title: Specific localization of nesprin-1-α2, the short isoform of nesprin-1 with a KASH domain, in developing, fetal and regenerating muscle, using a new monoclonal antibody.
abstract: Nesprin-1-giant (1008kD) is a protein of the outer nuclear membrane that links nuclei to the actin cytoskeleton via amino-terminal calponin homology domains. The short nesprin-1 isoform, nesprin-1-α2, is present only in skeletal and cardiac muscle and several pathogenic mutations occur within it, but the functions of this short isoform without calponin homology domains are unclear. The aim of this study was to determine mRNA levels and protein localization of nesprin-1-α2 at different stages of muscle development in order to shed light on its functions.
  
  
  
  mRNA levels of all known nesprin-1 isoforms with a KASH domain were determined by quantitative PCR. The mRNA for the 111 kD muscle-specific short isoform, nesprin-1-α2, was not detected in pre-differentiation human myoblasts but was present at significant levels in multinucleate myotubes. We developed a monoclonal antibody against the unique amino-terminal sequence of nesprin-1-α2, enabling specific immunolocalization for the first time. Nesprin-1-α2 protein was undetectable in pre-differentiation myoblasts but appeared at the nuclear rim in post-mitotic, multinucleate myotubes and reached its highest levels in fetal muscle. In muscle from a Duchenne muscular dystrophy biopsy, nesprin-1-α2 protein was detected mainly in regenerating fibres expressing neonatal myosin. Nesprin-1-giant was present at all developmental stages, but was also highest in fetal and regenerating fibres. In fetal muscle, both isoforms were present in the cytoplasm, as well as at the nuclear rim. A pathogenic early stop codon (E7854X) in nesprin-1 caused reduced mRNA levels and loss of protein levels of both nesprin-1-giant and (unexpectedly) nesprin-1-α2, but did not affect myogenesis in vitro.
  
  
  
  Nesprin-1-α2 mRNA and protein expression is switched on during myogenesis, alongside other known markers of muscle differentiation. The results show that nesprin-1-α2 is dynamically controlled and may be involved in some process occurring during early myofibre formation, such as re-positioning of nuclei.
authors.affiliation: Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, SY10 7AG, UK. ian.holt@rjah.nhs.uk.|Institute for Science and Technology in Medicine, Keele University, Keele, ST5 5BG, UK. ian.holt@rjah.nhs.uk.
authors.forename: Ian
authors.initials: I
authors.lastname: Holt
authors.affiliation: Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, SY10 7AG, UK.|Institute of Genome Research (IGR), Vietnam Academy of Science and Technology (VAST), Hanoi, Vietnam.
authors.forename: Nguyen Thuy
authors.initials: NT
authors.lastname: Duong
authors.affiliation: Cardiovascular Division, James Black Centre, King's College, London, SE5 9NU, UK.
authors.forename: Qiuping
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, SY10 7AG, UK.
authors.forename: Le Thanh
authors.initials: le T
authors.lastname: Lam
authors.affiliation: Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, SY10 7AG, UK.|Dubowitz Neuromuscular Centre, Institute for Child Health and Great Ormond Street Hospital, London, WC1 1EH, UK.
authors.forename: Caroline A
authors.initials: CA
authors.lastname: Sewry
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS974, CNRS FRE3617, Center for Research in Myology, 47 Boulevard de l'hôpital, 75013, Paris, France.
authors.forename: Kamel
authors.initials: K
authors.lastname: Mamchaoui
authors.affiliation: Cardiovascular Division, James Black Centre, King's College, London, SE5 9NU, UK.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Shanahan
authors.affiliation: Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry, SY10 7AG, UK.|Institute for Science and Technology in Medicine, Keele University, Keele, ST5 5BG, UK.
authors.forename: Glenn E
authors.initials: GE
authors.lastname: Morris
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D003598:Cytoskeletal Proteins; D005260:Female; D005333:Fetus / Q000378:metabolism*; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D024510:Muscle Development / Q000235:genetics; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D018482:Muscle, Skeletal / Q000196:embryology* / Q000378:metabolism*; D020388:Muscular Dystrophy, Duchenne / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D032446:Myoblasts / Q000378:metabolism; D009419:Nerve Tissue Proteins; D010455:Peptides / Q000378:metabolism; D000072417:Protein Domains; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D012038:Regeneration; D055815:Young Adult
chemical_list: D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D020033:Protein Isoforms; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C415465:SYNE1 protein, human
keywords: Cardiomyopathy; Emery-Dreifuss muscular dystrophy; Lamin A/C; Monoclonal antibody; Nuclear membrane; SYNE1