The role of cytoskeletal tropomyosins in skeletal muscle and muscle disease Nicole Vlahovich This thesis is submitted in fulfilment of the requirements for the degree of Doctor of Philosophy The Muscle Development Unit Children’s Medical Research Institute And The School of Natural Sciences University of Western Sydney April 2007 Acknowledgements Firstly I would like to thank Dr Edna Hardeman for providing me with the opportunity to be a member of the MDU at CMRI and a part of the muscle Tm project. I very much appreciated her help and support as a supervisor throughout my time as a PhD student, thankyou for all the amazing opportunities. I would like to thank Dr Anthony Kee for all his help as a co-supervisor and also Professor Peter Gunning, the Tm guru, for much advice and an amazing amount of enthusiasm, which always made me feel like my results were groundbreaking! Thankyou to Dr Galina Schevzov for a great amount of help on the project and a calming nature that was very much appreciated. Also to Emma Kettle whose amazing work on the immunogold EM was invaluable and her friendship cherished. To our collaborators: Rob Parton and Delia Hernandez from IMB QLD, Kathy North and Bili Ilkovski at the NGU at the Children’s Hospital Westmead and David James and Greg Cooney at the Garvan Institute for all your help and advice. Also to Ross Boadle from Westmead Hospital, who allowed me to use the fantastic EM facility and for many helpful discussions. To all of the MDU, past and present, whose technical advice and friendship over the years has got me though this PhD particularly Lini, Enoch, Mai-Anh and Majid who taught me my precious techniques and Nicole, along with Emma, whose company on coffee breaks was so helpful in times of stress. Also to all of the CMRI, I could not have asked for a better place to complete a PhD; fantastic staff, amazing facilities and a great atmosphere. I am grateful for all the opportunities I have been presented with. Thankyou to the animal house staff for looking after my mice, especially Ben Tuckfield and Shelley Dimech and also Tina Borovina from the ORU, CHW. And thankyou to the admin girls for lunchtime relaxation where I could get away from science for 45 mins, it was a mental health saviour. A big thankyou to Dr Mark Jones, his advice and encouragement steered me into a PhD in the first place and allowed me to think outside the box. His advice, right from his first class at UWS when I was in second year has been instrumental to my career. Finally to my family and friends who have made this whole experience possible. I want to thank my extremely supportive family: Carmel, Elie, Jeff, Eileen and Susy. Their efforts have kept me sane (at least partially) and I appreciate how much of my stress they have put up with over the last three years (well more like 10 really!). And to Greg Mitchell, from day one he has been supportive, patient and the best friend I could ask for. Thankyou for believing in me. ii Declaration: The work presented in this thesis is, to the best of my knowledge and belief, original except as acknowledged in the text. I hereby declare that I have not submitted this material, either in full or in part, for a degree at this or any other institution. _____________________________ Nicole Vlahovich iii Table of Contents Section One: General Introduction 1 Chapter One: Literature review and research objectives 2 1.1 Cytoskeletal filament systems 2 1.1.1 Microtubules 2 1.1.2 Intermediate filaments 5 1.1.3 Actin microfilaments 8 Myosin motor proteins 12 Tropomyosins 14 1.2 Muscle cytoarchitecture 19 1.2.1 Filamentous proteins of the sarcomere 21 1.2.2 Cytoskeletal structures in muscle costameres and the Z-LAC 25 Costameres 25 Z-line Associated Cytoskeleton (Z-LAC) 27 1.2.3 The sarcoplasmic reticulum and the T-tubule system 29 1.2.4 Neuromuscular junction (NMJ) and actin 30 1.3 Significant functions of skeletal muscle 32 1.3.1 Muscle contraction 32 1.3.2 The transport of glucose in skeletal muscle 33 1.4 Muscle fibre formation 37 1.4.1 Muscle development 36 1.4.2 The regeneration of muscle fibres 41 1.5 Muscle Disease 42 1.5.1 Muscular dystrophies 43 1.5.2 Congenital myopathies with affected filaments 48 Nemaline myopathy (NM) 48 Actin myopathy (AM) 50 1.6 Research Objectives 52 Section Two: The roles of cytoskeletal tropomyosins in muscle 54 Chapter Two: Cytoskeletal tropomyosins form functionally distinct 55 filaments in skeletal muscle 2.1 Introduction 55 2.2 Materials and Methods 57 2.2.1 Specific materials 57 2.2.2 Animal strains 57 2.2.3 Primary antibodies 57 2.2.4 Secondary antibodies 58 2.2.5 Preparation of tissue samples for western analysis of protein 58 2.2.6 Western blotting analysis 59 2.2.7 Preparation of tissue samples for cryomicrotomy 60 2.2.8 Preparation of tissue samples for semi-thin cryomicrotomy 60 2.2.9 Immuno-staining of muscle sections 61 2.2.10 Immuno-gold labeling and electron microscopy (EM) analysis 61 2.2.11 Muscle fibre isolation and analysis 62 2.2.12 Isolation of membrane components 62 2.2.13 Processing of isolated membranes for protein analysis 63 iv 2.2.14 Processing of isolated triads fro EM 64 2.2.15 Processing of isolated triads for EM and immuno-labelling 64 2.3 Results 66 2.3.1 Tms are differentially expressed in skeletal muscles 66 2.3.2 Tm isoforms define filaments associated with organelles in muscle fibres 68 2.3.3 Tm4 and Tm5NM1 define discrete actin filament populations at the 72 Z-LAC 2.3.4 Tm4 is associated with the sarcoplasmic reticulum 77 2.4 Discussion 81 2.4.1 Various cytoskeletal Tm isoforms are expressed in skeletal muscle 81 2.4.2 Tm5NM1 and Tm4 define distinct membrane associated structures 82 adjacent to the Z-line in muscle fibres Chapter Three: Tropomyosin 4 indicates repair/remodeling in skeletal 85 muscle disease 3.1 Introduction 85 3.2 Materials and Methods 88 3.2.1 Specific materials 88 3.2.2 Animal strains 88 3.2.3 Human muscle samples 88 3.2.4 Primary antibodies 89 3.2.5 Secondary antibodies 89 3.2.6 Western blotting of human muscle samples 89 3.2.7 Protein preparations to enrich for Tms 89 3.2.8 Immunohistochemistry of human muscle biopsy samples 89 3.2.9 Notexin induced muscle regeneration 90 3.2.10 Mouse hindlimb immobilization 90 3.3 Results 91 3.3.1 Cytoskeletal Tm4 defines two cytoskeletal compartments in normal 91 skeletal muscle 3.3.2 Longitudinal structures defined by Tm4 are evident during myofibrillar 95 assembly and remodeling 3.3.3 Tm4 is an indicator of muscle disease 99 3.4 Discussion 105 3.4.1 Tm4-defined longitudinal filaments reflect the processes of skeletal 105 muscle regeneration and repair 3.4.2 A Tm4/actin cytoskeleton plays a role in the repair of skeletal muscle 106 fibres Chapter Four: The altered expression of Tm5NM1 in skeletal muscle 108 affects membrane morphology and metabolic pathways 4.1 Introduction 108 4.2 Materials and Methods 111 4.2.1 Specific materials 111 4.2.2 Animal strains 111 4.2.3 Primary antibodies 112 4.2.4 Secondary antibodies 112 4.2.5 Oligonucleotides used for RT-PCR 112 4.2.6 Ruthenium Red staining of isolated muscle fibres 112 4.2.7 RNA extraction from muscles for microarray analysis 113 4.2.8 Affymetrix gene chip analysis 113 4.2.9 RNA extraction from muscles from muscles for RT-PCR 114 v 4.2.10 Transcription of RNA to cDNA 114 4.2.11 Agarose gel electrophoresis 114 4.2.12 Preparation of GAPDH standards for quantitative PCR 115 4.2.13 Quantitative real time PCR 115 4.3 Results 117 4.3.1 Ablation and over-expression of Tm5NM1 does not impact on levels or 117 localisation of other Tm isoforms 4.3.2 A lack of Tm5NM1 in skeletal muscle causes abnormalities in T-tubule 126 and caveolae morphology 4.3.3 Tm5NM1 knockout and transgenic mice have alterations in gene 130 expression in soleus muscle 4.4 Discussion 138 4.4.1 Tm isoforms from different genes are independently regulated 138 4.4.2 Tm5NM1 plays a role in the organisation of membrane morphology and 139 cellular metabolism Chapter Five: Tropomyosin 5NM1 is involved in glucose transport and 143 adipose tissue proliferation 5.1 Introduction 143 5.2 Materials and Methods 146 5.2.1 Specific materials 146 5.2.2 Animal strains 146 5.2.3 Primary antibodies 146 5.2.4 Secondary antibodies 146 5.2.5 Solubilisation of muscle and adipose tissue in RIPA buffer 146 5.2.6 In vitro analysis of glucose uptake in adipose tissue 147 5.2.7 Wortmannin inhibition of glucose uptake 147 5.2.8 Glucose tolerance testing 148 5.2.9 Analysis of fat pad mass 147 5.3 Results 149 5.3.1 Tm5NM1 co-localises with proteins involved in glucose uptake 149 5.3.2 De-regulation of Tm5NM1 causes changes in glucose uptake and glucose 151 tolerance and knockout and transgenic mice 5.3.3 Tm5/52 transgenic mice have increased body fat 159 5.4 Discussion 161 5.4.1 Tm5NM1-defined actin filaments play a role in insulin-mediated glucose 161 uptake 5.4.2 Tm5NM1 impacts on adipose tissue 163 Section Three: General discussion and future directions 164 Chapter Six: General Discussion 165 6.1 Cytoskeletal Tms segregate to form functionally distinct 167 compartments in skeletal muscle 6.1.1 Tm-defined filament populations segregate with organelles and 167 membrane structures 6.1.2 Tm isoforms are involved in the specification of γ-actin filaments in 168 skeletal muscle 6.1.3 Tm5NM1 plays a unique non-essential role defining γ-actin filaments in 171 association with the T-tubules and sarcolemma 6.1.4 Cytoskeletal Tm filaments are proposed to associate with other actin 174 binding proteins in skeletal muscle vi 6.2 A role for Tm4 in the regeneration and repair of muscle