Development and Characterization of a Red Fluorescent Protein-Based Sensor RZnP1 for the Detection of Cytosolic Zn²⁺

Abstract

Fluorescent sensors have been developed to record Zn2+ dynamics and measure Zn2+ 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 Zn2+ sensor, Red Zinc Probe (RZnP1), which can detect intracellular concentrations of Zn2+. RZnP1 demonstrates a sensitive response to cytosolic Zn2+ (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 Zn2+ over other biologically relevant metal cations. RZnP1 offers a way to image Zn2+ with multiple intracellular ions in tandem. We demonstrate the simultaneous recording of Zn2+ and Ca2+ using RZnP1 alongside the Ca2+ sensor GCaMP5G in HeLa cells. We also use RZnP1 with mito-GZnP2, a green fluorescent protein (GFP)-based mitochondrial Zn2+ sensor, to track Zn2+ dynamics in the cytosol and mitochondria concurrently in rat primary neuron culture. Our work not only expands the toolbox of Zn2+ sensors but also demonstrates techniques for imaging Zn2+ dynamics along with other cations and between multiple subcellular compartments simultaneously.

Fluorescent Protein-Based Sensors for Detecting Essential Metal Ions across the Tree of Life

“…Aside from ZnRed, two other red-shifted single fluorescent protein-based Zn 2+ sensors have been reported. 63,64 RZnP1, like the GZnP series, utilizes the Zap zinc fingers but with cpmApple in place of cpGFP. 63 RZnP1 was applied alongside green sensors for Ca 2+ (GCaMP5G) and Zn 2+ (mito-GZnP2) to concurrently detect different ions or the same ion in different subcellular compartments in cultured or primary mammalian cells.…”

“…63,64 RZnP1, like the GZnP series, utilizes the Zap zinc fingers but with cpmApple in place of cpGFP. 63 RZnP1 was applied alongside green sensors for Ca 2+ (GCaMP5G) and Zn 2+ (mito-GZnP2) to concurrently detect different ions or the same ion in different subcellular compartments in cultured or primary mammalian cells. A far-red fluorescent indicator for synaptic Zn 2+ (FRISZ) was recently developed using a similar approach to ZnGreen2, whereby the pf Rad50 Zn hook peptides were fused to cpmMaroon.…”

“…Not long after the development of the first FRET-based sensor for Ca 2+ , the first single fluorescent protein-based sensor for Ca 2+ , Camgaroo, was developed by inserting CaM into the sequence of enhanced yellow fluorescent protein (EYFP) such that CaM is close to the chromophore, and the conformational change induced by Ca 2+ binding influences the chromophore, resulting in an increase in fluorescence signal. , A more widely used and highly optimized platform for Ca 2+ sensing is the GCaMP series. − Here, circularly permuted GFP is fused with CaM and RS20 (a CaM-binding peptide similar to M13) at different termini, such that Ca 2+ binding modulates fluorescence. Similar approaches have been more recently used to generate new zinc-sensing fluorescent proteins. − These sensors have seen widespread use in the calcium field, with cellular and in vivo applications in organisms ranging from mice to worms, zebrafish, flies, plants, and microbes. , Fluorescent protein-based Zn 2+ sensors have been frequently applied to mammalian cells, but other applications have been more limited. Most single fluorescent protein-based sensors use extrinsic metal ion-binding domains such as CaM for Ca 2+ or zinc fingers for Zn 2+ .…”

“…These sensors also have low affinities and showed lower dynamic ranges but were successfully applied in dual-color imaging experiments involving the simultaneous detection of glucose-stimulated Zn 2+ release at the cell surface and cytosolic increase of Ca 2+ with R-GECO1. Aside from ZnRed, two other red-shifted single fluorescent protein-based Zn 2+ sensors have been reported. , RZnP1, like the GZnP series, utilizes the Zap zinc fingers but with cpmApple in place of cpGFP . RZnP1 was applied alongside green sensors for Ca 2+ (GCaMP5G) and Zn 2+ (mito-GZnP2) to concurrently detect different ions or the same ion in different subcellular compartments in cultured or primary mammalian cells.…”

Revealing the elevation of Zn2+ in the brain of depressed mice by a ratiometric fluorescent probe with dual near-infrared emissions

“…1 Zn 2+ is a ubiquitous trace element in the human body, and is an important coenzyme factor and signaling molecule. 2 In addition, Zn 2+ is also linked with mitochondrial oxidative stress, as it can work as an antioxidant indirectly at a certain concentration range. However, it shows prooxidant effects at abnormal levels in mitochondria.…”