Development and Characterization of a Red Fluorescent Protein-Based Sensor RZnP1 for the Detection of Cytosolic Zn.

Fluorescent sensors have been developed to record Zn dynamics and measure Zn concentrations within the cell. Most previous efforts on developing single-wavelength sensors are focused on green sensors. Here, we engineer a genetically encoded, single red fluorescent protein-based Zn sensor, Red Zinc Probe (RZnP1), which can detect intracellular concentrations of Zn. RZnP1 demonstrates a sensitive response to cytosolic Zn ( = 438 pM), decent brightness (quantum yield (QY) = 0.15), good in situ dynamic range (/ = 4.0), and specificity for Zn over other biologically relevant metal cations. RZnP1 offers a way to image Zn with multiple intracellular ions in tandem. We demonstrate the simultaneous recording of Zn and Ca using RZnP1 alongside the Ca sensor GCaMP5G in HeLa cells. We also use RZnP1 with mito-GZnP2, a green fluorescent protein (GFP)-based mitochondrial Zn sensor, to track Zn dynamics in the cytosol and mitochondria concurrently in rat primary neuron culture. Our work not only expands the toolbox of Zn sensors but also demonstrates techniques for imaging Zn dynamics along with other cations and between multiple subcellular compartments simultaneously.