Anisotropy Imaging of Apollo-NADP+ In Vivo to Track NADPH Dynamics in the Pancreatic Beta Cells of Living Zebrafish
Bui, Cindy 1, 2 ; 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 genetically-encoded homoFRET sensor to measure NADPH/NADP+ redox state based on homo-oligomerization of inactive glucose-6-phosphate dehydrogenase (Apollo-NADP+). We now aim to use Apollo-NADP+ to study in vivo NADPH dynamics during pancreatic beta cell redox stress. Redox stress induces beta cell failure, ultimately resulting in Type 2 Diabetes. We previously found that light-scattering in cell aggregates caused a shift in absolute fluorescence anisotropy values, but the relative differences between samples were maintained. We are now investigating zebrafish as a low-scatter alternative platform for 3D, in vivo fluorescence anisotropy imaging. Zebrafish are an ideal platform for this work due to their optical transparency for imaging, genetic tractability, and ability to model many pathological diseases. Using the tol2 transposon system, we generated transgenic zebrafish with stable Apollo-NADP+ expression targeted to the insulin-secreting beta cells of the zebrafish pancreas. Both 2-photon and light-sheet microscopy will be explored to track cellular NADPH dynamics in live transgenic zebrafish. Overall, this project aims to determine whether homoFRET-based sensors such as Apollo-NADP+ can be translated to study living tissue.