Direct comparison of a genetically encoded sensor and small molecule indicator: Implications for quantification of cytosolic Zn²⁺

Fluorescent sensors are powerful tools for visualizing and quantifying molecules and ions in living cells. A variety of small molecule and genetically encoded sensors have been developed for studying intracellular Zn²⁺ homeostasis and signaling, but no direct comparisons exist, making it challenging for researchers to identify the appropriate sensor for a given application. Here we directly compare the widely used small molecule probe FluoZin-3 and a genetically encoded sensor, ZapCY2. We demonstrate that, in contrast to FluoZin-3, ZapCY2 exhibits a well-defined cytosolic localization, provides estimates of Zn²⁺ concentration with little variability, does not perturb cytosolic Zn²⁺ levels, and exhibits rapid Zn ²⁺ response dynamics. ZapCY2 was used to measure Zn²⁺ concentrations in 5 different cell types, revealing higher cytosolic Zn ²⁺ levels in prostate cancer cells compared to normal prostate cells (although the total zinc is reduced in prostate cancer cells), suggesting distinct regulatory mechanisms.