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

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 (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 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.