Dataset Information

Monoclonal antibodies against muscle actin isoforms: epitope identification and analysis of isoform expression by immunoblot and immunostaining in normal and regenerating skeletal muscle.

ABSTRACT: Higher vertebrates (mammals and birds) express six different highly conserved actin isoforms that can be classified in three subgroups: 1) sarcomeric actins, ?-skeletal (?-SKA) and ?-cardiac (?-CAA), 2) smooth muscle actins (SMAs), ?-SMA and ?-SMA, and 3) cytoplasmic actins (CYAs), ?-CYA and ?-CYA. The variations among isoactins, in each subgroup, are due to 3-4 amino acid differences located in their acetylated N-decapeptide sequence. The first monoclonal antibody (mAb) against an actin isoform (?-SMA) was produced and characterized in our laboratory in 1986 (Skalli et al., 1986) . We have further obtained mAbs against the 5 other isoforms. In this report, we focus on the mAbs anti-?-SKA and anti-?-CAA obtained after immunization of mice with the respective acetylated N-terminal decapeptides using the Repetitive Immunizations at Multiple Sites Strategy (RIMMS). In addition to the identification of their epitope by immunoblotting, we describe the expression of the 2 sarcomeric actins in mature skeletal muscle and during muscle repair after micro-lesions. In particular, we analyze the expression of ?-CAA, ?-SKA and ?-SMA by co-immunostaining in a time course frame during the muscle repair process. Our results indicate that a restricted myocyte population expresses ?-CAA and suggest a high capacity of self-regeneration in muscle cells. These antibodies may represent a helpful tool for the follow-up of muscle regeneration and pathological changes.

SUBMITTER: Chaponnier C

PROVIDER: S-EPMC4893938 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Monoclonal antibodies against muscle actin isoforms: epitope identification and analysis of isoform expression by immunoblot and immunostaining in normal and regenerating skeletal muscle.

Chaponnier Christine C Gabbiani Giulio G

F1000Research 20160330

Higher vertebrates (mammals and birds) express six different highly conserved actin isoforms that can be classified in three subgroups: 1) sarcomeric actins, α-skeletal (α-SKA) and α-cardiac (α-CAA), 2) smooth muscle actins (SMAs), α-SMA and γ-SMA, and 3) cytoplasmic actins (CYAs), β-CYA and γ-CYA. The variations among isoactins, in each subgroup, are due to 3-4 amino acid differences located in their acetylated N-decapeptide sequence. The first monoclonal antibody (mAb) against an actin isoform ...[more]

PMID: 27335638

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Project description:BackgroundNesprin-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.ResultsmRNA 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.ConclusionsNesprin-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.

Dataset Information

Monoclonal antibodies against muscle actin isoforms: epitope identification and analysis of isoform expression by immunoblot and immunostaining in normal and regenerating skeletal muscle.

Publications

Monoclonal antibodies against muscle actin isoforms: epitope identification and analysis of isoform expression by immunoblot and immunostaining in normal and regenerating skeletal muscle.

Similar Datasets

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