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Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.
Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.
Zinc is an essential trace element in the cellular milieu. It incorporates one-third of all proteins and is involved in various cellular processes, such as catalysis1, transcription2, and structural motifs3. However, despite being redox-inert, high zinc concentrations are toxic to the cell, which is why no mammalian organism has survived without the presence of mechanisms regulating zinc homeostasis. In mammals, three mechanisms are responsible for this process: (1) metallothioneins, which are cytosolic cysteine-rich proteins that bind zinc at a high affinity, thus preventing excess free cytosolic zinc4; (2) Zrt/Irt-like proteins (ZIPs), which are zinc transporters responsible for zinc influx into the cytosol through the plasma membrane or from intracellular organelles4,5,6,7,8; and (3) ZnTs, which are a mammalian subset of the ubiquitous cation diffusion facilitator (CDF) family and are zinc transporters, as they extrude zinc from the cytosol across the plasma membrane or into the intracellular organelles4,5,6,7,8,9. Due to the importance of zinc to cellular metabolism, it is vital to understand cellular zinc dynamics.
Previous methods to assess zinc dynamics depended on assessing the expression levels of mRNA under different zinc conditions by correlating them with cellular zinc measurements of fixed tissues or cells10,11,12. These methods include chemical detection and immunohistochemistry staining. However, these methods yield only indirect measures and, thus, determine only an offline correlation between intracellular zinc concentration and the expression of zinc transporters. Consequently, these methods cannot infer any parameters requiring high temporal resolution.
A more direct measurement of Zn2+ transport uses radioactive isotopes of zinc13. This method relies on the measurement of radiolabeled Zn2+ to monitor zinc transport and its kinetics. However, due to the importance of zinc to cellular homeostasis, multiple cellular processes regulate intracellular zinc concentration. Among these are extracellular binding and several transport systems that work in concert to maintain tight control of intracellular Zn2+ levels. The combination of these processes creates considerable background noise, which makes it difficult to test individual zinc-related transport functions.
This article demonstrates a method to directly monitor the zinc transport rate by measuring the intracellular free zinc concentration using a zinc-specific fluorescent dye, FluoZin-3. The dye has high specificity for Zn2+ and little interference from other divalent cations, such as calcium. In addition, in its ester form, it enters the cells by nonionic diffusion and is then trapped due to the activity of intracellular di-esterase. Thus, its fluorescence is correlated primarily with the free cytosolic zinc concentration. These experiments were conducted to study the structure-function relationship of zinc transporter 1 (ZnT1), a member of the ZnT family.
1. Cell transfection
2. Microscope preparation
3. Sample preparation
4. Measurement preparation
5. Experimental procedure
6. Data export
7. Data analysis
ZnT1 is a mammalian zinc transporter located on the cell plasma membrane13. It is a member of the cation diffusion facilitator (CDF) protein family that extrudes zinc from the cytosol to the extracellular millieu14. ZnT1 has a two-domain architecture: the transmembrane domain, which transports the ions across the membrane, and a C-terminal domain14. Unlike other known CDF proteins, ZnT1 has an extended unstructured C-terminal domain (USCTD). The role...
The above-described method allows for the direct measurement of the intracellular zinc concentration with high temporal resolution. Compared to other methods, this method involving monitoring changes in intracellular Zn2+ can substantially decrease background noise. In addition, the dye's selectivity for zinc eliminates potential cross-interactions with other metal cations18,19. Finally, its lack of immediate cytotoxicity enables the testing of liv...
The authors declare no conflicts of interest.
Raz Zarivach is supported by the Israel Science Foundation (grant no. 163/22). Tomer Eli Ben Yosef and Arie Moran are supported by the Israel Science Foundation (grant no. 2047/20). We would like to thank Daniel Gitler and his group at Ben-Gurion University for their cooperation, support, and expertise.
Name | Company | Catalog Number | Comments |
10 cm plate | greiner bio-one | 664160 | |
12-well cell culture plate | greiner bio-one | 665180 | |
13 mm coverslips | Superior Marienfeld | 111530 | |
22 mm cover slides | Superior Marienfeld | 101050 | |
6-well culture plate | greiner bio-one | 657160 | |
Bovine serum albumin | bioWorld | 22070008 | |
Calcium chloride anhydrous, granular | Sigma Aldrich | C1016 | Concentration in Ringer solution: 1 mM |
D-(+)-Glucose | Glentham Life Science | GC6947 | Concentration in Ringer solution: 10 mM |
Dubelco’s Modified Eagle Media (DMEM) | Sartorius | 01-055-1A | |
Eclipse Ti inverted microscope | Nikon | TI-DH | Discontinued. Replaced by Eclipse Ti2 |
Fetal Bovine Serum (FBS) | Cytiva | SH30088.03 | |
Fine tweezers | Dumont | 0203-55-PS | |
Fluozin-3AM | Invitrogen | F24195 | |
HyClone Penicillin-Streptomycin 100x solution | Cytiva | SV30010 | |
LED illumination system | CoolLED | pE-4000 | |
L-glutamine | Biological Industries | 03-020-1B | |
Magnesium chloride hexahydrate | Merck | 1.05833 | Concentration in Ringer solution: 0.8 mM |
N[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES) | Formedium | HEPES10 | Concentration in Ringer solution: 10 mM |
Neo 5.5 sCMOS camera | ANDOR | DC-152Q-FI | |
NIS-Elements imaging software | Nikon | AR | |
Pluronic acid F-127 | Millipore | 540025 | |
Pottasium chloride | Bio-Lab | 163823 | Concentration in Ringer solution: 5.4 mM |
Pyrithione | Sigma Aldrich | H3261 | Concentration in Ringer zinc solution: 7 μM |
Silicone Grease Kit | Warner Instruments | W4 64-0378 | |
Sodium chloride | Bio-Lab | 190305 | Concentration in Ringer solution: 120 mM |
Zinc sulfate | Concentration in Ringer zinc solution: 7 μM | ||
Sigma Aldrich | 31665 |
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