THE ROLE OF DYSTONIN AND MACFl IN SKELETAL MUSCLE by: SOPHIE JOANISSE A thesis submitted as a partial fulfillment of the requirement for the degree of Maitrise en activite physique (M.A.P) School of Graduate Studies Laurentian University Sudbury, Ontario © Sophie Joanisse, 2011 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington OttawaONK1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre r6ference ISBN: 978-0-494-82027-8 Our file Notre r&terence ISBN: 978-0-494-82027-8 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 1*1 Canada iii Abstract Dystonin and microtubule actin cross linking factor 1 (MACF 1) are crosslinking proteins that are members of the plakin family. Plakin crosslinking proteins are believed to play a crucial role in maintaining the structural and mechanical integrity of contractile cells. The specific functions of dystonin and MACFl in skeletal muscle are not yet thoroughly understood. To gain further insights into the role of dystonin and MACFl in contractile cells, the subcellular localization of these plakin proteins in longitudinal sections of mouse hindlimb skeletal muscle was determined by double immunofluorescence experiments. Furthermore, the impact of dystonin-deficiency on specific cytoskeletal networks was assessed. The localization of dystonin and MACFl was compared to that of other proteins that localize to specific compartments of the muscle fiber namely the sarcomere (myosin/actin), the Z-disc (a-actinin/desmin), the subsarcolemmal area (desmin) and the neuromuscular junction (rapsyn/acetylcholine resceptors). In parallel, the expression profile of dystonin and MACFl in type I (slow-twitch) and type II (fast-twitch) fibers was assessed by immunofluorescence. The expression of dystonin and MACFl was also determined in soleus and gastrocnemius muscle by Western blot. In longitudinal sections and single muscle fibers of hindlimb muscle immunofluorescence staining of both dystonin and MACFl were observed at the Z-disc. Dystonin staining was also present at the subsarcolemmal area and at the perinuclear region while MACFl staining was present at the neuromuscular junction. iv Serial cross-sections of muscle fibers from mouse soleus and gastrocnemius muscles were stained for type I myosin heavy chain (labelling slow-twitch fibers) and either dystonin or MACFl. These immunofluorescence experiments revealed no difference in staining intensity for both dystonin and MACFl in either fast- or slow- twitch fibers. Howevere, dystonin and MACFl protein levels were significantly higher (1.5- and 2-fold respectively) in fast-twitch gastrocnemius muscle versus slow-twitch soleus muscle as revealed by Western blotting. Taken together these experiments suggest that dystonin and MACFl are more highly expressed in predominantly fast-twitch muscle. The protein expression profile of MACFl, plectin and cytoskeletal proteins actin, a-tubulin, desmin and a-actinin were compared between hindlimb skeletal muscles excised from dystonin-deficient (dt) and wild-type (wt) mice from the dystonia musculorum Tg4 (dt g ) transgenic mouse line. The results suggest that while the microfilament and microtubule network appears to be largely unaffected in dystonin- deficient muscle, the expression levels of the intermediate filament desmin is down- regulated in skeletal muscle of dt mice whereas the levels of a-actinin, MACFl and plectin are up-regulated. Collectively, our findings suggest that dystonin and MACFl exert their respective functions in specific subcellular compartments of the muscle fiber based on our localization studies and that predominantly fast-twitch fibers express greater levels of these crosslinking proteins. When skeletal muscles lack dystonin the other muscle plakins (MACFl and plectin) appear to undergo a compensatory up-regulation that is likely V sufficient to stabilize the microtubule and microfilament networks but not the desmin or a-actinin cytoarchitecture. vi Acknowledgements First I would like to extend my sincere gratitude to my supervisor Dr. Boudreau-Lariviere for her direction, assistance and guidance from the preliminary to the concluding stages of the project. Dr. Boudreau-Lariviere has given me the opportunity to advance my knowledge and skill as a researcher and enabled me to develop a thorough understanding of the subject. I am also greatful to all lab co-workers in particular Tyler Kirwan for his help, insight and technical assistance, Paul Micheal for his advice, as well as as my committee members Drs. Dorman and Ritz for their helpful suggestions and comments. Additionally, I would like to acknowledge Chris Blomme and Nicole Paquette for ensuring the proper care of all mice used during the course of the study. Lastly, I would like to thank my family for their constant support and encouragement during these past years. vii List of abbreviations ABD; Actin binding domain ACF7; Actin crosslinking factor 7 ACh; Acetylcholine AChRs; Acetylcholine receptors ACTA1; a-skeletal actin gene ACTC; a-cardiac actin gene Bpagl; Bullous pemphigoid antigen 1 CH; Calponin homology CRP; Cysteine-rich protein DCM; Dilated cardiomyopathy DRM; Desmin-related myopathies dt; Dystonia musculorum dtTg4; Dystonia musculorum transgenic line 4 (insertional mutation) EBS-MD; Epidermolysis bullosa simplex-with muscular dystrophy ECM; Extracellular matrix FATZ; Filamin-, a-actinin- and telethonin-binding protein of the Z-disc viii GAR; Gas2-related GAS; Gastrocnemius muscle GSR; Glycine-serine-arginine rich HCM; Hypertrophic cardiomyopathy IF; Intermediate filaments IFBD; Intermediate filament binding domain IFBD2; Intermediate filament binding domain of the dystonin-b muscle isoform IHC; Immunohistochemical MACFl; Microtubule actin cross-linking factor 1 mATPase; Myofibrillar actomyosin adenosine triphosphatase MD; Muscular dystrophy MF; Microfilaments MHC; Myosin heavy chain MT; Microtubules MTBD; Microtubule binding domain NMJ; Neuromuscular junction PCR; Polymerase chain reaction ix PLECl; Human plectin gene PRD; Plakin repeat domain SOL; Soleus muscle SR-rods; Spectrin repeat rods wt; Wild-type X Table of Contents Abstract iii Acknowledgments vi List of abbreviations vii Table of contents x List of Figures xii CHAPTER 1: Introduction 1 1.0 Introduction 1 2.0 Basic structure of the skeletal muscle fiber 3 2.1 TheZ-disc 4 2.2 The Costamere 4 2.3 The Neuromuscular Junction 6 3.0 Slow- and fast-twitch muscle fibers 7 4.0 Involvement of cytoskeletal proteins in myopathies 9 5.0 The Cvtoskeleton 11 5.1 Microfilaments 11 5.2 Intermediate filaments 12 5.3 Microtubules 14 6.0 Plakin family of cross-linking proteins 15 6.1 Plakins in skeletal muscle 16 6.2 Plectin gene and plectin muscle isoforms 17 6.2.1 Plectin and disease 18 6.3 Dystonin 19 6.3.1 Dystonin gene 20 6.3.2 Dystonin isoforms 20 6.3.3 Dystonia musculorum (dt) mice 24 6.3.4 Abnormalities in dt g striated muscle 25 6.3.5 Dystonin and disease 26 6.4MACF1 28 6.4.1 MACF1 gene 28 6.4.2 MACF1 isoforms 29 xi 6.4.3 MACF1 and disease 30 7.0 Statement of the problem 31 8.0 Specific hypotheses 32 References 33 CHAPTER 2: Manuscript 47 Abstract 48 Introduction 50 Materials and Methods 53 Results 57 Discussion 74 References 82 CHAPTER 3: General discussion 88 1.0 Plakin protein localization in skeletal muscle fibers 88 2.0 Protein expression profile of plakin proteins in slow-twitch versus fast-twitch muscle fibers 93 3.0 Cytoskeletal protein alterations in dystonin-deficient skeletal muscle 94 4.0 Conclusion 96 References 97 XII List of Figures Figure 1.1 Schematic representation of the cytoskeleton 2 Figure 1.2 Schematic representation of the skeletal muscle fiber 5 Figure 1.3 Schematic representation of protein structure of plakin proteins 17 Figure 1.4 Schematic representation of the three alternatively spliced dystonin iso forms 24 Figure 2.1 Localization of dystonin-b in skeletal muscle relative to desmin,