Visualization of Peroxynitrite-Induced Changes of Labile Zn[superscript 2+] in the Endoplasmic Reticulum with Benzoresorufin-based Fluorescent Probes

Abstract

Zn[superscript 2+] plays essential roles in biology, and the homeostasis of Zn[superscript 2+] is tightly regulated in all cells. Subcellular distribution and trafficking of labile Zn[superscript 2+], 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 Zn[superscript 2+], 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 [N[subscript 3]O] binding motif that resembles that of previously reported fluorescein-based sensors of the Zinpyr family, reproducing well their binding capabilities and yielding comparable K[subscript d] values in the sub-nanomolar and picomolar ranges. The ZBR sensors display up to 8.4-fold emission fluorescence enhancement upon Zn[superscript 2+] 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[superscript 2+] thus providing evidence for a direct connection between ER stress and ER Zn[superscript 2+] homeostasis.