Organelle-Level Labile Zn²⁺ Mapping Based on Targetable Fluorescent Sensors

Although many Zn²⁺fluorescent probes have been developed, there remains a lack of consensus on the labile Zn²⁺concentrations ([Zn²⁺]) in several cellular compartments, as the fluorescence properties and zinc affinity of the fluorescent probes are greatly affected by the pH and redox environments specific to organelles. In this study, we developed two turn-on-type Zn²⁺fluorescent probes, namely, ZnDA-2H and ZnDA-3H, with low pH sensitivity and suitable affinity (K_d= 5.0 and 0.16 nM) for detecting physiological labile Zn²⁺in various cellular compartments, such as the cytosol, nucleus, ER, and mitochondria. Due to their sufficient membrane permeability, both probes were precisely localized to the target organelles in HeLa cells using HaloTag labeling technology. Using an in situ standard quantification method, we identified the [Zn²⁺] in the tested organelles, resulting in the subcellular [Zn²⁺] distribution as [Zn²⁺]_ER< [Zn²⁺]_mito< [Zn²⁺]_cyto∼[Zn²⁺]_nuc.