Targeting Apollo-NADP⁺ to image NADPH generation in pancreatic beta-cell organelles

ABSTRACT NADPH/NADP + redox state supports numerous reactions related to cell growth and survival; yet the full impact is difficult to appreciate due to organelle compartmentalization of NADPH and NADP + . To study glucose-stimulated NADPH production in pancreatic beta-cell organelles, we targeted the Apollo-NADP + sensor by first selecting the most pH stable version of the single colour sensor. We subsequently targeted mTurquoise2-Apollo-NADP + to various organelles and confirmed activity in the cytoplasm, mitochondrial matrix, nucleus, and peroxisome. Finally, we measured the glucose- and glutamine-stimulated NADPH responses by single and dual colour imaging of the targeted sensors. Overall, we developed multiple organelle-targeted Apollo-NADP + sensors to reveal the prominent role of beta-cell mitochondria in determining NADPH production in the cytoplasm, nucleus, and peroxisome.