Assessing C. elegans innexin location phenotypes in touch-sensitive neurons

Gap junctions are intercellular channels made up of hexameric units, connexins in vertebrates or innexins in invertebrates, that allow ions and small molecules to pass between adjacent cells. Although gap junctions are essential to the survival and function of organisms, relatively little is understood about them. To understand more about gap junctions, we used TurboID, a proximity biotinylation enzyme fused to a neuronal innexin, UNC-7, folowed by HPLC Mass Spectrometry to identify proteins of interest. We generated single gene deletion strains, guided by the TurboID data to gauge its effect, if any, on innexin localization. The effect the proteins of interest have on innexin regulation was assessed by introducing deletion alleles into a strain that labels an innexin closely associated with UNC-7, UNC-9, with GFP and expressed in a single pair of touch-sensitive neurons in the animal’s tail, the PLM neurons. PLM neurons form gap junctions with several other important neurons in C. elegans, including the PVC, PVR, and LUA neurons. These neurons should appear as bright GFP puncta on the PLM axon. Using this system, we found that the deletion of syg-1, unc-116, vab-1, and src-1 lead to phenotypes in the localization of the GFP labeled UNC-9 innexin. When looking at these genes, we sought to find out whether the deletions affected function – forming nonfunctional gap junctions, interrupting cell-cell communication, or not forming gap junctions – trafficking, or positioning at the correct synaptic face.