Exploring Gap Junction Synthesis and Dynamics through C. elegans

My research at the Yan Lab involves investigating gap junctions in the neurons of C. elegans. Gap junctions are an electrical synapse, allowing for two adjacent cells to transmit information by permitting ions to pass through. These ions are important for various functions, including cell growth and differentiation, neural development, and embryo development. They are composed of two hexameric subunits arranged around a central pore. Their monomers are connexin proteins in vertebrates and innexin proteins in invertebrates, which is what my research focuses on. Although gap junctions are found in nearly all cell types – with the exception of striated tissue – my research focuses on gap junctions found in the PLM neurons of C. elegans. These neurons are found near the tail of the worm and are associated with movement of the posterior half of the body. Despite being so crucial for the survival and functionality of cells and an organism, relatively little is known about gap junctions. Conversely, chemical synapses, which is another form of intercellular communication, have been studied in much more detail. Among the many things unknown about gap junctions include the exact composition of the innexin gap junction channels in C. elegans, how gap junction and gap junction proteins are turned over in a healthy brain, and how gap junction channels are oligomerized in the E.R. and assembled in the Golgi apparatus. To try to answer these questions, certain genes are knocked out in the worms to see what the effects are on the worm’s phenotype. For example, I have been working on crossing mutant worms carrying various loss of function mutations. After crossing, I observe the PLM neurons of the worms under a compound fluorescent microscope. While viewing worms, I compare the location and number of dots, or puncta (seen through GFP tagging), in the cell body of the PLM neurons as well as the puncta anterior to the cell body. In a wild type worm, there are supposed to be two distinct puncta in the cell body as well as two to four puncta anterior to the cell body. Any variations to this phenotype would indicate some sort of abnormality with the gap junctions in the PLM neurons of the worms. From there, I have to perform genotyping – either through PCR or Sanger Sequencing – to confirm that the mutant worms are homozygous rather than heterozygous for the mutation. Finally, I perform behavioral assays (like lightly touching the worm to see if it responds or dropping the plate from 1-2 cm off the ground to see if the worms respond) to begin attributing the phenotypes observed in the microscope to the actual function of the gap junctions.