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Zn²⁺ is required by thousands of cellular proteins where it has structural, catalytic and regulatory functions . Zn²⁺ regulates a widerange of molecular processes that can profoundly affect cellular and organismal biology [1]. The total concentration of Zn²⁺ within mammalian cells is on the order of hundreds of micromolar, while readily exchangeable or labile Zn²⁺ is maintained on the order of hundreds of picomolar. Multiple studies have demonstrated that in a wide range of mammalian cell types cytosolic Zn²⁺ levels are near 100 pM and the labile Zn²⁺ pool can fluctuate in response to extracellular stimuli.Tools and techniques that are capable of differentiating the labile and bound Zn²⁺ pools, manipulating Zn²⁺ levels in media and in cells, and proteomic approaches for profiling the Zn²⁺ proteome are critical to our understanding of Zn²⁺ biology [2].

Illuminating Zn²⁺ in living cells with fluorescent sensors

In contrast with elemental mapping techniques which can provide valuable information about total Zn²⁺ distribution in cells fluorescent Zn²⁺ sensors can specifically report on the labile Zn²⁺ pool. Fluorescent Zn²⁺ sensors come in two main flavors: genetically encoded and smallmolecule sensors. Both classes contain a metal-binding group and at least one fluorophore that absorbs and emits light in the visible region of the electromagnetic spectrum. Zn²⁺ binding results in a change in the overall structure or electronic configuration of the sensor, resulting in a change in fluorescence that can be measured using a fluorescence microscope.

When selecting a Zn²⁺ sensor, one must consider characteristics inherent to the fluorophore, such as absorption and emission properties, photostability, pH-sensitivity and brightness, as well as metal-binding properties, like selectivity, kinetics and affinity. Fluorescent sensors are most sensitive to changes in Zn²⁺ concentrations that are near their apparent dissociation constant for Zn²⁺ binding (Kd'). The Kd' is defined as the labile Zn²⁺ concentration at which the sensor is half-saturated and can be determined by performing an in vitro or in situ Zn²⁺ titration. Another important characteristic of fluorescent sensors is the dynamic range, which is determined by the fluorescence signal in the Zn²⁺- bound state versus the fluorescence signal in the Zn²⁺-unbound state. In general, a sensor with a high dynamic range can provide both sensitivity and accuracy for estimating Zn²⁺ levels. Ultimately, selecting a sensor will depend on the intended application. For instance, genetically encoded Zn²⁺ sensors can be selectively targeted to subcellular compartments as a means to measure Zn²⁺ within organelles or detect Zn²⁺ release at the cell surface the in situ dynamic range, Kd' and targeted cellular compartments of the current repertoire of genetically encoded Zn²⁺ sensors. In contrast, small-molecule sensors are difficult to direct to a particular compartment, but they often offer a large dynamic range making them particularly sensitive to Zn²⁺ dynamics. Hybrid, or chemigenetic, sensors are a promising new set of tools that leverage small-molecule Zn²⁺ sensors and take advantage of a genetically encoded component to target organelles. In this review, we cover recent advances in the development and application of fluorescent Zn²⁺ sensors. We also refer readers to excellent reviews that cover earlier work in the field [3].

Zn²⁺ in cellular regulation

It is widely known that Zn²⁺ is necessary for cell proliferation, as Zn²⁺ deficient cells fail to divide and proliferate and one of the symptoms of Zn²⁺ deficiency is stunted growth. However, the molecular mechanisms of how Zn²⁺ deficiency leads to cell cycle arrest have remained elusive. Recently, new tools to study both Zn²⁺ and the cell cycle have begun to provide insight into the role of Zn²⁺. Cell cycle studies typically rely on serum starvation of cells to synchronize the cell cycle phases across a population. While serum starvation removes essential growth factors such as mitogens, it also removes essential vitamins and minerals, including Zn²⁺. Furthermore, cell synchronization can induce stress response pathways, making it difficult to correlate findings to naturally cycling cells. Advances in fluorescent reporters, high throughput microscopy, and quantitative image analysis have made it possible to monitor cell cycle phases in naturally cycling cells to characterize cells’ temporary exit from the cell cycle, termed quiescence [4].

In an asynchronously-dividing population of cells, Lo and colleagues were able to track cells throughout the cell cycle and determine which aspects of the cell cycle are dependent on Zn²⁺. Cells were grown in media with chelex-treated serum to create a minimal media Zn²⁺ condition (1.46 μM Zn²⁺, measured by ICP-MS), and experimental conditions were either supplemented with 30 μM Zn²⁺ (Zn²⁺ replete) or further Zn²⁺-restricted by the addition of 2–3 μM TPA. The metal in cells was then quantified using the genetically encoded sensor ZapCV2, which demonstrated that media Zn²⁺ and TPA manipulation result in changes in labile cellular Zn²⁺, and that these manipulations are not toxic to cells for the duration of the experiment. The Zn²⁺ deficient condition was found to either induce cellular quiescence or cell cycle stall in S-phase, with insufficient DNA replication. The cellular response to Zn²⁺ deficiency was shown to be dependent on when in the cell cycle Zn²⁺ deficiency was induced. Interestingly, DNA damage was increased in the Zn²⁺-deficient cells that continued through the cell cycle, but not their quiescent neighbors, suggesting that while Zn²⁺ is necessary for DNA replication and repair, its role in the proliferation/ quiescence decision is independent of DNA damage. Furthermore, resupply of Zn²⁺ promotes cell cycle re-entry, but the mechanism by which proteins and signaling pathways mediate cell cycle re-entry has not been identified.

Zn²⁺ in cell signaling

Another major push in the field of Zn²⁺ biology is understanding systems in which Zn²⁺ can act as a cellular signal. A variety of cell systems have been shown to exhibit dynamic Zn²⁺ behavior, including neurons and oocytes. It has long been known that specific regions of the brain are Zn²⁺-rich and that the Zn²⁺ transporter, ZnT3, imports Zn²⁺ into synaptic vesicles. Along with ZnT3 knockout mice, the extracellular Zn²⁺ chelator ZX1 has led to insights about the role of vesicular Zn²⁺ in neurons. Zn²⁺ has been shown to modulate signaling through neurotransmitter receptors, including the inhibition of NMDA glutamate receptors. New electrophysiology studies show that synaptically-released Zn²⁺ from hippocampal mossy fiber neurons contributes to long term potentiation, or strengthening of neuronal synaptic connections Recently, Sanford and colleagues quantified Zn²⁺ dynamics upon stimulation by KCl in cultured hippocampal neurons and demonstrated that over 900 genes exhibit changes in expression in a Zn²⁺-dependent manner in response to subnanomolar fluctuations of Zn²⁺. Many of these transcriptional changes involve synaptic plasticity pathways Furthermore, in vivo animal studies have demonstrated that Zn²⁺ may play a role in fear conditioning, long-term and spatial memory, and audition. These in vivo phenotypes are subtle, suggesting that synaptic Zn²⁺ may play more of a role in fine-tuning neuronal connections or that there are compensatory mechanisms during development that mask synaptic Zn²⁺ deficiency phenotypes.

While it has long been recognized that Zn²⁺ is concentrated in mossy fiber neurons in the hippocampus and released with synaptic activity through studies in brain slices and animals, cellular models of Zn²⁺ dynamics are far less clear. In dissociated hippocampal neuron culture, stimulation with glutamate/glycine or KCl has been shown to increase intracellular Zn²⁺, and this Zn²⁺ signal has important downstream signaling consequences. While it was routinely assumed that this intracellular Zn²⁺ derived from synaptic release, more recent studies suggest that this Zn²⁺ may arise from an intracellular source. In particular, it was suggested that neuronal acidification upon glutamate treatment was responsible for Zn²⁺ mobilization. Sanford and Palmer recently quantified Zn²⁺, Ca2+, and pH changes using a series of fluorescent sensors during stimulation of dissociated hippocampal neurons in culture. Both KCl and glutamate stimulation led to increases in cytosolic Zn²⁺, and the signal was comparable in the presence of ZX1, suggesting that in dissociated neuron culture Zn²⁺ was released from intracellular stores. Although the pH decreased upon neuronal stimulation, the magnitude and timing of the changes in pH did not correlate with the magnitude and timing of Zn²⁺ changes, suggesting that Zn²⁺ release from intracellular stores might be due to Ca2+ dynamics or reactive oxygen species (ROS) production [5].

Another cellular system that experiences Zn²⁺ signals is the developing oocyte. Recently, Zn²⁺ accumulation during oocyte maturation and release at fertilization were rigorously quantified to better understand the source of the “Zn²⁺ spark” at fertilization. Que and colleagues, used the fluorescent Zn²⁺ probe ZincBY-1, elemental mapping, and extracellular FluoZin-3 Zn²⁺ dye to show that oocytes contain thousands of vesicles loaded with approximately 1 million Zn²⁺ ions each, and that these vesicles undergo exocytosis when the oocyte becomes fertilized, resulting in the extracellular Zn²⁺ spark. Subsequent research demonstrated that the Zn²⁺ released from oocytes upon fertilization is linked to the hardening of the zona pellucida, the oocyte’s glycoprotein extracellular matrix, which prevents subsequent fertilizations and maintains the viability of the newly formed zygote . Furthermore, larger Zn²⁺ sparks have been shown to be indicative of future embryo quality, suggesting that Zn²⁺ could potentially be used as a biomarker for fertility treatments. These recent discoveries in oocyte biology suggest that Zn²⁺ regulation and dynamics can play multiple roles in cell development and physiology.

Acknowledgement:

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Conflict of Interest:

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