The long-term objectives of this proposed research are to understand the functions of members of the UNC-7/EAT-5 family of proteins in C. elegans. These proteins appear to be required for proper cell-cell coupling through gap junctions and eventually their role in specificity of neuronal connections, neuronal function, and embryonic development will be determined. unc-7 and related genes are postulated to encode invertebrate proteins that are functionally analogous to vertebrate connexins, gap junction channel proteins. This hypothesis will be tested by determining the subcellular structures with which these proteins associate and by examining the effect that family members have on gap junctional connections between cells. The location of the EMP protein, an embryonically expressed family member, will be examined by immunoelectron microscopy. Whether expression of unc-7 or emp can alter the properties of gap junction channels between insect cells in culture will be analyzed. Alternatively, whether UNC-7 or EMP protein can mediate dye-coupling in vertebrate tissue culture cells or in C. elegans oocytes will be tested. Additionally, whether there is loss of dye-coupling in cells of emp mutant embryos will be examined. The unc-7 gene is required in C. elegans for coordinated locomotion and is an additional focus of the investigations proposed here. unc-7 appears to affect several properties of neurons including the specificity of gap junctional connections between particular interneurons and motor neurons. The function of unc-7 will be investigated by analyzing which cells express unc-7, the location within these cells of the UNC-7 protein and the genetic lesions present in unc-7 mutants. Whether ectopic gap junctions observed in the nervous system of unc-7 mutants are responsible for the unc-7 uncoordinated phenotype will be tested by laser killing experiments. Genes whose products may interact with UNC- 7 or modulate UNC-7 function will be identified by isolating and beginning to characterize extragenic suppressors of unc-7 mutations. The expression of genes of the unc-7/eat-5 family will also be analyzed to have, eventually, a complete knowledge of the temporal and spatial expression pattern of all family members. The analysis will include, at this time, only family members expressed during early embryogenesis.
