Development of genetically encoded sensors based on PKM2
Chang, Huntley1; Rocheleau, Jonathan1, 2, 3
1. Institute of Biomaterials & Biomedical Engineering, University of Toronto; 2. Toronto General Research Institute, University Health Network; 3. Department of Physiology, University of Toronto
Our lab recently developed a family of genetically encoded sensors to track NADPH/NADP+ redox state and the pentose phosphate pathway (PPP) in real time (termed Apollo-NADP+). These sensors provide colour-independent ratiometric readouts based on fluorescence anisotropy, making them ideal for multiparametric and high-throughput imaging. NADPH critically supplies reducing equivalents to biosynthetic pathways and for the scavenging of reactive oxygen species (ROS). In most cells, NADPH is produced primarily by shunting the glycolytic intermediate glucose-6-phosphate (G6P) to the PPP. In contrast, β-cells lack major PPP activity and instead generate NADPH more slowly through mitochondrial metabolism and pyruvate cycling. However, β-cells are also very heterogeneous with varying levels of glucose-stimulated insulin secretion. We believe that variations in β-cell function signify variance in the conventional metabolism of glucose, including glycolysis and upregulation of the PPP. To enable simultaneous tracking of glycolysis and the PPP, we are developing another Apollo sensor based on pyruvate kinase M2 (PKM2) that can be measured simultaneously with Apollo-NADP+. We have so far tagged PKM2 with Venus fluorescent protein and measured anisotropy using two-photon homoFRET imaging. In these studies, the peptide linker between PKM2 and Venus was varied to achieve the maximum anisotropy change. Initial experiments will be performed in pancreatic β-cell lines using various metabolites and metabolic inhibitors to measure links between glycolytic flux and the PPP. These studies will also easily translate to ex vivo mouse and human pancreatic islets using adenoviral transduction under the rat insulin promoter. Ultimately, we will simultaneously image Apollo-PKM2 and Apollo-NADP+ to map the metabolic heterogeneity of beta cells within living islets.