Monitoring cytosolic and ER Zn2+ in stimulated breast cancer cells using genetically encoded FRET sensors

Hessels, Anne M., Taylor, Kathryn Mary ORCID: https://orcid.org/0000-0002-9576-9490 and Merkx, Maarten 2016. Monitoring cytosolic and ER Zn2+ in stimulated breast cancer cells using genetically encoded FRET sensors. Metallomics 8 , pp. 211-217. 10.1039/C5MT00257E

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Abstract

The Zn2+-specific ion channel ZIP7 has been implicated to play an important role in releasing Zn2+ from the ER. External stimulation of breast cancer cells has been proposed to induce phosphorylation of ZIP7 by CK2α, resulting in ZIP7-mediated Zn2+ release from the ER into the cytosol. Here, we examined whether changes in cytosolic and ER Zn2+ concentrations can be detected upon such external stimuli. Two previously developed FRET sensors for Zn2+, eZinCh-2 (Kd = 1 nM at pH 7.1) and eCALWY-4 (Kd = 0.63 nM at pH 7.1), were expressed in both the cytosol and the ER of wild-type MCF-7 and TamR cells. Treatment of MCF-7 and TamR cells with external Zn2+ and pyrithione, one of the previously used triggers, resulted in an immediate increase in free Zn2+ in both cytosol and ER, suggesting that Zn2+ was directly transferred across the cellular membranes by pyrithione. Cells treated with a second trigger, EGF/ionomycin, showed no changes in intracellular Zn2+ levels, neither in multicolor imaging experiments that allowed simultaneous imaging of cytosolic and ER Zn2+, nor in experiments in which cytosolic and ER Zn2+ were monitored separately. In contrast to previous work using small-molecule fluorescent dyes, these results indicate that EGF–ionomycin treatment does not result in significant changes in cytosolic Zn2+ levels as a result from Zn2+ release from the ER. These results underline the importance of using genetically encoded fluorescent sensors to complement and verify intracellular imaging experiments with synthetic fluorescent Zn2+ dyes.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Pharmacy
Publisher:	Royal Society of Chemistry
ISSN:	1756-5901
Date of First Compliant Deposit:	30 March 2016
Date of Acceptance:	22 December 2015
Last Modified:	02 May 2023 19:24
URI:	https://orca.cardiff.ac.uk/id/eprint/84325

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