Abstract
The Caenorhabditis elegans unc-60 gene encodes two functionally distinct isoforms of ADF/cofilin that are implicated in myofibril assembly. Here, we show that one of the gene products, UNC-60B, is specifically required for proper assembly of actin into myofibrils. We found that all homozygous viable unc-60 mutations resided in the unc-60B coding region, indicating that UNC-60B is responsible for the Unc-60 phenotype. Wild-type UNC-60B had F-actin binding, partial actin depolymerizing, and weak F-actin severing activities in vitro. However, mutations in UNC-60B caused various alterations in these activities. Three missense mutations resulted in weaker F-actin binding and actin depolymerizing activities and complete loss of severing activity. The r398 mutation truncated three residues from the COOH terminus and resulted in the loss of severing activity and greater actin depolymerizing activity. The s1307 mutation in a putative actin-binding helix caused greater activity in actin-depolymerizing and severing. Using a specific antibody for UNC-60B, we found varying protein levels of UNC-60B in mutant animals, and that UNC-60B was expressed in embryonic muscles. Regardless of these various molecular phenotypes, actin was not properly assembled into embryonic myofibrils in all unc-60 mutants to similar extents. We conclude that precise control of actin filament dynamics by UNC-60B is required for proper integration of actin into myofibrils.
Highlights
IN striated muscles, actin and myosin are precisely aligned in a striated pattern so that they coordinately generate contractile forces
Sequence analyses of the genomic DNA from the unc60 mutants showed that mutations in the unc-60B but not in the unc-60A coding region cause the Unc-60 phenotypes (Fig. 1 B). s1309, m35, and s1307 have missense mutations within a region homologous in sequence to the ␣-helix that contains the putative actin-binding surface of the members of the ADF/cofilin family for which structures have been solved (Yonezawa et al, 1991; Hatanka et al, 1996; Fedorov et al, 1997; Leonard et al, 1997; Van Troys et al, 1997). e723, s1310, and s1331 have the same mutation as m35
This was concluded from three major findings: mutations in UNC-60B, one of the two ADF/cofilin isoforms encoded by the unc-60 gene, were sufficient to cause disorganization of myofibrils; these mutations resulted in abnormal actin-regulating activities of UNC-60B; and UNC-60B was primarily expressed in body wall muscle cells during embryonic stages
Summary
IN striated muscles, actin and myosin are precisely aligned in a striated pattern so that they coordinately generate contractile forces. CapZ, a barbed end actin-capping protein is involved in myofibril assembly (Schafer et al, 1995), which probably terminates actin assembly (Eddy et al, 1997) and regulates the length of actin filaments (Xu et al, 1999). In chicken embryonic skeletal muscle, profilin (Ohshima et al, 1989), actin depolymerizing factor (ADF1) (Abe and Obinata, 1989), and cofilin (Abe et al, 1989) have been identified as G-actin binding proteins that are postulated to regulate actin assembly into myofibrils. Overexpression of cofilin in Dictyostelium enhances cell motility (Aizawa et al, 1996) These observations show that ADF/cofilin regulates dynamic aspects of the actin cytoskeleton in vivo. Our results provide the first evidence of an isoform-specific requirement of ADF/cofilin for assembly of a differentiated actin cytoskeleton
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