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The Role of MEGF10 in Skeletal Muscle Myopathy

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The Role of MEGF10 in Skeletal Muscle Myopathy The role of MEGF10 in skeletal muscle myopathy. Thesis by Ruth Elizabeth Hughes Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Faculty of Biological Sciences School of Molecular & Cellular Biology October 2016 -ii- The candidate confirms that the work submitted is his/her own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. © 2016 The University of Leeds and Ruth Elizabeth Hughes -iii- Acknowledgements I am indebted to the Medical Research Council for their funding, and the Faculty of Biological Sciences for making my PhD possible. I would like to thank my supervisors Michelle Peckham and Colin Johnson for their patience, tireless enthusiasm and sage advice throughout the ups and downs of the project. I am grateful to Sara Cruz Migoni from the Borycki lab (Sheffield) for teaching me the single fibre isolation technique and Stuart Egginton and Roger Kissane for their help in setting up the hyperplasia model. I am thankful to Clare Logan and Gabrielle Wheway for their help with setting up and analysing the RNAseq data. Protein identification by mass spectrometry was performed by James Ault in the University of Leeds Mass Spectrometry Facility. Also thanks to the University of Leeds Bio-imaging facility for use of the DeltaVision and LSM880 microscopes. I am indebted to the past and present members of the Johnson and Peckham labs for their camaraderie and invaluable support. In particular Adriana, Alexa, Alistair, Brendan, Charlie, Fran, Glenn, Kasia, Marcin, Marta, Matt, it’s been an adventure. I am so grateful to my friends, my second family, here in Leeds for helping me to keep work and life in perspective. Finally, the support and encouragement of my family; Mum, Dad, David (Dr Geezer-Features), Rachael & Evangeline has been amazing. -iv- Abstract Mutations in the transmembrane protein MEGF10 (multiple epidermal growth factor-like protein 10) cause early-onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD), an autosomal recessive congenital myopathy, characterized by reduced muscle fibre size. MEGF10 is reportedly expressed in satellite cells, playing a role in regulating cell proliferation and differentiation. The pattern of expression and the function of MEGF10 have not been clearly described. Cultured myoblasts, isolated single fibres and a muscle hyperplasia model were used to investigate changes in MEGF10 expression. Western blotting, immunofluorescence and RNAseq showed that MEGF10 was expressed at low levels in cultured myoblasts, suggesting that these cells are a poor model for studying endogenous MEGF10. Overexpression of GFP-tagged wildtype MEGF10 and MEGF10 containing pathogenic mutations decreased myoblasts cell motility and fusion. Isolated single fibres showed the presence of quiescent (Pax7+), committed and activated (MyoD+ or myogenin+) satellite cells after 2 - 4 days in culture. However, these cells did not consistently express MEGF10. A muscle hyperplasia model showed no MEGF10 expression in CD34+/Pax7+ or MyoD+ positive satellite cells, but MEGF10+/CD34- cells were present on these fibres. Finally, the expressed and purified extracellular domain (ECD) of MEGF10 was heavily post- translationally modified and facilitated myoblast attachment to a non- adherent surface. Pathogenic mutations in the ECD did not have a significant effect on myoblast fusion, whilst the wildtype ECD inhibited fusion. In conclusion, the choice of skeletal muscle model system is important in obtaining meaningful results. The discovery of MEGF10 expression in, as yet, unidentified CD34- cells on isolated fibres requires further study. MEGF10 is likely to respond to differing cues within the complex muscle environment to regulate myogenesis through cellular changes affecting differentiation and motility. Whilst EMARDD is a rare condition, insights into MEGF10 function may give insight into mechanisms of pathogenesis in a range of skeletal muscle diseases. -v- Table of Contents Acknowledgements.................................................................................... iii Abstract....................................................................................................... iv Table of Contents........................................................................................ v List of Tables .............................................................................................. xi List of Figures ........................................................................................... xii 1 Introduction. ........................................................................................ 1 1.1 Early-onset Myopathy Areflexia Respiratory Distress and Dysphagia (EMARDD) .................................................................. 1 1.2 Multiple epidermal growth factor-like domains protein 10 (MEGF10) ..................................................................................... 4 1.2.1Disease mutations................................................................. 4 1.2.2The MEGF Family................................................................. 7 1.3 The Structure of MEGF10 ........................................................... 10 1.3.1EMI domain......................................................................... 10 1.3.2EGF domains ...................................................................... 10 1.3.3Post-translational modification of EGF-like domains........... 14 1.3.4Intracellular domain............................................................. 17 1.3.5The Function of MEGF10.................................................... 21 1.3.6The function of MEGF10 in engulfment and apoptosis ....... 21 1.3.7The neuronal function of MEGF10. ..................................... 23 1.4 The role of MEGF10 in skeletal muscle ...................................... 25 1.4.1Renewal of the satellite stem cell pool ................................ 34 1.5 Satellite cell populations.............................................................. 35 1.6 MEGF10 and myogenic cells ...................................................... 35 1.7 Hypothesis and Aims .................................................................. 41 2 Materials and Methods...................................................................... 43 2.1 Chemicals and Enzymes............................................................. 43 2.2 Strains and Genotypes................................................................ 43 2.3 Growing Escherichia coli (E.coli)................................................. 43 2.4 Plasmids...................................................................................... 44 2.5 General Procedures.................................................................... 46 2.6 Working with DNA ....................................................................... 46 2.6.1Spectrophotometric quantification of nucleic acids.............. 46 -vi- 2.6.2Precipitation and concentration of nucleic acid ................... 46 2.6.3‘Miniprep’ DNA preparations ............................................... 46 2.6.4‘Maxiprep’ DNA preparations .............................................. 47 2.6.5Restriction endonuclease digestions of plasmid DNA ......... 48 2.6.6Separation of DNA fragments by agarose gel electrophoresis.................................................................... 48 2.6.7Extraction of DNA from an agarose gel............................... 49 2.6.8Transformation of competent cells with plasmid DNA ......... 50 2.6.9Amplification of DNA by the polymerase chain reaction...... 50 2.6.9.1 Cloning primers..................................................... 51 2.6.9.2 Sequencing primers.............................................. 51 2.6.9.3 Colony PCR .......................................................... 51 2.6.9.4 Phusion® High-Fidelity PCR.................................. 52 2.6.10 InFusion Cloning ......................................................... 52 2.6.11 DNA sequencing ......................................................... 55 2.6.12 Site directed mutagenesis........................................... 55 2.7 Mammalian Cell Culture.............................................................. 57 2.7.1C1F myoblasts .................................................................... 57 2.7.2Differentiation of C1F myoblasts ......................................... 58 2.7.3Growth of Ad293 and HEK-293 Cells.................................. 58 2.7.4Recovery of cells from liquid nitrogen storage..................... 59 2.7.5Passaging cells ................................................................... 59 2.7.6Storage of cells ................................................................... 59 2.7.7Preparation of coverslips..................................................... 59 2.7.8Seeding cells onto coverslips.............................................. 60 2.7.9Transfection of mammalian cells......................................... 60 2.7.10 Calcium chloride transfection ...................................... 60 2.7.11 Lipid-based transfection (FuGene®)............................61 2.8 Immunocytochemistry ................................................................. 61 2.8.1Fixation of cells ..................................................................
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