Visualization of peroxynitrite-induced changes of labile Zn²⁺ in the endoplasmic reticulum with benzoresorufin-based fluorescent probes

Zn²⁺ plays essential roles in biology, and the homeostasis of Zn²⁺ is tightly regulated in all cells. Subcellular distribution and trafficking of labile Zn²⁺, and its inter-relation with reactive nitrogen species, are poorly understood due to the scarcity of appropriate imaging tools. We report a new family of red-emitting fluorescent sensors for labile Zn²⁺, ZBR1-3, based on a benzoresorufin platform functionalized with dipicolylamine or picolylamine-derived metal binding groups. In combination, the pendant amines and fluorophore afford an [N₃O] binding motif that resembles that of previously reported fluorescein-based sensors of the Zinpyr family, reproducing well their binding capabilities and yielding comparable K_d values in the sub-nanomolar and picomolar ranges. The ZBR sensors display up to 8.4-fold emission fluorescence enhancement upon Zn²⁺ binding in the cuvette, with similar responses obtained in live cells using standard wide-field fluorescence microscopy imaging. The new sensors localize spontaneously in the endoplasmic reticulum (ER) of various tested cell lines, allowing for organelle-specific monitoring of zinc levels in live cells. Study of ER zinc levels in neural stem cells treated with a peroxynitrite generator, Sin-1, revealed an immediate decrease in labile Zn ²⁺ thus providing evidence for a direct connection between ER stress and ER Zn²⁺ homeostasis.