unc-43 encodes the C. elegans CaMKII homologue. CaMKII is a protein kinase responsible for orchestrating many cellular responses to the second messenger Ca 2+ . CaMKII has been extensively studied biochemically and many potential targets have been identified by in vitro kinase assays. However, due to the lack of specificity of these assays, it remains unclear what proteins are phosphorylated by CaMKII in vivo . We determined the localization of UNC-43 by in situ antibody staining and GFP fusion analysis. A monoclonal antibody to rat CaMKII a stains the nerve ring and the ventral and dorsal nerve cords of wild-type worms. An
unc-43 promoter- gfp fusion is expressed in ventral cord neurons, a variety of sensory neurons and interneurons throughout the animal and the HSN motorneurons. Expression is also observed in body-wall and vulval muscles and in the intestine. This expression pattern suggests that
unc-43 functions in neuronal regulation throughout the animal consistent with the diverse phenotypes seen in
unc-43 mutants. A kinase-activating mutation causes defects in egg laying, defecation expulsion and timing, locomotion and body muscle tone. Loss of function mutations cause generally opposite phenotypes in each case. We are currently determining the site of action for each phenotype using tissue specific expression of wild-type or kinase-activated
unc-43 cDNAs in an
unc-43 null background. We are also testing a peptide inhibitor of CaMKII for the ability to cause loss of function phenotypes when expressed in vivo .
unc-43(lf) mutants have a repetition in their defecation motor program that we refer to as the "echo" phenotype with a consistent latency of 11 seconds. Double mutants between
unc-43 and defecation cycle ( dec ) mutants were constructed to test whether they would alter the latency of the echo. Instead, strong suppression of the echo was observed for all double mutants examined (4 in total). Two dec mutants (
flr-1 and
flr-4 ) have been cloned and both are expressed in the intestine, suggesting that the location of the defecation "clock" may be in the intestine. Experiments are currently underway to test if the observed expression of
unc-43::gfp in the intestine is responsible for its regulation of the defecation clock.