Organelle-Targeting of Apollo-NADP+ Requires Careful Selection of Fluorescent Proteins to Maintain pH Independence and Reveals NADPH Compartmentalization
Cameron, William 1 ; Rocheleau, Jonathan 1, 2
1. Institute of Biomaterials and Biomedical Engineering, University of Toronto; 2. Toronto General Research Institute, University Health Network
We recently developed a spectral family of genetically encoded homoFRET sensors to measure NADPH/NADP+ redox state based on changes in anisotropy/polarization due to oligomerization of glucose-6-phosphate dehydrogenase (Apollo-NADP+). In cells such as insulin-secreting beta-cells, the NADPH/NADP+ redox state supports the scavenging of the reactive oxygen species H2O2 by the glutathione/thioredoxin antioxidant pathway. A loss of beta-cell mass due to oxidative stress leads to type 2 diabetes. A major source of H2O2 production is the mitochondrial electron transport chain (ETC), with overproduction a characteristic of many metabolic disorders including type 2 diabetes. Although H2O2 can cross cell membranes through facilitated diffusion, the NADPH/NADP+ redox is compartmentalized within organelles. We therefore explored targeting Apollo-NADP+ to various organelles including: the mitochondria, nucleus, plasma membrane, peroxisomes, Golgi apparatus, and endoplasmic reticulum. Unlike the cytoplasm, however, the pH of organelles such as the mitochondria are dynamic and therefore require pH-independent sensors. Here we found that pH significantly affects the anisotropy of fluorescent proteins with high pKa (ex. Venus), while fluorescent proteins with low pKa values (ex. Cerulean3, Turquoise2) maintained stable anisotropy values across a wider range of pH values (4.0-8.0). We also found that dimeric Turquoise2-tagged Apollo-NADP+ was stable from pH = 5.0-8.0, making it suitable for use in most cellular compartments. We are now investigating the ability to simultaneously measure the NADPH/NADP+ redox state in various organelles of beta-cells as well as using these sensors in high throughput screening. Overall, this project demonstrates how homoFRET-based sensors may be adapted for specific organelles while revealing a novel use of the intensity-independence property of homoFRET towards simultaneous single-colour measurements and high-throughput assays.