The role of the cell adhesion molecule CHL1 and its interaction partners in cerebellar development Dissertation with the aim of achieving a doctoral degree at the Faculty of Mathematics, Informatics and Natural Sciences Department of Biology of Universität Hamburg Submitted by Jelena Katic 2016 in Hamburg Day of Oral Defense: The following evaluators recommended the admission of the dissertation: Prof. Dr. Melitta Schachner Prof. Dr. Christian Lohr TABLE OF CONTENT 1 INTRODUCTION .............................................................................. 10 1.1 Development of the cerebellum ...................................................................................................... 11 1.1.1 Tissue organization in the adult cerebellar cortex .............................................................................. 15 1.2 Cell adhesion molecule close homolog of L1‐ CHL1 ........................................................................... 17 1.2.1 The role of CHL1 in the cerebellum ..................................................................................................... 20 1.3 Sonic hedgehog signaling pathway .................................................................................................. 21 1.4 Extracellular matrix molecules – vitronectin and plasminogen activator inhibitor 2 .......................... 25 2 AIMS OF THE STUDY ..................................................................... 28 3 MATERIALS ..................................................................................... 29 3.1 Animals ........................................................................................................................................... 29 3.2 Bacterial strains and cell lines .......................................................................................................... 29 3.3 Plasmid vectors ............................................................................................................................... 30 3.4 Primers ........................................................................................................................................... 31 3.5 Recombinant protein constructs and peptides ................................................................................. 32 3.6 Solutions and buffers ...................................................................................................................... 34 3.7 Cell culture media, buffers and reagents .......................................................................................... 41 3.8 Bacterial media and reagents .......................................................................................................... 43 3.9 Antibodies ...................................................................................................................................... 44 3.10 Chemicals and supplies.................................................................................................................... 46 4 METHODS ........................................................................................ 47 4.1 Biochemical methods ...................................................................................................................... 47 4.1.1 Preparation of cerebellar homogenates and cell lysates .................................................................... 47 4.1.2 Determination of the protein concentration ....................................................................................... 47 4.1.3 Sodium Dodecyl Sulfate‐Polyacrylamide Gel Electrophoresis (SDS‐PAGE) ......................................... 47 4.1.4 Western blot analysis .......................................................................................................................... 48 4.1.5 Coomassie staining of polyacrylamide gels ......................................................................................... 48 4.1.6 Immunoprecipitation ........................................................................................................................... 48 4.1.7 RhoA activation assay .......................................................................................................................... 49 4.1.8 Caspase‐3 assay ................................................................................................................................... 49 4.1.9 Caspase‐9 assay ................................................................................................................................... 50 4.1.10 Enzyme‐Linked Immuno‐Sorbent Assay (ELISA) .................................................................................. 50 4.1.11 Label‐free binding assay (BIND assay) ................................................................................................. 51 4.1.12 RNA isolation from brain homogenates .............................................................................................. 51 4.1.13 RNA isolation from cell lysates ............................................................................................................ 52 4.2 Molecular biology methods and cloning techniques ......................................................................... 53 4.2.1 Reverse transcription ........................................................................................................................... 53 4.2.2 PCR primer design and general principles of the In‐Fusion cloning technique ................................... 53 4.2.3 Polymerase chain reaction (PCR) ......................................................................................................... 53 4.2.4 DNA agarose gel electrophoresis ......................................................................................................... 54 4.2.5 PCR fragment purification ................................................................................................................... 55 4.2.6 DNA fragment extraction from agarose gels ....................................................................................... 55 4.2.7 Restriction digestion ............................................................................................................................ 55 4.2.8 In Fusion cloning reaction .................................................................................................................... 55 4.2.9 Production of competent bacteria ...................................................................................................... 56 4.2.10 Transformation of plasmid DNA into bacteria ..................................................................................... 56 4.2.11 Small scale plasmid isolation (Miniprep) ............................................................................................. 57 4.2.12 Large scale plasmid isolation (Maxiprep) ............................................................................................ 57 4.2.13 Sequencing of DNA .............................................................................................................................. 57 4.2.14 Site‐directed mutagenesis ................................................................................................................... 57 4.2.15 Expression and purification of recombinant proteins from E. coli ...................................................... 58 4.3 Real‐time PCR ................................................................................................................................. 59 4.4 Histological methods and microscopy .............................................................................................. 60 4.4.1 Tissue preparation and sectioning ....................................................................................................... 60 4.4.2 Immunohistochemistry ........................................................................................................................ 60 4.4.3 Proximity ligation assay ....................................................................................................................... 61 4.4.4 In situ cell death detection assay (TUNEL assay) ................................................................................. 62 4.4.5 Microscopy and image processing ....................................................................................................... 63 4.4.6 Co‐localization analysis ........................................................................................................................ 63 4.4.7 Stereological analysis ........................................................................................................................... 64 4.5 Cell culture methods and assays ...................................................................................................... 64 4.5.1 Primary culture of dissociated cerebellar neurons .............................................................................. 64 4.5.2 Neurite outgrowth assay ..................................................................................................................... 65 4.5.3 Microexplant culture of cerebellar neurons ........................................................................................ 66 4.5.4 Maintenance and long‐term storage of HEK cells ............................................................................... 67 4.5.5 Transient transfection of HEK cells .....................................................................................................