Our previous studies suggest that
unc-82 is involved in a novel pathway required to maintain the organization of the body-wall muscle contractile apparatus during growth and/or contractile activity. Mutant homozygotes move normally but slowly, become Egl, and exhibit defects in thick filament morphology and arrangement within the body-wall muscle (Waterston et al, 1980). In previous studies, our lab determined that UNC-82 is a putative serine/threonine kinase encoded by the predicted gene B0496.3. Our current studies are designed to elucidate UNC-82 function in muscle and nonmuscle cells. In one study, we have undertaken a non-complementation screen to identify temperature-sensitive and other new
unc-82 alleles, using GFP::myosin to score muscle phenotype in F1 animals. Although UNC-82 is predicted to contain 1600 amino acids, both available alleles contain mutations within the kinase domain, and no recognizable motifs are found in the remainder of the molecule. Mutations recovered in regions outside the kinase domain may aid in defining other areas of the protein important for function. The isolation of temperature-sensitive mutations will allow analysis of the onset of the
unc-82 phenotype in older animals using antibody-staining and ultrastructural techniques. Our second study addresses the role of
unc-82 function outside muscle. UNC-82::GFP is present in a punctate pattern in adult body-wall muscle, pharynx, and other structures. In body wall, the GFP pattern overlaps that of the known M-line protein, UNC-89. However, in the pharynx, the UNC-82::GFP signal lies outside the UNC-89-positive muscle cells, apparently localized within marginal cells. The GFP signal in these and other nonmuscle cells suggests that UNC-82 may be associated with structures containing intermediate filaments. To assess a possible role for
unc-82 in organizing intermediate filaments, we have undertaken antibody staining experiments and a TEM analysis of mutant animals. We have generated additional epitope-tagged constructs, as well as antisera raised against fusion proteins, to further define UNC-82 localization within the contractile apparatus and in nonmuscle cells.