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.

Fig. 1. (A and B) Determination of the free cytosolic Zn²⁺ concentration in wild-type MCF-7 (A) and TamR cells (B) using eZinCh-2. Responses to the addition of 50 μM TPEN, followed by the addition of excess 100 μM Zn²⁺/ 5 μM pyrithione. All traces in A and B represent the average of at least four cells after normalization of the emission ratio at t = 0 s. Error bars represent the standard error of the mean (SEM). (C) False-colored ratiometric images of an MCF-7 cell expressing eZinCh-2 in resting state (resting), after perfusion with 50 μM TPEN (+TPEN), and 100 μM ZnCl₂/5 μM pyrithione (+Zn²⁺/pyr). Scale bar, 10 μM.

Fig. 2. Zn²⁺ imaging using ER-targeted eZinCh-2 in both MCF-7 and TamR cells. (A) Schematic representation of eZinCh-2 containing an N-terminal signal sequence (PPI) and a C-terminal retention sequence (KDEL). (B) False-colored image showing MCF-7 cells expressing ER-eZinCh-2. (C and D) Responses of MCF-7 (C) and TamR (D) cells expressing ER-eZinCh-2 to the addition of 50 μM TPEN, and the subsequent addition of excess Zn²⁺ together with pyrithione (100 μM/5 μM). All traces represent the average of four cells after normalization of the emission ratio at t = 0 s. Error bars represent SEM.

Fig. 3. Zn²⁺ imaging using ER-targeted eCALW-4 in both MCF-7 and TamR cells. (A) Schematic representation of eCALWY-4 containing a PPI and a KDEL sequence. (B) False-colored image showing a TamR cells expressing ER-eCALWY-4. (C and D) Responses of MCF-7 (C) and TamR (D) cells expressing ER-eCALWY-4 to the addition of 50 μM TPEN, and the subsequent addition of Zn²⁺/pyrithione. All traces in represent the average of four cells after normalization of the emission ratio at t = 0 s. Error bars represent SEM.

Fig. 4. (A and B) Responses of MCF-7 cells loaded with FluoZin-3 AM to the addition of 20 μM Zn²⁺ together with the Zn²⁺-specific ionophore pyrithione, monitored in real time. (C and D) Responses of TamR cells loaded with FluoZin-3 AM to the addition of 20 μM Zn²⁺ together with the Zn²⁺-specific ionophore pyrithione, monitored in real time. Traces in B and D represent the average of four and three cells, respectively, after normalization at t = 0 s. Error bars represent SEM.

Fig. 5. (A) Responses of MCF-7 cells expressing ER-eZinCh-2 to the addition of 20 μM Zn²⁺/10 μM pyrithione. (B) Responses of TamR cells expressing ER-eCALWY-4 to the addition of 20 μM Zn²⁺/10 μM pyrithione. Traces in A and B represent the average of three cells after normalization at t = 0 s. Error bars represent SEM.

Fig. 6. (A) Responses of MCF-7 cells co-expressing redCALWY-4 and ER-eZinCh-2 to the addition of 10 ng mL^–1 EGF together with 500 nM ionomycin. (B) Responses of TamR cells co-expressing redCALWY-4 and ER-eCALWY-4 to the addition of 10 ng mL^–1 EGF together with 500 nM ionomycin. Traces in A and B represent the average of three cells after normalization at t = 0 s. Error bars represent SEM.

Fig. 7. Responses of MCF-7 cells expressing cytosolic eCALWY-4 (A) and TamR cells expressing cytosolic eZinCh-2 (B) to the addition of 10 ng mL^–1 EGF together with 500 nM of the calcium ionophore ionomycin. Responses of MCF-7 cells expressing ER-eZinCh-2 (C) and TamR cells expressing ER-eCALWY-4 (D) to the addition of 10 ng mL^–1 EGF together with 500 nM of the calcium ionophore ionomycin. All traces represent the average of three cells after normalization at t = 0 s. Error bars represent SEM.