Manipulating Gap Junction Assembly and Communication in CHO Cells

Andrea Vines, Biology Faculty Mentor: Professor Derek Beahm, BiologyGap junction channels bridge the cytoplasmic compartments of neighboring cells, allowing for metabolic cooperation and synchronization of electrical activity. Almost all animal cell types express gap junction proteins and form channels with neighboring cells. However, different cell types can express similar protein levels but form very different numbers of channels. Chinese Hamster Ovary (CHO) cells are known to be gap junction competent but assembly inefficient. I examined effects of drugs that target different aspects of the gap junction assembly process to try and enhance gap junction communication between CHO cells. Cell-cell communication was measured by observing the transfer of a small fluorescent dye between cells. Lectin and neuraminidase treatments were used to reduce the density of large surface complexes that could possibly interfere with gap junction formation, but these treatments were ineffective. A proteasome inhibitor, ALLN, was used to extend the half-life of the gap junction protein Cx43 in the plasma membrane which has been shown to enhance gap junction formation in CHO cells. I also had positive results with ALLN, suggesting my protocols and technique were good. The most dramatic increase in gap junction formation resulted from treatments with forskolin, which stimulates cAMP production and is known to enhance the trafficking and assembly of gap junctions in other cell types. An unexpected finding was that gap junction assembly could only be stimulated in newly formed cell monolayers and not in older monolayers, suggesting a possible time-dependent down regulation of the gap junction protein in CHO cell monolayers. These studies expand on our general knowledge and understanding of mechanisms involved in gap junction formation.https://digitalcommons.buffalostate.edu/srcc-sp20-physgeosci/1026/thumbnail.jp