The C. elegans
unc-60 gene encodes two functionally distinct isoforms of ADF/cofilin. We have recently shown that one of the gene products, UNC-60B, is specifically required for proper assembly of actin into myofibrils. Biochemical analysis of UNC-60B has suggested that it enhances actin filament turnover by accelerating both polymerization and depolymerization and by severing actin filaments. However, during myofibril assembly, many other factors are involved and coordinately regulate the assembly of highly organized functional structures. We present our genetic and biochemical analysis of candidate modulators for UNC-60B. The
sup-12 is a recessive suppressor of
unc-60 . Therefore, myofibrils of
sup-12;
unc-60 double mutants appear normal. Although
sup-12 itself does not show an obvious phenotype in muscle, we found that
sup-12(
st89) causes a remarkable reduction in the protein level of UNC-60B but not of UNC-60A. In wild-type muscle, UNC-60B was found in the myofibrils, while, in
sup-12 mutant, UNC-60B was largely dissociated from the myofibrils. These results suggest that the
sup-12 gene product is a modulator of the function of UNC-60B or other actin-binding proteins. In addition to the currently available
sup-12 alleles,
st89 and
st203 , we have recently isolated 4 new alleles of
sup-12 that should help understand the function of the
sup-12 gene. The identification of the
sup-12 gene by transgenic rescue is in progress. Tropomyosin (TM) has been reported to compete with ADF/cofilin for actin binding in other systems. Since TM is a major component of thin filaments of myofibrils, we tested the possibility of competition between UNC-60B and TM in vitro . Using actin and TM from wild-type C. elegans , we found that TM tightly bound to F-actin and inhibited binding of UNC-60B to F-actin. However, partial depolymerization of F-actin by UNC-60B was not inhibited by TM. These results suggest that, in the presence of TM, access of UNC-60B to the side of F-actin is inhibited, while UNC-60B can enhance actin dynamics from the filament ends. We are in the process of characterizing the effects of mutations in the TM gene,
tmy-1/lev-11 on Unc-60 phenotypes.