The septin cytoskeleton is associated with distinct myelin structures of the central and peripheral nervous system Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Andres Buser aus Känerkinden (BL) Basel, 2008 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Nicole Schaeren-Wiemers Prof. Dr. Markus Rüegg PD. Dr. Thomas Meier Basel, den 24. Juni 2008 Prof. Dr. Hans-Peter Hauri To my parents Table of Contents Table of contents 1 Acknowledgments ............................................................................................... 7 2 Abbreviations....................................................................................................... 8 3 Summary........................................................................................................... 11 4 Introduction........................................................................................................ 12 4.1 From polarized transport to the mature myelin sheath ............................... 12 4.2 Myelin......................................................................................................... 12 4.2.1 Myelination.......................................................................................... 12 4.2.2 Myelinating cells.................................................................................. 14 4.2.2.1 Oligodendrocytes ......................................................................... 15 4.2.2.2 Schwann cells .............................................................................. 16 4.3 Myelin structure and function...................................................................... 17 4.3.1 The nodes of Ranvier.......................................................................... 19 4.3.2 Myelin proteins.................................................................................... 21 4.3.2.1 MBP, PLP and P0 ........................................................................ 21 4.3.2.2 MAG and MOG ............................................................................ 22 4.3.2.3 MAL ............................................................................................. 23 4.4 Polarized cells ............................................................................................ 24 4.4.1 Membrane rafts ................................................................................... 25 4.4.2 Polarized sorting and trafficking mechanisms ..................................... 26 4.4.3 MAL in polarized epithelial cells .......................................................... 28 4.5 Cytoskeleton elements in myelinating cells ................................................ 29 4.5.1 Schwann cells ..................................................................................... 29 4.5.2 Oligodendrocytes ................................................................................ 30 4.6 The septins................................................................................................. 31 4.6.1 Septins in yeast................................................................................... 31 4.6.2 Septin complex formation and structure.............................................. 33 4.6.3 Septins in the mammalian central nervous system ............................. 36 4.6.4 Septins in oligodendrocytes and Schwann cells.................................. 37 4.7 The contribution of cytoskeleton elements to subdomain formation in myelinating glial cells ................................................................................. 37 5 Motivation and Aim of the Work......................................................................... 39 6 Material and Methods........................................................................................ 40 6.1 Custom peptide antibodies......................................................................... 40 6.1.1 Peptide sequences.............................................................................. 40 6.1.2 Peptide synthesis and immunization ................................................... 40 6.1.3 Antibody purification............................................................................ 40 4 Table of Contents 6.1.4 Western blot analysis with custom-made anti-septin antibodies ......... 41 6.2 Other antibodies......................................................................................... 41 6.3 Yeast two-hybrid screen............................................................................. 42 6.4 Western blot analysis ................................................................................. 42 6.5 Immunohistochemistry ............................................................................... 43 6.5.1 Teased peripheral nerve fibers............................................................ 43 6.5.2 Cryostat sections of fresh frozen human femoralis nerve tissue ......... 43 6.5.3 Cell cultures ........................................................................................ 43 6.6 Electron microscopy ................................................................................... 44 6.7 Immobilization of IgG for Immunoprecipitation ........................................... 44 6.7.1 SeizeX ProteinA Immunoprezipitation kit (Pierce, USA)...................... 44 6.7.2 ProFound Co-Immunoprecipitation kit (Pierce, USA) .......................... 44 6.7.3 Affi-Gel Hz Immunoaffinity kit (Biorad, USA) ....................................... 45 6.8 Immunoprecipitation................................................................................... 45 6.8.1 Sample preparation............................................................................. 45 6.8.2 Immunoprecipitation............................................................................ 46 6.9 GST pull down assays................................................................................ 46 6.9.1 Sept6 with MAL(1-55)-GST................................................................. 46 6.9.2 Sept2/ 6/ 7 trimer with MAL(1-24)-GST ............................................... 47 6.9.2.1 Generation of the MAL(1-24)-GST construct................................ 47 6.9.2.2 Expression and purification of recombinant GST fusion proteins. 48 6.9.2.3 GST pull down assay ................................................................... 48 6.10 Light cycler quantitative real-time PCR ...................................................... 49 6.10.1 Primer pairs for real-time PCR ............................................................ 49 6.10.2 Standard curve generation for real-time PCR ..................................... 51 6.10.3 Total RNA isolation from sciatic nerve tissue ...................................... 51 6.10.4 Total RNA isolation from cultured cells................................................ 51 6.10.5 Reverse transcription reaction............................................................. 52 6.10.6 Quantitative real-time PCR.................................................................. 52 6.11 Cell culture ................................................................................................. 52 6.11.1 Standard cell culture ........................................................................... 52 6.11.2 Primary cell culture.............................................................................. 52 6.11.2.1 DRG/ Schwann cell co-cultures ................................................... 52 6.11.2.2 Primary oligodendrocytes from neurospheres.............................. 53 6.12 RNA interference........................................................................................ 54 6.12.1 Target sequences ............................................................................... 54 6.12.2 Generation of retroviral RNAi constructs............................................. 55 6.12.2.1 RNAi hairpin oligonucleotide sequences...................................... 55 6.12.2.2 Annealing of hairpin oligonucleotides........................................... 55 6.12.2.3 Ligation and transformation.......................................................... 55 6.12.2.4 Production of retroviral stocks...................................................... 56 6.12.3 Retroviral infection .............................................................................. 56 6.13 Generation of mCherry tagged Sept2 constructs ....................................... 57 5 Table of Contents 6.13.1 Subcloning of the mCherry sequence into the retroviral pMX vector... 57 6.13.2 Subcloning Sept2 wild type into the pMX-mCherry vector................... 58 6.13.3 Subcloning the S51N Sept2 mutant sequence into the pMX-mCherry vector................................................................................................... 58 6.13.4 Mutation elimination in the pMX-S51N-mCherry construct.................. 59 6.13.5 Production of retroviral stocks ............................................................. 61 7 Results .............................................................................................................. 62 7.1 The cytoskeleton protein Sept6 interacts with the myelin protein MAL....... 62 7.1.1 Sept6 is a potential interaction partner of MAL.................................... 62 7.1.2 Verification of the Sept6 / MAL interaction by GST pull down ............