The Regulation of Beta- Dystroglycan Internalization Robert Piggott A thesis submitted to the University of Sheffield for the degree of Doctor of Philosophy Department of Biomedical Sciences, University of Sheffield July 2014 I TABLE OF CONTENTS CONTENTS………………………………………………………………………………II ABBREVIATIONS LIST…………………………………………………………....……VI ABSTRACT……………………………………………………………………………..VIII Chapter 1: Introduction .................................................................................. 1 1.1.1 The Dystroglycan Subunits ..................................................................... 2 1.1.2 Dystroglycan is an Essential Adhesion Protein......................................... 4 1.1.3 Dystroglycan in the Dystrophin-associated Glycoprotein Complex ............ 5 1.1.4 Dystroglycan in Other Complexes ......................................................... 10 1.1.5 Dystroglycan Complex Formation in Response to the Extracellular Matrix ................................................................................................................... 13 1.1.6 Summary: Why Dystroglycan is an Essential Protein.............................. 15 1.2.1 Duchenne Muscular Dystrophy ............................................................. 17 1.2.2 The mdx Mouse and Other Models ........................................................ 19 1.2.3 Dystroglycan Disruption and Other Muscular Dystrophies ...................... 22 1.2.4 Summary: The Loss and Restoration of β-Dystroglycan in DMD Pathology and Therapy ................................................................................................. 23 1.3.1 The H2Kb ts-A58 Myoblast Cell Line .................................................... 24 1.3.2 Chapter Summary ................................................................................ 26 Chapter 2: Materials and Methods ................................................................ 27 2.1 Bacterial Methods ................................................................................... 28 2.1.1 Preparation of Competent Bacteria..................................................... 28 2.1.2 Plasmid Purification ......................................................................... 28 2.1.3 Transformation of Competent Bacteria ............................................... 29 2.2 Protein Methods .................................................................................... 29 2.2.1 Growth and Induction of Recombinant Proteins ............................... 29 2.2.2 WW Domain GST Fusion Purification ............................................. 30 2.2.3 Multi-Dsk Protein Purification ........................................................ 30 2.2.4 Bradford Assay .............................................................................. 31 2.2.5 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis ........... 31 2.2.6 Electroblotting of Proteins .............................................................. 32 2.2.7 Western Blotting ............................................................................ 32 2.2.8 Peptide Arrays ............................................................................... 33 II 2.3 Cell Culture and Assays ......................................................................... 33 2.3.1 Growth of H2K Myoblasts .............................................................. 33 2.3.2 Preparation of Lysates .................................................................... 34 2.3.3 Immunoprecipitation ...................................................................... 34 2.3.4 Orthovanadate Treatment of H2K Myoblasts .................................... 34 2.3.5 Biotinylation Assay ........................................................................ 35 2.3.6 Transferrin Uptake Assay ............................................................... 35 2.3.7 Cholera Toxin Uptake Assay........................................................... 36 2.3.8 LysotrackerTM Staining and Ammonium Chloride Treatment ............. 36 2.3.9 H2K Myoblast Transfection ............................................................ 36 2.4 Microscopy Techniques.......................................................................... 37 2.4.1 Preparation of Cells for Microscopy ................................................ 37 2.4.2 Epifluorescence Microscopy ........................................................... 38 2.4.3 Confocal Microscopy ..................................................................... 38 2.5 Computer Aided Analysis of Data ........................................................... 38 2.5.1 Gel and Blot Analysis .................................................................... 38 2.5.2 Image Processing ........................................................................... 39 2.5.3 Co-localization Analysis ................................................................. 39 2.5.4 Statistical Analysis......................................................................... 39 2.6 Tables ................................................................................................... 40 2.6.1 Buffers and Solutions ..................................................................... 40 2.6.2 Antibodies ..................................................................................... 43 2.6.3 Gel Formulations ........................................................................... 44 2.6.4 Plasmids ........................................................................................ 44 Chapter 3: Y890 Phosphorylation and the Internalization of β -Dystroglycan 45 3.1.1 Introduction......................................................................................... 46 3.1.2 The Importance of the Interaction Between β-Dystroglycan and Dystrophin ................................................................................................................... 46 3.1.3 The Effect of Y890 Phosphorylation ..................................................... 48 3.1.4 Hypotheis: Y890 Phosphorylation is Required for the Internalization of β - Dystroglycan in H2K Myoblasts .................................................................... 48 3.1.5 1709 and MANDAG2: Specific Information on the Antibodies Used in This Study........................................................................................................... 50 3.2.1 Phosphorylation of Y890 is Linked to a Change in the Cellular III Localization of β-Dystroglycan ..................................................................... 51 3.2.2 Development of Biotinylation Assay: Optimization of Biotinylation Buffer ................................................................................................................... 53 3.2.3 Development of Biotinylation Assay: Optimization of Stripping Washes..56 3.2.4 Development of Biotinylation Assay: A Loading Control for Pellet Fractions .................................................................................................... 60 3.2.5 The pY890 Form of β-Dystroglycan is Selectively Internalized in H2K Myoblasts .................................................................................................... 62 3.2.6 Hyperphosphorylation is not Solely Responsible for the Modification of Internalized β-Dystroglycan .......................................................................... 65 3.3.1 Discussion ........................................................................................... 67 3.3.2 Y890 Phosphorylation Changes the Cellular Localization of β - Dystroglycan ............................................................................................... 68 3.3.3 Developing the Biotinylation Assay for Internalization in H2K Myoblasts ................................................................................................................... 69 3.3.4 The pY890 Form of β-Dystroglycan is Internalized from the Plasma Membrane ................................................................................................................... 70 3.3.5 Y890 Phosphorylation and β-Dystroglycan Internalization in Other Systems ................................................................................................................... 72 3.3.6 Summary ............................................................................................. 73 Chapter 4: Phosphorylation of Y890 and the Ubiquitylation of β-Dystroglycan ..... 74 4.1.1 Introduction......................................................................................... 75 4.1.2 Identifying a Candidate for the Post-Translational Modification of β- Dystroglycan ............................................................................................... 75 4.1.3 Monoubiquitylation and the Internalization of β-Dystroglycan ................ 78 4.1.4 Hypothesis: Y890 Phosphorylation is Required for the Ubiquitylation of β- Dystroglycan in H2K Myoblasts .................................................................... 79 4.2.1 Enrichment of Cellular Ubiquitin by Affinity Tagging ........................... 80 4.2.2 Enrichment of Cellular Ubiquitin with the MultiDsk Protein................... 80 4.2.3 Y890 Phosphorylation is Linked to the Ubiquitylation of β-Dystrolycan . 82 4.2.4 Identification of the Ligase Responsible for the Ubiquitylation of β - Dystroglycan ............................................................................................... 87 4.3.1 Discussion: Establishing the Use of the