thesis has not been submitted as part of another examination process neither in identical nor in similar form. Caroline Julia Velte April 2016 Göttingen, Germany V Publications Nicolas Snaidero*, Caroline Velte*, Matti Myllykoski, Arne Raasakka, Alexander Ignatev, Hauke B. Werner, Michelle S. Erwig, Wiebke Moebius, Petri Kursula, Klaus-Armin Nave, and Mikael Simons Antagonistic Functions of MBP and CNP Establish Cytosolic Channels in CNS Myelin, Cell Reports 18 *equal contribution (January 2017) E. d’Este, D. Kamin, C. Velte, F. Göttfert, M. Simons, S.W. Hell Subcortical cytoskeleton periodicity throughout the nervous system, Scientific Reports 6 (March 2016) K.A. Vassall, W. Bamm, A.D. Jenkins, C. Velte, D.R. Kattnig, J.M. Boggs, D. Hinderberger, G. Harauz Substitutions mimicking deimination and phosphorylation of 18.5-kDa myelin basic protein exert local structural effects that subtly influence its global folding, BBA – Biomembranes 6 (February 2016) S. Nawaz, P. Sánchez, S. Schmitt, N. Snaidero, M. Mitkovski, C. Velte, S. Brückner, (…), and M. Simons Actin filament turnover drives leading edge growth during myelin sheath formation in the central nervous system, Developmental Cell 2 (July 2015) VII Contents Affidavit ................................................................................................................................ V Publications ....................................................................................................................... VII Contents.............................................................................................................................. IX List of Figures .................................................................................................................. XIII List of Tables ..................................................................................................................... XV Abbreviations .................................................................................................................. XVII Acknowledgements .......................................................................................................... XXI Abstract .......................................................................................................................... XXIII 1. Introduction ...................................................................................................................1 1.1 Myelin – a speed-enhancing innovation ....................................................................1 1.2 Axonal insulation and support ...................................................................................1 1.3 The biogenesis of myelin ..........................................................................................3 1.4 The ultrastructure of myelin ......................................................................................5 1.5 Myelin composition ...................................................................................................6 1.6 Myelin: compaction versus de-compaction ...............................................................8 1.6.1 Intrinsically multitalented – Myelin Basic Protein (MBP) .....................................9 1.6.2 The mysterious multi-talent – 2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP)............................................................................................................... 12 1.6.3 Teamwork of CNP and MBP ............................................................................ 16 1.7 Neuron glia communication .................................................................................... 17 1.7.1 Schmidt-Lanterman Incisures in the PNS ......................................................... 18 1.7.2 Physiological activity of CNS myelin ................................................................ 20 1.7.3 Why should structures that fulfil essential functions in the periphery be missing in the CNS? ..................................................................................................... 21 1.8 Aim of this work ...................................................................................................... 23 IX 2 Materials and Methods ................................................................................................ 25 2.1 Materials ................................................................................................................. 25 2.1.1 General chemicals, consumables, and kits ...................................................... 25 2.1.2 General Buffers and Solutions ......................................................................... 26 2.1.3 Enzymes and Kits ............................................................................................ 26 2.1.4 Antibodies and dyes ......................................................................................... 27 2.1.5 Primers ............................................................................................................ 27 2.1.6 Chemicals for electron microscopy .................................................................. 28 2.1.7 Software .......................................................................................................... 29 2.2 Methods .................................................................................................................. 29 2.2.1 Molecular Cloning ............................................................................................ 29 2.2.2 Expression and purification of proteins............................................................. 30 2.2.3 Primary oligodendrocyte cell culture ................................................................ 31 2.2.4 Treatment of primary oligodendrocytes ............................................................ 32 2.2.5 Immunocytochemistry ...................................................................................... 33 2.2.6 Biomimetic supported lipid bilayer – giant unilamellar vesicle (SLB-GUV) assay ............................................................................................................... 34 2.2.7 Polymerization of actin for GUV bursting assay ............................................... 35 2.2.8 Interaction studies of CNP and actin ................................................................ 36 2.2.8.1 Actin purification .......................................................................................36 2.2.8.2 F-actin co-sedimentation ..........................................................................36 2.2.8.3 Actin bundling ..........................................................................................37 2.2.8.4 Actin polymerization assay .......................................................................37 2.2.8.5 Covalent crosslinking and mass spectrometry ..........................................37 2.2.9 Mouse lines...................................................................................................... 38 2.2.10 Genotyping ...................................................................................................... 39 2.2.11 Agarose gel electrophoresis ............................................................................. 40 2.2.12 Sample preparation of mouse CNS tissues by high-pressure freezing ............. 40 2.2.13 Sample processing for TEM ............................................................................. 41 X 2.2.14 Imaging ............................................................................................................ 43 2.2.14.1 Light microscopy ......................................................................................43 2.2.14.2 Stimulation Emission Depletion Microscopy (STED) ................................43 2.2.14.3 Transmission electron microscopy (TEM).................................................43 2.2.14.4 Image Processing and statistical analysis ................................................44 3 Results ......................................................................................................................... 45 3.1 Morphology of cytoplasmic channels in myelin sheaths of the CNS ........................ 45 3.1.1 Cytoplasmic channel distribution in thin-caliber axon ....................................... 45 3.1.2 Cytoplasmic channel distribution in myelinated axons of large caliber ............. 46 3.1.3 CNP & MBP – drive the biogenesis