Effect Of Loading Method On Metabolism Of Foreign Molecules In Intact Cells Of Dictyostelium Discoideum

Abstract

Loading foreign molecules inside living cells allows one to monitor cellular metabolism, track various metabolic pathways and alter activities of the cell. However, the effects of loading methods on the fate of these foreign molecules are poorly understood. In this experiment, we examined three promising loading methods including pinocytosis, electroporation and myristoylation to insert a peptide substrate reporter in cells of Dictyostelium discoideum. These methods were optimized to maximize cell viability, and factors such as loading efficiency, loading duration, localization and uniformity were characterized. Capillary electrophoresis was used to determine how each loading method affected the enzyme activity on the peptide substrate reporter. It was observed that these loading methods had strong effects on the stability and degradation of the peptide. Myristoylation was found to be the most effective method at transporting the peptide into cells while electroporation produced the most uniform loading within cells suggesting cytoplasmic loading. The peptide was degraded most rapidly after loading by myristoylation (half‐life = 12 ± 1 min), while pinocytic loading produced slightly slower degradation (half‐life = 19 ± 2 min). Phosphorylation of the peptide was only observed in cells loaded with electroporation. This experiment suggests that the choice of loading method influences the stability and metabolism of foreign molecules in cells. Our future work includes investigating the specificity of different peptide substrate reporters in Dictyostelium lysates.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.