title: Monitoring cytosolic and ER Zn(2+) in stimulated breast cancer cells using genetically encoded FRET sensors.
abstract: 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 (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 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.
authors.affiliation: Laboratory of Chemical Biology and Institute of Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands. M.Merkx@tue.nl.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Hessels
authors.affiliation: Breast Cancer Molecular Pharmacology Group, School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UK.
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Taylor
authors.affiliation: Laboratory of Chemical Biology and Institute of Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands. M.Merkx@tue.nl.
authors.forename: Maarten
authors.initials: M
authors.lastname: Merkx
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D061986:MCF-7 Cells; D015032:Zinc / Q000032:analysis / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D015032:Zinc