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The Role of the Cell Adhesion Molecule CHL1 and Its Interaction Partners in Cerebellar Development

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The Role of the Cell Adhesion Molecule CHL1 and Its Interaction Partners in Cerebellar Development 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 .....................................................................................................
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