The maintenance and organization of striated muscle in C. elegans requires the serine/threonine kinase UNC-82. Worms homozygous for
unc-82(0) exhibit bright patchy birefringence in the body wall muscles, a phenotype indicative of misplaced myosin filaments during cell shape changes in the developing embryo (Hoppe et al., 2010). Little is known about the targets of UNC-82 kinase, and the mechanism for its activation has yet to be determined. Antibody staining of
unc-82 mutants reveals abnormal accumulations of thick filament proteins myosin and paramyosin. Distinctive ectopic myosin B (
unc-54) and paramyosin (
unc-15) accumulations occur in the kinase domain missense mutant whereas in the presumptive null background, myosin B and paramyosin are present in amorphous patches; these data suggest a role for UNC-82 kinase in myosin B and paramyosin localization and incorporation into the thick filament.
unc-54(0) mutants expressing an UNC-82::GFP transgene exhibit distinctive accumulations of UNC-82 at the ends of muscle cells indicating that localization of UNC-82 to the contractile apparatus is dependent on myosin B. We explored physical interactions between UNC-82 and other M-line or thick-filament proteins by analyzing single and double mutant strains in which myosin, paramyosin or UNC-82 is abnormally localized within muscle cells, and determining which proteins are recruited to the aberrant structures. Our results suggest that UNC-82 physically interacts, either directly or indirectly, with both myosin B and paramyosin, but not myosin A or the UNC-98/Zn finger protein. In addition, we constructed double mutant strains to define possible signaling or assembly pathways involving UNC-82 and other M-line or thick filament proteins.
unc-98 mutants contain large accumulations of paramyosin at the ends of muscle cells, but have relatively normal myosin distribution. We found that
unc-15(0);
unc-98(0) double mutants are viable and exhibit a phenotype similar to that of
unc-15(0) alone, consistent with the proposal that the primary action of UNC-98/Zn finger, a potential chaperone, is on paramyosin (Miller et al., 2008). The
unc-15(0);
unc-82(0) double mutant, however, is not viable, consistent with UNC-82 acting to organize both myosin and paramyosin. The
unc-82(0);
unc-98(0) double is viable, and closely resembles the
unc-82 single mutant, suggesting that the two proteins do not have redundant functions in organizing other muscle components, and that the two proteins act in a single pathway to organize paramyosin. Our data are consistent with the model that UNC-82 kinase is required for regulation, localization and incorporation of myosin B and paramyosin into the thick filaments of the contractile apparatus.