A genetically encoded sensor based on PKM2 to measure glycolytic flux
Chang, Huntley (1); Rocheleau, Jonathan (1,2,3)
Institute of Biomaterials & Biomedical Engineering, University of Toronto
Departments of Physiology and Medicine, University of Toronto
Toronto General Research Institute, University Health Network
Our lab recently developed a family of genetically encoded sensors to track NADPH/NADP+ redox state 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 (proliferation) and for the scavenging of reactive oxygen species (survival). In most cells, NADPH is produced primarily by shunting the glycolytic intermediate glucose-6-phosphate (G6P) to the pentose phosphate pathway (PPP). In contrast, β-cells lack major PPP activity and instead generate NADPH more slowly through glycolytic flux and pyruvate cycling. To enable simultaneous tracking of glycolytic flux and NADPH generation, we are developing an Apollo sensor based on the rate-limiting enzyme of glycolysis (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 widefield and two-photon homoFRET imaging. Initial experiments will be performed in pancreatic β-cell lines using various metabolites and metabolic inhibitors to measure links between glycolytic flux and pyruvate cycling. 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 metabolism of beta cells relative to their proliferation and survival within living islets.