Anne M.
Hessels
a,
Kathryn M.
Taylor
b and
Maarten
Merkx
*a
aLaboratory of Chemical Biology and Institute of Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands. E-mail: M.Merkx@tue.nl
bBreast Cancer Molecular Pharmacology Group, School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UK
First published on 24th December 2015
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.
Whereas small molecule sensors continue to contribute valuable insights into the role of Zn2+ in numerous biological processes, their intracellular concentration and localization cannot be easily controlled. Therefore, we and others have developed fluorescent Zn2+ sensor proteins based on the modulation of Förster Resonance Energy Transfer (FRET) between a donor and acceptor fluorescent domain.6–8 These genetically-encoded sensors do not require cell-invasive procedures, their concentration and intracellular location can be tightly controlled, and the use of FRET allows for ratiometric detection. The eCALWY-sensors that were developed by our group consist of two small metal binding domains, ATOX1 and WD4, connected by a long flexible linker, with self-associating variants of cerulean and citrine fluorescent domains fused to the N- and C-terminus, respectively. A toolbox of 6 eCALWY variants has been developed that range in Zn2+ affinity between 2 pM and 5 nM. Of these, eCALWY-4 with a Kd of 630 pM was found to be optimal for measuring cytosolic Zn2+ concentrations, which typically reside in the 0.2–1 nM range, in a wide variety of cells.6,9 Replacement of cerulean and citrine by self-associating variants of mOrange and mCherry yielded the red-shifted redCALWY variants, whose spectral properties allowed them to be used in combination with CFP-YFP-based reporters.10 Recently, we also reported another versatile FRET sensor containing a de novo Cys2His2 Zn2+ binding pocket created directly on the surface of cerulean and citrine domains.11 This eZinCh-2 sensor displays a large increase in emission ratio upon binding Zn2+ at the interface of the two fluorescent domains, with an affinity that is similar to that of eCALWY-4. By default eCALWY-4 and eZinCh-2 reside in the cytosol, but both have been successfully targeted to the ER, mitochondria and secretory vesicles by the introduction of organelle specific targeting sequences.11
Because Zn2+ is unable to passively diffuse across cell membranes, active transport between the cytosol, the extracellular space and intracellular compartments is required to allow control over intracellular Zn2+ homeostasis. Two families of mammalian Zn2+ transporters are known that transport Zn2+ across cellular membranes. The ZnT (SLC30A) family exports Zn2+ from the cytosol into organelles or to the extracellular space.12 The ZIP family (SLC39A) acts as an importer protein and is responsible for Zn2+ import from either the extracellular space or from different cellular compartments.13 One of the ZIP family members, ZIP7, was found to localize on the membrane of the endoplasmic reticulum14,15 and is believed to play a role in the transportation of Zn2+ from the ER into the cytosol. Zn2+ transporters play a crucial role in maintaining the delicate balance between cell growth and cell death and aberrant functioning of Zn2+ transporters has been linked to several disease states including cancer. For example, Zn2+ was found to be increased in human breast tumors.16,17 A widely used model to investigate the development of breast cancer18 showed an up to 19-fold increase in total Zn2+ levels in mammary tumors in mice, rats and humans.19 One mechanism by which increased levels of cytosolic Zn2+ could enhance cell proliferation is by the inhibition of protein tyrosine phosphatases, which could lead to a net increase in phosphorylation of well-known cancer associated downstream effectors such as AKT and MAPK (mitogen-activated protein kinase).20,21
Taylor and coworkers have proposed that phosphorylation of ZIP7 might induce release of Zn2+ from the ER to the cytosol in Tamoxifen-resistant MCF-7 breast cancer cells (TamR).22 Using the membrane-permeable Zn2+-specific indicator Newport Green DCF a 2-fold higher fluorescence was observed in TamR cells compared to wild-type MCF-7 cells, suggesting a higher intracellular free Zn2+ concentration.22 Comparison of the expression levels of several Zn2+ importers (ZIPs) revealed a significant increase in expression level of ZIP7 in TamR cells compared to wild-type MCF-7 cells. Since ZIP7 is almost exclusively localized on the ER membrane,14 ZIP7 has been implicated to control the release of Zn2+ from the ER to the cytosol. Two types of triggers were used to study the involvement of ZIP7 in Zn2+ signaling in both wild-type MCF-7 and TamR cells: the addition of extracellular Zn2+ with pyrithione, and addition of EGF with ionomycin. Addition of external Zn2+ and pyrithione to MCF-7 and TamR cells loaded with the Zn2+-specific fluorescent indicator FluoZin-3 showed an increase in green fluorescence within a few minutes after stimulation, which was interpreted to be a result of ZIP7-dependent Zn2+ release from the ER.21,22 Immunoprecipitation experiments with a ZIP7 antibody in Zn2+ treated cells showed co-immunoprecipitation of CK2α, suggesting that this kinase may be responsible for phosphorylation of ZIP7. Consistent with this role, a peak in the physical association between protein kinase CK2α and ZIP7 was observed within two minutes after treatment with Zn2+/pyrithione. In addition, a proximity ligation assay, in which fluorescent dots appear when two molecules are in close proximity, showed significant association of ZIP7 and CK2α in the same timeframe.
Based on the above results a model was proposed whereby the phosphorylation of ZIP7 by CK2 results in ZIP7-mediated Zn2+ release from ER stores into the cytosol, resulting in subsequent activation of several downstream signaling pathways.22 However, the evidence for Zn2+ release from the ER upon external stimuli has remained indirect, because these studies did not directly measure ER Zn2+ levels. In addition, FluoZin-3 and the other synthetic fluorescent sensors that were employed to monitor intracellular Zn2+, are known to not only localize in the cytosol, but also to different cellular compartments, such as the Golgi, synaptic vesicles and the nucleus.23 Here, we report the use of two previously developed FRET sensors for Zn2+ (eZinCh-2 (Kd = 1 nM) and eCALWY-4 (Kd = 0.63 nM)) to test the hypothesis that Zn2+ is released from the ER into the cytosol upon external stimuli (Table 1). Unlike synthetic fluorescent sensors, these genetically encoded sensors can be targeted exclusively to either the cytosol or the ER. Moreover, the use of two different color variants allowed us to simultaneously monitor the free Zn2+ concentration in the cytosol and ER within the same cell. Using these sensors an immediate increase in both cytosolic and ER Zn2+ levels was observed upon treatment of MCF7 and TAMR cells with Zn2+/pyrithione, which is consistent with direct, pyrithione-mediated exchange of Zn2+ between the cell exterior and intracellular compartments. In contrast, addition of EGF/ionomycin did not induce significant changes in cytosolic or ER Zn2+ concentrations. The implication of these findings for the putative role of ZIP7 in controlled release of Zn2+ from the ER will be discussed.
Sensor | Fluorescent domains (donor/acceptor) | K d for Zn2+ (pH 7.1) (nM) | Cellular localization |
---|---|---|---|
a Cerulean: excitation maximum at 435 nm, emission maximum at 475 nm; citrine: excitation maximum at 510 nm, emission maximum at 535 nm. b mOrange2: excitation maximum at 549 nm, emission maximum at 565 nm; mCherry: excitation maximum at 587 nm, emission maximum at 610 nm. c ER-targeted sensor variants contained an N-terminal insulin signal peptide and a C-terminal KDEL retention sequence. | |||
eZinCh-211 | Cerulean/citrinea | 1.0 | Cytosol |
ER-eZinCh-211 | Cerulean/citrine | 1.0 | ERc |
eCALWY-46 | Cerulean/citrine | 0.63 | Cytosol |
ER-eCALWY424 | Cerulean/citrine | 0.63 | ERc |
redCALWY-410 | mOrange2/mCherryb | 0.24 | Cyotosol |
To explore a possible correlation between the increased ZIP7 expression and free ER Zn2+ levels in TamR cells,22 the ER-targeted FRET-based Zn2+ sensors ER-eZinCh-2 and ER-eCALWY-4 were expressed in both wild-type MCF-7 and TamR cells24 (Table 1 and Fig. 2 and 3). Colocalization studies showed exclusive localization of these sensors to the ER lumen. Using ER-eZinCh-2, addition of TPEN and excess Zn2+ yielded similar response curves as were found previously for HeLa and HEK293T cells,11 yielding free ER Zn2+ concentrations of 0.54 ± 0.27 nM for MCF-7 (Fig. 2C) and 0.75 ± 0.49 nM for TamR cells (Fig. 2D). Measurements using ER-targeted eCALWY-4 yielded similar ER Zn2+ concentrations of 0.39 ± 0.17 nM and 0.21 ± 0.05 nM for MCF-7 (Fig. 3C) and TamR cells (Fig. 3D), respectively.
The average free ER Zn2+ concentrations that were found are similar to those found in the cytosol, although the cell-to-cell variation in free Zn2+ concentration was found to be larger in the ER when compared to the cytosol. The latter may reflect less efficient buffering of the free Zn2+ concentration due to a lack of metallothionein in the ER.
This fast increase in fluorescence is consistent with direct pyrithione-mediated transfer of Zn2+ from the cell exterior into the cytoplasm, without the need to invoke intracellular release of Zn2+ from the ER. To directly assess the effect of stimulation with Zn2+/pyrithione on ER Zn2+ levels, we repeated these experiments in MCF-7 and TamR cells expressing either ER-ZinCh-2 or ER-eCALWY-4. Addition of 20 μM Zn2+/10 μM pyrithione to MCF-7 cells expressing ER-eZinCh-2 resulted in an immediate increase in emission ratio (Fig. 5A), consistent with an increase in the free ER Zn2+ concentration. Comparable responses were observed for TamR cells expressing ER-eZinCh-2 (Fig. S1, ESI†). The same experiments were performed on both MCF-7 (Fig. S2, ESI†) and TamR (Fig. 5B) cells expressing ER-eCALWY-4, resulting in a decrease in emission ratio upon Zn2+ addition, which again corresponds to an increase in free ER Zn2+ levels. The increase in ER Zn2+ is inconsistent with a specific release of Zn2+ from the ER to the cytoplasm, but is not unexpected given the fact that pyrithione can also mediate transfer of Zn2+ across the intracellular ER membrane.
To exclude the possibility that the lack of response to EGF/ionomycin was somehow a result of the multicolor imaging experiments, we repeated these experiments in single sensor experiments. In these experiments the classical cerulean/citrine-based probes can be used for cytosolic imaging. The higher dynamic range of these sensors compared to the redCALWY probes should make it easier to detect small changes in cytosolic Zn2+ levels. However, no changes in emission ratio were observed following addition of EGF and ionomycin in MCF-7 cells (Fig. 7A) or TamR cells (Fig. 7B) transfected with either eZinCh-2 or eCALWY-4. Similarly, EGF/ionomycin addition did not provoke any sensor response in either MCF7 or TamR cells expressing ER-eZinCh-2 or ER-eCALWY-4 (Fig. 7C and D).
An important difference between this work and previous reports is the use of genetically encoded sensors instead of small-molecule dyes. Using the Zn2+-specific fluorescent dye FluoZin-3, Taylor and coworkers did not observe an immediate increase in fluorescence after the addition of Zn2+/pyrithione, which is surprising because pyrithione is a Zn2+-specific ionophore that transfers Zn2+ across the plasma membrane. One possible explanation is that the fixation protocol that was used previously interfered with accurate and early detection of increases in Zn2+ concentration. E.g. fixation does not allow continuous monitoring of the free Zn2+ concentration in the same cell and cells on different coverslips were required for each time point after Zn2+ treatment. Finally, unlike genetically encoded FRET sensors that can easily be targeted to a specific place in the cell, FluoZin-3 has been shown to not be exclusively localized in the cytosol, but also be present in other subcellular compartments.23
In contrast to previous studies using Zinquin, no detectable changes in emission ratio were observed using our genetically encoded sensors in either MCF-7 or TamR cells following addition of EGF/ionomycin, neither in the cytosol nor when the sensors were targeted to the ER. Because the affinity of Zinquin that was used in previous studies is relatively low (Kd = 7 μM), increasing levels of cytosolic Zn2+ upon EGF/ionomycin treatment should have been easily detectable by the sensors used in this study, eZinCh-2 (Kd = 1 nM at pH 7.1) and eCALWY-4 (Kd = 0.63 pM at pH 7.1) (Table 1). We therefore conclude that addition of external EGF together with ionomycin does not trigger release of Zn2+ from the ER stores into the cytosol. Previous experiments with EGF/ionomycin were also performed on fixed cells, which holds the same limitations as discussed above. It may therefore be worthwhile to apply genetically encoded sensors also in other cell systems in which treatment with EGF/ionomycin has been reported to result in a transient increase in cytosolic Zn2+ levels such as the Zn2+ waves reported by Yamasaki and coworkers in mast cells.3
Having shown that addition of Zn2+ together with pyrithione results in an immediate increase in cytosolic and ER free Zn2+ concentrations, our results do not support the suggested Zn2+ release from the ER several minutes after this treatment. In addition, no changes in overall cytosolic or ER free Zn2+ levels were observed upon addition of EGF/ionomycin using our genetically encoded sensors. Other observations that were originally linked to the release of Zn2+ from the ER may therefore also need to be reconsidered. For example, the association of protein kinase CK2α with ZIP7, and the subsequent phosphorylation of ZIP7, could be a downstream consequence of the pyrithione-mediated increase in cytosolic Zn2+. Similarly, the reported activation of downstream signaling pathways may occur through phosphatase inhibition.20 Addition of 20 μM Zn2+ together with 10 μM pyrithione will increase the level of cytosolic free Zn2+ level to at least several nanomolar, which is sufficient to directly inhibit phosphatases.25 Since no changes in overall intracellular Zn2+ levels were observed upon treatment with EGF/ionomycin, another mechanism may need to be invoked to explain the increased levels of tyrosine phosphorylation observed after treatment with this trigger. Alternatively, phosphatase inhibition may result from transient increases in local Zn2+ concentrations that cannot be detected using the current sensor technology.
In conclusion, the present study using genetically encoded FRET sensors does not provide supportive evidence for ZIP7-mediated release of Zn2+ from ER stores into the cytosol in TamR cells. These unexpected results suggest that it may be worthwhile to apply the specific properties of genetically encoded FRET sensors to carefully reassess other examples of triggered Zn2+ release from intracellular stores.
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Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5mt00257e |
This journal is © The Royal Society of Chemistry 2016 |