Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations

1-1-2002

Molecular interactions among the intermediate filament proteins paranemin, synemin and desmin and their localization during skeletal muscle cell development

Stephanie Ann Lex Iowa State University

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Recommended Citation Lex, Stephanie Ann, "Molecular interactions among the intermediate filament proteins paranemin, synemin and desmin and their localization during skeletal muscle cell development" (2002). Retrospective Theses and Dissertations. 20141. https://lib.dr.iastate.edu/rtd/20141

This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Molecular interactions among the intermediate filament proteins paranemin, synemin and desmin and their localization during skeletal muscle cell development

by

Stephanie Ann Lex

A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE

Major: Biochemistry

Program of Study Committee: Richard M. Robson, Major Professor Elizabeth J. Huff-Lonergan Ted W. Huiatt Marvin H. Stromer

Iowa State University Ames, Iowa 2002 11

Graduate College Iowa State University

This is to certify that the master's thesis of

Stephanie Ann Lex has met the thesis requirements of Io\\ a State University

Signatures have been redacted for privacy ll1

TABLE OF CONTENTS

ABSTRACT IV

GENERAL INTRODUCTION Introduction Thesis Organization 2 Literature Review 2 References 14

MOLECULAR INTERACTIONS AMONG THE INTERMEDIATE FILAMENT 26 PROTEINS PARANEMIN, SYNEMIN AND DESMIN AND THEIR LOCALIZATION DURING SKELETAL MUSCLE CELL DEVELOPMENT

Abstract 26 Introduction 27 Materials and methods 30 Results 34 Discussion 41 Acknowledgements 45 References 46 Figures and tables 51

GENERAL CONCLUSIONS 81 General Discussion 81 Comprehensive Bibliography 82

ACKNOWLEDGEMENTS 95 lV

ABSTRACT

The intermediate filament (IF) proteins paranemin and synemin are unique members of the IF protein superfamily. Paranemin and synemin were originally identified as IF- associated proteins because they colocalize and copurify with desmin in avian muscle cells. The IFs wrap around the periphery of myofibrils at their Z-lines in adult skeletal muscle cells, connect adjacent myofibrils to one another and to any nearby mitochondria and nuclei, and connect the peripheral layer of myofibrils to the sarcolemma at costameres. We have followed the co localization of paranemin, synemin, and desmin during skeletal muscle myogenesis at different stages of development by immunofluorescence and immunoelectron microscopy. Localization of paranemin precedes that of desmin in cultured avian skeletal muscle presumptive myoblasts. Paranemin, synemin and desmin then colocalize throughout muscle cell development but, in relation to desmin and synemin, paranemin immunofluorescence is increased at the growth tips of elongating myotubes and decreased in areas of the cell where the myofibrils are in a more mature pattern of alignment. The IF proteins colocalized within longitudinal filaments dispersed around/between myofibrils, but there was no IF colocalization with the myofibrillar Z-line protein a-actinin until after myofibril alignment had occurred. At the ultrastructural level, IFs were observed near the ends of early nascent myofibrils and at cytoskeletal filament junctions. A direct interaction of paranemin with both desmin and synemin was demonstrated by in vitro binding assays. Furthermore, expression of GFP-tagged paranemin rod domain in cultured myotubes resulted in colocalization with, and eventual aggregation of, the desmin-containing IFs, but did not noticeably affect myofibril assembly and alignment. Rather than playing a direct role in myofibrillogenesis, it is likely that IFs play a role at the next level of cytoskeletal organization by preserving the overall shape and integrity of developing muscle cells as they grow into elongated myotubes. 1

GENERAL INTRODUCTION

Introduction

The research contained within this thesis focuses primarily upon the molecular interactions of the intermediate filament (IF) protein paranemin and its localization with the IF proteins synemin, desmin and vimentin during skeletal muscle cell development. It is our hypothesis that these IF proteins form heteropolymeric IFs in cells, and interact with other components of the cell cytoskeleton. The specific role(s) ofIFs in developing skeletal muscle, however, remains· unclear. The results of protein interaction and localization studies presented herein contribute to our understanding of the relationships among paranemin, synemin, and desmin and suggest a possible function for IFs during skeletal muscle cell development. A manuscript that will be submitted to the journal "Histochemistry and Cell Biology" for peer review and publication is included herein. This manuscript focuses upon the interactions among the IF proteins paranemin, synemin, and desmin and their localization within skeletal muscle cells during different stages of development. The first direct interactions in vitro between paranemin and synemin obtained by blot overlay assays are described. The direct interaction of paranemin as well as of the paranemin rod domain with desmin has also been shown by both blot overlay and cosedimentation assays. Additional interactions among the paranemin rod domain, synemin rod domain, vimentin, and desmin were identified by yeast two-hybrid analysis. Transfection of the cDNA construct for GFP- tagged paranemin rod domain into cultured avian skeletal muscle cells resulted in aggregation and disruption of the endogenous IF network. It is shown for the first time that paranemin is expressed before desmin in cultured avian presumptive myoblasts. Localization of paranemin, synemin, desmin, and the sarcomeric Z-line protein a-actinin in muscle cell cultures was observed by immunofluorescence and/or immunoelectron microscopy at different stages of development. The IFs were present throughout the developing skeletal muscle cells, with longitudinally dispersed IFs localized near the ends of nascent growing 2 myofibrils, especially within growth tips of elongating myotubes, between adjacent myofibrils, and between the peripheral layer of myofibrils and the sarcolemma.

Thesis Organization

This thesis has been prepared in the manuscript-based format with the inclusion of one paper. The manuscript within this thesis will be submitted to the journal "Histochemistry and Cell Biology". I was responsible for the design and implementation of almost all of the experiments described "'ithin this manuscript. Skeletal muscle cell lysates used for analysis of the IF protein content were prepared by Emily E. Helman, and the desmin and filamin used in the overlay assays were purified by Suzanne W. Sernett, both research associates in the lab. The references cited in the literature review and the manuscript have been formatted in the author/year style for easy reading and as per the requirements of the journal. The reference sections associated with the literature review and the manuscript, as well as the comprehensive bibliography for the entire thesis, include the titles of the papers for easy reference and as per the requirements of the journal.

Literature Review

Introduction

The cellular cytoskeleton of vertebrates typically consists of three unique, interconnecting filament networks, namely microfilaments, intermediate filaments (IFs) and microtubules (Robson 1989; Coulombe et al. 2000; Herrmann and Aebi 2000). In striated muscle cells there is an additional filament system, the myosin-containing thick filaments, which are - 15 nm in diameter and 1.5 µm long (Huxley 1957; Squire 1997). A conventional vertebrate striated muscle myosin molecule consists of a pair of small regulatory light chains, a pair of small essential light chains and two heavy chains, each with a helical rod domain 3 connected to a bi-lobed globular head with each lobe containing an ATPase site and an actin binding site (Rayment et al. 1993). The microfilaments (or thin filaments in muscle cells) are composed of tissue specific isoforms of actin and are - 6 nm in diameter, with lengths of several µm in typical non-muscle cells (Rubenstein 1990). In skeletal muscle, actin thin filament lengths are regulated and are - 1 µm long in vertebrates (Littlefield and Fowler 1998). The individual actin molecule has an ADP/ATP binding site flanked by binding sites for many other proteins, including other actin monomers with which it forms filamentous actin (F-actin) (Frankel and Mooseker 1996; Squire 1997). The microtubules, which have a backbone ofheterodimers of a and p tubulin, are - 23 nm in d;ameter and several µm long (Nogales 2001). The a and p tubulin isoforms have similar structures, each with a GTPase site, a Taxol-binding site and a motor protein binding site (Nogales et al. 1998). Isoforms of actin and a and p tubulin are highly conserved, both between isoforms and in different species (Fuchs and Weber 1994; Berg et al. 2002). Unlike the microfilaments and the microtubules, the - 10 nm diameter IFs, which are often several µm in length, consist of a large family ofIF proteins (Herrmann et al. 1999). The IF proteins typically have high sequence homology within each class of IF protein, especially within the characteristic - 310 amino acid a-helical rod domain (Fuchs and Cleveland 1998). Although each of these filament networks is essential and exciting in its own right, the studies contained within this thesis are focused upon IFs and their role during skeletal muscle cell development.

Intermediate filaments

There are six known classes/types of IF proteins and most of these IF proteins are tissue specific. The keratins, found in epithelial cells, are the largest group of IF proteins (> 30 different proteins) comprising Type I and Type II IF proteins. The keratins are obligate heteropolymers requiring the initial formation of heterodimers from an acidic, Type I keratin protein and a basic, Type II keratin protein (Fuchs and Weber 1994). The Type III IF proteins consist of four proteins: vimentin, which is found in various mesenchymal tissues; desmin, which is found primarily in muscle; glial fibrillary acidic protein (GF AP), which is 4 astrocyte-specific, and peripherin, which is specific to the peripheral neurons. Type III Ifs are capable of forming homopolymers in vitro, but are most likely heteropolymeric in vivo, associating with other Type III IF proteins and/or Type IV (e.g., a-intemexin) and Type VI IF proteins (e.g., nestin) (Steinert et al. 1999b; Coulombe et al. 2000). The Type IV IF proteins are found in neurons and consist of the heteropolymeric neurofilament triplet proteins, i.e., the low molecular weight neurofilament protein (NF-L), medium molecular weight neurofilament protein (NF-M), and high molecular weight neurofilament protein (NF- H), and a-intemexin. a-Intemexin and NF-Lare each capable of forming homopolymers in vitro, but neither NF-M nor NF-H are capable of forming filaments in the absence of NF-L (Carpenter and Ip 1996; Steinert et al. 1999a). Type V IF proteins, the lamins, are found in the nuclear lamina (Fuchs and Weber 1994). Finally, the Type VI IF proteins are unusually large members of the IF protein superfamily (Coulombe et al. 2000, 2001). Nestin (Sejersen and Lendahl 1993) and paranemin (i.e., transitin) (Price and Lazarides 1983; Yuan et al. 1997) are expressed predominately during development in various tissue types, such as muscle and throughout the nervous system, whereas synemin is expressed during development and at maturity in most muscle cells (Price and Lazarides 1983; Bilak et al. 1998), as well as avian lens cells (Granger and Lazarides 1984) and erythrocytes (Granger and Lazarides 1982). These large IF proteins are evidently incapable of forming filaments on their own, and presumably must form heteropolymers with Type III and/or Type IV IF proteins (Hemken et al. 1997; Bellin et al. 1999; Eliasson et al. 1999). Members of the IF protein family are identified by their overall molecular domain structure. Specifically, all cytoplasmic IF proteins have an -310 amino acid a-helical rod domain, responsible for oligomerization properties, and flanking N-terminal head and C- terminal tail domains of variable lengths (Heins and Aebi 1994). The exception to this rule is the nuclear lamins, which have an additional 42 amino acids within their rod domain (Fuchs and Weber 1994). The first step in IF assembly occurs via coiled-coil interactions between the a-helical rod domains of two polypeptides. Formation of these coiled-coils is dependent upon heptad repeats within the primary amino acid sequence of the rod, in which the first and fourth amino acid residues are usually nonpolar residues that form a hydrophobic strip along one side of the a-helix (Fuchs and Weber 1994). Two of these dimers then associate in an 5 anti-parallel fashion to one another, thereby forming a more stable tetramer (Herrmann and Aebi 1998). Exactly how these tetramers then assemble to form the very long, 10-nm diameter IFs is still not entirely clear. There are currently two major models for IF assembly, one based upon protofibril assembly to form IFs (Fuchs and Cleveland 1998; Parry et al. 2001) and one based upon assembly of"unit-length filaments" (ULFs) into IFs (Herrmann and Aebi 1998, 2000; Herrmann et al. 1999). The specific roles of the head and tail domains of IFs are still somewhat unclear. The head domain has been shown to be involved in assembly into filaments (Heins and Aebi 1994; Inagaki et al. 1996; Ching and Liem 1999). When the head (or most of the head domain) is missing, IF proteins are incapable of forming filaments on their own, but can associate with wild type IF proteins to form filaments (Herrmann et al. 1996). This may explain in part why Type VI IF proteins, which have very small head domains, are incapable of forming filaments on their own (Marvin et al. 1998). Also, covalent modification, such as phosphorylation (Inagaki et al. 1996) and ADP-ribosylation (Huang et al. 1993) of the head domains of specific IF proteins, has been shown to regulate filament assembly in vitro. The tail domains of IF proteins may help control filament diameter, and also may be involved in binding IFs to other cytoskeletal components (Heins and Aebi 1994; Herrmann et al. 1996; Bellin et al. 1999, 2001). The exact cellular function(s) ofIFs in cells is not clear. The IFs are believed to provide mechanical strength, and to help maintain the shape and plasticity of cells (Chou et al. 1997; Galou et al. 1997). Studies ofIF-related diseases have been useful in learning more about the functions of IFs in specific cell types. For example, epidermis bullosa simplex (EBS) is a genetic skin disease resulting from mutations in specific keratins. The severity of the EBS disease depends upon how much the genetic mutations disrupt IF assembly (Fuchs 1996). Desmin-related myopathies (DRM) have also been useful in addressing IF roles in muscle. Some DRMs result in defects in muscle contraction and/or abnormal aggregation of nonfunctional desmin (Abraham et al. 1998; Munoz-Marmol et al. 1998). Interestingly, one DRM results not from a mutation in the desmin gene, but from a point mutation in the gene for the small heat shock protein a.B-crystallin, where aggregates of desmin contain mutated a.B-crystallin (Vicart et al. 1998). Heat shock proteins act as molecular chaperones within 6 cells and may prevent unfavorable IF interactions during times of cytoskeletal rearrangeme~t, such as development (Benjamin et al. 1997) or cell stress (Collier et al. 1993 ). Production of transgenic mice has enabled researchers to study IF function in vivo. Interestingly, most types ofIF-null mice are viable and fertile (Evans 1998). This, however, does not mean that IFs have no important function in cells. Each type ofIF-null mouse has specific abnormalities. For example, mice lacking portions of, or entire, keratin proteins have abnormalities that range from skin lesions to epithelial abnormalities resulting in embryonic lethality, such as seen with keratin 8 (Galou et al. 1997; Fuchs and.Cleveland 1998). Furthermore, desmin-null mice were seemingly normal after birth and capable of reproducing nonnally, but died within about one year (Milner et al. 1996; Li et al. 1997). Ultrastructural analysis showed skeletal and cardiac muscle deterioration and misalignment of sarcomeres, as well as calcification of the heart, as the most obvious abnormalities (Li et al. 1996, 1997; Thornell et al. 1997). Contrary to the results with desmin, there seem to be very few obvious physical abnormalities observed with vimentin-null mice (Colucci-Guyon et al. 1994), which have neurological abnormalities similar to those of GFAP-null mice (Pekny et al. 1995; Eliasson et al. 1999).

Intermediate filament proteins in skeletal muscle

The IF protein vimentin is expressed in a variety of cell types; however, in skeletal muscle cells expression is primarily developmental in nature (Evans 1998). Vimentin, 54 kDa, is the first IF protein found in replicating presumptive skeletal myoblasts (Schultheiss et al. 1991). In postmitotic skeletal muscle cells, vimentin is co-expressed with desmin throughout myogenesis, but vimentin expression gradually decreases as the myotubes mature (Hill et al. 1986; Lin et al. 1994 ). Desmin is the primary IF protein present in muscle and is found in nearly all muscle cell types at all times of development. In skeletal muscle cells, expression of desmin (53 kDa) marks the initiation of definitive myoblast formation (Lin et al. 1994 ). In adult skeletal muscle cells, desmin-containing IFs wrap around the periphery of myofibrils at their Z-lines, 7 connect adjacent myofibrils to one another and to any nearby mitochondria and nuclei, and connect the peripheral layer of myofibrils to the sarcolemma (Richardson et al. 1981; Tokuyasu et al. 1983; Stromer 1990). Nestin, the first named member of the Type VI class oflFs, is one of a small group of large IF proteins, which characteristically have short head domains and extremely long tail domains. Nestin, which has a molecular mass of- 300 kDa by SDS-PAGE, is found in all types of developing mammalian muscle cells, as well as several cell types within the developing nervous system, and can form heteropolymeric IFs with vimentin and desmin in vivo (Sejersen and Lendahl 1993; Sjoberg et al. 1994). The expression ofnestin is down- regulated throughout development in must cell types (Sejersen and Lendahl 1993). Another large IF protein found in muscle cells is synemin (Becker et al. 1995; Bellin et al. 1999), which has a molecular mass of -230 kDa by SDS-PAGE, and was first found associated with desmin and vimentin in preparations of adult avian gizzard (Granger and Lazarides 1980). Thus, synemin was initially referred to as an IF-associated protein (IFAP) (Granger and Lazarides 1980). Synemin directly binds to desmin in vitro in blot overlay (solid-phase) assays (Bellin et al. 1999) and the synemin rod domain also interacts with desmin and vimentin in yeast two-hybrid assays (Bellin et al. 2001). Additionally, synemin binds to a-actinin and vinculin in blot overlay and yeast two-hybrid assays, suggesting that synemin may directly link the heteropolymeric intermediate filament network to skeletal muscle Z-lines and costameres, respectively (Bellin et al. 1999, 2001). Human synemin has recently been identified by two labs. Mizuno and associates (2001) identified a human protein with high sequence homology to synemin, referred to as desmuslin. The rod domain of desmuslin was pulled from a yeast two-hybrid library by the dystroglycan complex protein, a-dystrobrevin, and shown to interact with desmin by co- immunoprecipitation (Mizuno et al. 2001 ). Titeux and associates (2001) also cloned cDNA identical to that of desmuslin, calling it a human synemin because several regions in the rod and tail domain of their synemin were highly homologous to avian synemin. Furthermore, Titeux et al. (2001) found that human synemin had two splice variants, referred to as a- and P- synemin. The a-synemin, which is the larger splice variant and has the highest homology to avian synemin, is found predominately in developing muscle and adult smooth muscle, 8

whereas the smaller ~-synemin is enriched in human skeletal muscle cells (Titeux et al. 2001). Another large IF protein is paranemin, which has a molecular mass of-280 kDa by SDS-PAGE. Paranemin was first found to be colocalized with desmin and vimentin in developing chick skeletal muscle cells (Breckler and Lazarides 1982), and was originally identified as an IF AP (Breckler and Lazarides 1982). The avian paranemin protein has also been studied by other labs under the names IFAPa-400 (Cossette and Vincent 1991), EAP- 300 (Kelly et al. 1995), and transitin (Yuan et al. 1997) in a variety of embryonic muscle and neural tissues. In addition to localization in developing smooth, skeletal and cardiac muscle cells (Price and Lazarides 1983; Cossette and Vincent 1991 ), paranernin has been found in developing avian lens cells (S. A. Lex, unpublished observation) and radial glial cells of the developing retina, cerebellum, and spinal cord (Cole and Lee 1997). Like nestin, paranemin expression is down-regulated in most cells and tissues as they develop (Breckler and Lazarides 1982). The cDNA sequence of paranemin was published by two independent labs, namely Hemken et al. (1997) and Yuan et al. (1997), who referred to it as transitin. Analysis of the most complete cDNA sequence derived from the cDNA clones of Hemken et al. (1997) predicts a protein of - 193 kDa, with an acidic pl of - 4.0. The discrepancy between the molecular mass obtained from the sequence (- 193 kDa) and the molecular mass estimated by SDS-PAGE (-280 kDa) ofparanemin is likely due to the large number of acidic residues, which have been suggested to slow the rate of protein migration in SDS-PAGE (Hemken et al. 1997). Sequence analysis (Hemken et al. 1997; Yuan et al. 1997) also revealed that paranemin has the characteristic rod domain of IFs, thus establishing paranemin as a member of the IF protein superfamily, and not an IFAP. Comparison with other IF protein rod domains shows that the rod domains of frog tanabin (Hemmati-Brivanlou et al. 1992) and human nestin (Dahlstrand et al. 1992) are most similar to the paranemin rod domain, with - 60% identity; however, the overall sequence identities for the entire molecules are below 30% with no regions of homology existing within the long C-terminal tails. The rod domains of other IF proteins share at most only - 30% sequence homology with that of paranemin. 9

Napier et al. (1999) presented a genomic sequence for chicken paranemin/transitin, which classified paranemin as a type VI IF protein, along with nestin. Interestingly, the genomic sequence also showed that the entire pseudo-heptad repeat region found in the paranemin tail domain is contained within one exon, which is perplexing because cDNA clones from R T-PCR of the repeat region suggested some sort of alternative splicing was taking place (Yuan et al. 1997; Napier et al. 1999). Another interesting observation is that the sequence of paranemin/transitin has not been found within the human genome, or within any other mammalian genome to date (Hesse et al. 2001 ). Therefore, it has been suggested that paranemin/transitin is likely the avian ortholog of the mammalian protein nestin (Darenfed et al. 2001 ), even though the two proteins share no regions of homology outside of their rod domains. Recent studies involving stably, desmin-transfected SW13/cl.2 cells, which are an adenocarcinoma cell line that lacks an endogenous IF network, found that, in these and other non-muscle cells, desmin was incapable of forming extended filament networks on its own (Schweitzer et al. 2001 ). The desmin required the presence of an existing vimentin or keratin network or, interestingly, the coexpression ofparanemin, for formation of an extended IF network (Schweitzer et al. 2001 ). Coexpression of synemin or nestin in the stably, desmin- transfected cells did not result in formation of an extended desmin-containing IF network, although the synemin and nestin clearly colocalized with the desmin in these cells (Schweitzer et al. 2001 ). This suggests that paranemin may play a critical role in the attachments of desmin to other components of the cell cytoskeleton. An additional study involving localization of synemin, paranemin, and the IF AP plectin, utilized desmin-null transgenic mice (Li et al. 1997) for characterization of the localization of these proteins in the absence of desmin (Carlsson et al. 2000). In wild-type mice, paranemin, synemin, and plectin clearly localized with the IFs present between adjacent myofibrillar Z-lines and between Z-lines of the peripheral layer of myofibrils and the sarcolemma (Carlsson et al. 2000). However, in desmin-null mice, plectin remained near the Z-line, whereas both paranemin and synemin lost their Z-line association and were often localized near the sarcolemma (Carlsson et al. 2000). This differential localization in the absence of desmin suggests that the large IF proteins, such as paranemin and synemin, likely 10 play a different role than that of plectin and other IF APs as cytoskeletal linkers in muscle cells. Clearly, the relationship between these two sets of likely cytoskeletal cross-linkers needs to be studied in more detail.

Components of myofibrils and other components of the skeletal muscle cytoskeleton

The skeletal muscle sarcomere is a complex structure composed of numerous proteins encompassed within several distinct but interconnecting structural components, namely the A-band, I-band, H-zone, M-line and Z-line (for detailed reviews, see Huxley 1972, 2000; Littlefield and Fowler 1998; Gregorio et al. 1999). To preserve the internal focus of this literature review, the mechanisms of muscle contraction will not be included and only proteins with known IF interactions and key proteins involved in skeletal muscle cell myogenesis will be discussed. The sarcomeric A-band contains thick filaments composed primarily of myosin, which is the major protein found in mature muscle cells. Myosin molecules in each half of a thick filament are aligned antiparallel to those in the other half, creating a bare pseudo H- zone (Huxley 1972; Squire 1997). Within the pseudo H-zone is the M-line, where several proteins, including myomesin, M-protein, and the C-termini of titin molecules, act to connect adjacent myosin filaments (Obermann et al. 1996, 1997). The sarcomeric I-band contains a centrally located Z-line and consists of thin filaments composed primarily of actin, which is the second most abundant protein in muscle. Thin filaments on one side of a Z-line are anchored antiparallel to those on the other side of the same Z-line, and they extend, within their respective sarcomeres, into the A-band regions containing the thick filaments (Squire 1997). Tropomyosin and troponin, which act as regulatory protein molecules, are located along the entire length of the thin filaments (Squire

1997). Another component of skeletal muscle thin filaments is the large protein nebulin, ~ 800 kDa, which stretches from the free end of the thin filaments into the Z-line, where its C- terminus is firmly embedded (Moncman and Wang 1996). 11

Titin, another major sarcomeric protein, is the largest known single polypeptide with a molecular mass of-3,000 kDa and a length over 1 µm (Labeit et al. 1992, 1997; Stromer 1998). The C-terminal regions of two titin molecules coming from the two Z-lines defining a given sarcomere overlap in an antiparallel fashion in the M-line region (Obermann et al. 1996; Gregorio et al. 1999). The long titin molecule evidently interacts with thick and thin filaments along its entire length as it stretches from the Z-line, where its N-terminus is firmly anchored and overlaps with the N-termini of titin molecules extending into the adjacent sarcomere (Sorimachi et al. 1997; Squire 1997; Gregorio et al. 1999). Another recently identified titin-related protein is obscurin (- 800 kDa), which has also been found within sarcomeres of skeletal and cardiac muscle cells (Young et al. 2001). Interestingly, obscurin has been localized within or near both Z-lines and M-bands, although it is incapable of stretching that distance, suggesting a possible rearrangement in its localization from Z-lines in early developing sarcomeres to M-lines in mature sarcomeres (Bang et al. 2001; Young et al. 2001). The Z-lines of skeletal muscle sarcomeres are essential for muscle cell integrity, because they evidently absorb much of the force generated by muscle contraction (Young et al. 1998). The desmin-containing IFs surround the periphery of myofibrillar Z-lines and connect adjacent myofibrils to each other (Richardson et al. 1981; Tokuyasu et al. 1983; Stromer 1998). These desmin-containing IFs also connect the myofibrillar Z-lines to costameres, which are points of connection between the sarcolemma and the peripheral layer of myofibrils (Pardo et al. 1983b; Chou et al. 1997). A major protein component in costameres is vinculin (Pardo et al. 1983a, 1983b), which binds to cytoskeletal proteins such as actin (Johnson and Craig 2000), talin (Bass et al. 1999), and a-actinin (McGregor et al. 1994). The costamere presumably acts as a multi-protein bridge between the cell cytoskeleton and the extracellular matrix (Xu et al. 1998). Another protein likely to be involved in cytoskeletal linkages between costameres and the myofibrillar Z-line is the IF AP plectin (Hijikata et al. 1999; Schroder et al. 1999, 2000). Plectin, a member of the plakin family of adhesion plaque proteins, is an> 500 kDa protein believed to act as a cross-linker between different components of the cytoskeleton because it is capable of binding to IFs, microtubules, microfilaments, a-spectrin, and integrin P4 (Yang 12 et al. 1996; Geerts et al. 1999). Gene knockout studies of plectin have resulted in more severe defects than those seen with knockouts of most of the IF proteins. The mice died within three days from severe skin disorders and also had symptoms resembling the human disorder EBS with muscular dystrophy (Andra et al. 1997).

Cytoskeletal muscle proteins during myofibrillogenesis

The details of early skeletal muscle myogenesis have been studied extensively over many years, but there has been no clear consensus of the actual progression of protein expression and structural alignment, especially within a cell culture system. Most researchers agree that desmin is the first muscle cell-specific protein expressed after cells leave the pre-myoblast stage of cell replication (Lin et al. 1994 ). Myosin, actin, ti tin, nebulin and a-actinin are all expressed shortly thereafter, but their precise sequence of expression and structural alignment remain under debate. The differing views may result from several differences in the methods and systems used by researchers to study the early stages of myogenesis (e.g., see Moncman and Wang 1996 for discussion). First of all, there seem to be differences in the methods used for harvesting myoblasts, in that some labs pre-plate the cells to remove fibroblasts, possibly removing myogenic cells at the earliest stages of myogenesis (Hill et al. 1986; Colley et al. 1990). Second, myofibrillogenesis studies are being carried out by utilizing several species, specifically chicken (Lin et al. 1994), mouse (Babai et al. 1990), rat (Babai et al. 1990), or human (van der Ven et al. 1999). The third, and perhaps most likely, problem is differences in the antibodies used by different labs; this is especially true for the localization of the large proteins titin and nebulin, which stretch from the Z-lines into the A-band (Moncman and Wang 1996). Although these differences may present difficulties in the interpretation and comparison of results in different studies, cell culture remains a very good and widely used model system for studying the early stages of myogenesis. The majority of replicating presumptive myoblasts express vimentin, but not desmin, nor do they express myofibrillar proteins (Hill et al. 1986; Schultheiss et al. 1991 ). A subset 13 of these replicating presumptive myoblasts then begins producing desmin, in addition to vimentin (Hill et al. 1986; Lin et al. 1994). According to Moncman and Wang (1996) and others (e.g., Furst et al. 1989; van der Ven et al. 1993), desmin expression is followed sequentially by actin, myosin, titin and then nebulin. As stated earlier, the exact order of structural assembly during myofibrillogenesis is still not clear. One report (Moncman and Wang 1996) states that actin is first incorporated into nascent myofibrils, while myosin is the first protein to mature into a striated localization, followed by titin, nebulin, actin and, finally, desmin, after striated myofibrils are completely aligned. Another report (van der Ven et al. 1999) suggests that titin and myomesin are the first proteins with striated localization. Yet a different lab (Komiyama et al. 1992) suggested that nebulin is the first protein having a striated localization pattern. Localization of IFs has been followed in cultured avian skeletal muscle cells at several stages of development. Immunofluorescence localization studies by Granger and Lazarides (1980) showed that synemin, vimentin, and desmin colocalize within longitudinally-dispersed cytoplasmic filaments in five-day myotubes, but these filaments show no Z-line alignment at this stage. Paranemin and vimentin also colocalize within cytoplasmic filaments at four days in development by immunofluorescence (Breckler and Lazarides 1982; Lazarides et al. l 982a, l 982b ). At 9-10 days in development, paranemin, synemin, desmin and vimentin can be found at the Z-lines in salt- and Triton X-100-extracted cells (Granger and Lazarides 1980; Breckler and Lazarides 1982).

A role for intermediate filaments during development

Intermediate filaments do not seem to be directly associated with myofibrils until after the myofibrils have matured. Schultheiss et al. ( 1991 ), for instance, showed that disruption of the IF network by incorporation of truncated desmin and formation of aggregates of desmin did not interfere with myofibril assembly in cultured skeletal muscle cells. Furthermore, results of desmin knock-out studies (Milner et al. 1996; Li et al. 1997) indicate that myofibrils can assemble in the absence of desmin. Therefore, the role ofIFs in 14

the development of skeletal muscle remains uncertain. One possible role for IFs in development of skeletal muscle cells is maintenance of cell shape and indirect alignment of nascent myofibrils as the cells rapidly expand to form long myotubes (Stromer 1990). Other results suggest an interaction, or at least colocalization, of IFs and nascent myofibrils at the early stages of myofibril assembly. Holtzer et al. (1985) reported a correlation between desmin and vimentin localization and the localization of myofibrillar proteins in the growth tips of developing myotubes, which are areas of the cell undergoing active addition of new sarcomeres to the existing striated myofibrils (Ojima et al. 1999). Additionally, Yang and Makita (1996) showed by immunoelectron microscopy the presence of desmin-containing filaments associated with early developing myofibrils in human fotal skeletal muscle cells. Finally, van der Ven et al. (1993) found an interaction between desmin-containing IFs and titin in the earliest stages of development in human skeletal muscle cells. Apparently, the nascent titin interacts with IFs while in an aggregated form, before titin migrates to nascent myofibrils where it is then unraveled (van der Ven et al. 1993; van der Loop et al. 1996). The studies included within this thesis should corroborate and also clarify some of the information previously published regarding the role of IFs during skeletal muscle cell development. My studies utilize protein biochemistry to characterize interactions among the IF proteins paranemin, synemin, and desmin. Immunofluorescence and immunoelectron microscopy are used to characterize the localization of these IF proteins during skeletal muscle cell development.

References

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MOLECULAR INTERACTIONS AMONG THE INTERMEDIATE FILAMENT PROTEINS P ARANEMIN, SYNEMIN AND DESMIN AND THEIR LOCALIZATION DURING SKELETAL MUSCLE CELL DEVELOPMENT

A paper to be submitted to Histochemistry and Cell Biology

Stephanie A. Lex, Ted W. Huiatt, Marvin H. Stromer, and Richard M. Robson

Abstract

Paranemin and synemin are unique members of the intermediate filament (IF) protein family and are present with vimentin and/or desmin during skeletal muscle development. We have followed the colocalization of paranemin, synemin, and desmin during skeletal muscle myogenesis at different stages of development by immunofluorescence and immunoelectron microscopy. Localization of paranemin precedes that of desmin in cultured avian skeletal muscle presumptive myoblasts. Paranemin, synemin and desmin then colocalize throughout skeletal muscle cell development but, in relation to desmin and synemin, paranemin immunofluorescence comparatively increased at the growth tips of elongating myotubes and decreased in areas of the cell where the myofibrils are in a more mature pattern of alignment had occurred. The IF proteins colocalized within longitudinal filaments dispersed around/between myofibrils, but there was no IF colocalization with the myofibrillar Z-line protein a-actinin until after myofibril alignment. At the ultrastructural level, IFs were observed near the ends of early nascent myofibrils and at cytoskeletal filament junctions. A direct interaction of paranemin with both desmin and synemin was demonstrated by in vitro binding assays. Furthermore, expression of GFP-tagged paranemin rod domain in cultured myotubes resulted in colocalization with, and eventual aggregation of, the desmin-containing IFs, but did not noticeably affect myofibril assembly and alignment. Rather than playing a direct role in myofibrillogenesis, it is likely that IFs play a role at the next level of cytoskeletal organization by preserving the overall shape and integrity of developing muscle cells as they grow into elongated myotubes. 27

Keywords Intermediate filaments, Desmin, Synemin, Paranemin, Myogenesis

Introduction

There are three types of cytoskeletal filaments within the typical animal cell, namely actin-containing microfilaments, tubulin-containing microtubules and IFs. The IF protein superfamily consists of a large number of tissue- and cell- specific proteins with each containing a characteristic - 310 amino add rod domain that is responsible for the initial coiled-coil interactions involved in oligomerization and in eventual filament formation (Fuchs and Cleveland 1998). The IF proteins in skeletal muscle cells include desmin, vimentin, synemin, paranemin, nestin, and syncoilin (Sjoberg et al. 1994; Hemken et al. 1997; Bellin et al. 1999; Coulombe et al. 2001; Poon et al. 2002). The IFs in mature skeletal muscle cells surround the periphery of individual myofibrils at their Z-lines, connect adjacent myofibrils and nearby mitochondria and nuclei, and connect the peripheral layer of myofibrils to the sarcolemma (Richardson et al. 1981; Tokuyasu et al. 1983; Hijikata et al. 1999; Reipert et al. 1999). During early stages of skeletal muscle cell myogenesis, the IFs are predominately dispersed as longitudinal filaments running in parallel with the developing myofibrils (Hill et al. 1986; Schultheiss et al. 1991). The IFs do not migrate to the Z-lines until after the striated myofibrils are completely aligned within the cell (Lazarides et al. 1982b; Hill et al. 1986; Moncman and Wang 1996). Although the cellular function of IFs remains unclear, they are generally believed to provide mechanical strength and to help maintain the overall shape of cells (Chou et al. 1997; Galou et al. 1997). The precise role of IFs in striated muscle cells also remains unclear. Transgenic, desmin-null mice are viable, fertile and have relatively normal, aligned, striated myofibrils at birth (Li et al. 1997). Furthermore, abnormalities associated with the lack of desmin are not obvious until after birth, and then severe disruptions were observed only in weight-bearing and continuously used muscles, such as the soleus, diaphragm, and heart, thereby suggesting a role for IFs in the overall structural integrity of striated muscle cells (Li 28 et al. 1997). Schultheiss et al. ( 1991) also showed that introduction of truncated desmin into cultured skeletal muscle cells disrupted the endogenous IF network and formed IF aggregates, but had no obvious effect upon myofibrillogenesis. Thus, IFs may not have a direct role in myofibrillogenesis, but rather may be responsible for preserving the overall shape and integrity of cells as they grow into elongated myotubes, until the developing myofibrils become sufficiently rigid to contribute significantly to maintaining cellular shape (Tokuyasu et al. 1983; Stromer 1990). It also has been suggested (Schultheiss et al. 1991; van der Ven et al. 1993) that Ifs may play a role in the transport of myofibrillar proteins or their transcripts throughout myoblasts and myotubes in the earlv stages of myogenesis. The majority of replicating avian presumptive myoblasts express vimentin, but not desmin, nor myofibrillar proteins (Hill et al. 1986; Schultheiss et al. 1991 ). A subset of these replicating presumptive myoblasts then begins expressing desmin, along with the vimentin (Hill et al. 1986; Lin et al. 1994). Desmin expression is then followed by the expression of myofibrillar proteins, such as actin, myosin, titin, nebulin and a-actinin (Lin et al. 1994; Moncman and Wang 1996). Synemin (Granger and Lazarides 1980), paranemin (Breckler and Lazarides 1982), and nestin (Sejersen and Lendahl 1993) are present with desmin and vimentin in early stages of skeletal muscle cell myogenesis, but the expression levels of nestin and paranemin decrease as the cells mature. Synemin (- 230 kDa by SDS-PAGE) and paranemin (- 280 kDa by SDS-PAGE) were originally referred to as IF-associated proteins (IF APs) because of their co localization and copurification with desmin (Lazarides et al. 1982a, 1982b). Analysis of the cDNA sequence of synemin revealed synemin is in fact an IF protein because it contains the characteristic IF rod domain (Becker et al. 1995; Bellin et al. 1999). Paranemin was also shown to contain the characteristic IF rod domain simultaneously by our lab (Hemken et al. 1997) and under the name oftransitin by Yuan et al. (1997). A direct interaction between synemin and desmin has recently been shown by in vitro blot overlay and cosedimentation assays (Bellin et al. 1999). Additionally, the synemin rod domain interacts with both desmin and vimentin as shown by yeast two-hybrid analysis (Bellin et al. 2001 ). Our studies herein demonstrate for the first time a direct interaction of the paranemin rod domain with both desmin and synemin in vitro, as well as a direct interaction of the paranemin rod domain with 29 the synemin rod domain, desmin, and vimentin by yeast two-hybrid assays. These results suggest that heteropolymeric IFs containing all four IF proteins (vimentin, desmin, paranemin, and synemin) likely exist at some stages ofmyogenesis. The localization of synemin and paranemin has been examined in cultured avian skeletal muscle cells at a few stages of development. Granger and Lazarides ( 1980) reported that by immunofluorescence localization, synemin, vimentin, and desmin colocalize within longitudinally-dispersed cytoplasmic filaments in five-day myotubes, but do not exhibit Z- line alignment at this stage in development. Paranemin and vimentin also were shown to colocalize within cytoplasmic filaments at four days in development by immunofluorescence (Breckler and Lazarides 1982; Lazarides et al. 1982a, 1982b). At 9-10 days in culture, paranemin, synemin, desmin and vimentin were present at the Z-lines of salt- and Triton X- 100-extracted skeletal muscle cells (Granger and Lazarides 1980; Breckler and Lazarides 1982). One difficulty in interpretation of those studies is that the specific localization of the synemin and paranemin was not studied in direct comparison to myofibrillar proteins, although there have been several reports on the localization of desmin in comparison to myofibrillar proteins, such as a-actinin (Lazarides et al. 1982b ), nebulin (Moncman and Wang 1996) or myosin and titin (Hill et al. 1986). Therefore, localization of synemin and paranemin has not been clearly correlated with other events in myogenesis. The studies presented herein extend those ofBreckler et al. (1982) and Granger and Lazarides (1980) by examining the immunofluorescence localization of desmin, paranemin, synemin, and a- actinin within avian skeletal muscle cells at 2, 4, 8, and 12 days in culture. The immunofluorescence localization of vimentin, desmin, and paranemin in one day cultured skeletal muscle cells demonstrates for the first time that paranemin expression precedes expression of desmin in skeletal muscle presumptive myoblasts. Immunoelectron microscopy (IEM) has been used to more precisely show the ultrastructural localization of paranemin, synemin and desmin within cultured skeletal muscle cells. 30

Materials and methods

Cloning, protein expression and purification

The cDNAs for paranemin domain constructs were cloned, via specific PCR primers (produced by the Iowa State University DNA Sequencing and Synthesis Facility) with incorporated restriction enzyme recognition sites, into the pProEx HT prokaryotic 6X-His- tagged expression vector (Gibco BRL/Invitrogen) for bacterial expression. The cDNA constructs for full-length paranemin, paranemin rod domain, and sub-domains of the long paranemin tail were expressed in the BL21 CodonPlus strain of E. coli (Stratagene ). The bacterial cells were then spun down and resuspended in lysis buffer (50 mM sodium phosphate buffer, 6 M urea, 1 M NaCl, 10 mM P-mercaptoethanol, 10% glycerol, 0.5% Tween-20, pH 7.8). The cell lysate was stirred overnight at 4°C and then clarified by centrifugation at 100,000 x g for 1 h. The supernatant was then resuspended with Nickel NTA agarose resin (Qiagen), rotated slowly 12-16 hat 4°C, and loaded onto a column with the resin. The column was washed and eluted by a step-wise gradient of 7.8, 7.0, 6.0, and 5.0 pH buffers (50 mM sodium phosphate buffer, 6 M urea, 1MNaCl,10 mM P- mercaptoethanol, 10% glycerol, 0.5% Tween-20). Synemin (Bilak et al. 1998), desmin (Huiatt et al. 1980), and filamin (Shizuta et al. 1976) were purified from turkey gizzard by standard methods. Paranemin was purified from 14-day embryonic chick skeletal muscle as previously described (Hemken et al. 1997).

Antibodies

The polyclonal antibody (pAb) against gel-purified chicken gizzard desmin, and the paranemin pAb 3146 against purified, bacterially expressed paranemin rod domain were produced by the Iowa State University Cell and Hybridoma Facility. Antibodies were characterized by Western blotting of different tissue homogenates and found to bind specifically to desmin and paranemin, respectively. Further characterization of the 31 antibodies by irnrnunofluorescence labeling of cultured myoblasts showed labeling identical to that of other previously characterized antibodies against desmin and paranemin. The anti- synemin pAb 2856 (Bellin et al. 1999) and the an~i-paranemin monoclonal IgM antibody (mAb) 4D3 (Hemken et al. 1997) were described previously. Vimentin mAb AMF-17b (developed by Dr. A. B. Fulton) and desmin mAb D76 (developed by Dr. D. A. Fischman) were obtained from the Developmental Studies Hybridoma Bank. The mAbs against sarcomeric a-actinin (EA-53) and desmin (DE-U-10), as well as HRP-conjugated secondary antibodies for Western blotting, were purchased from Sigma. Secondary antibodies conjugated to Alexa Fluor 350, Alexa Fluor 488 and Alexa Fluor 546 dyes from Molecular Probes were used for immunofluorescence microscopy. AuroProbe 5 and 10 nm gold-conjugated secondary antibodies for IEM were from Amersham Pharmacia.

Western blotting, overlays and cosedimentations

Methods for SDS-PAGE and Western blots were essentially those described by Laemmli (1970) and Towbin et al. (1979), respectively. Western blots were detected by ECL according to the manufacturer's instructions (Amersham Pharmacia). Homogenates of embryonic chicken gizzard and of adult turkey gizzard for use in SDS-PAGE and Western blotting studies were produced by methods previously described (Bilak et al. 1998). Blot overlay assays for detection of protein interactions were done as described (Bellin et al. 1999). Samples of embryonic chicken gizzard homogenate, adult turkey gizzard homogenate, purified filamin, and purified desmin or synemin were subjected to SDS-PAGE and transferred to nitrocellulose. The nitrocellulose blots were then incubated with 5 µg of purified, bacterially expressed, full-length paranemin or of paranemin rod domain in IF- forming buffer (100 mM NaCl, 1mMMgCh,10 mM imidazole-HCl, pH 7.0), probed with the anti-paranemin rod pAb 3146, incubated with secondary antibody, and processed for Western blot detection. 32

Cosedimentation assays for testing the interaction of the paranemin rod domain and desmin in vitro were done by methods similar to those described in Bellin et al. (1999). An equal amount (25 µg) of the paranemin rod domain and of desmin were mixed under desmin IF forming conditions (100 mM NaCl, 1 mM MgCh, 10 mM imidazole-HCl, pH 7.0) and centrifuged at 100,000 x g for 20 min. Each protein was also individually subjected to the identical treatment to test its sedimentation behavior in the absence of the other protein. Ten µg of bovine serum albumen (BSA) was added to each mixture before centrifugation to show that desmin was not sedimenting proteins non-specifically. The supematants were analyzed by SDS-PAGE.

Yeast two-hybrid assays

The cDNA constructs coding for desmin, vimentin and specific domains of paranemin or synemin were cloned into both the Gal4 transactivation domain vector pPC 86 or the Gal4 DNA binding domain vector pPC 62 or pPC 97 (pPC 62 and pPC 97 are identical vectors differing only within their cloning sites) (Chevray and Nathans 1992). Pairs of cDNA constructs attached to both the DNA binding domain and the transactivation domain of Gal4 were then co-transfected into the PCY2 strain of yeast using a kit and protocol from Q-Biogene. Because pPC 97 carries the gene to produce leucine and pPC 86 carries the gene to produce tryptophan, the yeast cells were plated onto agar plates with media lacking leucine and tryptophan thereby permitting selection of only the yeast containing both vectors. The growth media and procedures utilized in the yeast two-hybrid interaction analyses were essentially those of Meng et al. ( 1996). Colonies were picked from plates representing each construct/vector combination and streaked onto plates with galactose and glycerol-containing media, thus permitting detection of the ~-galactosidase produced from a positive interaction between two protein constructs. For screening the yeast, Whatman #1 filter paper was used to lift replica yeast colonies off the plates. The yeast cells were then cracked open by immersion in liquid nitrogen and stained with X-gal. The intensity of the X- 33 gal was then examined qualitatively by the degree of blue staining that occurred with each yeast sample.

Cell culture, immunofluorescence and eukaryotic cell transfection

Primary skeletal muscle myoblasts were cultured from breast muscles of 12 day chick embryos by methods previously described (Hill et al. 1986). For transfection studies, paranemin cDNA constructs were cloned into pEGFP vectors (Clonetech) for expression in eukaryotic cells controlled by the CMV promoter. Two µg of cDNA encoding for GFP- tagged paranemin rod domain was transfected into primary myoblasts with Fugene 6 (Roche Applied Science) eukaryotic transfection agent two days after cell plating. The pEGFP vector was also transfected into cells and the expressed GFP served as a control for non- specific protein expression. The transfected myoblasts and myotubes were then grown on collagen-coated coverslips for an additional 4 days before fixation. For immunofluorescence detection, transfected and non-transfected cells grown on coverslips were fixed with 3% paraformaldehyde in PBS at pH 7.2 for 10 min, washed with PBS and permeablized with 0.1 % Triton X-100 for 20 min. The coverslips were then blocked with 5% goat serum at 37°C for 30 min before incubation with primary antibodies at 3 7°C for 1 h. After washing in PBS the cells were incubated with secondary antibodies for 45 min, washed in PBS again and mounted on microscope slides with Gelvatol mounting media. Slides were visualized with either a Zeiss Photomicroscope III and SPOT RT Slider digital camera (Diagnostic Instruments, Inc.) or a Zeiss Axioplan 2. For preparation oflysates, 2, 4, 8, and 12 day cultured skeletal muscle cells were scraped from plates and suspended in lysis buffer (50 mM Tris, pH 7.5, 6 M urea, 100 mM NaCl, 2 mM each ofEGTA, PMSF, Leupeptin, and Aprotinin). The cell lysates were vortexed extensively before centrifugation at 13,000 x g at 4°C for 20 min. For analysis of cell lysates, 15 µg of total protein lysate from each sample was loaded onto gels for SDS- p AGE and then transferred to nitrocellulose for Western blotting. The nitrocellulose blots were probed with desmin mAb D76, paranemin pAb 3146, and synemin pAb 2856. 34

Immunoelectron microscopy (IEM)

For IEM, primary myoblasts were cultured in sterile Permanox 4-well Sonic Seal slides (Nalge-Nunc) for 4, 8, or 12 days. Methods for fixation and labeling were based primarily upon a combination of methods described by Colucci-Guyon et al. (1994) and Kouchi et al. (1993). Cells were washed briefly in PBS prior to fixation with 3% paraformaldehyde in PBS at pH 7.2 for 10 min, and then washed again before being permeabilized with 0.1 % Triton X-100 for 30 min. The cells were then blocked with 5% goat serum at 25°C for 1 h before incubation with primary antibody overnight at 4°C. After thoroughly washing in PBS, the cells were incubated with 5- and/or 10- nm diameter gold- conjugated secondary antibodies for 4 hat 25°C. As a secondary antibody control, cells were treated with secondary antibody only. The cells were then subjected to a secondary fixation with 2% glutaraldehyde in PBS for 15 min, followed by incubation with 1% osmium tetroxide in PBS for 30 min. The cells were then dehydrated in a series of acetone washes before infiltration and embedding in Epon-Araldite resin. Polymerized resin blocks were removed from the Sonic Seal slides, cut into blocks, and sectioned with a Reichart Ultracut S ultramicrotome. Sections were cut from myotubes in a longitudinal orientation. After staining with uranyl acetate and lead citrate, sections were examined with a JEOL JEM- lOOCX II. Negatives were scanned into the computer on an Agfa Arcus II scanner and image contrast was enhanced with Adobe Photoshop.

Results

Expression and purification of cDNA constructs and antibody characterization

A schematic of the paranemin molecule (Fig. 1 A) and a list of the cDNA constructs corresponding to individual domains and subdomains of paranemin produced by PCR (Fig. 1 B) are shown in Fig. 1. The individual paranemin constructs were ligated into bacterial, yeast two-hybrid, and GFP-tagged eukaryotic expression vectors. Purification of the 35 bacterially expressed domains and subdomains of paranemin required denaturing conditions for the solubilization of inclusion bodies. SDS-PAGE of purified paranemin rod domain is shown in Fig. 2 A (lanes 3 and 7) and purified paranemin tail domain and subdomains are shown in Fig. 2 B (lanes 1-4). The paranemin rod domain pAb 3146 was produced to permit specific immunological localization/identification of the rod domain in Western blotting, blot overlays, and immunocytochemistry. As shown in Fig. 2 A, the pAb 3146 bound specifically to bacterially expressed paranemin rod, partially purified paranemin, and to paranemin in the 12 day embryonic chick gizzard homogenates. The pAb 3146 did not interact with other IF or cytoskeletal proteins in homogenates of either adult turkey or 12 day embryonic chick gizzards (Fig 2 B, lanes 5 and 8, respectively). The paranemin mAb 4D3 described previously (Hemken et al. 1997) was shown to specifically interact with paranemin by Western blotting and immunofluorescence localization. The mAb 4D3 has now been further characterized and found to bind specifically to the Pn Tail II subdomain (i.e., the pseudo heptad repeat region) within the tail domain of paranemin (Fig. 2 B, lane 7) and not to Pn Tail I (lane 6) or to Pn Tail III (lane 8).

Blot overlays and cosedimentation assays

Blot overlay analysis was used to determine whether co localization of paranemin and desmin observed previously within cells (Breckler and Lazarides 1982; Hemken et al. 1997) reflects a direct interaction between the paranemin and desmin rod domains. Bacterially expressed full-length paranemin overlaid onto a nitrocellulose blot containing SDS-PAGE transferred purified desmin, bound strongly and specifically to purified desmin and to the desmin present in adult turkey gizzard homogenate (Fig. 3 B, lanes 2 and 4, respectively). The overlaid paranemin bound neither to the purified filamin (lane 3) nor to the other negative control proteins ( e. g., myosin heavy chains and actin) within the adult turkey gizzard homogenates (lane 4). The paranemin pAb 3146, which was used to probe for the overlaid paranemin, did not bind to any protein other than paranemin on the nitrocellulose 36 blots, as shown in the control blots (not overlaid) in Fig. 3 C and E (lane 4). Bacterially expressed paranemin rod domain overlaid onto these nitrocellulose blots also bound specifically to desmin (compare Fig. 3 D and E; lane 2). The full-length paranemin and the paranemin rod both bind to the small amount of vimentin found, which migrates slightly above desmin in adult turkey gizzard homogenate (Fig. 3 A, Band D; lane 4). The desmin and vimentin bands in the 12 day embryonic chicken gizzard homogenate (Fig. 3, lane 1) do not show labeling because the amount of protein loaded on the gel was kept very low to demonstrate the high degree of specificity of the paranemin pAb 3146. Expressed full-length paranemin and the expressed rod domain of paranemin, were also overlaid onto nitrocellulose blots from transferred SDS-PAGE gels containing purified synemin. The full-length paranemin did not bind to purified synemin on the nitrocellulose blot (Fig. 4 B, lane 2). However, the paranemin rod domain did bind to purified synemin (Fig. 4 D, lane 2). The latter results suggest that the full-length paranemin and synemin molecules may interact. The very large tail domains of paranemin and synemin may stearically hinder the interactions between the two rod domains because the synemin was denatured and bound to nitrocellulose before being overlaid by full-length paranemin. Overlays of the full-length paranemin, paranemin rod, and subdomains of the paranemin tail domain onto nitrocellulose blots containing a-actinin and vinculin did not clearly demonstrate specific interaction of paranemin with these two proteins (results not shown). The interaction of paranemin with desmin was also analyzed by cosedimentation of bacterially expressed paranemin rod with purified desmin under IF-forming conditions (Fig. 5). Desmin by itself formed filaments under IF-forming conditions and essentially all of it sedimented when centrifuged at high speeds (Fig. 5, panel 1). Approximately 60% of the paranemin rod domain sedimented by itself under IF-forming conditions after high speed centrifugation (Fig. 5, panel 2). However, over 90% of the paranemin sedimented with the desmin after high speed centrifugation when paranemin rod and desmin were mixed under IF-forming conditions (Fig. 5, panel 3). Similar results from cosedimentation assays of the synemin rod domain and desmin under IF forming conditions were observed by Bellin et al. (1999). The results in Fig. 5 demonstrate that paranemin and desmin also interact in vitro. 37

Yeast two-hybrid analysis

The yeast two-hybrid system was used to probe for interactions among paranemin, synemin, desmin, and vimentin. The cDNA constructs used in the IF yeast two-hybrid studies are shown in Table 1 A. The results in Table 1 B summarize the interactions between IF proteins or their protein domains within the yeast two-hybrid system. The paranemin rod domain interacted with itself, the synemin rod domain, and with desmin and vimentin. The synemin rod domain interacted with itself, the paranemin rod domain, and with desmin and vimentin. In contrast, the tail domains of paranemin and synemin did not interact with any domain of paranemin, synemin, or with desmin or vimentin within the yeast two-hybrid assays. The lack of detection of positive interactions involving the full-length paranemin and synemin tail domains may be due to difficulties in the two-hybrid system with use of large proteins (i.e., the produced products are 1,290 amino acids). The yeast two-hybrid system was also used to probe for interactions of different domains/subdomains of paranemin with domains ofvinculin and a-actinin. Neither the paranemin rod domain, nor any domain/subdomain of the paranemin tail domain was shown to interact with those two proteins (results not shown).

Immunofluorescence localization of IF proteins in cultured skeletal muscle cells at different stages of development

One day cultured skeletal muscle myoblasts were triple labeled with paranemin mAb 4D3 (lgM), vimentin mAb AMF-17b (IgG), and desmin pAb, to determine which IF proteins were expressed first (Fig. 6). All cells observed were labeled with the vimentin mAb. A subset of the cells was labeled with both the vimentin and paranemin mAbs, and another subset of the cells was labeled with antibodies to vimentin, paranemin, and desmin. These results suggest an additional step exists in the expression of IF protein during myogenesis, in which presumptive myoblasts express vimentin and paranemin before expression of desmin. 38

The localization of paranemin, synemin, desmin, and a-actinin in cultured avian skeletal muscle cells was examined by immunofluorescence at 2, 4, 8, and 12 days in culture. At two days (Fig. 7), paranemin and desmin were colocalized within longitudinally-dispersed cytoplasmic filaments present throughout the desmin-containing myoblasts and early developing myotubes (Fig. 7 A-C). As shown at one day in culture in Fig. 6, some cells express paranemin, but not desmin, at two days in culture (Fig. 7 C). Paranemin and synemin also colocalized at two days in culture (Fig. 7 D-F). Unlike colocalization ofIF proteins, synemin did not directly colocalize with the sarcomeric a-actinin, which was present in a striated pattern (Fig. 7 G-I). At four days in culture, paranemin and desmin (Fig. 8 A-C), as well as paranemin and synemin (Fig. 8 D-F), were colocalized within longitudinally-dispersed cytoplasmic filaments throughout the entire myotube, and at this stage the IFs were not visibly associated with myofibrils. At the growth tips (Fig. 8 A-F), which are at or near the ends of elongating myotubes where cells are undergoing active addition of new sarcomeres to existing striated myofibrils (Ojima et al. 1999), the levels of immunofluorescence ofparanemin, synemin and desmin were all increased in comparison to the elongated regions of the myotubes where the myofibrils are more mature. Furthermore, the level of paranemin immunofluorescence was proportionally much greater than the corresponding levels of desmin and synemin immunofluorescence within the growth tip areas, as was especially apparent in the merged images (Fig. 8 C and F). Near the outer edges of the growth tip regions, the levels of immunofluorescence of each of the three IF proteins were proportionally more equal. The synemin and a-actinin were not colocalized at this stage of development, as the synemin labeling appeared to surround the striated a-actinin labeling of the myofibrils (Fig. 8 G-I). At eight days in culture (Fig. 9), paranemin, synemin and desmin began to localize at Z-lines, but the IF proteins also still clearly localized within the cytoplasmic areas between and around developing myofibrils and at growth tips. Although synemin did not colocalize with a-actinin at the growth tips where myofibrils were still actively assembling, synemin did colocalize with a-actinin at Z-lines in regions of the myotube where adjacent myofibrils were aligned (Fig. 9 G-I). At 12 days in culture (Fig. 10), the synemin localizes strongly at the Z-lines (Fig. 10 E and G) and the level ofparanemin expression has decreased in 39 comparison to that of desmin (Fig. 10 A-C) and of synemin (Fig. 10 D-F). Desmin and paranemin also localize at the Z-lines (Fig. 10 A-C), but seemingly less strongly than does synemin. In cells double labeled with synemin and paranemin, the synemin is more closely associated with the Z-lines than is the paranemin (Fig. 10 D-F). At this stage, there is still cytoplasmic localization of IF proteins between adjacent myofibrils, as well as between the peripheral layer of myofibrils and the sarcolemma. Synemin clearly co localizes with a- actinin at Z-lines during these late stages of development (Fig. 10 G-I).

W estem blot analysis of cell lysates

Lysates of skeletal muscle cells cultured for 2, 4, 8, and 12 days were analyzed by SDS-PAGE (Fig. 11 A) and Western blotting (Fig. 11 B-D). An equal quantity of protein for each cell lysate was run on gels. The amount of desmin within the cell lysates increased from two to four days in culture, remained relatively constant at eight days, and decreased slightly at 12 days in culture. In contrast, the amounts of paranemin and synemin present in the cell lysates increased from two to four days in culture, and then decreased considerably during development as observed in the lysates from the 8 and 12 day cultures. The synemin in each of these skeletal muscle cell lysates (Fig. 11 D) appears as a doublet, where at two days in culture (lane 1) both bands label weakly but the lower band appears to predominate; at four and eight days (lanes 2 and 3) both bands are clearly visible, and at twelve days (lane 4) the upper band predominates. The reason for two bands is not clear but possibilities include differences in posttranslational covalent modification, such as phosphorylation (Inagaki et al. 1996) or ADP-ribosylation (Yuan et al. 1999), the presence of two isoforms as observed in human synemin (Titeux et al. 2001 ), or from differing degrees of proteolysis (Bilak et al. 1998). 40 lmmunoelectron microscopy localization of IFs during development

The IFs can clearly be seen to be localized as longitudinally dispersed cytoplasmic filaments at the ultrastructural level in early developing skeletal muscle cells. The ultrastructural detail achieved with pre-embed labeling of the cultured skeletal muscle cells shows labeling of individual IFs in the cytoplasm with antibodies to paranemin, synemin, and desmin (Figs. 12 and 13). Paranemin-containing IFs are clearly found within longitudinally- dispersed cytoplasmic filaments in the sarcoplasm (Fig. 12 A and C) and along side myofibrils in the process of assembly and alignment (Fig. 12 D). Labeling ofparanemin is also observed in groups of 5 nm gold particles on aggregates associated with IFs, as shown by arrows in Fig. 12 A and at higher magnification in Fig. 12 B. Synemin and desmin labeling of longitudinally-dispersed IFs along side of nascent myofibrils is also evident in Fig. 13 A, where the IFs are located just below a myofibrillar Z-body, i.e., a developmental precursor to Z-lines. As shown in Fig. 13 Band C, a high level of desmin labeling is also observed in the growth tips of actively elongating myotubes. The desmin-containing IFs are spread throughout the growth tip (Fig. 13 C), but there is no labeling of desmin within the nascent myofibrils themselves. The paranemin and synemin labeling in Fig. 12 A, and the desmin labeling in Fig. 13 D, suggests IFs may associate with the growing ends of early, nascent myofibrils.

Paranemin rod transfections

A cDNA construct encoding GFP-tagged paranemin rod domain was transfected into two day cultured skeletal muscle cells. The cells were then grown for an additional four days and then fixed and labeled with desmin pAb. At low levels of expression, the GFP-tagged rod domain colocalized with desmin in longitudinally-dispersed cytoplasmic filaments (Fig. 14 A-C, bottom myotube). At higher levels of expression, the GFP-tagged rod domain accumulated into granular appearing aggregates present throughout the cytoplasm, with the quantity and size of the aggregates appearing to enlarge as the level of protein expression 41

increases within the cell (Fig. 14 A, top myotube). The endogenous desmin-containing Ifs appear to be disrupted and the desmin was colocalized with the aggregates containing the expressed GFP-tagged rod domain (Fig. 14 D-G). Aggregated and unaggregated GFP-tagged rod domain and the endogenous desmin are still present at the Z-line in mature myotubes (Fig. 14, D-G, arrows). The aggregates oflF proteins do not affect localization of sarcomeric a-actinin at the Z-lines until after the aggregates enlarge and completely fill the interior of the cells (data not shown).

Discussion

The unusually large IF proteins paranemin and synemin are an integral part of the heteropolymeric desmin-containing Ifs in muscle. We are exploring the role of these large IF proteins in skeletal muscle cells. Based upon recent studies in our lab (Bellin et al. 1999, 2001 ), which demonstrated that synemin interacts with the IF proteins desmin and vimentin, and with the cytoskeletal proteins a-actinin and vinculin, it is likely that synemin may help link the Ifs to the Z-lines of adjacent myofibrils and the peripheral layer of myofibrils to the costameric sites along the sarcolemma. In the studies presented herein, we show that paranemin interacts with desmin and vimentin, and also via its rod domain with the synemin rod domain. However, we have not identified a clear interaction of paranemin with either a- actinin or vinculin, thus it seems likely that paranemin plays a different, unique role of its own within muscle cells. Supporting evidence for a special role for paranemin in cells was demonstrated by Schweitzer et al. (2001) in SW13/cl.2 cells stably transfected with desmin. In the IF-free SW13/cl.2 cells, desmin did not form normally extended IF networks in the absence of other IF networks. However, expression oftransfected paranemin, but not of synemin or of nestin, resulted in the formation of an extended desmin/paranemin-containing IF network (Schweitzer et al. 2001). These results also suggest that paranemin may have yet to be discovered interaction partners at the cell membrane that are different from those of synemm. 42

Although it is uncertain whether paranemin and synemin form heterodimers in vivo, the protein interaction studies presented herein show that paranemin interacts with desmin and vimentin and also, via its rod domain, with the synemin rod domain. In vitro interactions involving synemin or the synemin rod domain with desmin have been described previously (Bellin et al. 1999, 2001). The results of yeast two-hybrid assays presented herein involving interactions of the synemin rod and tail domains with either desmin or vimentin are consistent with our previous results (Bellin et al. 2001). The interaction in vitro of paranemin with both desmin and vimentin was previously suggested by overlays with IF APa- 400, a protein with a nearly identical partial cDNA sequence (Duval et al. 1995) to that of paranemin, and now considered the same protein as paranemin (Hemken et al. 1997). Our results of protein interactions from blot overlays, cosedimentation assays, and yeast two- hybrid analysis all clearly demonstrate that paranemin interacts via its rod domain with desmin and vimentin. Additional evidence ofparanemin rod/desmin interaction was obtained herein which shows that expression of the paranemin rod domain is capable of not only binding to, but also of disrupting, endogenous desmin-containing IFs in cultured skeletal muscle cells. The results of protein interactions of paranemin and synemin with desmin and vimentin all indicate that these proteins could form heteropolymeric IFs in vivo. We have for the first time herein presented a complete set of studies of the co localization of synemin and paranemin with either desmin or vimentin in avian myogenic cell cultures. Synemin, paranemin, desmin and vimentin colocalize in embryonic avian striated and smooth muscle cells (Price and Lazarides 1983). Synemin, paranemin, vimentin, and desmin are also all expressed early in cultured avian skeletal muscle cells (Lazarides et al. 1982a, 1982b ), but the expression of synemin and paranemin has not heretofore been carefully described in detail throughout development in cultured cells. In agreement with results of Granger and Lazarides (1980) and Breckler and Lazarides (1982), synemin, paranemin and desmin colocalize at early stages of skeletal muscle cell development. We show, however, that expression of paranemin precedes desmin expression in skeletal muscle presumptive myoblasts. It appears, therefore, that paranemin may be the first muscle specific IF protein expressed in cultured skeletal muscle presumptive myoblasts. Double labeling of synemin 43 and paranemin revealed differences in labeling with development, especially within the growing ends or growth tips of myotubes where the level of paranemin immunofluorescence is comparatively higher than the levels of either synemin or desmin. It was previously reported that the IFs migrate from a longitudinally-dispersed localization pattern around maturing myofibrils to a Z-line alignment pattern, after the myofibrils have completely aligned (e.g., Hill et al. 1986; Moncman and Wang 1996). The results of double labeling with paranemin and synemin herein suggest that in the later stages of development synemin either migrates more rapidly to the myofibrillar Z-lines, or has a stronger association with Z- lines, than does paranemin. The latter results are based upon the observations that synemin has a more distinct Z-line localization at 12 days in culture than does paranemin, which exhibits a much more diffuse localization around the Z-lines. We report herein the first ultrastructural localization results of the IF proteins synemin and paranemin in developing skeletal muscle. Pre-embed labeling preserved much of the ultrastructure of the developing myotubes, while simultaneously maintaining orientation within the sample and incorporation of antibodies. Individual filaments were clearly visible and, although the amount of label with IEM is decreased in comparison to post-embedding techniques, the label is very specific. Tokuyasu and associates (1985) showed that in the early and intermediate stages of myogenesis the IFs are localized in longitudinally-dispersed networks between adjacent developing myofibrils and between the outer layer of developing myofibrils and the sarcolemma. Similar to these findings, we found that the overall number ofIFs per volume of the myotube decreased as myofibrils matured. Thus, the concentration of IFs was highest in the early developing myotubes containing few nascent myofibrils, and in the growth tips of myotubes undergoing extension where myofibrillogenesis was actively occurring. We also found increased IF localization at the ends of nascent growing myofibrils in the developing cultured myotubes, which is in keeping with findings by Yang and Makita (1996) in studies on human fetal skeletal muscle. We found that the labeling of paranemin and synemin often occurred in areas of cytoskeletal junctions between converging IFs and possibly other filament networks, which is consistent with the hypothesis that the large IF proteins act as cytoskeletal cross-linkers between different components of the cell cytoskeleton (Bellin et al. 2001 ). Paranemin labeling was 44 also clearly visible in aggregates near IF junctions. Similar appearing aggregates of titin were found associated with IFs in developing myotubes (van der Ven et al. 1993), but it is unknown whether these are the same type of aggregates. A homolog to the avian IF protein paranemin is not present within the draft sequence of the human genome (Hesse et al. 2001). The closest related ortholog of paranemin is human nestin, which has 48.5% identity within its rod domain to the rod domain of paranemin (Hemken et al. 1997). However, paranemin and nestin exhibit only an overall sequence identity of~ 25%, and exhibit no regions of homology within their huge C-terminal tail domains (Hemken et al. 1997). Analysis of the genomic sequence of chicken transitin, a protein almost identical (~ 99%) in sequence and therefore presumably the same protein as paranemin, suggests that transitin/paranemin and nestin have similar gene structure and are related evolutionarily (Napier et al. 1999; Darenfed et al. 2001). Synemin, in contrast, has been identified within the human genome by Titeux et al. (2001) and under the name desmuslin by Mizuno et al. (2001). Chicken synemin and human synemin share 46% identity within their rod domains. Furthermore, the overall sequence of human synemin, which has two isoforms (larger a and smaller~ synemin) resulting from a deletion within the tail domain, exhibits 3 7% identity for a synemin and 29% identity for ~ synemin to chicken synemin (Titeux et al. 2001). Unlike paranemin and nestin, chicken and human synemin orthologs contain regions of significant homology outside of the characteristic IF rod domain. The results presented herein indicate in toto that IFs do not play a direct role in myofibrillogenesis, but do play an important role in the next higher level of cytoskeletal organization in development of skeletal muscle cells. The IFs fill areas in early developing skeletal muscle cells, especially the growth tips, which are subsequently filled with myofibrils. As the assembling myofibrils undergo elongation, the IFs present at the ends of the nascent myofibrils may function by helping to guide insertion/addition of myofibrillar proteins into existing myofibrils, or possibly by helping in transporting components necessary for myofibrillogenesis, as previously suggested (Schultheiss et al. 1991; van der Ven et al. 1993). It is also very likely that, following myofibrillogenesis, the IFs help maintain overall cytoskeletal integrity within the muscle cell. 45

The IFs in developing avian skeletal muscle cells are very likely heteropolymeric in composition, containing desmin, vimentin, synemin, and paranemin. The large IF proteins paranemin and synemin, with their long extended tail domains, are likely vital in cell development. This is most clearly the case for synemin, which may enable the IF network to link adjacent myofibrils and the peripheral layer of myofibrils to the sarcolemma. Elucidating the role for paranemin has been more difficult, because, as of yet, no direct non- IF protein interaction partners have been clearly established. Paranemin is expressed with vimentin before the expression of desmin in cultured skeletal muscle presumptive myoblasts. We have demonstrated for the first time an interaction between paranemin and vimentin within a cellular environment by yeast two-hybrid assays. We also have shown clearly in blot overlay assays, cosedimentation assays, and yeast two-hybrid analysis that paranemin, via its rod domain, binds desmin, and that when the paranemin rod domain is transfected into cultured skeletal muscle cells, it colocalizes with the desmin and disrupts the endogenous desmin-containing IF network. We also have shown for the first time that paranemin, via its rod domain, interacts directly with synemin in vitro. Taken in toto, we have demonstrated interactions among the four muscle cell IF proteins, characterized their specific localization through development in culture, and increased our understanding of the function of the unique IF proteins paranemin and synemin within developing skeletal muscle cells.

Acknowledgments

We thank Robert Bellin and Diana Walker for discussions pertaining to this research, Suzanne Semett for assistance with protein purification and conducting the paranemin/desmin cosedimentation assays, Emily Helman for preparation and Western blotting of muscle cell lysates, and Mary Sue Mayes for help and discussions regarding digital imaging, immunofluorescence imaging and immunoelectron microscopy. We also thank the Bessy Microscopy Facility, Iowa State University, for use of the Zeiss Axioplan 2 microscope. The Developmental Studies Hybridoma Bank was developed under the auspices 46 of the NICHD and is maintained by the Department of Biological Sciences, University of Iowa, Iowa City, IA 52242.

References

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Inagaki M, Matsuoka Y, Tsujimura K, Ando S, Tokui T, Takahashi T, Inagaki N (1996) Dynamic property of intermediate filaments: regulation by phosphorylation. Bioessays 18:481-487 Kouchi K, Takahashi H, Shimada Y (1993) Incorporation of microinjected biotin-labeled actin into nascent myofibrils of cardiac myocytes: an immunoelectron microscopic study. J Muscle Res Cell Motil 14:292-301 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 Lazarides E, Gard DL, Granger BL, O'Connor CM, Breckler J, Danto SI (1982a) Regulation of the assembly of the Z-disc in muscle cells. Prog Clin Biol Res 85:317-340 Lazarides E, Granger BL, Gard DL, O'Conner CM, Breckler J, Price M, Danto SJ (1982b) Desmin- and vimentin-containing filaments and their role in the assembly of the Z disk in muscle cells. Cold Spring Harb Symp Quant Biol 46:351-378 Li Z, Mericskay M, Agbulut 0, Butler-Browne G, Carlsson L, Thornell LE, Babinet C, Paulin D ( 1997) Desmin is essential for the tensile strength and integrity of myofibrils but not for myogenic commitment, differentiation, and fusion of skeletal muscle. J Cell Biol 139:129-144 Lin Z, Lu MH, Schultheiss T, Choi J, Holtzer S, DiLullo C, Fischman DA, Holtzer H (1994) Sequential appearance of muscle-specific proteins in myoblasts as a function of time after cell division: evidence for a conserved myoblast differentiation program in skeletal muscle. Cell Motil Cytoskeleton 29: 1-19 Meng JJ, Khan S, Ip W (1996) Intermediate filament protein domain interactions as revealed by two-hybrid screens. J Biol Chem 271:1599-1604 Mizuno Y, Thompson TG, Guyon JR, Lidov HG, Brosius M, Imamura M, Ozawa E, Watkins SC, Kunkel LM (2001) Desmuslin, an intermediate filament protein that interacts with alpha-dystrobrevin and desmin. Proc Natl Acad Sci US A 98:6156-6161 Moncman CL, Wang K (1996) Assembly ofnebulin into the sarcomeres of avian skeletal muscle. Cell Motil Cytoskeleton 34:167-184 Napier A, Yuan A, Cole GJ (1999) Characterization of the chicken transitin gene reveals a strong relationship to the nestin intermediate filament class. J Mol Neurosci 12:11-22 49

Ojima K, Lin ZX, Zhang ZQ, Hijikata T, Holtzer S, Labeit S, Sweeney HL, Holtzer H (1999) Initiation and maturation of I-Z-I bodies in the growth tips oftransfected myotubes. J Cell Sci 112:4101-4112 Poon E, Howman EV, Newey SE, Davies KE (2002) Association of syncoilin and desmin: linking intermediate filament proteins to the dystrophin-associated protein complex. J Biol Chem 277:3433-3439 Price MG, Lazarides E (1983) Expression of intermediate filament-associated proteins paranemin and synemin in chicken development. J Cell Biol 97:1860-1874 Reipert S, Steinbock F, Fischer I, Bittner RE, Zeold A, Wiehe G (1999) Association of mitochondria with plectin and desmin intermediate filaments in striated muscle. Exp Cell Res 252:479-491 Richardson FL, Stromer MH, Huiatt TW, Robson RM (1981) Immunoelectron and immunofluorescence localization of desmin in mature avian muscles. Eur J Cell Biol 26:91-101 Schultheiss T, Lin ZX, Ishikawa H, Zamir I, Stoeckert CJ, Holtzer H (1991) Desmin/vimentin intermediate filaments are dispensable for many aspects of myogenesis. J Cell Biol 114:953-966 Schweitzer SC, Klymkowsky MW, Bellin RM, Robson RM, Capetanaki Y, Evans RM (2001) Paranemin and the organization of desmin filament networks. J Cell Sci 114:1079-1089 Sejersen T, Lendahl U (1993) Transient expression of the intermediate filament nestin during skeletal muscle development. J Cell Sci 106:1291-1300 Shizuta Y, Shizuta H, Gallo M, Davies P, Pastan I (1976) Purification and properties of filamin, an actin binding protein from chicken gizzard. J Biol Chem 251 :6562-6567 Sjoberg G, Jiang WQ, Ringertz NR, Lendahl U, Sejersen T (1994) Colocalization ofnestin and vimentin/desmin in skeletal muscle cells demonstrated by three-dimensional fluorescence digital imaging microscopy. Exp Cell Res 214:447-458 Stromer MH (1990) Intermediate (10-nm) filaments in muscle. In: Goldman RD, Steinert PM (eds) Cellular and Molecular Biology of Intermediate Filaments. Plenum Pub Corp, New York, pp 19-36 50

Titeux M, Brocheriou V, Xue Z, Goa J, Pellissier JF, Guicheney P, Paulin D, Li Z (2001) Human synemin gene generates splice variants encoding two distinct intermediate filament proteins. Eur J Biochem 268:6435-6449 Tokuyasu KT, Dutton AH, Singer SJ (1983) Immunoelectron microscopic studies of desmin ( skeletin) localization and intermediate filament organization in chicken skeletal muscle. J Cell Biol 96: 1727-1735 Tokuyasu KT, Maher PA, Singer SJ (1985) Distributions ofvimentin and desmin in developing chick myotubes in vivo. II. Immunoelectron microscopic study. J Cell Biol 100:1157-1166 Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U SA 76:4350-4354 van der Ven PF, Schaart G, Croes HJ, Jap PH, Ginsel LA, Ramaekers FC (1993) Titin aggregates associated with intermediate filaments align along stress fiber-like structures during human skeletal muscle cell differentiation. J Cell Sci 106:749-759 Yang Y, Makita T ( 1996) Immunocytochemical localization of desmin in human fetal skeletal muscle. J Electron Microsc (Tokyo) 45:401-406 Yuan J, Huiatt TW, Liao CX, Robson RM, Graves DJ (1999) The effects ofmono-ADP- ribosylation on desmin assembly-disassembly. Arch Biochem Biophys 363:314-322 51 Fig. 1 (A) A schematic of the paranemin molecule showing that it contains a short N- terminal head and an extremely long C-terminal tail, as well as the a-helical rod domain characteristic of all IF proteins. (B) A map of the cDNA constructs created by PCR of each of the individual domains. Pn 24 is a full-length clone of paranemin. Pn rod and Pn tail constructs were made from Pn 24. The tail domain has been further split into Pn Tail I, Pn Tail II (the repeat region), and Pn Tail III. Numbers at the right of each construct correspond to the amino acid residues coded for by the cDNA construct, with the N-terminus of Pn 24 as residue number 1. A

N Repeat c

B

Pn 24 (1-1750) Pn Rod (16-323) Pn Tail (324-1750) PnTail I (324-751) PnTail II (752-1134) Pn Tail Ill (1135-1750) 53 Fig. 2 Characterization of paranemin antibodies pAb 3146 and mAb 4D3. (A) The paranemin pAb 3146 was tested for specificity by Western blotting. Lanes 1-4, Coomassie stained gel and lanes 5-8, corresponding Western blot of 12 day embryonic chick gizzard homogenate (lanes 1 and 5), partially purified paranemin from embryonic skeletal muscle containing some desmin and vimentin contamination (lanes 2 and 6), purified bacterially expressed paranemin rod domain (lanes 3 and 7), and adult turkey gizzard homogenate (lanes 4 and 8). As shown in lane 8, there is essentially no paranemin in the adult gizzard homogenate. (B) Lanes 1-5, Coomassie stained gel and lanes 6-10, corresponding Western blot of purified bacterially expressed Pn Tail I (lanes 1 and 6), bacterially expressed Pn Tail II (lanes 2 and 7), bacterially expressed Pn Tail III (lanes 3 and 8), bacterially expressed, full- length Pn Tail (lanes 4 and 9), and partially purified paranemin from embryonic skeletal muscle (lanes 5 and 10). The paranemin mAb 4D3 specifically recognizes the Tail II subdomain of the paranemin tail domain, which is a region specifically containing several unique pseudo-heptad repeats. 54

A

B 1 2 3 4 5 6 7 8 9 10 ----· ...... - 55 Fig. 3 Blot overlay analysis of the interactions of bacterially expressed full-length paranemin and of bacterially expressed paranemin rod domain with desmin. (A) Coomassie blue-stained SDS-PAGE gel and (B-E) duplicate Western blots, transferred from gels like that shown in panel A for each overlay and its corresponding antibody control blot are shown. Lane 1, 12 day-chick embryo gizzard homogenate; Lane 2, purified desmin; Lane 3, purified filamin (a negative control protein); Lane 4, adult turkey gizzard homogenate. (B) Bacterially expressed full-length paranemin overlaid on a nitrocellulose blot and probed with the paranemin pAb 3146; (C) Blot treated identically to B, but overlaid with buffer only and probed with the pAb 3146; (D) Bacterially expressed paranemin rod domain overlaid on a nitrocellulose blot and probed with pAb 3146; (E) Blot treated identically to D, but overlaid with buffer only and probed with pAb 3146. The asterisks (*) in panels A, B, and D correspond to the faint 54 kDa vimentin band which migrates slightly above the 53 kDa desmin band in adult turkey gizzard homogenate which are bound by paranemin and paranemin rod in overlays. The 280 on the left side of A marks the migration position ofparanemin. The 250 corresponds to the migration position of filamin, 205 to the myosin heavy chain, 53 to desmin, and 42 to actin. 56

,.. I J .

,.. I • c______,Cl

,..L______,o

,..c______,m g ~ C\J C\J C\J ~ ~ ~;,tt

M i t t ., ·- 57 Fig. 4 Blot overlay analysis of the interactions of bacterially expressed full-length paranemin and of bacterially expressed paranemin rod domain with purified synemin. (A) Coomassie blue-stained SDS-PAGE gel and (B - E), duplicate Western blots transferred from gels like that shown in panel A for each overlay and its corresponding antibody control blot are shown. Lane 1, 12 day-chick embryo gizzard homogenate; Lane 2, purified synemin; Lane 3, purified filamin (a negative control protein); Lane 4, adult turkey gizzard homogenate. (B) Bacterially expressed full-length paranemin overlaid on a nitrocellulose blot and probed with the paranemin pAb 3146; (C) Blot treated identically to B, but overlaid with buffer only and probed with the pAb 3146; (D) Bacterially expressed paranemin rod domain overlaid on a nitrocellulose blot and probed with pAb 3146; (E) Blot treated identically to D, but incubated with buffer only and probed with pAb 3146. The 280 and the 230 on the left side of A mark the migration positions of paranemin and synemin, respectively. The 250 corresponds to the migration position of filamin, 205 to the myosin heavy chain, 53 to desmin, and 42 to actin. 58

•

j .. L_~~~_:__·~-~._~ - --~c

I

T-Lt =----=-----~=-("')~C\J;-CC LO ~ 59 Fig. 5 Cosedimentation of bacterially expressed paranemin rod domain with purified desmin. Coomassie blue-stained SDS-PAGE gel of results from cosedimentation of bacterially expressed paranemin rod (Pn Rod) and purified desmin under IF forming conditions (100 mM NaCl, 1 mM MgClz, 10 mM imidizole-HCl, at pH 7.0). An excess of BSA was added to all samples to show the specificity of the protein cosedimented with desmin and to also show that essentially no unbound protein (e.g. paranemin rod) was simply trapped within the volume of the pellet(s). Panel 1 is desmin alone; panel 2 is paranemin rod alone; panel 3 is paranemin rod and desmin mixed; S, supernatant; P, pellet. 60

_j_ _2_ _3_ s p s p s p

BSA._ Des min Pn Rod - - 61 Fig. 6 IF proteins in one day skeletal muscle cell cultures. Panels A - C, triple labeled with (A) paranemin mAb 4D3, (B) vimentin mAb AMF-17b, and (C) desmin pAb. The asterisks(*) correspond to cells with vimentin and paranemin labeling, but no desmin labeling. Bar = 20 µm.

63 Fig. 7 IF proteins in two day skeletal muscle cell cultures. Panels A - C, double labeled with (A) paranemin mAb 4D3 and (B) desmin pAb. Note that there are cells with paranemin labeling, but no desmin labeling. (C) merged image of paranemin and desmin labeling. Panels D - F, double labeled with (D) paranemin mAb 4D3 and (E) synemin pAb 2856; (F) merged image of paranemin and synemin labeling. Panels G - I, double labeled with (G) synemin pAb 2856 and (H) a-actinin mAb EA53; (I) merged image of synemin and a-actinin labeling. Bar= 20 µm.

65 Fig. 8 IF proteins in four day skeletal muscle cell cultures. Panels A - C, double labeled with (A) paranemin mAb 4D3 and (B) desmin pAb; (C) merged image ofparanemin and desmin labeling. Panels D- F, double labeled with (D) paranemin mAb 4D3 and (E) synemin pAb 2856; (F) merged image of paranemin and synemin labelin5. Panels G - I, double labeled with (G) synemin pAb 2856 and (H) a-actinin mAb EA53; (I) merged image of synemin and a-actinin labeling. Bar= 20 µm.

67 Fig. 9 IF proteins in eight day skeletal muscle cell cultures. Panels A - C, double labeled with (A) paranemin mAb 4D3 and (B) desmin pAb; (C) merged image of paranemin and desmin labeling. Panels D- F, double labeled with (D) paranemin mAb 4D3 and (E) synemin pAb 2856; (F) merged image ofparanemin and synemin labeling. Panels G- I, double labeled with (G) synemin pAb 2856 and (H) a-actinin mAb EA53; (I) merged image of synemin and a-actinin labeling. Bar= 20 µm.

69 Fig. 10 IF proteins in twelve day skeletal muscle cell cultures. Panels A - C, double labeled with (A) paranemin mAb 4D3 and (B) desmin pAb; (C) merged image of paranemin and desmin labeling. Panels D - F, double labeled with (D) paranemin mAb 4D3 and (E) synemin pAb 2856; (F) merged image of paranemin and synemin labeling. Panels G - I, double labeled with (G) synemin pAb 2856 and (H) a-actinin mAb EA53; (I) merged image of synemin and a-actinin labeling. Bar= 20 µm.

71 Fig. 11 Western blot analysis of IF proteins from skeletal muscle cell lysates. (A) Coomassie blue-stained SDS-PAGE gel and (B-D) duplicate Western blots of skeletal muscle cell lysates from a 2 day lysate (lane 1), a 4 day lysate (lane 2), an 8 day lysate (lane 3), and a 12 day lysate (lane 4). (B) Desmin mAb D76 labeled Western blot oflysates. (C) Paranemin pAb 3146 labeled Western blot oflysates. (D) Synemin pAb 2856 labeled Western blot oflysates. 72

~

"l M •• N ~ ,.,.. c

I,_ I ~ M l' N I ,.,.. I_ J()

~ \ M N ' ,.,.. J I{) m 0 C") C\I I{)' I ~ 1 ~ M • N ,.,.. !.._____ Jc:( 73 Fig. 12 Paranemin and synemin localization in skeletal muscle cell cultures by IEM. (A) Paranemin mAb 4D3 (5 nm gold) and synemin pAb 2856 (10 nm gold) double labeled IFs associated with the growing end of a nascent myofibril in a four day myotube. Arrows represent groupings of paranemin label associated with aggregates along IFs. (B) High magnification image of paranemin mAb 4D3 (5 nm gold) and synemin pAb 2856 (10 nm gold) labeled IFs dispersed longitudinally near a Z-body (Z) in a nascent myofibril in a four day myotube. The paranemin label is grouped and associated with an aggregate (long arrow) along an IF near a nascent myofibril. Paranemin and synemin labels are also found near a cytoskeletal filament junction (short arrow). (C) Paranemin pAb 3146 (10 nm gold) labeled longitudinally dispersed IFs in an eight day myotube. (D) Paranemin pAb 3146 (10 nm gold) labeled IFs surrounding an extending/assembling myofibril in an eight day myotube. Bars, 0.2 µm (A, C and D); 0.1 µm (B).

75 Fig. 13 Desmin and synemin localization in skeletal muscle cell cultures. (A) Desmin mAb DE-U-10 (5 nm gold) and synemin pAb 2856 (10 nm gold) labeling ofIFs near a Z-body (Z) of a nascent myofibril in a four day myotube. (B) Desmin pAb ( 10 nm gold) labeled growth tip of an eight day myotube. Box corresponds to area of image represented in C. (C) Higher magnification of desmin pAb (10 nm gold) labeled IFs in the growth tip of an eight day myotube. (D) Desmin pAb (10 nm gold) labeled IFs surrounding the growing end of a nascent myofibril in an eight day myotube. Bar= 0.2 µm (A); 0.5 µm (B-D).

77 Fig. 14 Immunofluorescence localization of GFP-tagged paranemin rod transfected into cultured skeletal muscle cells. Skeletal muscle cells grown in culture for two days were transfected with cDNA encoding GFP-tagged paranemin rod domain and examined four days later. (A and D) GFP fluorescence associated with expressed paranemin rod domain with some present in aggregates; (B and E) Desmin pAb labeling; (C and F) Merged image of GFP-paranemin rod and desmin labeling. In earlier developing myotubes with low expression levels, the GFP-tagged paranemin rod colocalizes with desmin (Panels A- C, bottom myotube). Aggregates of expressed paranemin rod are localizated with desmin at Z-lines (arrow). At high expression levels, the GFP-tagged rod domain containing aggregates completely abolish normal desmin localization (Panels A- C, top myotube ). Bar = 20 µm.

79 Table 1 Yeast two-hybrid protein interactions studies involving synemin, paranemin and other IF proteins. (A) The cDNA constructs utilized in the yeast two hybrid assays. The vectors pPC 97 and pPC 62 differ only in their cloning sites. Murine desmin (GenBank™ accession number 122550); avian paranemin (Pn) (GenBank™ accession number U59287); avian synemin (Syn) (GenBank™ accession number U28143); murine vimentin (GenBank™ accession number M24849). (B) Summary of a series of yeast two-hybrid assays to test for interactions among synemin, paranemin, desmin and vimentin. The + signifies a positive interaction; the - signifies no interaction detected. 80

A Gal4 Transactivation Gal 4 DNA Binding Domain Vectors Domain Vectors Desmin - pPC 86 Desmin - pPC 62 ~ Pn Rod - pPC 86 Pn Rod - pPC 97 Pn Tail- pPC 86 Pn Tail - pPC 97 Syn Rod - pPC 86 Syn Rod - pPC 62 Syn Tail - pPC 86 Syn Tail- pPC 97 Vimentin - pPC 86 Vimentin - pPC 62 pPC86 pPC97 B Pn Rod Syn Rod Desmin Vimentin Pn + + + + Rod Pn Tail -- - - Syn + + + + Rod Syn Tail - - - - 81

GENERAL CONCLUSIONS

General Discussion The major goal of the research presented within this thesis was to provide a better understanding of the role(s) of the unique IF proteins paranemin and synemin within developing skeletal muscle cells. Specifically, I studied the in vitro protein interactions and the localization of the IF proteins paranemin, synemin, and desmin within cultured skeletal muscle cells. In the process, several tools were developed that will be useful in future studies on paranemin. A paranemin rod domain specific pAb was produced for use in both protein overlay assays and immunocytochemical localization studies. The cDNA constructs for different subdomains of paranemin were produced by PCR and ligated into vectors for use in (1) His-tagged protein expression within bacteria, (2) selective expression in yeast for yeast two-hybrid assays, and (3) GFP-tagged protein expression in eukaryotic cells. In addition, a method was developed for the pre-embed labeling of cultured skeletal muscle cells, which increased the overall ultrastructure detail achieved during immunolabeling of the samples for IEM. The protein interaction studies presented herein focused upon the interactions of paranemin and of the paranemin rod domain with desmin, synemin, and vimentin. In protein blot overlays, paranemin interacted with desmin, and the paranemin rod domain interacted with both desmin and synemin. In cosedimentation assays, the paranemin rod domain cosedimented with desmin under IF-forming conditions. The paranemin rod domain also interacted with desmin, vimentin, and the synemin rod domain by yeast two-hybrid analysis. Furthermore, the expression of GFP-tagged paranemin rod domain within cultured skeletal muscle cells showed that the paranemin rod domain colocalized with desmin, and formed aggregates with desmin that resulted in the eventual disruption of the endogenous IF network. These results in toto suggest that paranemin, synemin, vimentin, and desmin form heteropolymeric IFs within skeletal muscle cells during development. The localization studies presented herein focused upon co localization of paranemin with either desmin or synemin, as well as the localization of synemin in comparison to the myofibrillar Z-line protein, a-actinin, within cultured skeletal muscle cells. Additionally, we 82 show herein for the first time that paranemin localization precedes localization of desmin in some presumptive myoblasts, suggesting that paranemin may in fact be the first muscle specific IF protein expressed within early developing skeletal muscle cells. In most cases, paranemin colocalized with both desmin and synemin throughout development. The paranemin, synemin, and desmin were initially localized within longitudinally dispersed IFs. Examination by IEM showed the longitudinally dispersed IFs were found throughout the cultured skeletal muscle cells and often appeared to be associated with the growing ends of myofibrils in elongating myotubes, especially within the growth tips at the ends of the myotubes. Upon further development, after the myofibrils became aligned consistent with the pattern observed in mature muscle cells, foe IF proteins became localized at and along the myofibrillar Z-lines. Thus, the synemin (and other IF proteins) did not colocalize with the myofibrillar Z-line protein, a-actinin, until late in skeletal muscle cell development. The IFs clearly must play a role in skeletal muscle myogenesis, but the precise role is unclear. It appears unlikely that IFs are responsible for lateral alignment of myofibrils within developing skeletal muscle cells. The IFs more likely play a role in linking components of the cytoskeleton and in helping to preserve the overall integrity of the muscle cell as they grow into elongated myotubes. The IF proteins may also play a role in the transport of components necessary for myofibrillogenesis (e.g., proteins and/or their transcripts) to the actively extending, growing ends of myofibrils. In toto, the results herein suggest that, rather than playing a very direct role in myofibrillogenesis, the IFs play a role at the next level of cytoskeletal organization by preserving the overall shape and integrity of developing muscle cells as they grow into elongated myotubes.

Comprehensive Bibliography

Abraham SC, DeNofrio D, Loh E, Minda JM, Tomaszewski JE, Pietra GG, Reynolds C (1998) Desmin myopathy involving cardiac, skeletal, and vascular smooth muscle: report of a case with immunoelectron microscopy. Hum Pathol 29:876-882 83

Andra K, Nikolic B, Stoeber M, Drenckhahn D, Wiehe G (1997) Targeted inactivation of plectin reveals essential function in maintaining the integrity of skin, muscle, and heart cytoarchitecture. Genes Dev 11 :3143-3156 Babai F, Musevi-Aghdam J, Schurch W, Royal A, Gabbiani G (1990) Coexpression of alpha- sarcomeric actin, alpha-smooth muscle actin and desmin during myogenesis in rat and mouse embryos I. Skeletal muscle. Differentiation 44: 132-142 Bang ML, Centner T, FomoffF, Geach AJ, Gotthardt M, McNabb M, Witt CC, Labeit D, Gregorio CC, Granzier H, Labeit S (2001) The complete gene sequence of ti tin, expression of an unusual approximately 700-kDa titin isoform, and its interaction with obscurin identify a novel Z-line to I-band linking systerr,. Circ Res 89: 1065- 1072 Bass MD, Smith BJ, Prigent SA, Critchley DR (1999) Talin contains three similar vinculin- binding sites predicted to form an amphipathic helix. Biochem J 341 :257-263 Becker B, Bellin RM, Semett SW, Huiatt TW, Robson RM (1995) Synemin contains the rod domain of intermediate filaments. Biochem Biophys Res Commun 213:796-802 Bellin RM, Huiatt TW, Critchley DR, Robson RM (2001) Synemin may function to directly link muscle cell intermediate filaments to both myofibrillar Z-lines and costameres. J Biol Chem 276:32330-32337 Bellin RM, Semett SW, Becker B, Ip W, Huiatt TW, Robson RM (1999) Molecular characteristics and interactions of the intermediate filament protein synemin. Interactions with alpha-actinin may anchor synemin-containing heterofilaments. J Biol Chem 274:29493-29499 Benjamin IJ, Shelton J, Garry DJ, Richardson JA (1997) Temporospatial expression of the small HSP/alpha B-crystallin in cardiac and skeletal muscle during mouse development. Dev Dyn 208:75-84 Berg JM, Tymoczko JL, Stryer L (2002) Biochemistry, 5th edn. W. H. Freeman and Co, New York, pp 951-569 Bilak SR, Semett SW, Bilak MM, Bellin RM, Stromer MH, Huiatt TW, Robson RM (1998) Properties of the novel intermediate filament protein synemin and its identification in mammalian muscle. Arch Biochem Biophys 355:63-76 84

Breckler J, Lazarides E (1982) Isolation of a new high molecular weight protein associated with desmin and vimentin filaments from avian embryonic skeletal muscle. J Cell Biol 92:795-806 Carlsson L, Li ZL, Paulin D, Price MG, Breckler J, Robson RM, Wiehe G, Thornell LE (2000) Differences in the distribution of synemin, paranemin, and plectin in skeletal muscles of wild-type and desmin knock-out mice. Histochem Cell Biol 114:39-47 Carpenter DA, Ip W (1996) Neurofilament triplet protein interactions: evidence for the preferred formation ofNF-L-containing dimers and a putative function for the end domains. J Cell Sci 109:2493-2498 Chevray PM, Nathans D (1992) Protein interaction cloning in yeast: identification of mammalian proteins that react with the leucine zipper of Jun. Proc Natl Acad Sci US A 89:5789-5793 Ching GY, Liem RK (1999) Analysis of the roles of the head domains of type IV rat neuronal intermediate filament proteins in filament assembly using domain- swapped chimeric proteins. J Cell Sci 112:2233-2240 Chou YH, Skalli 0, Goldman RD (1997) Intermediate filaments and cytoplasmic networking: new connections and more functions. Curr Opin Cell Biol 9:49-53 Cole GJ, Lee JA (1997) Immunocytochemical localization of a novel radial glial intermediate filament protein. Brain Res Dev Brain Res 101:225-238 Colley NJ, Tokuyasu KT, Singer SJ (1990) The early expression ofmyofibrillar proteins in round postmitotic myoblasts of embryonic skeletal muscle. J Cell Sci 95: 11-22 Collier NC, Sheetz MP, Schlesinger MJ (1993) Concomitant changes in mitochondria and intermediate filaments during heat shock and recovery of chicken embryo fibroblasts. J Cell Biochem 52:297-307 Colucci-Guyon E, Portier MM, Dunia I, Paulin D, Poumin S, Babinet C (1994) Mice lacking vimentin develop and reproduce without an obvious phenotype. Cell 79:679-694 Cossette LJ, Vincent M (1991) Expression of a developmentally regulated cross-linking intermediate filament-associated protein (IF APa-400) during the replacement of vimentin for desmin in muscle cell differentiation. J Cell Sci 98:251-260 85

Coulombe PA, Bousquet 0, Ma L, Yamada S, Wirtz D (2000) The 'ins' and 'outs' of intermediate filament organization. Trends Cell Biol 10:420-428 Coulombe PA, Ma L, Yamada S, Wawersik M (2001) Intermediate filaments at a glance. J Cell Sci 114:4345-4347 Dahlstrand J, Zimmerman LB, McKay RD, Lendahl U (1992) Characterization of the human nestin gene reveals a close evolutionary relationship to neurofilaments. J Cell Sci 103 :589-597 Darenfed H, Ma X, Davis L, Juge N, Savard PE, Cole GJ, Vincent M (2001) Molecular polymorphism of the intermediate filament protein transitin. Histochem Cell Biol 116:397-409 Duval M, Ma X, Valet JP, Vincent M ( 1995) Purification of developmentally regulated avian 400-kDa intermediate filament associated protein. Molecular interactions with intermediate filament proteins and other cytoskeleton components. Biochem Cell Biol 73:651-657 Eliasson C, Sahlgren C, Berthold CH, Stakeberg J, Celis JE, Betsholtz C, Eriksson JE, Pekney M (1999) Intermediate filament protein partnership in astrocytes. J Biol Chem 274:23996-24006 Evans RM (1998) Vimentin: the conundrum of the intermediate filament gene family. Bioessays 20:79-86 Frankel S, Mooseker MS (1996) The actin-related proteins. Curr Opin Cell Biol 8:30-37. Fuchs E (1996) The cytoskeleton and disease: genetic disorders of intermediate filaments. Annu Rev Genet 30:197-231 Fuchs E, Cleveland DW (1998) A structural scaffolding of intermediate filaments in health and disease. Science 279:514-519 Fuchs E, Weber K (1994) Intermediate filaments: structure, dynamics, function, and disease. Annu Rev Biochem 63:345-382 Furst DO, Osborn M, Weber K (1989) Myogenesis in the mouse embryo: differential onset of expression of myogenic proteins and the involvement of titin in myofibril assembly. J Cell Biol 109:517-527 86

Galou M, Gao J, Humbert J, Mericskay M, Li Z, Paulin D, Vicart P (1997) The importance of intermediate filaments in the adaptation of tissues to mechanical stress: evidence from gene knockout studies. Biol Cell 89:85-97 Geerts D, Fontao L, Nievers MG, Schaapveld RQ, Purkis PE, Wheeler GN, Lane EB, Leigh IM, Sonnenberg A (1999) Binding of integrin alpha6beta4 to plectin prevents plectin association with F-actin but does not interfere with intermediate filament binding. J Cell Biol 147:417-434 Granger BL, Lazarides E (1980) Synemin: a new high molecular weight protein associated with desmin and vimentin filaments in muscle. Cell 22:727-738 Granger BL, Lazarides E ( 1982) Structural associations of synemin and vimentin filaments in avian erythrocytes revealed by immunoelectron microscopy. Cell 30:263-275 Granger BL, Lazarides E (1984) Expression of the intermediate-filament-associated protein synemin in chicken lens cells. Mol Cell Biol 4: 1943-1950 Gregorio CC, Granzier H, Sorimachi H, Labeit S ( 1999) Muscle assembly: a titanic achievement? Curr. Opinion Cell Biol. 11: 18-25 Heins S, Aebi U (1994) Making heads and tails of intermediate filament assembly, dynamics and networks. Curr Opin Cell Biol 6:25-33 Hemken PM, Bellin RM, Semett SW, Becker B, Huiatt TW, Robson RM (1997) Molecular characteristics of the novel intermediate filament protein paranemin. Sequence reveals EAP-300 and IF APa-400 are highly homologous to paranemin. J Biol Chem 272:32489-32499 Hemmati-Brivanlou A, Mann RW, Harland RM (1992) A protein expressed in the growth cones of embryonic vertebrate neurons defines a new class of intermediate filament protein. Neuron 9:417-428 Herrmann H, Aebi U ( 1998) Intermediate filament assembly: fibrillogenesis is driven by decisive dimer-dimer interactions. Curr Opin Struct Biol 8: 177-185 Herrmann H, Aebi U (2000) Intermediate filaments and their associates: multi-talented structural elements specifying cytoarchitecture and cytodynamics. Curr Opin Cell Biol 12:79-90 87

Herrmann H, Haner M, Brettel M, Ku NO, Aebi U ( 1999) Characterization of distinct early assembly units of different intermediate filament proteins. J Mol Biol 286: 1403-1420 Herrmann H, Haner M, Brettel M, Muller SA, Goldie KN, Fedtke B, Lustig A, Franke WW, Aebi U ( 1996) Structure and assembly properties of the intermediate filament protein vimentin: the role of its head, rod and tail domains. J Mol Biol 264:933-953 Hesse M, Magin TM, Weber K (2001) Genes for intermediate filament proteins and the draft sequence of the human genome: novel keratin genes and a surprisingly high number of pseudogenes related to keratin genes 8 and 18. J Cell Sci 114:2569-2575 Hijikata T, Murakami T, Imamura M, Fujimaki N, Ishikawa H (1999) Plectin is a linker of intermediate filaments to Z-discs in skdetal muscle fibers. J Cell Sci 112:867-876 Hill CS, Duran S, Lin ZX, Weber K, Holtzer H (1986) Titin and myosin, but not desmin, are linked during myofibrillogenesis in postmitotic mononucleated myoblasts. J Cell Biol 103:2185-2196 Holtzer H, Forry-Scheaudies S, Dlugosz A, Antin P, Dubyak G (1985) Interactions between IFs, microtubules, and myofibrils in fibrogenic and myogenic cells. Ann NY Acad Sci 455:106-125 Huang HY, Graves DJ, Robson RM, Huiatt TW (1993) ADP-ribosylation of the intermediate filament protein desmin and inhibition of desmin assembly in vitro by muscle ADP- ribosyltransferase. Biochem Biophys Res Commun 197:570-577 Huiatt TW, Robson RM, Arakawa N, Stromer MH (1980) Desmin from avian smooth muscle. Purification and partial characterization. J Biol Chem 255 :6981-6989 Huxley HE (1957) The double array of filaments in cross-striated muscle. J Biophys Biochem Cytol. 3 :631-648 Huxley HE (1972) Molecular basis of contraction in cross-striated muscles. In: Bourne G (ed) The Structure and Function of Muscle, 2nd edn. Academic Press, New York, pp 301-341 Huxley HE (2000) Past, present and future experiments on muscle. Phil Trans R Soc Lond B 355:539-543 88

Inagaki M, Matsuoka Y, Tsujimura K, Ando S, Tokui T, Takahashi T, Inagaki N (1996) Dynamic property of intermediate filaments: regulation by phosphorylation. Bioessays 18:481-487 Johnson RP, Craig SW (2000) Actin activates a cryptic dimerization potential of the vinculin tail domain. J Biol Chem 275:95-105 Kelly MM, Phanhthourath C, Brees DK, McCabe CF, Cole GJ (1995) Molecular characterization ofEAP-300: a high molecular weight, embryonic polypeptide containing an amino acid repeat comprised of multiple leucine-zipper motifs. Brain Res Dev Brain Res 85:31-47 Komiyama M, Zhou ZH, Maruyama K, Shimada Y (1992) Spatial relationship of nebulin relative to other myofibrillar proteins during myogenesis in embryonic chick skeletal muscle cells in vitro. J Muscle Res Cell Motil 13:48-54 Kouchi K, Takahashi H, Shimada Y (1993) Incorporation of microinjected biotin-labeled actin into nascent myofibrils of cardiac myocytes: an immunoelectron microscopic study. J Muscle Res Cell Motil 14:292-301 Labeit S, Gautel M, Lakey A, Trinick J (1992) Towards a molecular understanding oftitin. Embo J 11 : 1711-1 716 Labeit S, Kolmerer B, Linke WA ( 1997) The giant protein ti tin. Emerging roles in physiology and pathophysiology. Circ Res 80:290-294 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 Lazarides E, Gard DL, Granger BL, O'Connor CM, Breckler J, Danto SI (1982a) Regulation of the assembly of the Z-disc in muscle cells. Prog Clin Biol Res 85:317-340 Lazarides E, Granger BL, Gard DL, O'Conner CM, Breckler J, Price M, Danto SJ (1982b) Desmin- and vimentin-containing filaments and their role in the assembly of the Z disk in muscle cells. Cold Spring Harb Symp Quant Biol 46:351-378 Li Z, Colucci-Guyon E, Pincon-Raymond M, Mericskay M, Pournin S, Paulin D, Babinet C (1996) Cardiovascular lesions and skeletal myopathy in mice lacking desmin. Dev Biol 175:362-366 89

Li Z, Mericskay M, Agbulut 0, Butler-Browne G, Carlsson L, Thornell LE, Babinet C, Paulin D (1997) Desmin is essential for the tensile strength and integrity of myofibrils but not for myogenic commitment, differentiation, and fusion of skeletal muscle. J Cell Biol 139:129-144 Lin Z, Lu MH, Schultheiss T, Choi J, Holtzer S, DiLullo C, Fischman DA, Holtzer H (1994) Sequential appearance of muscle-specific proteins in myoblasts as a function of time after cell division: evidence for a conserved myoblast differentiation program in skeletal muscle. Cell Motil Cytoskeleton 29: 1-19 Littlefield R, Fowler VM (1998) Defining actin filament length in striated muscle: rulers and caps or dynamic stability? Annu Rev Cell Dev Biol 14:487-525 Marvin MJ, Dahlstrand J, Lendahl U, McKay RD (1998) A rod end deletion in the intermediate filament protein nestin alters its subcellular localization in neuroepithelial cells of transgenic mice. J Cell Sci 111: 1951-1961 McGregor A, Blanchard AD, Rowe AJ, Critchley DR (1994) Identification of the vinculin- binding site in the cytoskeletal protein alpha-actinin. Biochem J 301 :225-233 Meng JJ, Khan S, Ip W (1996) Intermediate filament protein domain interactions as revealed by two-hybrid screens. J Biol Chem 271:1599-1604 Milner DJ, Weitzer G, Tran D, Bradley A, Capetanaki Y (1996) Disruption of muscle architecture and myocardial degeneration in mice lacking desmin. J Cell Biol 134:1255-1270 Mizuno Y, Thompson TG, Guyon JR, Lidov HG, Brosius M, Imamura M, Ozawa E, Watkins SC, Kunkel LM (2001) Desmuslin, an intermediate filament protein that interacts with alpha-dystrobrevin and desmin. Proc Natl Acad Sci US A 98:6156-6161 Moncman CL, Wang K (1996) Assembly ofnebulin into the sarcomeres of avian skeletal muscle. Cell Motil Cytoskeleton 34:167-184 Munoz-Marmol AM, Strasser G, Isamat M, Coulombe PA, Yang Y, Roca X, Vela E, Mate JL, Coll J, Fernandez-Figueras MT, Navas-Palacios JJ, Ariza A, Fuchs E (1998) A dysfunctional desmin mutation in a patient with severe generalized myopathy. Proc Natl Acad Sci US A 95:11312-11317 90

Napier A, Yuan A, Cole GJ (1999) Characterization of the chicken transitin gene reveals a strong relationship to the nestin intermediate filament class. J Mol Neurosci 12:11-22 Nogales E (2001) Structural insights into microtubule function. Annu Rev Biophys Biomol Struct 30:397-420 Nogales E, Wolf SG, Downing KR (1998) Structure of the alpha beta tubulin dimer by electron crystallography. Nature 391: 199-203 Obermann WM, Gautel M, Steiner F, van der Ven PF, Weber K, Furst DO ( 1996) The structure of the sarcomeric M band: localization of defined domains of myomesin, M- protein, and the 250-kD carboxy-terminal region of titin by immunoelectron microscopy. J Cell Biol 134: 1441-1453 Obermann WM, Gautel M, Weber K, Furst DO (1997) Molecular structure of the sarcomeric M band: mapping of titin and myosin binding domains in myomesin and the identification of a potential regulatory phosphorylation site in myomes.in. Embo J 16:211-220 Ojima K, Lin ZX, Zhang ZQ, Hijikata T, Holtzer S, Labeit S, Sweeney HL, Holtzer H (1999) Initiation and maturation ofl-Z-I bodies in the growth tips of transfected myotubes. J Cell Sci 112:4101-4112 Pardo N, Siliciano JD, Craig SW (1983a) Vinculin is a component of an extensive network of myofibril-sarcolemma attachment regions in cardiac muscle fibers. J Cell Biol 97:1081-1088 Pardo N, Siliciano JD, Craig SW (1983b) A vinculin-containing cortical lattice in skeletal muscle: transverse lattice elements ("costameres") mark sites of attachment between myofibrils and sarcolemma. Proc Natl Acad Sci US A 80:1008-1012 Parry DA, Marekov LN, Steinert PM (2001) Subfilamentous protofibril structures in fibrous proteins: cross-linking evidence for protofibrils in intermediate filaments. J Biol Chem 276:39253-39258 Pekny M, Leveen P, Pekna M, Eliasson C, Berthold CH, Westermark B, Betsholtz C ( 1995) Mice lacking glial fibrillary acidic protein display astrocytes devoid of intermediate filaments but develop and reproduce normally. Embo J 14:1590-1598 91

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ACKNOWLEDGEMENTS

I would like to thank my major professor Dr. Richard Robson for the research opportunities I had while working in his lab. I'd also like to thank the past and present members of my POS committee, Dr. Richard Robson, Dr. Marvin Stromer, Dr. Ted Huiatt, Dr. Elizabeth Huff-Lonergan, Dr. Donald Graves, and Dr. Andy Norris, for their continual support and advice throughout my project. I would like to especially thank Dr. Harry Horner, Dr. Marvin Stromer, and Mary Sue Mayes for sharing their knowledge and teaching me the art of electron microscopy. I would also like to thank several past and present members of the Muscle Biology Group who have supported and helped me with ideas and research techniques. Dr. Robert Bellin guided me through much of my project and taught me much of what I know about molecular biology. Dr. Diana Walker also gave valuable insight and instruction. Suzanne Sernett and Emily Helman both helped with cell culture, SDS-PAGE, and Western blotting for various projects. Jo Phillips helped with cell culture and preparation of reagents for cell culture. Mary Sue Mayes has helped tremendously with digital imaging and microscopy techniques and troubleshooting. I would like to thank my family and friends for their support while I've been in graduate school. I'd like to thank my parents, who have always believed in my abilities and have given continued love and support throughout the years. Finally, I'd like to thank my husband Kevin, who has continually supported me and my decisions and has helped me to stay focused upon what is really important in life.