In a screen for enclosure defective mutants, the partially penetrant, maternal-effect cold-sensitive allele
jc11 was identified. 82 and 19 percent of
jc11 animals display mutant phenotypes at 15 and 25 degrees, respectively, and a single maternal or zygotic copy of wild type
jc11 is sufficient for gene function. Using 4D microscopy
jc11 embryos are observed to arrest at various developmental stages including pre-morphogenesis, enclosure, and elongation. Animals that successfully complete elongation often have morphogenetic defects including tail and snout abnormalities.
jc11 was genetically inseparable from the
lin-7 allele used in the genetic screen and a minimal genomic region containing the neighboring gene R06A4.8 was sufficient for the rescue of
jc11 phenotypes. RNAi against R06A4.8 phenocopies
jc11. R06A4.8 is predicted to encode glycogen debranching enzyme (
agl-1). The R06A4.8 locus in
jc11 animals was completely sequenced and a four base pair deletion was found in the c-terminus of
agl-1. This deletion is predicted to result in a frame shift and early termination, resulting in the truncation the final 253 residues of the protein, adding 22 extraneous residues prior to a stop codon. The
jc11 mutation is predicted to eliminate the glycogen binding domain of debranching enzyme.
jc11 was also phenocopied by treatment with MOR-14 (N-Methyl-1-Deoxynojirimycin), an inhibtor that specifically targets the a-1,6-glucosidase activity of glycogen-debranching enzyme. Overexpression of AGL-1::GFP results in increased debranching activity based on reduced glycogen staining with carminic acid in both embryos and adults expressing AGL-1::GFP. Recently, debranching enzyme was shown to interact with the
b1 subunit of the energy sensor AMP-activated kinase, while binding of AMPK
b1 directly to glycogen depends on the a-1,6-branch; AMPK binding to glycogen branches in turn functions to inhibit AMPK activity [1, 2]. Loss of
agl-1 function in C. elegans is predicted to result in the formation of a ''limit dextrin'' glycogen molecule in which outer glucose chains are digested by phosphorylase to within four glucose residues of the a-1-6 branch, resulting in a glycogen molecule likely to bind to and inhibit AMPK activity. The prediction that AMPK activity is inhibited in
agl-1 mutants is supported by initial experiments indicating that
jc11 animals surviving to adulthood have shorter life spans, similar to animals lacking AMPK activity. The role of glycogen debranching enzyme in the integration of energy and lifespan signals via AMPK is being further investigated. 1.Sakoda, H., et al.,. Am J Physiol Endocrinol Metab, 2005. 289(3): p. E474-81. 2.McBride, A., et al., Cell Metab, 2009. 9(1): p. 23-34.