THE ROLE of DYSTONIN and Macfl in SKELETAL MUSCLE By

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

395 Wellington Street 395, rue Wellington OttawaONK1A0N4 Ottawa ON K1A 0N4 Canada Canada

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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, a-actinin and plectin 59 Figure 2.2 Localization of dystonin-b in skeletal muscle relative to actin, ai-tubulin and myosin 60 Figure 2.3 Localization of MACFl in skeletal muscle relative to desmin, a-actinin and plectin 62 Figure 2.4 Localization of MACFl in skeletal muscle relative to actin and a-tubulin.... 63 Figure 2.5 Localization of MACFl at the neuromuscular junction 64 Figure 2.6 Plakin protein expression in slow- and fast-twitch muscle fibers 66 Figure 2.7 Plakin protein expression levels in SOL and GAS muscle 67

Figure 2.8 Distribution pattern of plectin, MACFl, a-actinin and desmin in wt and dt skeletal muscle 70 Figure 2.9 Distribution pattern of actin and a-tubulin in wt and dt skeletal muscle 71

Figure 2.10 Effect of dystonin-deficiency on plectin and MACFl protein expression in skeletal muscle 72 Figure 2.11 Effect of dystonin-deficiency on oactinin, desmin, actin and a-tubulin protein expression in skeletal muscle 73

Figure 3.1 Schematic representation of dystonin-b and MACFl protein localization in skeletal muscle 91

Figure 3.2 Simplified schematic representation of the NMJ and its components 92

Figure 3.3 Schematic highlighting the differences between wt and dt skeletal muscle... 95 1

CHAPTER 1: Introduction

1.0 Introduction

The cytoskeleton plays a crucial role in maintaining the structural integrity of a variety of eukaryotic cell types including but not limited to neuronal, immune and muscle cells. For example the morphogenesis and the structural plasticity of neuronal cells rely on changes in the shape of the underlying cytoskeleton1. More specifically the actin microfilaments (MFs) in neuronal cells drive axon and dendrite growth, guidance and branching which are essential in creating synaptic connections1. The response of T cells to a foreign antigen in cell-mediated immunity also depends upon the cytoskeleton . For instance, the cortical actin of T cells is initially re-organized to help establish interaction with antigen presenting cells2. Finally in striated muscle the intricate design of the cytoskeleton allows this tissue to withstand substantial mechanical stress during cycles of muscle contraction and relaxation . Furthermore, the cytoskeleton of muscle cells is responsible for strategically positioning and anchoring contractile proteins that generate mechanical force and allow movement of both the cell membrane and organelles within the cell's cytoplasm . Elements of the cytoskeleton within these eukaryotic cells and others therefore play a crucial role in regulating a number of physiological processes.

The cytoskeleton is composed of MFs, intermediate filaments (IFs) and microtubules (MTs)3'4 (Fig 1.1). Crosslinking proteins are another group of cytoskeletal proteins that are thought to mediate interactions between MFs, IFs and MTs5'6.

Crosslinking proteins belonging to the plakin and spectraplakin families have been the focus of a number of studies aimed at defining their functions in a variety of cell types6.

In muscle cells, understanding the role of these crosslinking proteins is incomplete. 2

Therefore the purpose of the current study is to examine the role of dystonin and microtubule actin cross-linking factor 1 (MACF1) in skeletal muscle. In particular, the objectives were: to describe the localization of these proteins in skeletal muscle; to determine the fiber-type specific expression of dystonin and MACF1; and to assess the impact of dystonin-deficiency on the expression profile of key cytoskeletal and plakin proteins in skeletal muscle.

The following review of the literature aims to provide sufficient background details in two primary areas. Sections 2.0, 3.0 and 4.0 describe the phenotype of skeletal muscle by focusing on the structure and function of muscle cells. While sections 5.0 and

6.0 provide details of key cytoskeletal components focusing on the structure and function of plakin crosslinking proteins. 3

Figure 1.1

Schematic representation of the interaction between the 3 cytoskeletal filament systems and plakin crosslinking proteins, MACF1, dystonin (Bpagl)-e and plectin in epithelial cells. The diagram highlights the interaction that exists between MACF1 and MFs and

MTs linking these two filament systems. The interaction between plectin and the MFs and the IFs is also highlighted. Although only representing the interaction between plakin crosslinking proteins and the cytoskeleton in epithelial cells similar interactions exist in other tissues like striated muscle and neuronal tissue among many others.6

Abreviations are: a6p4: integrin; BPAG: bullous pemphigoid antigen; DP : desmoplakin;

Dsg: desmoglein; Dsc: desmocollins; Pg: plakoglobin; PKP : plakophilins; MTOC: micortubule organizing center.

2.0 Basic structure of the skeletal muscle fiber

Skeletal muscle fibers are structured to withstand significant mechanical stress during cycles of contraction and relaxation. This is in part owing to their remarkable structural architecture composed of an intricate network of anchoring and scaffolding proteins strategically positioned within specific subcellular compartments of the muscle fiber. These subcompartments include the Z-discs (or Z-lines), the costameres and the neuromuscular junction3.

The sarcomere, which is the basic contractile unit of the muscle fiber, is composed of the I-band, which composed of actin thin filaments and the A-band, which contains think myosin filaments3 (Fig 1.2). Titin and nebulin, which are large sarcomeric proteins, also contribute to the structural organization of the filament system in skeletal muscle .

Titin helps maintain the position of the A-band during force generation7, whereas nebulin 4 is thought to control the specific lengths of the thin filaments as a lack of nebulin in rodents results in a significant elongation of thin filaments .

2.1 The Z-disc

The sarcomere is delimited by Z-discs, consisting of anchoring sites for titin, nebulin and actin filaments3'9. The Z-disc is home to a variety of proteins, including for instance a-actinin which is a well characterized actin crosslinking protein ' ' (Fig 1.2).

Alpha-actinin crosslinks actin and titin filaments of neighbouring sarcomeres as well as a variety of other proteins, which include but are not restricted to cysteine-rich protein

(CRP) family members, zyxin, calsarcin-2/myozenin, telethonin and myotilin ' .

Furthermore, several signalling molecules are localized at the Z-disc. For example FATZ

(filamin-, oactinin- and telethonin-binding protein of the Z-disc) is thought to anchor the signalling protein calcineurin at the Z-disc. Calcineurin is involved in initiating the hypertrophic response in skeletal muscle12. Thus the anchoring of calcineurin at the Z- disc by FATZ may play a role in regional regulation of the hypertrophic response seen in skeletal muscle ' ' . The Z-disc is therefore regarded as an important subcellular compartment of the muscle fiber playing a crucial role in force generation, maintenance of structural stability and in cell signalling pathways.

2.2 The Costamere

The costamere is a group of proteins spanning the overlying area of each Z-disc to the sarcolemma. Costameric proteins include vinculin, talin, Y-filamin, a-actinin, pi integrins, sarcoglycans, dystroglycans and desmin among others13'14 (Fig 1.2).

Collectively, components of the costamere play an essential role in linking the contractile apparatus of muscle fiber to the extracellular matrix (ECM) and thereby contribute to 5 maintaining the structural stability of the skeletal muscle fiber. Interestingly, costameres

are highly dynamic structures 5' . For instance previous work suggests that the

expression of the costameric protein y-filamin is up-regulated in skeletal muscle of mice

and of humans that lack dystrophin, another costameric protein16. This highlights the

adaptability of the costamere in skeletal muscle. In particular, a weakness in the muscle

fibers ability to generate adequate mechanical forces due to dystrophin-deficiency, leads

to a compensatory increase in y-filamin expression at the costamere16.

7-lme Z-lme

Figure 1.2

Schematic representation of the specific localization of cytoskeletal proteins in different

compartments of a skeletal muscle fiber. At the Z-line plectin Id interacts with IF

desmin. At the costameric region plectin 1/1 f interacts with IF desmin and the dystrophin

glycoprotein complex. Although only some cytoskeletal proteins are represented to 6 simplify the schematic, the diagram highlights the highly organized structure of the muscle fiber which is dependent upon accurate protein localization. The importance of the interactions between the cytoskeleton and other proteins in maintaining structural stability of the muscle fiber is also shown.13

2.3 The Neuromuscular Junction

The neuromuscular junction (NMJ) is a chemical synapse between an axon

1 7 terminal of a motor neuron and a motor endplate . The motor endplate represents a discrete region of the muscle fiber membrane where a number of specialized proteins involved in muscle excitation, such acetylcholine receptors (AChRs) and acetylcholinesterase, are clustered. The electrical depolarization of the muscle fiber will allow for the excitation-contraction process to ensue and ultimately generate tension in the recruited motor units. The transmitter at the NMJ is acetylcholine (ACh) and the appropriate localization of AChRs on the surface of the muscle fiber is necessary to allow for adequate signal transduction 7. Cytoskeletal proteins localized at the NMJ include rapsyn, utrophin, dystroglycan, agrin, lamin and others18'13. Rapsyn is a scaffolding protein that localizes to the NMJ and binds directly to AChRs in the muscle fiber19'20.

Rapsyn's role in the postsynaptic compartment involves stabilizing and promoting AChR clustering and assuring accurate signal transduction via the AChRs . Rapsyn forms a complex with utrophin, (3-dystroglycan and AChR at the NMJ. This complex further stabilizes AChRs localization at the NMJ . Contributions of other cytoskeletal proteins, such as plakin crosslinking proteins involved in establishing and maintaining the NMJ have not been extensively studied. Due to the number of cytoskeletal proteins required in 7 this area to maintain adequate AChR clustering, it is plausible that plakin proteins contribute to the structural stability of the NMJ by crosslinking other proteins.

3.0 Slow- and fast-twitch muscle fibers

Muscle contraction results from the recruitment of numerous muscle fibers that display phenotypic variations in terms of their contractile and metabolic properties. As reviewed by Pette and Staron (1990), two types of fibers, originally referred to as "red" and "white" fibers, were first identified in 1873 by Ranvier21. The red color of certain fibers is due to the high concentration of myoglobin and a higher vascularisation, the lack thereof giving rise to the white appearance of other fibers. The classification terminology was later modified from red and white fibers to slow-twitch and fast-twitch fibers respectively. More specifically, there exists subtypes of slow-twitch or type I muscle fibers and of fast-twitch or type II muscle fibers. Subtypes of fast-twitch fibers include type Ha, type lid (or type IIx) and type lib while slow-twitch fibers have only one subtype, type IC fibers '23. Slow-twitch fibers are chronically recruited producing small amounts offeree whereas fast-twitch fibers are required to produce large amounts of force in short periods of time.

Each fiber type can be readily visualized from a thin muscle section using a number of classic histological approaches that include measuring myofibrillar actomyosin adenosine triphosphatase (mATPase) activity and by determining the activity levels of specific metabolic enzymes such as succinate dehydrogenase or by immunolabelling fibers using antibodies that specifically target the various type I and II myosin heavy chains isomers. Each skeletal muscle is in fact composed of various proportions of type I and type II fiber subtypes that collectively dictate whole muscle contractile properties. 8

In addition to metabolic differences between slow-twitch and fast-twitch muscle fibers, structural differences have also been documented for these two muscle fiber

9 1 types . For instance, slow-twitch fibers have smaller costameres, smaller intercostamenc regions and thicker Z-discs compared to fast-twitch fibers24. Some components of the cytoskeleton have also been shown to be differentially expressed in slow- and fast-twitch fibers. For example, a-tubulin, a MT subunit, is more highly expressed in the soleus muscle (primarily slow-twitch fibers) than the vastus-lateralis muscle (primarily fast- twitch fibers) as assessed by western blotting, though the localization pattern is similar for both fiber types as determined by immunofluorescence . Similarly, plectin which is a plakin crosslinking protein appears to be enriched in slow-twitch versus fast-twitch muscle fibers26. Conflicting results have been reported in regards to the expression of dystrophin in fast- and slow-twitch fibers. In a study by Bonilla et ol. (1988), it was shown through immunostaining that no differences exist in dystrophin expression between fast-twitch and slow-twitch fibers . In contrast, Ho-Kim & Rogers (1992) found higher dystrophin expression in primarily slow-twitch muscle compared to primarily fast- 9R twitch muscle at the protein but not the transcript level . Due to the differences in the contractile properties of slow- and fast-twitch fibers it is expected that cytoskeletal protein expression may differ between fiber types. Slow-twitch muscles are chronically recruited as they play a key role in maintaining posture and it has therefore been argued that they require a more solid architecture to withstand continuous activation in comparison to fast-twitch muscle which undergo periodic burst of activity during dynamic movements21. Whether this argument can be supported by reports showing more elevated expression of several cytoskeletal proteins in slow-twitch versus fast-twitch 9 muscle remains an open question as evidenced by the contradictory results reported in the literature for dystrophin.

4.0 Involvement of cytoskeletal proteins in myopathies

Mutations in genes encoding muscle specific proteins account for a number of human myopathies. These myopathies affect people worldwide regardless of gender or race. Of all myopathies muscular dystrophies (MD) are the most common. Muscular dystrophies are categorized as dystrophinopathies as there is a lack or attenuated

97 expression of the cytoskeletal protein dystrophin in the skeletal muscle . Dystrophin is a cytoplasmic protein which is essential in maintaining the structural stability of the myo fiber. When dystrophin is not expressed the function of the skeletal muscle is 97 affected . More specifically, MDs are characterized by progressive muscle wasting and weakness. Duchenne and Becker's MDs are the two most prevalent forms of muscular dystrophy characterized by a general progressive muscle wasting observed in the legs, arms and chest. Most people suffering from Duchenne MD will be wheelchair bound by 9Q early adolescence and die in their 20s . Becker MD is very similar to the Duchenne type but less severe as disease onset usually occurs in early adulthood. People living with this form of MD will experience general muscle weakness and lose the ability to walk but 9Q may survive well into middle age . The world health organization (WHO) states that 1 in

3500 males will be born with Duchenne MD. In 2007, statistics Canada reported 135 deaths caused by MD and another 9 deaths caused by congenital myopathies.

Skeletal muscle myopathies such as nemaline myopathy and certain forms MD such as Limb Girdle, Duchenne, Beckers and Epidermolysis bullosa simplex with MD

(EBS-MD) are caused by the lack of expression of proteins normally localized at the 10 sarcomere, the sarcolemma or the Z-disc3,29'6. Specific Z-disc diseases that manifest themselves in humans include myotilinopathy, zaspopathy, filaminopathy and telethoninopathy30. These diseases are caused by mutations in the genes encoding proteins localized at the Z-disc and are characterized by muscle weakness presenting at different stages in postnatal life and with varying severities30. For instance, in people suffering from myotilinopathies the sarcomeric Z-disc associated protein myotilin appears as deposits covering the muscle fiber with multiple aggregates in the cytoplasm or subsarcolemmal area31. The altered distribution of myotilin ultimately causes an irregular distribution of the Z-disc.

Interestingly certain myopathies differentially affect the protein expression pattern in fast- and slow-twitch fibers. For example, muscles from patients suffering from

Duchenne and Becker's MD, both dystrophinopathies, have a higher expression of plectin at the sarcolemma in type II fast twitch fibers; this may represent a compensatory response by the muscle fibers . Slow-twitch fibers are reportedly less severely affected in these dystrophinopathies. It is hypothesized that this is because they are less susceptible to damage associated to dystrophinopathies than fast-twitch fibers2 . As described in section 2.2, slow-twitch fibers have thinner costameres and may have a lower expression of dystrophin (though this point is debatable). Their reduced susceptibility to damage in dystrophinopathies may be because dystrophin, a costameric protein, may not be as highly expressed in this fiber type and therefore its reduced expression in MD would have less of a deleterious impact on slow-twitch fibers. This essentially indicates that a normally lower expression of this protein in slow-twitch fibers may be protective in dystrophinopathies. Altered expressions of cytoskeletal proteins in myopathies 11 underscore the importance of key cytoskeletal proteins in maintaining the form and function of muscle fibers.

5.0 The Cytoskeleton

The cytoskeleton is composed of a network of filaments including: actin MFs; IFs; and MTs5 (Fig 1.1). It maintains the cell's structural integrity, allows movement of the cell membrane and of molecules within the cytoplasm. The structural filaments that make up the cytoskeleton interact with each other and organelles strengthening the cellular architecture. Additional architectural proteins are necessary to mediate these filament interactions5.

5.1 Microfilaments

Actin is a globular monomere composed of four domains; one of which binds the myosin head; this binding is essential for skeletal muscle contraction3. When an actin monomer is combined to other actin molecules a polymer chain (F-actin) is formed. Actin filaments are helical arrays of two polymer chains ' . Actin also plays a role in cell motility and cell division (cytokinesis). This ubiquitously expressed protein exists in several similar isoforms. These isoforms are subdivided into three classes: oc, /?; and y.

Alpha-skeletal actin and ocardiac actin are both expressed in skeletal muscle . Mutations in the oskeletal actin gene (ACTA1) cause actin myopathy and nemaline myopathy in humans . Mutations in the a-cardiac actin (ACTC) causes dilated cardiomyopathy

(DCM)34 and hypertrophic cardiomyopathy (HCM)35 due to alterations in actin protein conformation. 12

Alpha-skeletal actin-deficient mice have been generated by disrupting the gene coding for this protein through homologous recombination . The a-skeletal actin- deficient mice die early in development (between day 1 and 9 postnatal), have a lower body weight than wild-type (wt) and heterozygous littermates and often develop scoliosis by day 4 postnatal which is a sign of muscle weakness . Interestingly skeletal muscles from these knockout mice express higher levels of osmooth muscle actin and a-cardiac actin and the overall total actin content is similar to that of wt and heterozygous littermates. This finding suggests a compensatory up-regulation of the other actin iso forms in response to the silencing of the a-skeletal actin gene . Accordingly, it has been proposed that the increase in production of other actin iso forms could potentially provide a therapeutic route for patients suffering from myopathies involving a-skeletal actin defects . Taken together this emphasizes the important role that MFs play in maintaining the normal structure of striated muscle.

5.2 Intermediate filaments

There are six known classes (I - VI) of IFs . The cytoskeleton of mature skeletal muscle fibers is predominantly composed of type III IFs such as desmin, vimentin, paranemin and synemin . Other IFs expressed in muscle include nestin, syncoilin, lamins, and cytokeratins40. Vimentin is the predominant type III IF expressed in myoblasts. As myoblasts differentiate, vimentin expression is down-regulated whereas the expression of desmin increases ' ' . Desmin is the key IF in differentiated myotubes, thus it plays an important role in skeletal, cardiac and smooth muscle and is also used as a marker of muscle differentiation4 '44. Desmin is localized at the Z-disc43'39'45'38'40, around the nuclei, at the costamere45, at the myotendinous junctions and at the NMJs46. This type 13

III IF plays a role in maintaining the cytoarchitecture in the muscle by creating a scaffold around the Z-disc, by linking adjacent myofibrils43, and by connecting components of the sarcomere to the subsarcolemmal area . Since desmin interacts with a variety of cytoskeletal proteins, impaired expression of desmin can have a negative impact on muscle as described in the following section.

A mutation in the desmin gene in humans leads to the accumulation of abnormal desmin in muscle fibers 7. More specifically there is an aggregation of poorly polymerized desmin in the muscle tissue impacting the organization and function of the muscle45. The myopathies resulting in the accumulation of abnormal desmin in human muscle are deemed desmin-related myopathies (DRM) . DRMs are first characterized by weakness of the lower extremities with proximal, cardiac and respiratory muscles eventually becoming myopathic. These symptoms are usually manifested in early to middle adulthood46'47.

To further understand the complex role of desmin in muscle, desmin knockout mouse models have been generated. All muscle types of the desmin null mice are myopathic. Knockout mice are viable and fertile but, are less tolerant to physical activity thereby fatiguing quickly following exercise and have a reduced lifespan compared to healthy wt littermates . The fibers of highly used muscles (i.e. soleus) of the desmin null mouse are characterized by a disorganization of myofibrils, some sarcolemmal disruption and Z-disc streaming ' 7. Interestingly, the expression levels of vimentin 7, another type

III IF, and plectin , a plakin protein, are not up-regulated in desmin null mice which suggest no compensatory role of these proteins in the absence of desmin. Furthermore, muscle fibers do not regenerate normally in these mice indicating that desmin likely plays 14 a role in the muscle regeneration process . It has also been reported that the spacing of

MFs is wider in desmin-deficient soleus muscle (predominantly slow-twitch) compared to healthy soleus muscle. In contrast, the spacing of MFs in both desmin-deficient and normal psoas muscle (predominantly fast-twitch) is nearly identical. As a whole these results suggest that desmin may play a more significant role in maintaining the structural integrity of slow-twitch muscle fibers.

5.3 Microtubules

Much less is known about MTs compared to both MFs and IFs. MTs are heterodimers of both o and /3- tubulin iso forms49. They also play a role in the transport of organelles within the cell as well as cell motility and mitosis3. In the skeletal fiber, MTs are localized between the thin and thick filaments of actin and myosin, at the sarcolemma, the Golgi apparatus and also within the perinuclear region3. The staining pattern of MTs has been reported to be significantly different between slow-twitch and fast-twitch muscle and has been shown to be altered in response to changes in neuromuscular activation patterns thereby highlighting the dynamic nature of MT organization in skeletal muscle fibers50.

Protein levels of otubulin are higher in slow-twitch muscle compared to fast- twitch muscle25. Slow-twitch fibers generate and maintain force for extended periods of time potentially explaining the need for additional structural support (i.e. higher MT content) in this fiber type25. Interestingly it is believed that MTs regulate the distribution of the Golgi complex in skeletal muscle. In predominantly slow-twitch muscles the Golgi apparatus is primarily located within the subsarcolemmal region of the fibers whereas in 15 predominantly fast-twitch muscle the Golgi apparatus is evenly distributed throughout the fibers51.

MT knockout rodent models or muscle-specific MT knockout animal models have not been reported in the literature, likely because such animals would not be viable even in the earliest stages of embryogenesis. The disruption of MTs by pharmacological interference however has been investigated. For example colchicine, which inhibits polymerization of MTs by binding to tubulin, can induce colchicine myopathy in patients using this drug for cancer therapy52. Colchicines myopathy is characterized by vacuolar accumulation, muscle weakness and myofibrillar disorganization . These data suggest that MTs are an essential part of the muscle cytoskeleton and that any defects in the MT scaffold can hinder skeletal muscle structure and function.

Taken together, it is clear that the primary filament systems described above, namely MFs, IFs and MTs, are important contributors to the structure and function of skeletal muscle fibers. Crosslinking proteins that mediate interactions between these filament systems are also increasingly recognized as playing a substantial role in regulating the architecture and physiology of skeletal muscle fibers. A more detailed description is provided in the next section.

6.0 Plakin family of cross-linking proteins

Crosslinking proteins belonging to the plakin family mediate filament interactions of the cytoskeleton. The plakin family of proteins includes desmoplakin, plectin, dystonin

(bullous pemphigoid antigen 1-Bpagl), envoplakin, periplakin, MACF1 (ACF7) and epiplakin. These proteins localize to IFs and to filament attachment sites at the plasma 16 membrane55'58'6. Plakin proteins provide architectural stability to the cell and are commonly expressed in skin and muscle; both of which are tissues that must withstand substantial mechanical stress53.

Plakin proteins are characterized by a plakin domain and/or a plakin repeat domain (PRD) ' (Fig 1.3). The plakin domain consists of six a-helical segments organized in an antiparallel manner. The PRD is composed of 4 to 8 spectrin repeats; for this reason, plakin proteins: dystonin; and MACFl (or actin crosslinking factor 7-ACF7) have been further categorized as the spectraplakins54'5'57'6. Although there is much speculation regarding the possible function of the PRD, its precise role is still poorly understood54"56.

6.1 Plakins in skeletal muscle

There are three plakin proteins predominantly expressed in skeletal muscle: plectin, dystonin and MACFl ' . Generally, plakins expressed in muscle tissue are composed of an actin binding domain (ABD) near the N-terminal region, followed by a plakin domain and in some cases an additional PRD that has been referred to as an intermediate filament binding domain ' . These muscle plakins also encompass spectrin repeat rods (SR-rods), and a C-terminal composed of a microtubule-binding domain

(MTBD). The MTBD consists of EF hands, a Gas2-related (GAR) domain and a glycine- serine-arginine rich (GSR) domain ' (Fig 1.3). The highly organized structure of the muscle plakins enables us to predict their possible role and localization in skeletal musclethrough potential interacting proteins. The role of plectin in skeletal 17 muscle has been extensively described in the literature whereas, the specific functions of

MACF1 and dystonin in skeletal muscle are not as thoroughly understood.

ABO €B PI akin domain • *»»"• SH3 domain 0 SR's 4E9 SR-ROO IX.-ROD (BHHB PRO e Linker region % EF-hands i GAR domain # GSR domain • Plakin repeat •

Figure 1.3

Schematic representation of plakin crosslinking proteins expressed in skeletal muscle.

Plectin, dystonin (Bpagl)-b and MACFla/b are expressed in skeletal muscle. All three proteins expressed in muscle have an ABD at their N-terminal region which is followed by a plakin domain, a plakin repeat domain (believed to interact with IFs) and in some instances spectrin repeat rods. Both dystonin and MACF1 have a MTBD (thought to interact with MTs) at their C-terminal, composed of EF hands, a GAR domain and a GSR rich domain. (Modified from6) 18

6.2 Plectin gene and plectin muscle iso forms

The plectin gene (PLEC1), is composed of 33 exons, spans over 26 kb and is located on chromosome 8 in humans and on chromosome 15 in mice5 . There are more than eight iso forms of plectin generated through alternative splicing of the plectin gene that are expressed in a tissue and cell type specific manner60'61. The four skeletal muscle specific plectin iso forms include: plectin 1; lb; Id; and If51. It is believed that each of these iso forms exerts its function in specific compartments of the muscle fiber. Plectin is localized at the Z-disc62, the sarcolemma61 and the costamere63. Plectin is known to link

IF desmin to the Z-disc64. It has been demonstrated that plectin 1 and If localize to the sarcolemma whereas plectin Id localizes to the Z-disc . All three plectin iso forms appear to play a crucial role in maintaining and organizing the structure of the costamere by linking the Z-disc to the sarcolemma through desmin IF interactions64,61. It has also been reported that plectin lb links the mitochondria to the IF network " . Lastly, plectin may interact with the other filament systems of the cytoskeleton (MFs and MTs)53. Due to the similarity in the protein structure of plakins, the role of dystonin and MACF1 in skeletal muscle may be similar to that of plectin.

6.2.1 Plectin and disease

In humans there exist genetic and autoimmune diseases associated with plectin defects. A homozygous 8-bp deletion in exon 32 in the PLEC1 gene leads to the genetic disease EBS-MD59'68. This disease is characterized by blistering of the skin, of various mucous membranes and the cornea beginning at birth with progressive onset of muscular dystrophy in adulthood68. Auto-antibodies targeting plectin cause the autoimmune diaseases: paraneoplastic pemphigus and certain cases of Bullous pemphigoid associated 19 with skin blistering6. Paraneoplastic pemphigus is characterized by erosions of the lips and oral cavity and by skin blistering of the torso in patients with lymphoproliferative neoplasms . It was later described that patients with tumors other than lymphoproliferative neoplasms, like thymoma, sarcoma and lung carcinomas can also develop paraneoplastic pemphigus69. In plectin null mice, generated by homologous recombination, mice die between postnatal day one and three and display abnormalities in the skin, cardiac and skeletal muscle70'6. More specifically, plectin null mice have an increased number of necrotic fibers and have disrupted sarcomeres, Z-discs and myofibrils. Muscle membrane fragility was also noted in skeletal muscle of plectin- deficient mice processed for electron microscopy70.

To further explore the role of plectin in the skeletal muscle, viable muscle-specific conditional plectin knockout mice have been generated7 . This conditional knockout model was exploited to investigate the longer-term impacts of plectin-deficiency in muscle because muscle samples could be harvested from older mice. The importance of plectin in the skeletal muscle was again highlighted as plectin-deficient muscle showed increased necrotic fibers, damage to the subsarcolemmal area leading to detachment of the sarcolemma from the contractile apparatus, misalignment of Z-discs and loss of mitochondria at the Z-disc7 . Along with the many structural defects, IF and costameric cytoskeletal protein localization and expression were also altered in the conditional plectin knockout model71. As a whole, our understanding of plectin's structure, localization, function and the impact of plectin-deficiency in skeletal muscle has improved significantly. In comparison, much less advancement has been made in our knowledge of the role of MACF1 and dystonin in skeletal muscle. 20

6.3 Dystonin

Dystonin is expressed in the heart, skeletal muscle, brain and spinal cord and is therefore thought to play a role in maintaining the cytoarchitecture of muscle cells ' , epithelial cells75'76 and neural cells74'72'77. Studies involving mice show that dystonin- deficiency leads to degradation of sensory neurons and disruption of the cytoskeletal 78 architecture of muscle cells . These dystonin-deficient mice are known as dystonia 70 musculorum (dt) mice . The following sections will further describe our current understading of dystonin. 6.3.1 Dystonin gene

The dystonin gene has been cloned by two research groups. Brown et al. (1995), who termed the gene dystonin80 and Guo et al. (1995), who named it BPAGl81. For the purpose of this study we have chosen to refer to the gene as dystonin as this is the name given to this gene in Genbank (Gene ID: 13518). The gene encoding dystonin is very large; in mice it spans nearly 400 kb and includes 107 exons . The locus of the dystonin gene has been mapped to chromosome 6pl 1-12 in humans and to chromosome 1 in

84 * 89 mice .The large size of the gene increases its potential for alternative splicing .

6.3.2 Dystonin isoforms

There exists several isoforms of dystonin generated from the same gene. They include: dystonin-a/n (the neural isoforms); dystonin-b (the muscle isoform); and dystonin-e (the epithelial isoform)6 (Fig 1.3). Furthermore, both dystonin-a/b exist as

f\ 8S three alternatively spliced isoforms termed dystonin-al/a2/a3 and dystonin-bl/b2/b3 '

(Fig 1.4). The differences between isoforms 1, 2 and 3 are found within the N-terminal or region of the dystonin-a and dystonin-b molecules (Fig 1.4). 21

Leung et al. (2001), demonstrated that dystonin-b is expressed abundantly in cardiac and skeletal muscle72. In dystonin-deficient mice the lack of dystonin-b is thought to be the underlying cause of skeletal muscle abnormalities78'53. The dystonin-b isoform is composed of an ABD, a plakin domain, PRD and a MTBD6 (Fig 1.4), and is the largest of all known dystonin isoforms with an approximate molecular weight of 834 kDa7 .

Originally it was predicted that the PRD of dystonin-b could potentially interact with the

IF network . More recent work however suggests that the PRD of dystonin-b is unable to biochemically bind to the IFs desmin, vimentin, synemin-H or to desmin/synemin heterodimers. In addition, co-transfection studies using the dystonin-b PRD and full length desmin, vimentin and synemin-H demonstrated that the IF network and the PRD recombinant protein do not co-localize in KEB-3D and HEK 293T cells . Further experimentation is needed to determine the PRD binding partners.

Dystonin-a is the main neural isoform expressed in both the brain and the spinal cord72. Its predicted molecular weight is approximately 615 kDa72. Dystonin-n is a

77 smaller neuronal isoform expressed at very low levels . Dystonin-a is very similar in structure to that of dystonin-b but is lacking the PRD, while dystonin-n is nearly identical to dystonin-e (see below) except that it also encompasses an ABD at its N-terminal region ' ' ' (Fig 1.3). Dystonin-a is expressed primarily in the pituitary and in the dorsal root ganglia both within the central nervous system . Dystonin-a is thought to play a role in maintaining proper organization of IFs, specifically neurofilaments, and actin 1A. 87 8^ 8.8

MFs in the nervous system ' ' , along with stabilizing the structure of Schwann cells .

Therefore, it is suspected that the lack of dystonin-a is responsible for the degeneration of 22 neurons observed in dt mice . Deficiencies in dystonin-a are also thought to lead to defects in retrograde axonal transport89'90.

Dystonin-e is the epithelial iso form; it is one of the targeted antigens in the auto immune skin blistering disease Bullous pemphigoid6. In this disease, antibodies target dystonin-e and prevent it from anchoring intermediate filaments (keratin) at hemidesmosomes resulting in blisters6. Dystonin-e, the smallest of all dystonin iso forms

77 with a molecular weight of 302kDa , is composed of a plakin domain at its N-terminus followed by a coiled-coil rod domain and two sets of PRDs at its C-terminus ' (Fig 1.3).

This particular PRD region, also known as the IFBD1 of the epithelial iso form is of particular interest as it interacts with keratin IFs linking them to the hemidesmosomes ' .

To further understand the need for three N-terminal variants of both the dystonin- a/b iso forms (al, a2, a3 and bl, b2, b3 see Fig 1.4), studies to examine the localization of these iso forms in varying cell lines have been performed. Although only having slight differences at the N-terminal region the dystonin isoforms 1, 2 and 3 are targeted to different areas of the cell, hence they are anticipated to have varying roles in different compartments of the muscle cell. The N-terminus of iso form 1 is composed of an ABD

70 formed of two calponin homology domains in tandem (CHI, CH2) (Fig 1.4). It was initially described by Young et al. (2003), that N-terminal iso form 1 and actin MFs co- Q9 localize in C2C12 cells transfected with an iso form 1 expression construct . These results were later confirmed in COS-7 cells transfected with an iso form 1 expression construct. In COS-7 cells the resulting recombinant dystonin iso form 1 protein localized at the cell membrane and also co-localized with MFs throughout the cytoplasm93.

Fluorescently tagged full length dystonin-al fusion protein expressed in COS-1 cells was 23 found to localize throughout the cytoplasm and also at the cell perimeter thus creating a staining pattern similar to MTs . These studies highlight the important role that the dystonin N-terminal isoform 1 likely plays in organizing both the actin MF and MT cytoskeletal networks in the cell.

The dystonin N-terminal isoform 2 is characterized by a transmembrane domain followed by an ABD composed of two CH domains (CH1,CH2)85 and is more predominantly expressed in neural tissue94 (Fig 1.4). In COS-1 cells, recombinant protein composed of the transmembrane domain of isoform 2 co-localizes with the membranous structures surrounding the nucleus7 . Staining near the nucleus and surrounding the Golgi apparatus was observed within C2C12 myoblast cells treated with a dystonin isoform 2 specific antibody indicating that endogenous dystonin isoform 2 localizes to these areas of the cell. Similar staining patterns were also obtained in COS-1 cells transfected with a construct expressing the full length dystonin-a2 fusion protein79. The N-terminal isoform

2, has been proposed to bundle actin MFs in the vicinity of the nucleus in COS-7 cells transfected with an expression construct containing a truncated version of isoform 293.

When the MT network of C2C12 cells is disrupted with nocodazole, an anti-neoplastic agent, the staining pattern of dystonin isoform 2 is unaltered indicating that the localization of isoform 2 to actin MFs is not dependent on MTs92.

The N-terminus of dystonin isoform 3 is composed of a myristoylation motif followed by one CH domain (CH2), which in effect represents an incomplete ABD85 (Fig

1.4). The myristoylation motif targets isoform 3 mainly to the plasma membrane as it was described in COS-7 cells transfected with a truncated construct of isoform 393. Since isoform 3 is lacking the CHI domain and therefore has an incomplete ABD, it is likely unable to interact with actin MFs.

To summarize, both N-terminal isoforms 1 and 2 seem to co-localize with actin

MFs. Isoform 1 also co-localizes with MTs while isoform 2 localizes to the perinuclear region. Finally, N-terminal isoform 3 lacks a complete ABD and is cortically expressed specifically at the plasma membrane. Taken together the results demonstrate that the N- terminal region will dictate where dystonin-a/b iso forms are targeted in a cell.

ABD , , men plskin isoform 1 aJXl>--HHHHBBiHHHHHaHHHy Brown et ai, 1995 rM isoform 2 §HHCir>-HMnHBHnHnnHHHMy Young et ai., 2006 myr isoform 3 !•••_>••—^••••^•^••M*/ Jefferson etal., 2006

Figure 1.4

A schematic representation of the three alternatively spliced N-terminal iso forms of dystonin-a/b. Isoform 1 is composed of an ABD and is therefore able to interact with actin MFs and other actin crosslinking proteins. Isoform 2 is composed of a transmembrane domain; which is believed to be responsible for its perinuclear localization followed by and ABD which also makes it capable of interacting with actin

MFs. Isoform 3 is composed of a myristoylation motif; this targets this isoform to the plasma membrane, and is also composed of an incomplete ABD that likely impairs interactions with actin MFs. (Modified from85)

6.3.3 Dystonia musculorum (dt) mice

Duchen et al. (1962), initially described dystonia musculorum (dt), as a mouse neurodegenerative disease95. The dt mouse was initially described as being a model of an 25 autosomal recessive hereditary neuropathy95'96. There are several allelic variants of the dt mouse disease, most of which occur as spontaneous mutations owing to the large size of the dt locus .

The dtTg4 dystonin-deficient transgenic mouse line was first established in 1988.

Researchers were trying to create a transgenic mouse expressing an hsp68 promoter-

Escherichia coli lacZ hybrid gene to study heat shock regulation . In the process, a large integration complex of 70 kb representing 15 to 20 tandem copies of the transgene was inadvertently inserted into the dt locus which resulted in the simultaneous deletion of 45 on na kb of what would be later identified as the dystonin gene ' . When the hemizygote mice were mated 25 % of the offspring, by the age of 10 to 12 days postnatal, displayed the severe limb incoordination phenotype of the dt mouse originally described by Duchen in 07 the 1960s. This line of mice was at that time labelled Tg4 . Further analyses of the insertional deletion revealed the exon encoding the N-terminal isoform 1 and the last exon encoding N-terminal isoform 2 were lost80. This deletion results in the lack of expression of dystonin-a/b in the dtTg4 mouse . Mice belonging to the dtTg4 as well as other allelic variants exhibit signs of limb in coordination that progress as the mice age.

They are also smaller than their healthy wt littermates and die shortly after weaning of unknown causes ' 7.

6.3.4 Abnormalities in dt s4 striated muscle

Disruption of the dystonin gene in dtTg4 mice is associated with an intrinsic muscle defect. Abnormalities have been observed in both the skeletal muscle and in cultured • 7X myotubes from dystonin-deficient animals . In particular, electron microscopy data revealed regionalized disruption of the skeletal muscle cytoarchitecture characterized by 26 thickening of Z-discs, partial disassembly of myofibrils, shortening of sarcomere length and accumulation of mitochondria at the periphery of the muscle fiber . Abnormalities were also seen in cultured myotubes harvested from dystonin-deficient mice of the dtTg line. These myotubes have fewer myofibrils, incompletely formed Z-discs and an

no abnormal distribution of mitochondria . Interestingly, the earlier stages of myoblast

•JO proliferation and differentiation do not appear to be affected in the absence of dystonin

. Collectively, these data suggest that although dystonin does not seem necessary in establishing normal cytoarchitecture of the skeletal muscle fiber it is important in maintaining it.

Since dystonin is also expressed in cardiac muscle it may be assumed that abnormalities exist in the cytoskeletal network and at the ultrastructural level of the hearts of dystonin-deficient mice. In dtTg4 animals increased transcript levels were observed for atrial natriuretic factor, /3-myosin heavy chain and decreased levels of sarcoplasmic reticulum calcium pump isoform 2A when compared to wild-type animals; indicating the presence of cardiac muscle stress in the dystonin-deficient mice . Both morphological and histological data from dtTg cardiac tissue were normal therefore, that the absence of dystonin in this muscle type does not significantly impact cytoskeletal organization, at least within the first two weeks of post-natal development73. More specifically no alterations were observed in MF, IF and MT localization and protein expression. In contrast to what had been previously reported in skeletal muscle no differences were seen in the organization and structural integrity of Z-discs in cardiac muscle . It is worth noting that the mRNA transcript levels of desmoplakin, another plakin family member highly expressed in cardiac muscle and enriched at the intercalated discs, is increased in 27 dtTg4 cardiac tissue73. Desmoplakin may therefore be compensating to some extent in dystonin-deficient heart tissue.

6.3.5 Dystonin and disease

In humans the underlying cause of some forms of Bullous pemphigoid, a skin blistering disease, are attributable to auto-antibodies targeting the epithelial isoform of dystonin. In healthy tissue dystonin-e is associated with the hemidesmosome", and can also interact with keratin IFs, BPAG2 and /34-integrin both components of the hemidesmosomal plaque . Bullous pemphigoid is characterized by the separation of the basal cell lamina and the epithelial tissue sometimes caused by the disruption of the interaction between dystonin-e and elements of the hemidesmosome thus resulting in skin blistering6.

Reports of genetic diseases linked to mutations within the dystonin gene are rare.

Recently it has been demonstrated that a homozygous nonsense mutation (p.Ginl 124X) in the dystonin gene results in trauma-induced skin blistering in an adult male . This specific mutation is located within the region encoding the coiled-coil domain of dystonin-e1 °. This patient also displayed neurological symptoms such as headaches, periodoc collapsing and episodes of left arm weakness which may be attributed to the cellular effects caused by mutation of the dystonin gene100. In another case study, the patient had a 6; 15 chromosome translocation that affected both muscle and neural dystonin isoform expression10 . This patient had numerous symptoms such as oesophageal atresia, non-progressive encephalopathy, severe motor and mental retardation and delayed visual development . Early onset mild muscle hypotonia and increased tendon reflex were present and by age 4 gross motor in-coordination and 28 disorganized gait pattern were also observed101. Unlike the first case study this patient did not present with the skin blistering phenotype as the mutation did not occur in the area coding for the coiled-coil region specific to the epithelial isoform of dystonin. Given the paucity of information available on genetic diseases associated with dystonin mutations in humans, most of our understanding of the function of dystonin has been generated by studying the dystonin-deficient mouse model.

6.4 MACF1

The domain organization of MACF1 is similar to that of dystonin (Fig 1.3).

Although MACF1 is ubiquitously expressed it is most abundant in the nervous system, striated muscle, lungs, adrenal glands , and in the skin . MACF1 mRNA levels are detected as early as day 7.5 in embryogenesis suggesting an important role early in mouse embryonic development104.

6.4.1 MACF1 Gene

MACF1 was first discovered in 1995 from a polymerase chain reaction (PCR)- mediated screening exercise aimed at identifying novel proteins containing an ABD.

Specifically, degenerate primer-mediated PCR was used to identify partial clones. In the process, a cDNA encoding a novel actin crosslinking protein, characterized by the presence of an ABD, was found and initially termed ACF7105.

The ACF7 gene localizes to chromosome 1 in humans 5 and to chromosome 4 in the mice103. When the mouse ACF7 gene was further cloned and characterized, high homology with the ABD of dystonin was observed103. Later, the full length of the ACF7 murine gene was cloned and found to be 17.3 kb. At this time ACF7 was referred to as the MACF1 gene because expression of specific domains found either at the N-terminal 29

(ABD) and the C-terminal (MTBD) regions of the cDNA in COS-7 cells resulted in the protein products being associated with either the actin MFs or the MT networks respectively102.

6.4.2 MACFl isoforms

Like dystonin and plectin, the MACFl gene is thought to generate a variety of isoforms through alternative splicing of the gene ' 3. Two main isoforms have been identified and are referred to as MACFla, b 106. The calculated molecular mass of 608 kDa reported for MACFl by Leung et al. (1999), is likely that of MACFla m. According to the predicted domain organization of MACFl b, its estimated molecular mass is likely to be greater than that of MACFl a and similar to that of dystonin-b (>800 kDa). Like other plakin crosslinking proteins MACFl is composed of an ABD at its N-terminus followed by a plakin domain, SR-rods and a MTBD at its C-terminus 6 (Fig 1.3). Due to the domain organization of MACFl, it is possible to hypothesize its localization and function in cells based on plausible interacting proteins (MFs and MTs). MACFlb is the larger of both isoforms as it has PRDs after its plakin domain. It has been shown to interact with the Golgi apparatus through the region found between the plakin domain and the PRDs106'6.

Both MACFla and b are thought to exist as three alternatively spliced N-teminal isoforms nearly identical to those of dystonin-a/b which have been previously

1 0"^ 1 07 107 1 OR described ' ' ' (Fig 1.4). These N-teminal alternatively spliced isoforms (i.e. al, a2, a3 / bl, b2, b3) have distinct structures; isoform 1 and 2 both contain a complete ABD at their 5' end while isoform 3 encompasses half of the ABD103. Isoform 1 transcripts are more highly expressed in skin, kidney, and stomach while isoform 2 and 3 expression is 30 highest in the brain and spinal cord104. Isoform 1 and 2 are both expressed in skeletal muscle but to a lesser extent than the tissues stated above104.

Of the three skeletal muscle plakins (plectin, dystonin and MACFl), the molecular and cell biology of MACFl is the least well characterized. The ABD of

MACFl has been shown to co-localize with actin MFs, while the C-terminus of MACFl containing a putative MTBD has been found to associate to and stabilize MTs in COS-7 cells transfected with MACFl-ABD and MACFl-MTBD expression constructs102. It was also demonstrated through biochemical assays that the ABD of MACFl can interact with rapsyn, a scaffolding protein expressed in the postsynaptic compartment of the NMJ and thought to bind and stabilize AChRs109. MACFl may therefore play a structural role in maintaining AChR clusters at the sarco lemma of the muscle fiber, through its direct interaction with rapsyn.

6.4.3 MACFl and disease

Although mutations in the MACFl gene have not yet been reported to play a role in any diseases or myopathies in humans, it is apparent that MACFl plays a critical function in the development and maintenance of proper cytoskeletal architecture. For example, in mouse embryonic cell lines derived from MACFl-deficient animals generated through vector mediated targeted disruption of the MACFl gene, the actin MF cytoskeleton is normal but the MT network displays irregular trajectories and its dynamic nature appears to be altered110. Furthermore, studies of MACFl-deficient mice highlight its importance early on in development as these mice die during embryonic growth, specifically at embryonic day 11.5 of the gastrulation stage ' . Unlike dystonin,

MACFl clearly plays a role in the early development of tissues and without its expression 31 mice die early in development. To further explore the role of MACFl-deficiency, the generation of tissue specific knockout (conditional knockout model) models would be necessary thus allowing us to better understand the role of MACFl in different tissues most notably skeletal muscle.

7.0 Statement of the problem

Dystonia and MACFl are both plakin crosslinking proteins that are expressed in skeletal muscle. These proteins are thought to play a critical role in maintaining the organized structure of the cytoskeleton as highlighted in studies conducted using tissues from dystonin and MACFl knockout mouse models. The specific functions of dystonin and MACFl in skeletal muscle however are still poorly understood. For instance, our knowledge of the subcellular placement of dystonin and MACFl in skeletal muscle fibers is lacking. Determining the localization pattern of dystonin and MACFl will provide important information about their respective putative functions within specific compartments of the muscle fiber. Furthermore, the impact of dystonin-deficiency on the cytoarchitecture of the major filament systems namely MFs, desmin IFs and MTs in the skeletal muscle fiber has not been thoroughly investigated. Availability of the dystonin- deficient model dtTg4 will allow skeletal muscle from these animals to be analyzed to determine whether MF, desmin IF and MT networks are affected. As a whole, the findings of the present research are anticipated to further our understanding of the role of dystonin and MACFl in skeletal muscle. 32

8.0 Specifc hypotheses

The four hypotheses tested are:

1) That dystonin will be localized near the Z-disc and will co-localize with plectin

and ce-actinin.

2) That MACFl will be localized near the Z-disc and at the NMJ.

3) That dystonin and MACFl will be more highly expressed in slow-twitch versus

fast-twitch fibers.

4) That dystonin-deficiency will destabilize the MF, IF and MT networks possibly

leading to a down-regulation in o;-tubulin, desmin and actin.

5) That dystonin-deficiency will affect the expression profile of Z-disc protein a-

actinin.

6) That dystonin-deficiency will lead to a compensatory increase in the expression of

plectin and MACFl. 33

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CHAPTER 2% Manuscript

Co-Authorship and Author Contributions

Chapter 2 has been presented as a manuscript ready for submission to a peer-reviewed journal {Muscle and Nerve). The following is the proposed paper with contributing authors:

THE LOCALIZATION AND EXPRESSION OF DYSTONIN AND MACF1 IN SKELETAL MUSCLE AND THE EFFECTS OF DYSTONIN-DEFICIENCY ON CYTOSKELETAL PROTEIN EXPRESSION AND ORGANIZATION

Joanisse, S., Kirwan, T., Boudreau-Lariviere, C.

The research work completed in Chapter 2 was supported by the Laurentian University Research Fund (LURF), the Natural Science and Engineering Research Council of Canada (NSERC) and the Northern Ontario Heritage Fund Corporation (NOHFC). All phases of data collection and analysis were led by S. Joanisse in consultation with Dr. Boudreau-Lariviere. T. Kirwan was involved in optimizing the methodology used in Chapter 2, specifically the high molecular western blots and the collection of single muscle fibers. 48

Abstract

Introduction: The specific functions of plakin proteins MACFl and dystonin in skeletal muscle are not thoroughly understood. To gain insights into the role of MACFl and dystonin their subcellular localization in skeletal muscle fibers, and the impact of dystonin-deficiency on select cytoskeletal elements in skeletal muscle fibers was determined.

Methods: Double immunofluorescence experiments were performed to determine the distribution of MACFl and dystonin relative to microtubules as well as sarcomeric and Z- disc proteins in longitudinal sections of mouse soleus muscle and in singles fibers isolated from gastrocnemius muscle. The expression profile of these plakin proteins in slow- versus fast-twitch muscle were compared. In addition, the impact of dystonin-deficiency on sarcomeric actin, microtubules, MACF and Z-disc proteins a-actinin, desniin and plectin was determined by western blot and by immunofluorescence.

Results: Dystonin localizes to the Z-disc, the subsarcolernmal and perinuclear regions while MACFl localizes to the Z-disc and to the post-synaptic region of the neuromuscular junction. Dystonin and MACFl expression was approximately 2-fold higher in fast-twitch, compared to slow-twitch muscle. In dystonin-deficient muscle, the

MF and MT networks are largely unaffected whereas the a-actinin, MACFl and plectin expression levels were up-regulated and expression levels of desmin were reduced

(p<0.05).

Discussion. Dystonin and MACFl exert their respective functions in specific subcellular compartments of the muscle fiber as predicted by their structural make-up. Dystonin- 49 deficiency is associated with alterations in the expression profile of Z-disc proteins a- actinin, desmin, MACFl and plectin. 50

Introduction

The cytoskeleton is composed of microfilaments (MFs), intermediate filaments

(IFs) and microtubules (MTs)1,2. Crosslinking proteins are another set of cytoskeletal proteins that are thought to mediate the interactions between MFs, IFs and MTs3'4. Plakin proteins provide architectural stability to the cell and are commonly expressed in skin and muscle which are tissues that must withstand substantial mechanical stress . There are three plakin proteins predominantly expressed in skeletal muscle: plectin, MACF1 and dystonin4'6. The highly organized structure of these proteins enables us to predict their possible role and localization in skeletal muscle through potential interacting proteins.

The role of plectin in muscle has been more extensively described in the literature7"12 whereas, the specific functions of MACF1 and dystonin in skeletal muscle are not as thoroughly understood.

The gene encoding dystonin is very large; in mice it spans nearly 400 kb and includes 107 exons . The large size of the gene increases the potential for production of numerous iso forms generated by alternative splicing . These isoforms include the neural iso forms (dystonin-a/n), the muscle iso form (dystonin-b) and the epithelial iso form

(dystonin-e)4. Furthermore, both dystonin-a/b exist as three alternatively spliced isoforms termed dystonin-al/a2/a3 and dystonin-bl/b2/b34'14 (Fig. 4). The differences between isoforms 1, 2 and 3 are found within the N-terminal region of the dystonin-a and dystonin-b molecules14. The dystonin-b iso form is composed of an actin binding domain

(ABD), a plakin domain, plakin repeat domain (PRD) and a microtubule binding domain

(MTBD) consisting of EF hands, a Gas2-related (GAR) domain and a glycine-serine- 51 arginine rich (GSR) domain5,4. Dystonin-b is the largest of all known dystonin isoforms with an approximate molecular weight of 834 kDa15.

Duchen et al. (1962), initially described dystonia musculorum (dt), as a mouse neurodegenerative disease16. The dtTg4 dystonin-deficient transgenic mouse line was first established in 1988 and is characterized by a 45kb deletion within the 5' end of the dystonin gene ' . This deletion virtually abolishes the expression of dystonin-a/b in the dfs mouse1 . Disruption of the dystonin gene in dtTg4 mice is associated with an intrinsic muscle defect characterized by abnormalities in both the skeletal muscle and in cultured myotubes . In particular, electron microscopy data revealed regionalized disruption of the skeletal muscle cytoarchitecture characterized by thickening of Z-discs, partial disassembly of myofibrils, shortening of sarcomere length and accumulation of mitochondria at the periphery of the muscle fiber . Interestingly, the earlier stages of myoblast proliferation and differentiation do not appear to be affected in the absence of dystonin1 'l . Collectively, these data suggest that although dystonin does not seem necessary in establishing normal cytoarchitecture of the skeletal muscle fiber it is required to maintain the fiber's structural integrity.

The domain organization of MACFl is similar to that of dystonin. Although

MACFl is ubiquitously expressed it is most abundant in the nervous system, striated muscle, lungs, adrenal glands 20, and in the skin21. MACFl mRNA levels are detected as

99 early as day 7.5 in embryogenesis suggesting an important role in early development .

MACFl is composed of an ABD at its N-terminus followed by a plakin domain, SR-rods and a MTBD at its C-tenninus4. Based on the structure of MACFl it can potentially interact with actin MFs or other actin-binding proteins through its ABD at the N-terminus 52 and MTs through its MTBD at the C-terminus. It is at this moment unknown which type of proteins the plakin domain can establish interactions with. Two main isoforms have been identified and are referred to as MACFl a and b23. Leung et al. (1999), reported a molecular mass of 608 kDa for MACFl, based on protein structure it is likely that of

MACFl a20. MACFlb is the larger of both isoforms as it has PRDs after its plakin domain. Its estimated molecular mass is likely to be greater than that of MACFl a and similar to that of dystonin-b (>800 kDa). Both MACFl a and b are thought to exist as three alternatively spliced N-teminal isoforms nearly identical to those of dystonin-a/b

(i.e. MACFl al, a2, a3 /bl, b2, b3) 21>20>24'25.

It is apparent that MACFl plays a critical function in the development and maintenance of proper cytoskeletal architecture. For example, in mouse embryonic cell lines derived from MACFl-deficient animals generated through vector mediated targeted disruption of the MACFl gene, the actin MF cytoskeleton is normal but the MT network

ralar trajectories and its -51-——•-- -~<—~ ~»~—~ *~ •*— „i*~-~»26 studies of MACFl-deficient mice highlight its importance early on in development as these mice die during embryonic growth, specifically at embryonic day 11.5 of the gastrulation stage 7' . Unlike dystonin, MACFl clearly plays a role in the early development of tissues and without its expression mice die early in development. MACFl may also be implicated in stabilizing elements within the neuromuscular junction. In particular, biochemical interactions between the ABD of MACFl and rapsyn, a scaffolding protein expressed in the postsynaptic compartment of the NMJ and thought to bind and stabilize AChRs have been reported . Of the three skeletal muscle plakins

(p lectin, dystonin and MACFl), the molecular and cell biology of MACFl is the least 53 well characterized in muscle cells. The current study aims to improve our understating of the role of dystonin and MACF1.

Material and Methods

Mice and Tissue Excision.

The transgenic mouse model of dystonia musculorum (dt) used in the present study is the dtTgA line originally obtained from Dr. R. Kothary (University of

Ottawa/Ottawa Health Research Institute). This transgenic mouse model is characterized by an insertional mutation within the dystonin gene . Mice were housed at Laurentian

University's Animal Care Facility until anaesthetized on postnatal day 14. Mice were anaesthetized with ketamine/rompun (O.lmg/g, 0.01 mg/g) through intraperitoneal injection and sacrificed immediately following tissue excision by cervical dislocation.

Gastrocnemius (GAS) and soleus (SOL) muscles were excised from dt and wild-type (wt) mice (N=8 GAS and N=8 SOL Western blot; N=8 dt and N=8 wt Western blot; N=5 dt and N=5 wt for immunofluorescence). If muscles were to be used for protein isolation they were immediately flash frozen in liquid nitrogen. Muscles to be used for immunoflorescence experiments were embedded in Histo Prep (Fisher Scientific

International Inc., Hampton, NH) and frozen in pre-cooled isopentane. Mice were cared for according to the Canadian Council on Animal Care (CCAC) guidelines. All procedures were approved by the Laurentian University Animal Care Committee.

Antibodies.

Primary polyclonal rabbit antibodies were generated (Sigma Genosys), to target either the PRD2 (intermediate filament binding domain 2, IFBD2) (anti-dystonin- 54 b(PRD2)) in the muscle isoforms of dystonin (dystonin-b) or to target the plakin domain common to all known dystonin iso forms (anti-dystonin(plakin) Sigma Genosys). The

PRD2 antibody was designed to target peptide sequence EREDEENIQKGPSV whereas the plakin domain antibody was designed to target peptide sequence

DNRLRDLEGIGKSL. Preimmune sera were also obtained in order to test the specificity of the dystonin antibodies. Other commercially available antibodies used were: rabbit polyclonal anti-MACFl (Santa Cruz Biotechnology, Inc., Santa Cruz, CA. USA); mouse monoclonal anti-plectin (Santa Cruz Biotechnology, Inc.); mouse monoclonal anti-desmin

(Covance, Princeton, NJ. USA); mouse monoclonal anti-a-actinin-2 (Abeam, Cambridge,

MA. USA); and mouse monoclonal anti-MHCI (Sigma-Aldrich, St. Louis MO. USA).

The secondary antibodies used were: Alexa Fluor 488 conjugated goat anti-rabbit IgG

Alexa Fluor 596 conjugated goat anti-rnouse IgG and horseradish peroxidase (HRP)- coupled anti-rabbit and anti-mouse IgG (Invitrogen, Carlsbad, CA. USA).

Immuno fluorescence.

For immunofluorescence microscopy longitudinal and cross sections of GAS and

SOL muscle from wt and dt muscle were collected at a thickness of Sum on the same slide using a Leica CM 3050 S cryostat set at -22°C. In addition, single muscle fibers were collected from GAS muscles by incubating the muscle for 45 minutes in a solution of DMEM supplemented with 0.2% collagenase (Sigma-Aldrich). Muscles were then placed in 4% paraformaldehyde (PFA) in IX phosphate buffered saline (PBS) for 20 minutes; then single fibers were teased apart. All muscles (longitudinal and cross sections and single fibers) were then stored at -80°C. Tissue sections and single fibers were fixed in 4% PFA in IX PBS for 10 minutes then permeabilized by incubating sections in 55 glycine buffer prepared using IX PBS and blocked with 5% normal goat serum in PBS for 20 minutes. To determine the localization of dystonin-b longitudinal sections of wt muscle tissue were incubated overnight with primary antibodies targeting dystonin-b

(1:50, PRD2 antibody, see above) and either plectin (1:50, Santa Cruz Biotechnology,

Inc.), desmin (1:50, Covance) or af-actinin-2 (1:50, Abeam). To determine the differential expression of plakin proteins in slow- and fast-twitch muscle fibers serial cross sections of wt muscle tissue were incubated overnight with either MHCI (1:600, Sigma-Aldrich), dystonin-b (1:50, PRD2 rabbit), MACF1 (1:25, Santa Cruz Biotechnology, Inc.) or plectin (1:50, Santa Cruz Biotechnology, Inc.). To determine if cytoskeletal protein expression and localization was altered in dt mice, longitudinal sections of wt and dt muscle were incubated overnight with either MACF1 (1:25, Santa Cruz Biotechnology,

Inc.), plectin (1:50, Santa Craz Biotechnology, Inc.), desmin (1:50, Covance) or a- actinin-2 (1:50, Abeam). The overnight incubation was followed by 3 rinses in IX PBS followed by a 1 hour incubation with secondary antibodies either Alexa 488 conjugated goat anti-rabbit IgG or Alexa Fluor 596 conjugated goat anti-mouse IgG (Invitrogen).

Sections were then rinsed 5 times in IX PBS, dried then mounted in VectaShield

(Cedarlane Laboratories USA Inc., Burlington, NC. USA). Nuclei were visualized using a

Hoechst stain DNA binding compound (Sigma-Aldrich). Visualization and documentation were performed using a Zeiss Axiovert 200 M fluorescent inverted deconvolution microscope and photographed using an AxioCam HR camera and the Axio

Vision 4.2 computer software program. Muscle tissue sections from wt and dt animals were processed in parallel on the same slides. Images were obtained from the same slide and exposure times were identical between genotypes. 56

Protein Isolation and Western Blot Analysis.

Protein extracts from wt and dt SOL and GAS muscles were prepared by homogenizing the tissues in RIPA lysis buffer (150 mM NaCl, 10 mM Tris pH 8.0, 1%

Triton-X 100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (lug/ml Leupeptin, l|ag/ml Aprotinin, 5 fJ-g/ml PMSF, 10 mM NaF, ImM

NaOAv). Protein concentration was determined using the Bradford protein assay (Bio-

Rad Laboratories, Hercules, CA. USA). Protein samples were resolved on a 4% SDS-

PAGE gel when probing for high molecular weight proteins (dystonia, MACF1 and plectin) and a 7% SBS-PAGE gel when probing for all other proteins. Proteins were then transferred from the gel to a polyvinylidene fluoride (PVDF) membrane. Membranes were then blocked for 1 hour in 5% non-fat milk in Tris buffered saline with 0.1%

Tween-20 (TBS-T). Membranes were incubated overnight with primary antibodies

(against dystonin(plakin) 1:1000, MACF1 1:100, plectin; 1:200 and desniin, a-actinin=23 actin, ^tubulin 1:1000, MHO 1:4000) followed by three 15 minute washes in TBS-T, followed by secondary detection using the HRP conjugated secondary antibodies followed by enhanced chemiluminescence (ECL) detection (GE healthcare, Life

Sciences, Piscataway, NJ. USA).

Densitometry analysis was performed using the FluroChem software (Alpha

Innotech Corporation). The membranes were then stained with Coomassie blue stain

(50% MeOH, 10% Acetic Acid, .05% Brilliant Blue (Sigma-Aldrich)) then scanned using the GS-800 Imaging Densitometer (Bio-Rad). Protein expression was then normalized to total protein per lane as assessed with the Coomassie staining. Because the expression levels of tubulin, actin and GAPDH were thought to potentially be affected in dt muscles, 57 these routinely used internal controls were not selected to normalize protein expression data.

Statistical Analysis.

All normalized protein expression data were subjected to the Shapiro-Wilk test to determine whether the data was normally distributed. All data were found to be normally distributed and an independent t-test was used to establish differences between dt and wt mice in regards to the difference in protein expression. The same test was used to determine differences between SOL and GAS muscle plakin protein expression.

Statistical analysis was performed using SPSS 12.0 and the significance level was set to p

<0.05.

Results

Dystonin localizes to the Z-disc the subsarcolemmal area and at the perinuclear region in skeletal muscle fibers

To analyze the localization of dystonin-b in skeletal muscle fibers we performed immunofluorescence experiments on longitudinal sections collected from the SOL skeletal muscle of 14-day old mice and on single muscle fibers isolated from GAS muscle. The anti-dystonin-b(PRD2) serum revealed a staining pattern characterized by cross-striations seen throughout the muscle fiber, as well as subsarcolemmal and perinuclear staining (Fig 2.1). Faint longitudinally oriented filamentous structures were also observed (Fig 2.1). Immunofluorescence signals were not observed from muscle sections processed in parallel using the pre-immune serum (data not shown). Double immunofluorescence studies using the dystonin-b(PRB2) antibody and antibodies 58 targeting known Z-disc proteins such as plectin, a-actinin and desmin, confirmed that dystonin-b is localized to the Z-disc. This expression pattern was characterized by partial co-localization with both plectin and a-actinin and to a lesser extent with desmin (Fig

2.1). Dystonin-b did not co-localize with either sarcomeric proteins actin or myosin or with the MT network (Fig 2.2). Additionally, we demonstrate a partial co-localization of dystonin with desmin within the subsarcolemmal area and at the pemiculear region (Fig

2.1). Dystonin-b Desmin Dvstonin-b/ Desmin

Dystonin-b a-Actinin Dvstonin-b / a-Actinin

Dystonin-b Plectin Dystonin-b' Plectin

Figure 2.1

Localization of dystonin-b (A, D, G), relative to desmin (B), a-actinin (E) and plectin (H) in longitudinal sections of SOL muscle from 14 day old mice. The blue staining in the merged images (C, F31) represent the nuclei. Dystonin-b staining is characterized by a cross-striation pattern, with sub-sarcolemmal and perinuclear staining (A, D, G). Fine 60 longitudinally oriented filamentous structures corresponding to dystonin-b are also observed (see long arrows). Dystonin-b co-localizes with Z-disc proteins desmin, a- actinin and plectin (see arrow heads) (C, F, I), and also co-localized with desmin at the sub-sarcolemmal area and at the perinuclear region (see small arrow) (C). Bar: 20jUm.

Dvstonin-b Actin Dvstonin-b Actin

Dvstonin-b Myosin Dystonin-b / Myosin 61

Figure 2.2

Localization of dystonin-b (A, D,G) relative to, actin (B), o-tubulin (E) and myosin (H) in longitudinal sections from SOL muscle (A,B,C) and single fibers from GAS muscle

(D-I) of 14 day old mice. Dystonin-b staining is similar in both single fibers (D,G) and longitudinal muscle sections (A). Dystonin-b staining does not overlap with that of actin

(C), otubulin (F) or myosin (I). Bar: 20/xm.

MACFl localizes to the Z-disc and at the NMJ in skeletal muscle fibers

The overall staining pattern of MACFl in longitudinal sections of mouse SOL muscle was very similar to that of dystonin-b. MACFl cross-striations were consistently observed through the muscle fiber, and light subsarcolemmal staining was also visualized

(Fig 2.3). Double immunofluorescence experiments confirmed partial co-localization of

MACFl with plectin, a-actinin and desmin at the Z-disc and with desmin at the subsracolemmal area (Fig 2.3). MACFl did not co-localize with sarcomeric protein actin or with the MT network (Fig 2.4). MACFl staining was also evident at the NMJ as revealed by the overlapping signal observed in both GAS muscle sections and single fibers stained with the MACFl antibody and with rhodamine-conjugated a-bungarotoxin

(Fig 2.5). MACFl Desnmi MACFl/ Desniiii C

bi^&JttxU&ujx rvLZ&lC?

MACFl a-Actmin MACFl *' a-Actinin

• •*.**• '-si

/*».!***&• ••" m MACFl Plectin MACFl/Plectiu

Figure 2.3

Distribution of MACFl (A, D, G) relative to, desmin (B), a-actinin (E) and plectin (H) in longitudinal sections from SOL muscle of 14 day old mice. The blue staining in the merged images (C, F, I) represent the nuclei. MACFl staining is characterized by cross- striations, with light sub-sarcolemmal staining (see arrow heads) (A, D, G). Fine longitudinally oriented filamentous structures corresponding to MACFl are also observed

(see long arrows). MACFl co-localizes with desmin, a-actinin and plectin at the cross- striations (see arrowheads) (C, F, I), and co-localized with desmin at the subsarcolemmal (C). Bar: 20/ttn.

MACFl Act in MACFl/Actin

MACFl a-Tubuliii MACFl / a-Tiibulin

Figure 2.4

Localization of MACFl (A, D) relative to, actin (B) and a?-tubulin (E) in longitudinal sections of SOL muscle from 14 day old mice. Overlapping signals were not observed (C,

F). Bar: 20pm. 64

MACFl NMJ

Figure 2o

MACFl clusters at the NMJ of mouse SOL muscle fibers. The NMJs were visualized with rhodamine-conjugated a-bungarotoxin (B, D). MACFl is more highly expressed the NMJ as observed from a muscle fiber oriented either longitudinally (A) and from muscle fiber cross-sections (C) (see arrows). Bar: 20/zm. 65

Plakin proteins are more highly expressed in predominantly fast- versus slow-twitch muscle

To compare the expression levels of dystonin and MACFl in slow-twitch versus fast-twitch fibers, immunofluorescence experiments were performed using serial cross- sections of GAS muscle stained for MHCI to label slow-twitch fibers and either dystonin- b or MACFl. Similar experiments were also conducted to target plectin. Our experiments revealed that whereas there appears to be no appreciable difference in the staining intensity of dystonin and MACFl is slow- versus fast-twitch muscle fibers, plectin is more highly expressed in fast-twitch muscle fibers (Fig 2.6). Immunoblotting experiments were also performed using protein lysates from the SOL and the GAS muscles to determine the protein expression levels in predominantly slow-twitch versus fast-twitch muscle. Immunoblotting of dystonin and MACFl revealed two bands over the

500 kDa marker probably indicating isoforms, which have predicted molecular masses greater than 500 kDa, of each protein expressed in muscle tissue. (Fig 2.7, B and C). The

MACFl, dystonin and plectin protein expression levels were 2-fold, 1.5-fold and 3-fold greater respectively in GAS muscle compared to SOL muscle (Fig 2.7). Collectively, these data suggest that fast-twitch muscle have a greater plakin protein content compared to slow-twitch muscle. Figure 2.6

Plakin protein expression in slow- versus fast-twitch fibers from serial cross-sections of

GAS muscle from 14 day old mice; nuclei are visualized in blue. Circles indicate same fibers in consecutive serial cross-sections (A and B, C and D3 E and F). Serial sections were stained for MACFl (A), dystonin-b (C), plectin (E) and for MHCI (B, B, F) to label type I slow-twitch muscle fibers (red fibers in panels B, D, F). MACFl and dystonin expression patterns are similar in slow- and fast-twitch muscle fibers, whereas plectin expression is greater in type II fast-twitch fibers. Bar: 20/mx 67

A SOL GAS 50 Plectm % d f« f40

£3 0

B.2 0 Jio 00 MHCI SOL GAS

B SOL GAS

Dystomn 25 '4 s20 -i

8*15 **>€ 11 0 N ,^««W ^ Jo5 00 gi.$$W?fe MHC: SOL GAS

SOL GAS 3 0 i MACF1 \%f t«» <* §25 8.2 0 -' » I |15 -i

fgs* is f-io - ."•Siit I J05 i MHCI ! 00 SOL GAS

Figure 2.7

Plakin protein expression levels in primarily slow-twitch SOL and primarily fast-twitch

GAS muscles from two week old mice, (data are means ± SEM, N=8 per muscle type. 68

Asterisks (*) indicate significance, p<.05. Arrow indicates the location of the 500 kDa molecular weight marker). Plectin (A), dystonin (B) and MACF1 (C) protein levels are higher in GAS versus SOL muscles. Membranes were stripped and re-probed for MHCI

(specific to slow-twitch fibers) (MHCI band is approximately at 200 kDa). Results from densitometric analyses are presented in the bar charts to the right.

Skeletal muscles from dystonin-deficient mice display altered expression of cvtoskeletal proteins

Based on the domain organization of dystonin-b (i.e. the presence of an ABD, a

PRD and a MTBD) and on the localization pattern of dystonin-b in muscle fibers, we hypothesized that the expression of certain cytoskeletal and plakin proteins would be affected in dystonin-deficient muscle. Therefore the protein expression levels of actio, a- tubulin, desmin, a-actinin, plectin and MACF1 as well as the localization of these proteins in wt versus dt (dystonin-deficient) skeletal muscle were compared.

Immunofluorescence experiments revealed a more intense signal for a-actinin in the dt muscle (Fig 2.8). Z-disc oactinin staining also appeared to be thicker in dt versus wt skeletal muscle (Fig 2.8). Immunofluorescence experiments demonstrated a consistent staining pattern between dt and wt muscle tissue for both MACF1 and plectin, but the signal intensity was greater in dt muscle suggesting higher protein expression (Fig 2.8). In contrast, the staining intensity for desmin was found to be lower in dt muscle compared to wt muscle (Fig 2.8). Immunoblotting results suggest that oactinin protein expression is up-regulated 1.5-fold whereas desmin protein levels are down-regulated 2-fold in dt skeletal muscle (p <0 .05) (Fig 2.11). In addition, protein expression levels of both plectin and MACF1 were up-regulated 1.8- and 2-fold respectively in dt skeletal muscle (Fig 69

2.10). In comparison the protein expression levels (Fig 2.11) and muscle staining pattern of actin and a-tubulin (Fig 2.9) were not altered in dystonin-deficient muscle. 70 71

Figure 2.8

Distribution pattern of plectin (A), MACF1 (B), oactinin (C), and desmin (D) cytoskeletal and plakin proteins in longitudinal sections of SOL muscle froml4 day old wt and dt mice. Bar: 20/mi.

Figure 2.9

Localization of actin (A) and otubulin (B) cytoskeletal proteins in longitudinal sections of SOL muscle from two week old mice of 14 day old wt and dt mice. The staining intensity and pattern was nearly identical in wt and dt muscle. Bar: 20/im. 72

A wt dt

Plectin 30 s-ii) §25 m VI &2 0 SI 5 |io 1 I05 Coomassie % < 00 Stain tfh- ,«*«», wt ofe

B wt dfe

MACF1 30 - M\ T \ |25 \ |2 0 1 31 5 - |l 0 - I 05 J; l 00 - Coomassse wt Stain '$

Figure 2.10

Effect of dystonin-deficiency on plectin (A) and MACFl (B) protein expression in hindlimb skeletal muscle from 14 day old mice (data are means ± SEM, N=8 per genotype). Asterisks (*) indicate significance, p<0.05. Arrow indicates 500 kDa molecular weight marker). Also shown is a protein band detected by Coomassie staining that corresponds to approximately 220 kDa). Results from densitometric analyses are presented in the bar charts to the right. 73

A 2.0 wt dt ss 8 1.5

Xa a-Actinin ^waLs**^/ <&"-..

Coomassie Stain o.o B 1.2 wt <& .a 1-0 i « j D esmin I 0.8 - J 0.6 -1 Coomassie 4 Stain I °- I °-2 0.0 1.4 wt dt S 1-2 .a | 1.0 - r Actin **»q*f y 08 J .Ia °6 " 4 Coomassie | °- 1 Stain I 0.2 -, 0.0 -

1.2 - wt dt * 10 - .s lUy J a-Tubulin .Ms °-6 - ^ §. 0.4 -] Coomassie Stain 1 0-2 i £ 0.0 - wt dt 74

Figure 2.11

Effect of dystonin-deficiency on protein expression of a-actinin (A), desmin (B), actin

± SEM, N=8 per genotype). Asterisks (*) indicate significance, p<.05). Also shown are

Coomassie stained bands to verify protein loading. Results from densitometric analyses are presented in the bar charts to the right.

Discussion

Dystonin is localized at the Z-disc, the sub-sarcolemrnat area and the perinuclear region in the skeletal muscle fiber

One of the primary objectives of this study was to determine the localization of dystonin in the skeletal muscle fiber. We therefore performed double immunofluorescence experiments aimed at comparing the localization of the muscle isoform dystonin-b relative to other proteins expressed in specific compartments of the muscle fiber. We hypothesized that dystonin would localize to the Z-disc structure in skeletal muscle as previous work has demonstrated that Z-discs appear to be thicker in dystonin-deficient animals as revealed by electron microscopy analyses of skeletal muscle

1 S from dt animals . Our results demonstrate a partial co-localization of dystonin with plectin, desmin and a-actinin all known Z-disc proteins. This co-localization indicates that dystonin likely exerts part of its functions, likely structural in nature, at the Z-disc. A partial co-localization of dystonin with desmin was also observed in the subsarcolemmal area and at the perniculear region. These results are in accordance with those from two recent studies demonstrating partial co-localization of dystonin-b with plectin, oactinin 75 and to a lesser extent desmin in both skeletal and cardiac muscle29'30. The longitudinally oriented filamentous structures also obsereved with dystonin-b staining are in accordance with a recent study . The exact functions of these longitudinal filaments are for the moment not known.

The biochemical interactions between the ABD of dystonin-b isoform 1, plectin and oactinin have recently been documented . This same study also found that the PRD of dystonin-b located in the center of the molecule, was surprisingly unable to bind IFs desmin, vimentin or synemin . It had been hypothesized that the PRD of dystonin-b (i.e.

IFBD2) would be able to interact with IFs based on its sequence similarity with the C- terminal PRD of the epithelial dystonin isoform (i.e. IFBD1) that has been demonstrated to bind keratin IFs expressed in the skin. The first two PRD repeats within the PRD region of MACFl-b have been shown to be important for directing MACFl-b to the

Golgi apparatus independent of IF interactions . Therefore, although we demonstrate some degree of overlap between dystonin and desmin immunostaining, it remains to be determined whether these proteins directly interact via regions other than the PRD. For the moment, it would appear that dystonin-b localizes to the Z-disc via interactions with plectin and a-actinin . Future studies are needed to determine whether additional molecular interactions take place between dystonin-b and Z-disc proteins other than plectin and a-actinin.

One limitation in the current study is that we were unable to distinguish the subcellular placement of the dystonin-b subvariants 1, 2 and 3 because our antibody targeted a region common to all dystonin b isoforms (i.e. the centrally located

PRD/IFBD2). However, previous QPCR experiments conducted in our laboratory have 76 revealed that dystonin-b3 is the most abundantly expressed of all iso forms in skeletal and cardiac muscle as well as in C2C12 myogenic cells at least at the mRNA level followed by dystonin-bl then dystonin-b231. Dystonin bl, b2 and b3 are expected to distribute to specific compartments of the muscle fiberjus t as the plectin iso forms 1, lb, Id and If that are predominantly expressed in skeletal muscle have been shown to be shuttled to distinct regions of the muscle fiber32'10.

In vitro experiments using cell lines have revealed that fusion protein comprised of the dystonin N-terminal iso form 3 domain are confined to the cortical region of COS-7 cells33. This cortical localization may be mediated by a putative lipid modification signal contained within the unique N-terminal domain of dystonin iso form 333. Dystonin b3 may be expected to localize near the muscle membrane and may therefore contribute to the subsarcolemmal staining observed in the present investigation. The N-terminal region of plectin iso form 1 has been shown to interact with a-dystrobrevin which is a subsarcolemmal protein of the dystrophin glycoprotein complex (DGC) . Whether dystonin interacts with elements of the DGC remains to be determined. Dystonin N- terminal iso form 2 harbors a transmembrane domain that has been purported to anchor this iso form to membranous structures around the nucleus of COS-7 and C2C12 myogenic cells ' 5. Accordingly, the perinuclear staining detected in skeletal muscle fibers in the present study may depict the distribution of dystonin-b2. In COS-1 cells, dystonin iso form 2 is thought to be connected to the cell nuclei via interactions with nesprin 3-a, a nuclear membrane protein . This molecular interaction may also be the case in muscle cells though further experimentation is required to ascertain this notion .

Expressing the dystonin N-terminal iso form 1 domain in C2C12 cells results in co- 77 localization of this fusion protein with cytoplasmic actin . The cross-striated pattern of dystonin-b coinciding with Z-discs may be a reflection of the distribution of either dystonin-bl or b2 as both of these iso forms harbour an ABD that has been shown to

•5 A biochemically interact with Z-disc proteins plectin and o<-actinin .

Furthermore, dystonin-bl and b2 may play a role in anchoring sarcomeric actin at the Z-disc owing to the presence of a functional ABD that has been demonstrated to bind and stabilize MF33'35. Future experiments to determine the precise localization of the three alternatively spliced dystonin iso forms (bl, b2 and b3) in skeletal muscle fibers as opposed to an in vitro setting (i.e, cell culture) will provide further detail of the function of dystonin in the various subcellular compartments of the fiber.

MACFl is localized at the Z-disc and at the NMJ in the skeletal muscle fiber

The contributions of MACFl to muscle function and structure has not been as extensively studied compared to plectin and dystonia. In a research study published in

1989, a novel spectrin-repeat protein was reported to aggregate within the postsynaptic compartment of the NMJ of rat myotubes and rat diaphragm muscle37. This spectrin- repeat protein clustered at the NMJ was more recently confirmed to be MACFl28.

Interestingly the ABD of MACFl has been shown shown to interact biochemically with rapsyn, an AChR-associated scaffolding protein found at the post-synaptic compartment of the NMJ . In the present study, we have also confirmed that MACFl is enriched within the post-synaptic compartment of the NMJ as evidenced by brighter MACFl staining overlapping with that of rhodamine conjugated a-bungarotoxin thereby supporting results from previous studies37. The contributions of MACFl to the 78 development and maintenance of the NMJ remain to be elucidated. A potentially useful model to help further delineate the role of MACFl in skeletal muscle and in the establishment of the NMJ would be a MACFl skeletal muscle-specific knockdown mouse model.

Not previously reported in the literature are our findings that MACFl co-localizes with plectin, desmin and o;-actinin at the Z-disc and appears to be present to some extent within the subsarcolemmal region of muscle fibers. MACFl was also represented as light longitudinally oriented filamentous structure. Again, the exact function of these structures is currently unknown. Because of the high homology in protein configuration between dystonin and MACFl, it is possible to envision that molecular interactions between

MACF and plectin, desmin or a-actinin Z-disc proteins occur. MACFl also has three alternatively spliced iso forms two of which have an ABD (iso forms 1 and 2) and the third that has an incomplete ABD (isoform 3) . Whether there is redundancy in the types of molecular interactions between dystonia or MACFl and Z-disc elements or whether

MACFl attaches to other Z-disc components remains to be determined. Contrary to previous studies using cell lines, MACFl perinuclear staining was not observed in the present study suggesting that MACFl distribution near the nucleus is cell-type specific.

Plakin proteins expression profile in predominantly fast- versus slow-twitch muscle

Asides from metabolic and enzymatic differences between fast- and slow-twitch muscle fibers, structural differences are also well documented. For example, compared to fast-twitch fibers, slow-twitch fibers have thicker Z-discs , have smaller costameres and

in intercostameric regions . In the present study, we determined whether the expression 79 profile of dystonin and MACFl plakin proteins is different between slow- and fast-twitch fibers. Although we did not observe any notable differences in the staining intensity of either dystonin or MACFl in type 1 versus type II fibers from cross-sections of hindlimb skeletal muscle, we did find significantly higher dystonin and MACFl protein levels in predominantly fast-twitch (GAS) compared to slow-twitch (SOL) hindlimb muscles.

Unexpectedly, our immunofluorescence and immunoblotting results suggest that plectin is more highly expressed in fast-twitch fibers thus contradicting results from the previous study4 . Reasons for this discrepancy are not known but could be explained by the fact that our analyses were performed using hindlimb muscles from young animals as opposed to human muscle. As a whole, our results suggest an overall higher expression level of plakins in fast-twitch compared to slow-twitch muscle.

Plectin is known to localize at the costamere and dystonin and MACFl may also localize in this area. The costamere is reported to be smaller in slow-twitch fibers and may in part explain the lower expression of costameric proteins (i.e. plakin proteins) in this fiber type. Fast-twitch fibers have a larger diameter than slow-twitch fibers thus implying that they have a larger sarcolemmal area. The expression of plakin proteins in this region may also contribute to the observation made here that plakins are more highly expressed in predominantly fast-twitch fibers. Although fast-twitch muscle fibers are not recruited as often as slow twitch fibers, they are required to generate greater amounts of force in a shorter amount of time. A more elaborate structural scaffold (i.e. more plakins) to withstand larger mechanical forces may therefore be a requirement of fast-twitch muscle fibers. 80

Impact of dvstonin-deficiency on the expression profile of select cytoskeletal elements

We studied hindlimb skeletal muscles from dystonin-deficient mice to determine whether lack of dystonin would disrupt or alter specific cytoskeletal elements selected primarily based on our current knowledge of dystonin-interacting proteins and on the domain organization of dystonin. Although the MF and MT networks appeared to be largely unaffected in skeletal muscle lacking dystonin, enhanced expression of a-actinin, plectin and MACF1 and reduced expression of desmin were observed in dt compared to wt muscle. Given that our localization experiments and those of others29,30 suggest that dystonin is likely a Z-disc protein, it was not unexpected that Z-disc elements such as o- actinin, desmin, plectin and MACF1 were found to be differentially expressed in muscle lacking dystonin whereas other cytoskeletal networks such as MF and MT that do not co- localize with dystonin were not overtly affected.

In the present study, the a-actinin staining pattern in muscle fibers was characterized by thicker and brighter striations and immiunoblotting experiments revealed an up-regulation in a-actinin protein expression levels. The thickened Z-disc structures in skeletal muscle of dt mice as revealed by electron microscopy and reported in an earlier study may be due in part to a-actinin aggregation. In contrast, desmin staining was significantly attenuated in dystonin-deficient skeletal muscle. Whether this reduction in desmin protein expression levels is directly related to a lack of dystonin or whether it is a secondary effect of the reduced mobility phenotype and therefore reduced muscle recruitment in dt mice remains unclear. Firstly, we have previously shown that the desmin network in the cardiac muscle from dt mice is not altered suggesting that the organization of this IF network is not impacted by the lack of dystonin in highly solicited muscle . 81

Secondly, a direct interaction between dystonin-b and desmin has yet to be reported. A recent study has demonstrated that the centrally located PRD in dystonin-b isoforms previously thought to interact with IF network does not bind to desmin IF30. Thirdly, plectin Id has been shown to be a key stabilizer of the desmin cytoskeletal network because lack of this particular plectin iso form leads to desmin aggregation in muscle fibers3 . The up-regulation of plectin and perhaps MACFl at the Z-discs of dt muscle fibers observed in the present investigation may represent a compensatory mechanism aimed at stabilizing Z-disc structures. Collectively, these findings suggest that lack of dystonin in skeletal muscle is unlikely to have a direct impact on the structural stability of the desmin network and would tend to indicate that the impact of dystonin-deficiency on the desmin network in skeletal muscle is more likely an indirect outcome of reduced muscle recruitment.

In summary our findings show that dystonin and MACFl likely exert their respective functions in specific subcellular compartments of the muscle fiber.

Specifically, dystonin is localized at the Z-disc, the subsarcolemmal area and the perinuclear region while MACFl is localized to the Z-disc the subsarcolemmal area and the NMJ. Predominantly fast-twitch muscles express greater levels of plakin proteins.

Dystonin-deficiency is associated with the altered expression of a-actinin and desmin in skeletal muscle. Future studies aimed at further elucidating the molecular interactions of dystonin and MACFl at the Z-disc as well as within the subsarcolemmal and perinuclear regions will provide important new insights into the functional roles these plakin crosslinking proteins exert in muscle cells. 82

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CHAPTER 3: General discussion

The functions of crosslinking proteins have been investigated in neuronal, epithelial and skeletal and cardiac muscle tissues " . However, much research is still required to fully understand the precise role and function of plakin proteins, specifically dystonin and MACF1, in skeletal muscle. The main purpose of the present study was therefore to 1) determine the subcellular distribution of dystonin and MACF1 in skeletal muscle fibers, 2) to assess the dystonin and MACF1 expression profile in slow- versus fast-twitch muscle fibers and 3) to investigate the impact of dystonin-deficiency on select architectural proteins in skeletal muscle. To achieve these objectives the localization of dystonin and MACF1 was compared to that of other proteins known to be expressed in specific compartments of the muscle fiber. The muscle fiber-type dependent expression of dystonin and MACF1 was determined by examining plakin protein expression in individual fast- and slow-twitch muscle fibers and in predominantly fast- (gastrocnemius) and slow- (soleus) twitch muscle. Lastly, a readily available dystonin-deficient mouse model was used to compare the expression profiles of MT, MF as well as select Z-disc proteins in the skeletal muscle of dt mice compared to that of healthy wt littermates.

1.0 Plakin protein localization in skeletal muscle fibers

Recently, the localization pattern of dystonin-b has been described in cardiac muscle by our research group and by others ' . In the present study, we determined the localization of dystonin-b in the skeletal muscle. Previous work has suggested that the Z- discs of muscle fibers are particularly disrupted in rodent muscles lacking dystonin6. A dystonin-deficiency was also shown to lead to reduced force production and an increase 89 in fatiguability of the diaphragm muscle in mice6. Therefore it was hypothesized that dystonin-b would likely localize to the Z-disc, as it likely plays a crucial role in stabilizing this structure. We confirmed this hypothesis as dystonin was found to localize at the Z-disc, the subsarcolemmal area and the perinuclear region. The staining pattern of dystonin-b was also characterized by occasional longitudinally oriented filamentous structures. Dystonin-b was found to co-localize with plectin, a-actinin and desmin at the

Z-disc and with desmin at the subsarcolemmal and the perinuclear regions (Fig 3.1). Our findings are in accordance to those of a recent study by Steiner-Champliaud et al. (2010) demonstrating a partial co-localization of dystonin-b with plectin, a?-actinin and to a lesser extent desmin in both skeletal and cardiac muscle . This study also confirmed biochemical interactions of both plectin. and o-actinin with dystonin-b isoform 1 through its ABD10. However, this study demonstrated that the PRD of dystonin-b, which was previously referred to as an intermediate filament binding domain (IFBD), is unable to bind IFs desmin, vimentin or synemin .

Although we were unable to determine the distribution of individual dystonin-b isoforms (bl, b2 and b3), we are able to make predictions based on previous studies conducted to determine the subcellular distribution of dystonin N-terminal fusion proteins expressed in cultured cells11"14. Dystonin-b 1 and -b2 both harbour and ABD and may therefore be expected to aggregate at the Z-disc via interactions between their ABD and

Z-disc proteins plectin and a-actinin. The unique N-terminal region of dystonin-b2 contains a transmembrane domain that is predicted to mediate interactions with the nuclear envelop and may therefore account for the perinuclear staining observed in muscle fibers in the current study and in cultured cells13. The dystonin-b2 isoform may 90 therefore play a role in stabilizing the nuclear envelop and/ or in positioning the nuclei within the muscle cell. Dystonin-b3 may be expected to aggregate at the sarcolemma as opposed to the Z-disc owing to its N-terminal myristolation site thought to mediate membrane interactions11 and due to its incomplete ABD which may hinder interactions with Z-disc proteins plectin and a-actinin. In order to determine the exact subcellular distributions of dystonin-bl, -b2, -b3, it will be necessary to generate isoform specific antibodies and process skeletal muscle fibers for immunofluorescence.

Our current study extends as well as offers new insights into the distribution of

MACFl in skeletal muscle fibers. Previous studies have suggested that MACF clusters at the NMJ 15. The ABD of MACFl has been shown to interact with rapsyn, an important

AChR-assoeiated scaffolding protein found within the post-synaptic compartnient of the

NMJ16. In the present study we have confirmed that MACFl clusters at the NMJ (Fig

3.2). Furthermore, we show that MACF 1 localizes at cross-striations coinciding with known Z-disc proteins plectin, desmin and a-actinin. Light subsarcolemmal staining and occasional light longitudinal staining was also observed (Fig 3.1). Although MACFl

1 7 perinuclear staining has been shown in other cell lines , we did not observe MACFl staining around the nucleus of skeletal muscle fibers. Although there are three known N- terminal MACFl isoforms produced by alternative splicing of the MACFl gene, the expression profile of these isoforms in muscle and a detailed analysis of the predicted binding motifs of the unique N-terminal regions has not been completed. Such analyses are required to understand how the N-terminal regions of MACFl dictate the subcellular distribution of this plakin family member in muscle cells. Future experiments to address these issues are required to better understand the functions of MACFl in regulating the 91 structural organization of the NMJ and other subcellular compartments of the contractile cell.

The Z-disc is regarded as an important subcellular compartment of the muscle fiber playing a crucial role in force generation, maintenance of structural stability and in cell signaling pathways. Our localization studies in skeletal muscle indicate both dystonin and MACF1 likely play an important role at the Z-disc by crosslinking and thereby stabilizing Z-disc components (i.e. a-actinin and plectin) though further studies are required to better understand their respective contributions in force generation and cellular signalling.

Figure 3.1

Schematic representation of dystonin-b and MACF1 protein localization in skeletal muscle. Our results demonstrate that dysonin-b is expressed at the Z-disc. Here, dystonin- bl biochemically interacts with both oactinin and plectin. Dystonin-b2 is depicted near 92 the nucleus whereas dystonin b3 is represented within the subsarcolemmal region.

MACFl is also shown to localize at the Z-disc. Potential interactions between MACFl and plectin, dystonin-bl and a-actinin are represented by hatched lines. (Modified from9)

»**(

5 ai col em in a

f*~ "Syha|«fcv" V^_ nucleus _^V

MFs C^MACFl custom n-bd. —\ ~w T 1L T

-t tczrr^B!Z^rs~s^z^xzzz "FT

•t 2-disc .-disc

Figure 3.2

Simplified schematic representation of the NMJ and its components. Our localization experiments have identified that MACFl is expressed in the post-synaptic compartment of the NMJ. MACFl interacts with rapsyn which in turn directly interacts with AChRs.

The indirect interaction of MACFl to AChRs via rapsyn highlights the likely role that

MACFl plays in maintaining AChR clustering at the NMJ. (Modified from9) 93

2.0 Protein expression profile of plakin proteins in slow-twitch versus fast-twitch muscle fibers

Skeletal muscles are composed of various proportions of slow-twitch and fast- twitch fiber subtypes displaying a spectrum of metabolic, structural and contractile features that collectively dictate whole muscle contractile properties. Slow-twitch fibers are continuously activated and generate low to moderate force whereas fast-twitch fibers produce substantial force levels in short bursts. In order to begin understanding the contributions of plakin proteins to the structure and function of slow-twitch versus fast- twitch fibers, we performed a set of simple experiments comparing the protein expression profile of dystonin-b and MACF1 in these fiber types. In parallel, we also determined the protein expression profile of plectin as previous studies have shown that plectin levels are greater in slow-twitch fibers . Our overall results suggest that the expression of plakin proteins is greater in fast-twitch muscle compared to slow-twitch fibers. We had originally thought that the slow-twitch fibers would require greater structural stability owing to their continuous recruitment. However, because these fibers generate low to moderate force, the level of mechanical stress is significantly less in slow-twitch fibers.

In comparison, fast-twitch fibers produce substantial forces in short bursts and must therefore withstand significant levels of mechanical stress. Our results suggests that plakins are required at higher levels in fast-twitch versus slow-twitch fibers and likely play a role in force generation, as they are expressed at the Z-disc which is a crucial structure in force production. 94

3.0 Cytoskeletal protein alterations in dystonin-deficient skeletal muscle

Given that dystonin-b is composed of an ABD, a MTBD and a PRD, it was hypothesized that a lack of dystonin in the muscle may alter the expression and localization of the primary cytoskeletal components (i.e. MFs, desmin IF and MTs). We have localized dystonin to the Z-disc of skeletal and cardiac muscle fibers9'10. It was therefore expected that Z-disc proteins, such as a-actinin, desmin and plectin, would be affected in dystonin-deficient skeletal muscle. Although the MF and MT networks appeared to be largely unaltered in skeletal muscle lacking dystonin, enhanced compensatory expression of a-actinin, plectin and JVLACF1 and reduced expression of desmin were observed in dt compared to wt muscle (Fig 3.3).

The staining pattern of a-actinin was highlighted by a thicker cross-striated pattern. This observation is in agreement with a previous study that demonstrated thicker

Z-discs in dt skeletal muscle as observed through electron microscopy . The thicker Z- discs may in part be due to the accumulation of a-actinin at this structure in dt skeletal muscle. Our results also demonstrated that the IF desmin expression is down-regulated in dt skeletal muscle (Fig 3.3). Dystonin-deficient animals are significantly less mobile than healthy wt animals. Therefore down-regulation of desmin is likely a result of hypo- mobility and a secondary rather than primary outcome of dystonin-deficiency.

MACF1 and plectin both are more highly expressed in dt skeletal muscle (Fig

3.3). The higher expression of these plakin proteins in dystonin-deficient muscle is likely to compensate for the lack of dystonin expression. This up-regulation could be sufficient in enabling the proper establishment and organization of the MF and MT cytoskeletal 95 filament networks explaining their normal expression profile in dystonin-deficient skeletal muscle.

Taking into account results from previous studies in conjunction with the data obtained in the present investigation, it is becoming increasingly clear that dystonin and

MACFl proteins play a crucial role in maintaining proper structural integrity of the skeletal muscle fiber. In dystonin-deficient muscle, the expression profiles of other cytoskeletal proteins, notably a-actinin, MACFl and plectin are altered. Also plakin proteins expressed in skeletal muscle seem to undergo a compensatory up-regulation to counteract the lack of dystonin expression in skeletal muscle (Fig 3.3).

Figure 3.3

Schematic highlighting the difference between protein expression between wt (A) and dt

(B) skeletal muscle. In dt skeletal muscle there is a down-regulation of IF desmin, whereas an up-regulation of Z-disc protein oactinin and plakin proteins MACFl and plectin is observed. A thickening of the Z-disc structure is also depicted in dt muscle.

(Modified from9) 96

4.0 Conclusions

Results from the present investigation offer new insights into the potential function of dystonin-b and MACFl within specific subcellular compartments of the skeletal muscle fibers and raise a number of research questions highlighted in the discussion requiring further exploration.

In summary:

1) Dystonin-b and MACFl localize to the Z-disc and show overlapping distribution

with known Z-disc proteins, a-actinin, desmnin and plectin.

2) Dystonin-b clusters in the perinuclear region whereas MACFl aggregates within

the postsynaptic compartment of the muscle fiber.

'stonin-

4) Dystonin-deficiency in skeletal muscle alters the expression profile of Z-disc

proteins a-actinin, desniin, plectin and MACFl wherease MF and MT networks

appear to be largely unaffected. A down-regulation of IF desmin observed in dt

muscle may be a secondary effect of reduced muscle activation in dt mice. The up-

regulation of a-actinin, MACFl and plectin may be a compensatory response to

dystonin-deficiency. 97

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