Apollo-Nadp D Expressed In The Pancreatic Beta-Cells Of Living Zebrafish Responds To Glucose And Diamide To Reveal Nadph Dynamics

We recently developed a family of genetically-encoded homoFRET sensors to measure NADPH/NADP+ redox state based on homo-dimerization of inactive glucose-6-phosphate dehydrogenase (Apollo-NADP+). Our aim is to use Apollo-NADP+ to study NADPH dynamics of pancreatic beta cells in vivo during redox stress. Redox stress induces beta cell failure, ultimately resulting in Type 2 Diabetes. We previously showed that light-scattering in cell aggregates caused a shift in absolute fluorescence anisotropy values, but the relative differences between samples were maintained. We now investigate 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. Here, we generated a transgenic zebrafish with stable Apollo-NADP+ expression targeted to the insulin-secreting beta cells of the zebrafish pancreas and show that Apollo-NADP+ responds to glucose and diamide treatments. Both 2-photon microscopy and microfluidics will be explored to track cellular NADPH dynamics and optimize imaging conditions for live transgenic zebrafish. Overall, this project aims to determine whether homoFRET sensors such as Apollo-NADP+ can be translated in vivo to study living tissue.