ORGANELLE-TARGETING OF APOLLO-NADP+ MAKES TRACKING NADPH/NADP+ REDOX POSSIBLE ACROSS MULTIPLE ORGANELLES
Cameron, William D.1; Rocheleau, Jonathan1, 2
1. Institute of Biomaterials and Biomedical Engineering, University of Toronto; 2. Toronto General Research Institute, University Health Network
With the advent of techniques such as machine learning, it is now possible to analyze and interpret enormous datasets containing complex, multivariate readouts. These multivariate readouts are particularly important in biology as they provide insight into complex cellular mechanisms. For example, we previously demonstrated that NADP+ based mechanisms must fail substantially before H2O2 accumulation can occur by simultaneously measuring NADP+ and H2O2 within the cell. Unfortunately, most genetically-encoded sensors are based on techniques that have large spectral requirements (ex. heteroFRET, circular permutations) and are therefore not well suited for this type of multiparametric imaging. To address this issue, we have adapted an alterative technique called homoFRET for use in genetically encoded sensors. Rather than detecting changes in colour or intensity, homoFRET sensors use changes in fluorescence polarization to provide a ratiometric readout.