Development and Characterization of a Red Fluorescent Protein-Based Sensor RZnP1 for the Detection of Cytosolic Zn 2 .

Fluorescent sensors have been developed to record Zn 2+ dynamics and measure Zn 2+ 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 2+ sensor, Red Zinc Probe (RZnP1), which can detect intracellular concentrations of Zn 2+ . RZnP1 demonstrates a sensitive response to cytosolic Zn 2+ ( K d = 438 pM), decent brightness (quantum yield (QY) = 0.15), good in situ dynamic range ( F max / F min = 4.0), and specificity for Zn 2+ over other biologically relevant metal cations. RZnP1 offers a way to image Zn 2+ with multiple intracellular ions in tandem. We demonstrate the simultaneous recording of Zn 2+ and Ca 2+ using RZnP1 alongside the Ca 2+ sensor GCaMP5G in HeLa cells. We also use RZnP1 with mito-GZnP2, a green fluorescent protein (GFP)-based mitochondrial Zn 2+ sensor, to track Zn 2+ dynamics in the cytosol and mitochondria concurrently in rat primary neuron culture. Our work not only expands the toolbox of Zn 2+ sensors but also demonstrates techniques for imaging Zn 2+ dynamics along with other cations and between multiple subcellular compartments simultaneously.