Abstract
Higher vertebrates 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 (α-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 mAb anti-α-SKA and anti-α-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 α-CAA, α-SKA and α-SMA by co-immunostaining in a time course frame during the muscle repair process. Our results indicate that a restricted myocyte population expresses α-CAA and suggest a high capacity of self-renewal in muscle cells. These antibodies may represent a helpful tool for the follow-up of muscle regeneration and pathological changes.
Highlights
Expression of actin isoforms in skeletal muscle at the mRNA and protein levels have been described since the 1980s using tissue extracts (Gunning et al, 1983; Hayward & Schwartz, 1986; Hayward et al, 1988; Minty et al, 1982; Ordahl, 1986; Paterson & Eldridge, 1984; Sassoon et al, 1988; Vandekerckhove et al, 1986)
These studies have indicated that sarcomeric actins (α-CAA and α-SKA) are expressed in fetal and regenerating skeletal muscle, whereas α-SKA becomes the predominant actin isoform in mature skeletal muscle
Identification of the epitopes recognized by the actin isoform antibodies Acetylated N-terminal peptide of different length (4–10 amino acids) and non-acetylated N-terminal decapeptide of α-SKA and α-CAA were tested for their blocking ability of the respective mAb (Figure 1)
Summary
Expression of actin isoforms in skeletal muscle at the mRNA and protein levels have been described since the 1980s using tissue extracts (Gunning et al, 1983; Hayward & Schwartz, 1986; Hayward et al, 1988; Minty et al, 1982; Ordahl, 1986; Paterson & Eldridge, 1984; Sassoon et al, 1988; Vandekerckhove et al, 1986) These studies have indicated that sarcomeric actins (α-CAA and α-SKA) are expressed in fetal and regenerating skeletal muscle, whereas α-SKA becomes the predominant actin isoform in mature skeletal muscle. Resting satellite cells in healthy adult muscle lack this isoform, whereas, when satellite cells are activated during the regeneration process, α-CAA is up-regulated In these two studies, a comparative α-SKA staining was not performed
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