Protocol for Measuring Labile Cytosolic Zn Using an in Situ Calibration of a Genetically Encoded FRET Sensor

Overview

Journal STAR Protoc

Publisher Cell Press

Specialties Biology
Biomedical Engineering
Science

Date 2024 Jun 13

PMID 38870018

Authors

Samuel E Holtzen

Ananya Rakshit

Amy E Palmer

Affiliations

Soon will be listed here.

Abstract

Zinc (Zn) plays roles in structure, catalysis, and signaling. The majority of cellular Zn is bound by proteins, but a fraction of total Zn exists in a labile form. Here, we present a protocol for measuring labile cytosolic Zn using an in situ calibration of a genetically encoded Förster resonance energy transfer (FRET) sensor. We describe steps for producing buffered Zn solutions for performing an imaging-based calibration and analyzing the imaging data generated to determine labile Zn concentration in single cells. For complete details on the use and execution of this protocol, please refer to Rakshit and Holtzen et al..

References

Janiszewski L, Minson M, Allen M, Dowell R, Palmer A . Characterization of Global Gene Expression, Regulation of Metal Ions, and Infection Outcomes in Immune-Competent 129S6 Mouse Macrophages. Infect Immun. 2021; 89(11):e0027321. PMC: 8519282. DOI: 10.1128/IAI.00273-21. View

Sanford L, Carpenter M, Palmer A . Intracellular Zn transients modulate global gene expression in dissociated rat hippocampal neurons. Sci Rep. 2019; 9(1):9411. PMC: 6598991. DOI: 10.1038/s41598-019-45844-2. View

Park J, Qin Y, Galati D, Palmer A . New sensors for quantitative measurement of mitochondrial Zn(2+). ACS Chem Biol. 2012; 7(10):1636-40. PMC: 3477250. DOI: 10.1021/cb300171p. View

Krezel A, Maret W . The biological inorganic chemistry of zinc ions. Arch Biochem Biophys. 2016; 611:3-19. PMC: 5120989. DOI: 10.1016/j.abb.2016.04.010. View

Marks P, Maxfield F . Preparation of solutions with free calcium concentration in the nanomolar range using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Anal Biochem. 1991; 193(1):61-71. DOI: 10.1016/0003-2697(91)90044-t. View

Rakshit A, Holtzen S, Lo M, Conway K, Palmer A . Human cells experience a Zn pulse in early G1. Cell Rep. 2023; 42(6):112656. PMC: 10592493. DOI: 10.1016/j.celrep.2023.112656. View

Carter K, Carpenter M, Fiedler B, Jimenez R, Palmer A . Critical Comparison of FRET-Sensor Functionality in the Cytosol and Endoplasmic Reticulum and Implications for Quantification of Ions. Anal Chem. 2017; 89(17):9601-9608. PMC: 6018025. DOI: 10.1021/acs.analchem.7b02933. View

Anson K, Corbet G, Palmer A . Zn influx activates ERK and Akt signaling pathways. Proc Natl Acad Sci U S A. 2021; 118(11). PMC: 7980380. DOI: 10.1073/pnas.2015786118. View

Qin Y, Miranda J, Stoddard C, Dean K, Galati D, Palmer A . Direct comparison of a genetically encoded sensor and small molecule indicator: implications for quantification of cytosolic Zn(2+). ACS Chem Biol. 2013; 8(11):2366-71. PMC: 4022295. DOI: 10.1021/cb4003859. View

10.

Andreini C, Banci L, Bertini I, Rosato A . Counting the zinc-proteins encoded in the human genome. J Proteome Res. 2006; 5(1):196-201. DOI: 10.1021/pr050361j. View

11.

Tsien R, Pozzan T . Measurement of cytosolic free Ca2+ with quin2. Methods Enzymol. 1989; 172:230-62. DOI: 10.1016/s0076-6879(89)72017-6. View

12.

Lo M, Damon L, Tay J, Jia S, Palmer A . Single cell analysis reveals multiple requirements for zinc in the mammalian cell cycle. Elife. 2020; 9. PMC: 7000218. DOI: 10.7554/eLife.51107. View

13.

Vinkenborg J, Nicolson T, Bellomo E, Koay M, Rutter G, Merkx M . Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis. Nat Methods. 2009; 6(10):737-40. PMC: 6101214. DOI: 10.1038/nmeth.1368. View

14.

Colvin R, Holmes W, Fontaine C, Maret W . Cytosolic zinc buffering and muffling: their role in intracellular zinc homeostasis. Metallomics. 2010; 2(5):306-17. DOI: 10.1039/b926662c. View

15.

Fiedler B, Van Buskirk S, Carter K, Qin Y, Carpenter M, Palmer A . Droplet Microfluidic Flow Cytometer For Sorting On Transient Cellular Responses Of Genetically-Encoded Sensors. Anal Chem. 2016; 89(1):711-719. PMC: 6019271. DOI: 10.1021/acs.analchem.6b03235. View