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Monitoring cytosolic and ER Zn(2+) in stimulated breast cancer cells using genetically encoded FRET sensors
The Zn(2+)-specific ion channel ZIP7 has been implicated to play an important role in releasing Zn(2+) from the ER. External stimulation of breast cancer cells has been proposed to induce phosphorylation of ZIP7 by CK2α, resulting in ZIP7-mediated Zn(2+) release from the ER into the cytosol. Here, w...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Royal Society of Chemistry
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756312/ https://www.ncbi.nlm.nih.gov/pubmed/26739447 http://dx.doi.org/10.1039/c5mt00257e |
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author | Hessels, Anne M. Taylor, Kathryn M. Merkx, Maarten |
author_facet | Hessels, Anne M. Taylor, Kathryn M. Merkx, Maarten |
author_sort | Hessels, Anne M. |
collection | PubMed |
description | The Zn(2+)-specific ion channel ZIP7 has been implicated to play an important role in releasing Zn(2+) from the ER. External stimulation of breast cancer cells has been proposed to induce phosphorylation of ZIP7 by CK2α, resulting in ZIP7-mediated Zn(2+) release from the ER into the cytosol. Here, we examined whether changes in cytosolic and ER Zn(2+) concentrations can be detected upon such external stimuli. Two previously developed FRET sensors for Zn(2+), eZinCh-2 (K (d) = 1 nM at pH 7.1) and eCALWY-4 (K (d) = 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 Zn(2+) and pyrithione, one of the previously used triggers, resulted in an immediate increase in free Zn(2+) in both cytosol and ER, suggesting that Zn(2+) was directly transferred across the cellular membranes by pyrithione. Cells treated with a second trigger, EGF/ionomycin, showed no changes in intracellular Zn(2+) levels, neither in multicolor imaging experiments that allowed simultaneous imaging of cytosolic and ER Zn(2+), nor in experiments in which cytosolic and ER Zn(2+) 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 Zn(2+) levels as a result from Zn(2+) 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 Zn(2+) dyes. |
format | Online Article Text |
id | pubmed-4756312 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Royal Society of Chemistry |
record_format | MEDLINE/PubMed |
spelling | pubmed-47563122016-03-03 Monitoring cytosolic and ER Zn(2+) in stimulated breast cancer cells using genetically encoded FRET sensors Hessels, Anne M. Taylor, Kathryn M. Merkx, Maarten Metallomics Chemistry The Zn(2+)-specific ion channel ZIP7 has been implicated to play an important role in releasing Zn(2+) from the ER. External stimulation of breast cancer cells has been proposed to induce phosphorylation of ZIP7 by CK2α, resulting in ZIP7-mediated Zn(2+) release from the ER into the cytosol. Here, we examined whether changes in cytosolic and ER Zn(2+) concentrations can be detected upon such external stimuli. Two previously developed FRET sensors for Zn(2+), eZinCh-2 (K (d) = 1 nM at pH 7.1) and eCALWY-4 (K (d) = 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 Zn(2+) and pyrithione, one of the previously used triggers, resulted in an immediate increase in free Zn(2+) in both cytosol and ER, suggesting that Zn(2+) was directly transferred across the cellular membranes by pyrithione. Cells treated with a second trigger, EGF/ionomycin, showed no changes in intracellular Zn(2+) levels, neither in multicolor imaging experiments that allowed simultaneous imaging of cytosolic and ER Zn(2+), nor in experiments in which cytosolic and ER Zn(2+) 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 Zn(2+) levels as a result from Zn(2+) 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 Zn(2+) dyes. Royal Society of Chemistry 2016-02-17 2016-01-07 /pmc/articles/PMC4756312/ /pubmed/26739447 http://dx.doi.org/10.1039/c5mt00257e Text en This journal is © The Royal Society of Chemistry 2016 http://creativecommons.org/licenses/by/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution 3.0 Unported License (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Chemistry Hessels, Anne M. Taylor, Kathryn M. Merkx, Maarten Monitoring cytosolic and ER Zn(2+) in stimulated breast cancer cells using genetically encoded FRET sensors |
title | Monitoring cytosolic and ER Zn(2+) in stimulated breast cancer cells using genetically encoded FRET sensors
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title_full | Monitoring cytosolic and ER Zn(2+) in stimulated breast cancer cells using genetically encoded FRET sensors
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title_fullStr | Monitoring cytosolic and ER Zn(2+) in stimulated breast cancer cells using genetically encoded FRET sensors
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title_full_unstemmed | Monitoring cytosolic and ER Zn(2+) in stimulated breast cancer cells using genetically encoded FRET sensors
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title_short | Monitoring cytosolic and ER Zn(2+) in stimulated breast cancer cells using genetically encoded FRET sensors
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title_sort | monitoring cytosolic and er zn(2+) in stimulated breast cancer cells using genetically encoded fret sensors |
topic | Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756312/ https://www.ncbi.nlm.nih.gov/pubmed/26739447 http://dx.doi.org/10.1039/c5mt00257e |
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