Scholarly article on topic 'Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis'

Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis Academic research paper on "Biological sciences"

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Academic research paper on topic "Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis"

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Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis

Jan L Vinkenborg1, Tamara J Nicolson2, Elisa A Bellomo2, Melissa S Koay1, Guy A Rutter2 & Maarten Merkx1

We developed genetically encoded fluorescence resonance energy transfer (FRET)-based sensors that display a large ratiometric change upon Zn2+ binding, have affinities that span the pico- to nanomolar range and can readily be targeted to subcellular organelles. using this sensor toolbox we found that cytosolic Zn2+ was buffered at 0.4 nM in pancreatic p cells, and we found substantially higher Zn2+ concentrations in insulin-containing secretory vesicles.

Zinc is important in many fundamental cellular processes, acting as a Lewis acid catalyst in many enzymes, having a structural function in DNA-binding proteins and acting as a modulator in neurotransmission1-3. At the same time, low nanomolar concentrations of free Zn2+ can be cytotoxic, rendering zinc homeostasis a delicate balance that is not well understood. Although synthetic fluorescent sensors have been used to monitor zinc fluctuations in live cells2,4, they typically lack control over subcellular localization and often have insufficient affinity to detect extremely low free Zn2+ concentrations. Genetically encoded fluorescence resonance energy transfer (FRET)-based sensor proteins can be used to overcome these limitations5,6 but their application for imaging transition metal homeostasis has thus far remained underdeveloped.

The FRET sensors reported here are based on a previously developed Zn2+ sensor (cALWY:CFP-Atox1-linker-WD4-YFP) that showed high Zn2+ affinity (dissociation constant (Kd) = 140 fM, pH 7.5), but suffered from a small change in emission ratio (15%)7. CALWY consists of two metal binding domains (Atoxl and domain 4 of ATP7B (WD4)) linked via a long flexible linker, with each domain providing two cysteines to form a single tetrahedral zinc binding pocket (Fig. 1a). First, we replaced the enhanced CFP and enhanced YFP domains with Cerulean and Citrine to improve brightness and reduce pH sensitivity of the fluorophores, respectively. Next, we improved the ratiometric response considerably by introducing mutations (S208F and V224L) on the surface of both fluorescent domains that are known to promote intramolecular complex formation8 (Fig. 1a-d). As a result, this enhanced CALWY

(eCALWY-1) displayed efficient energy transfer in the absence of Zn2+, but showed a large, 2.4-fold decrease in emission ratio upon Zn2+ binding (Fig. 1d,e). The Zn2+ affinity of eCALWY-1 (Kd = 2 pM at pH 7.1) was only tenfold lower than that of the CALWY sensor, showing that the interaction between the fluorescent domains was easily disrupted by zinc binding. Mutation of one of the zinc-binding cysteines in the WD4 domain (C416S) attenuated the Zn2+ affinity 300-fold, yielding eCALWY-4 with a Kd of 0.6 nM. This mutation also abrogated the binding of Cu+, which induces an interaction between the metal binding domains in eCALWY-1 (Supplementary Fig. 1). We subsequently fine-tuned Zn2+ affinity by shortening the linker between the metal binding domains, yielding sensors eCALWY-1-6 that span the picomolar to nanomolar ranges in Zn2+ affinity and display at least a twofold ratiometric change upon zinc binding (Fig. 1e).

Next we tested the performance of the eCALWY sensors to monitor free cytosolic Zn2+ levels in pancreatic P cells (INS-1(832/13)), a cell type known to contain high zinc levels in granules specialized in insulin storage9,10. All sensors were homogeneously expressed throughout the cytosol of the cells. We observed a large increase in emission ratio in cells expressing eCALWY-1 after addition of the membrane-permeant zinc chelator TPEN (N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine), indicative of a decrease in cytosolic zinc (Fig. 2a). Subsequent perifusion with 5 |M of the zinc ionophore pyrithione had little effect, but together with 100 |M ZnCl2 the emission ratio rapidly returned to the starting level. Addition of TPEN or Zn2+-pyrithione did not affect the emission ratio of a nonbinding sensor variant (Supplementary Fig. 2). Moreover, we observed a consistent trend for the emission ratio at the start of the experiment, changing from a fully saturated level for eCALWY-1 to nearly unsaturated for eCALWY-6 (Fig. 2a-g and Supplementary Fig. 3). We calculated the sensor occupancy as:

R — R

Occupancy = -^ x 100% (1)

Rmax — Rmin

in which R and R are the steady-state emission ratios

max mill 1

obtained after TPEN and zinc-pyrithione addition, respectively, and Rstart is the ratio at the start of the experiment. A plot of the sensor occupancy as a function of its Kd had a sigmoidal shape that is consistent with a free Zn2+ concentration of ~0.4 nM (Fig. 2h). Notably, repeating these experiments in HEK293 cells showed very similar responses (Supplementary Figs. 4,5 and Supplementary Video 1), suggesting that the ~0.4 nM level may be invariant among different cell lines. In fact, similar values have been reported using synthetic dyes in more indirect measurements on cell populations with FluoZin-3 (Invitrogen) in human colon cancer HT293 (~0.6 nM)11 and in fibroblastic L(TK)- cells with

laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands. 2Section of Cell Biology, Division of Medicine, Imperial College London, London, UK. Correspondence should be addressed to G.A.R. (g.rutter@imperial.ac.uk) or M.M. (m.merkx@tue.nl). received 29 MAY; AccEpTED 20 JuLY; puBLisHED oNLINE 30 AuGusT 2009; Doi:10.1038/NMETH.1368

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eCALWY-1

— Empty

-saturated

450 475 500 525 550 575 600 Wavelength (nm)

Kd (pM)

- eCALWY-1 2

- eCALWY-2 9

- eCALWY-3 45

- eCALWY-4 630

- eCALWY-5 1,850

- eCALWY-6 2,900

log (free Zn2+) (M)

Figure 1 | Design and properties of eCALWY sensors. (a,b) CALWY (a) and eCALWY-1 (b) sensor design. Atox1 and the fourth domain of ATP7B (WD4) were used as metal-binding domains. The CALWY sensor yielded a small FRET change owing to the presence of a distribution of conformations in the Zn2+-free state, whose average energy transfer efficiency was only slightly higher than the amount of energy transfer in the Zn2+-bound state. (c,d) Emission spectra of CALWY (c) and eCALWY-1 (d) before (empty) and after (Zn2+-saturated) addition of 0.9 mM Zn2+ in 1 mM HEDTA (c) or EGTA (d). (e) Zn2+ titrations of the eCALWY variants, showing the ratio of Citrine and Cerulean emission as a function of Zn2+ concentration using 420 nm excitation. Buffering systems such as EDTA and EGTA were used to reach free zinc concentration from the picomolar to the nanomolar range. Measurements were performed using ~1 |M protein in 150 mM Hepes, 100 mM NaCl, 10% (vol/vol) glycerol, 1 mM dithiothreitol (DTT) (pH 7.1) at 20 °C. The solid lines depict fits assuming single binding events and corresponding Kd values are listed for each variant.

Zinbo-5 (Merck Chemicals) (~1 nM)12. Previous studies in which researchers had tried to estimate free cytosolic Zn2+ concentrations using protein-based sensors did not use internal calibration and/or were done outside the sensors' detection range, resulting in either very high (180 nM) or very low (5 pM) values5,6.

The fact that the occupancies of all eCALWY variants could be described by a single concentration of free Zn2+ is important because it implies that the intracellular free Zn2+ concentration is strongly buffered and not perturbed substantially by the presence of low micromolar concentrations of the sensors. To test this buffering capacity, we grew INS-1(832/13) P-cells expressing eCALWY-4 overnight in medium containing either 100 |M EDTA or a Zn2+-buffer providing 5 |M of free Zn2+. Cells that we grew in the presence of 100 |M EDTA showed free cytosolic Zn2+ concentrations that were similar to those of cells grown in normal medium, but we observed nanomolar or higher free Zn2+ levels in cells grown in the presence of excess Zn2+ (Fig. 2i,j). Notably, when we perifused the latter cells with buffer containing 100 |M EDTA, cytosolic zinc levels rapidly decreased to ~0.4 nM, showing that these cells efficiently restore their intracellular Zn2+ levels once excess zinc is removed (Fig. 2i,j). We observed no decrease in cytosolic zinc for normally cultured cells after perifusion with 100 |M EDTA.

To determine whether the cytosolic environment affects the sensor Kd, we calibrated the eCALWY-4 sensor in situ by perifus-ing cells treated with a-toxin using buffers containing different free zinc concentrations. The in situ-determined Kd confirmed that the intracellular conditions were only weakly affecting the zinc affinity (Supplementary Fig. 6). Specificity of our sensors for Zn2+ over biologically relevant Ca2+ concentrations could be demonstrated by depolarizing INS-1(832/13) cells with KCl to

elicit Ca2+ influx. Despite a clear increase in cytosolic Ca2+ to 1 ||M, we observed no change in the emission in cells expressing eCALWY-5 (Supplementary Fig. 7).

As control over intracellular localization is a key advantage of genetically encoded sensors, we next targeted the eCALWY sensors to the insulin granules of INS-1(832/13) cells by fusing them to the C terminus of vesicle-targeted membrane protein 2 (VAMP2)13. As we anticipated higher free zinc concentrations in these vesicles9, we also used a low-affinity Zn2+ sensor (eZinCh; Kd = 8 |M at pH 7.1 and 250 |M at pH 6.0; Supplementary Fig. 8). eZinCh displayed a fourfold increase in emission ratio upon zinc binding and is similar to the previously reported ZinCh14, but contains Cerulean and Citrine as fluorescent domains. Localization studies of the vesicular-targeted Zn2+ sensors with a granule-localized neuropeptide Y-mCherry fusion protein showed that VAMP2-eCALWY-1, VAMP2-eCALWY-6 and VAMP2-eZinCh were indeed exclusively localized in insulin-containing granules (Fig. 3a and Supplementary Fig. 9). The low emission ratios observed for the eCALWY variants before stimulation suggested saturation with Zn2+ (Fig. 3b), whereas eZinCh appeared to be empty. No changes in emission ratio could be induced using either TPEN or Zn2+-pyrithione for VAMP2-eCALWY-1 and VAMP2-eZinCh, probably reflecting an inability of these agents to induce sufficient changes in the intravesicular free Zn2+ concentration (Supplementary Fig. 10). However, we observed robust and reversible ratiometric changes for VAMP2-eZinCh upon addition of monensin (Fig. 3b and Supplementary Video 2). This Na+/H+ exchanger increases the pH of granules from approximately pH 6.0 to pH 7.1, simultaneously increasing the affinity of eZinCh and inducing Zn2+ release from the insulin-Zn2+ complex. We could exclude changes owing to the pH sensitivity of the fluorescent domains because

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eCALWY-4 1 2 3

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figure 2 | Determination of cytosolic free Zn2+ concentration in INS-1(832/13) cells using eCALWY variants. (a-f) Responses of single cells expressing eCALWY-1-6 to perifusion with Krebs-Hepes-bicarbonate (KHB; 140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, 2 mM NaHCO3 and 3 mM glucose (pH 7.4)) buffer containing 50 |M TPEN (1), 5 |M pyrithione (2) or 5 |M pyrithione and 100 |M Zn2+ (3) analyzed using epifluorescence microscopy. (g) False-colored spinning disc confocal microscopy images of INS-1(832/13) cells expressing eCALWY-4 after perifusion with KHB (20 s), KHB with 50 |M TPEN (360 s) and

KHB with 5 |M pyrithione and 100 |M Zn2+ (400 s). Scale bar, 15 |m. (h) Sensor occupancy in INS-1(832/13) cells as a function of the sensor Kd for different eCALWY variants as determined from the traces in supplementary figure 3 using equation 1; error bars indicate the s.d. of 5, 12, 6, 9, 8 and 7 experiments for eCALWY-1-6, respectively. A nonlinear least-squares fit of the sensor occupancy as a function of sensor Kd yielded a free Zn2+ concentration of 0.46 ± 0.22 nM. The dashed lines depict the expected responses assuming free zinc concentrations of 0.05, 0.1, 0.2 (0.4, solid line), 0.8, 1.6 and 3.2 nM, respectively. (i) Effect of growth conditions on the ratiometric response in INS-1(832/13) cells expressing eCALWY-4 grown without zinc (100 |M EDTA), under normal conditions or in excess zinc (5 |M of buffered free Zn2+) for 20 h. During imaging, cells were perifused with KHB plus (25 mM instead of 2 mM NaHCO3 to prevent cytosolic pH changes from affecting fluorescence), to which 100 |M EDTA (1), 50 |M TPEN (2) or 5 |M pyrithione and 100 |M ZnCl2 (3) was added. (j) Occupancy of eCALWY-4 in the experiments depicted in i at the start of perifusion with KHB that mimics growth conditions, and after 10 min of perifusion with KHB containing 100 |M EDTA. Error bars, s.d. of 8, 16 and 12 experiments for cells grown with 100 |M EDTA, under normal conditions and in excess zinc, respectively.

Start 100 цМ EDTA Perifusion conditions

monensin addition did not induce similar ratiometric changes for a nonbinding variant of eZinCh (eZinCh-NB) or any of the eCALWY variants. These results imply that the VAM P2-e CALWY sensors were saturated with Zn2+ under normal conditions (Kd = 0.5 ||M at pH 6.0 for VAMP2-eCALWY-6; Supplementary Fig. 11) and that VAMP2-eZinCh was mostly Zn2+-free, thus suggesting that the free Zn2+ concentration in these vesicles was between 1 and 100 | M.

In conclusion, we developed a new generation of Zn2+ probes that can be used to image low concentrations of free

Zn2+ in single living cells. We found that cytosolic amounts of free Zn2+ were buffered at ~0.4 nM, which coincides with the Zn2+-buffering capacity of metallothioneins15. Cytosolic Zn2+ concentrations were maintained at a level sufficient to fully saturate native Zn2+ proteins (which typically have Kd values of 1-10 pM) but approximately tenfold below the low-nanomolar concentrations that have been reported to inhibit several cytosolic proteins3. We also demonstrated that these probes can be targeted to subcellular organelles, including secretory granules.

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Zn2+ probes to insulin-storing granules. (a) Confocal laser scanning microscopy images of INS-1(832/13) cells transfected with plasmids encoding VAMP2-eCALWY-1 (left) and neuropeptide Y-mCherry (middle). The VAMP2-eCALWY-1 emission was obtained using excitation at 440 nm, and excitation at 595 nm was used to image neuropeptide

Y-mCherry. Scale bar, 10 |im. (b) Ratiometric response of INS-1(832/13) cells expressing different VAMP2-eCALWY variants, VAMP2-eZinCh and VAMP2-eZinCh-NB to perifusion with 10 |M monensin followed by KHB plus buffer.

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nature methods | VOL.6 NO.10 | OCTOBER 2009 | 739

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METHODS

Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturemethods/.

Note: Supplementary information is available on the Nature Methods website.

ACKNOWLEDGMENTS

We thank S.M.J. van Duijnhoven for expressing and characterizing eZinCh, A. McDonald for assisting in the spinning disc confocal microscopy experiments, A. Tarasov for setting up the a-toxin incubation, H. Bayley (University of Oxford) for providing the a-toxin, C. Newgard (Duke University) for providing INS-1(832/13) cells, and L. Klomp and P. van den Berghe (University Medical Center Utrecht) and E.W. Meijer for their support at various stages of this research. M.M. and M.S.K. acknowledge support by the Human Frontier of Science Program (Young Investigator grant, (RGY)0068-2006). G.A.R. thanks the US National Institutes of Health for project grant RO1 DK071962-01, the Wellcome Trust for programme grants 067081/Z/02/Z and 081958/Z/07/Z, Medical Research Council (UK) for research grant G0401641, and the EU FP6 ("SaveBeta" consortium grant). T.J.N. and E.A.B. were supported by Imperial College divisional studentships.

AUTHOR CONTRIBUTIONS

J.L.V., G.A.R. and M.M. designed research; J.L.V., T.J.N., E.A.B. and M.S.K. conducted experiments, J.L.V., T.J.R., E.A.B., M.S.K., G.A.R. and M.M. analyzed data and J.L.V., G.A.R. and M.M. wrote the paper.

Published online at http://www.nature.com/naturemethods/.

Reprints and permissions information is available online at http://npg.nature.

com/reprintsandpermissions/.

1. Vallee, B.L. & Falchuk, K.H. Physiol. Rev. 73, 79-118 (1993).

2. Lim, N.C. et al. Chem. Eur. J. 11, 38-49 (2005).

3. Maret, W. Biometals 22, 149-157 (2009).

4. Domaille, D.W., Que, E.L. & Chang, C.J. Nat. Chem. Biol. 4, 168-175 (2008).

5. Dittmer, P.J., Miranda, J.G., Gorski, J.A. & Palmer, A.E. J. Biol. Chem. 284, 16289-16297 (2009).

6. Bozym, R.A., Thompson, R.A., Goddart, R.B., Stoddard, A.K. & Fierke, C.A. ACS Chem. Biol. 1, 103-111 (2006).

7. van Dongen, E.M. et al. J. Am. Chem. Soc. 129, 3494-3495 (2007).

8. Vinkenborg, J.L., Evers, T.H., Reulen, S.W.A., Meijer, E.W. & Merkx, M. ChemBioChem 8, 1119-1121 (2007).

9. Hutton, J.C., Penn, E.J. & Peshavaria, M. Biochem. J. 210, 297-305 (1983).

10. Hohmeier, H.E. et al. Diabetes 49, 424-430 (2000).

11. Krezel, A. & Maret, W. J. Biol. Inorg. Chem. 11, 1049-1062 (2006).

12. Taki, M., Wolford, J.L. & O'Halloran, T.V. J. Am. Chem. Soc. 126, 712-713 (2004).

13. Mitchell, K.J. et al. J. Cell Biol. 155, 41-51 (2001).

14. Evers, T.H., Appelhof, M.A.M., Graaf-Heuvelmans, P.T.H.M. de Meijer, E.W. & Merkx, M. J. Mol. Biol. 374, 411-425 (2007).

15. Krezel, A. & Maret, W. J. Am. Chem. Soc. 129, 10911-10921 (2007).

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ONLINE METHODS

Construction of expression plasmids. The expression vectors used for bacterial and mammalian expression are listed in Supplementary Table 1. All expression vectors for bacterial expression were modified variants of a pET28a vector (Novagen) containing an N-terminal polyhistidine tag and thrombin cleavage site for removal of the tag when necessary. A pUC59 vector containing a synthetic gene encoding for cerulean and citrine, connected by a flexible linker consisting of nine GGSGGS repeats (pUC-eZinCh-NB) was created synthetically by Genscript. Using Nhel and NotI digestion, the eZinCh-NB construct was cloned into a peCFP-C1-derived vector (Clontech) to generate peZinCh-NB. The bacterial expression vector pET28a-peZinCh-NB (Supplementary Fig. 12) was obtained via digestion of pUC57-eZinCh-NB and a pET28a-derived vector using NotI and NdeI, followed by ligation of the eZinCh-NB fragment into the pET28a vector. Site-directed mutagenesis was applied on pET28a-eZinCh-NB to create pET28a-eZinCh. Mutations Y39H and S208C were introduced simultaneously in both fluorescent domains using a QuikChange Multi site-directed mutagenesis kit (Stratagene; primers 1 and 2 for mutations Y39H and S208C, respectively (Supplementary Table 2)). peZinCh was obtained via double digestion of pET28a-eZinCh using AgeI and NotI and ligation into a pECFP-1 derived vector that was digested using the same enzymes.

To create the mammalian expression vector for CALWY, multiple cloning steps were required. First, a fragment encoding for Atox1 was obtained by digestion of pET28a-CALWY (Supplementary Fig. 13) using SacI and BspEI and ligated into a SacI and BspEI-digested peZinCh-NB to create pCer-Atox1-L9-Cit. Next, a fragment encoding for the WD4 domain obtained from pET28a-CALWY using digestion with SacII and KpnI and ligated into pCer-Atox1-L9-Cit via the same restriction sites, yielding pCALWY. This vector was then digested with NdeI and NotI to obtain a fragment encoding for CALWY that could be ligated into an NdeI and NotI digested pET28a-CLY9 vector to yield the bacterial expression vector pET28a-CALWY. QuikChange site-directed mutagenesis (Stratagene) via the method described in ref. 8 was used to introduce the S208F and V224L mutations in the Cerulean and Citrine domains of both pET28a-CALWY and pCALWY to create pET28a-eCALWY-1 and peCALWY-1, respectively. A QuikChange Multi site-directed mutagenesis kit (Stratagene) was used together with primer 3 (Supplementary Table 2) according to manufacturers' instructions to obtain pET28a-eCALWY-4 and peCALWY-4. Bacterial and mammalian expression vectors for eCALWY-2, eCALWY-3, eCALWY-5 and eCALWY-6 were obtained via partial digestion of the flexible peptide linker using BamHI followed by religation using the method described in ref. 16. The expression vector for the non-binding variant of eCALWY, pET28a-eCALWY-NB, was obtained using a QuikChange Multi site-directed mutagenesis kit (Stratagene) according to manufacturers' instructions (primer 4 for mutations C284S and C287S and primer 5 for mutations C413S and C416S (Supplementary Table 2)). The resulting vector was digested with AgeI and NotI, followed by ligation of the eCALWY-NB fragment into an AgeI-and NotI-digested peCALWY to obtain the mammalian expression vector peCALWY-NB.

To create the vesicle-targeted mammalian expression plasmids, a sequence encoding for VAMP2 was cloned N-terminally of the selected zinc sensor proteins (Supplementary Fig. 14). PCR was

used to amplify the sequence encoding VAMP2 and simultaneously add sequence encoding an N-terminal SpeI site and a C-terminal NheI-site (primers 6 and 7 respectively, Supplementary Table 2). The VAMP2 amplicon was digested using NheI and SpeI, followed by ligation into a mammalian NheI-digested expression vector encoding for one of the sensor constructs. The correct open reading frame for all expression vectors was confirmed by DNA sequencing (Baseclear).

Expression and purification of sensor proteins. All fusion proteins were expressed in Escherichia coli BL21(DE3) (Novagen) and purified according to a published method7. Expression was induced using 0.1 mM IPTG, and bacteria were subsequently grown at 15 °C for 4 h (eCALWY variants) or at 25 °C overnight (eZinCh and eZinCh-NB). Cells were collected and lysed using Bugbuster (Novagen). Next, the soluble protein fraction was purified using nickel affinity chromatography and histidine tags were subsequently removed by digestion with thrombin and a second round of nickel affinity chromatography. The cleaved product was additionally purified using size-exclusion chromatography (S200 Sephacryl column, GE) in 50 mM Tris, 100 mM NaCl and 1 mM DTT (pH 7.5). Fractions containing protein were analyzed using SDS-PAGE and fractions showing a single band corresponding to the expected molecular weight were pooled, resulting in >95% pure protein samples. Typical yields after the first round of nickel affinity chromatography were ~10 mg l-1 culture for eCALWY-1-3, ~35 mg l-1 culture for eCALWY-4-6 and ~150 mg l-1 for eZinCh and eZinCh-NB.

Zinc titrations. Zinc titrations for the eCALWY variants were performed in 150 mM Hepes, 100 mM NaCl, 1 mM DTT and 10% (vol/vol) glycerol (pH 7.1). At pH 6.0, measurements were performed using the same buffer, but using 150 mM MES instead of Hepes. DTT was used to prevent oxidation of the cysteines in the metal binding motif. For eZinCh, 150 mM MOPS, 100 mM NaCl, 10% (vol/vol) glycerol, 0.05% (vol/vol) Tween 20, 2 |M DTT and 0.5 mM TCEP, pH 7.1 was used. At pH 6.0, 150 mM MES was used instead of Hepes. Zn2+ titrations were done by mixing 0.1-0.9 mM of Zn2+ from a slightly acidic stock solution of ZnCl2 99.99% (Acros) with buffering systems consisting of 1 mM EGTA, 1 mM HEDTA, 1 mM EDTA, 1 mM NTA or 1 mM 1,3-diamino-2-hydroxypropane-N,N,N',N'-tetraacetic acid (DHPTA) (all from Sigma). For eCALWY-4, eCALWY-5 and eCALWY-6 titrations were also done by addition of 0.1-4.5 mM Zn2+ to 5 mM EGTA. These buffering systems are referred to as the EGTA, the HEDTA, the EDTA, the DHPTA and the NTA buffering systems, respectively. The free zinc concentrations were calculated using the program MaxChelator using the stability constants present within the program (Supplementary Tables 3,4). For DHTPA, calculations were done using log P values of 9.49, 6.96, 2.56 and 1.60 for H1, H2, H3 and H4, and log P values of 13.7 and 3.58 for the ML and MLH species, respectively17. All free zinc concentrations were calculated using T = 20 °C and ionic strength (I) = 0.1 M. Fluorescence spectra and emission anisotropy were recorded on a Varian Cary Eclipse spectrometer. Protein concentrations were determined by measuring the citrine absorbance at 515 nm using an extinction coefficient of 77,000 M-1 cm-1 (ref. 18). The Citrine/Cerulean (Cit/Cer) emission ratio was calculated by dividing the emissions at 527

doi:10.1038/nmeth.1368

NATURE METHODS

and 475 nm, respectively. Dissociation constants were obtained by fitting the titration curves using

Ratio(Cit/Cer) = _ Pl[Zn. }+ + P2

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Kd + [Zn2+] ~ (2)

(in which P1 is defined as the ratiometric change upon zinc binding [Zn2+] as the concentration of free zinc in moles per liter, Kd as the dissociation constant in moles per liter and P2 as the Ratio(Cit/Cer) in the absence of zinc) and the nonlinear fitting procedure of GraphPad Prism.

Copper titrations. Stock solutions of (Cu+(CH3CN)4)(PF6) were freshly prepared in acetonitrile. Copper titrations were performed in 100 mM MOPS, 100 mM NaCl and 10% (vol/vol) glycerol (pH 7.0). All manipulations and sample preparation were performed under an inert atmosphere and the protein samples were sealed in a cuvette with a rubber septum for fluorescence measurements. Copper aliquots were added to the protein sample via a gas-tight Hamilton syringe. For eCALWY-1 and eCALWY-4, the samples were pretreated with excess DTT and EDTA for 1 h to reduce all cysteine residues and to chelate any Zn2+ bound, respectively. The protein samples were then passed through a PD10 desalting column (GE Healthcare), pre-equilibrated with anaerobic MOPS buffer to remove all DTT. The protein was eluted and 1 mM EDTA added to the protein sample to inhibit Zn2+ binding. Protein concentrations were determined by measuring the citrine absorbance at 515 nm using an extinction coefficient of 77,000 M-1 cm-1 (ref. 18). The Citrine/Cerulean ratio was calculated by dividing the emission intensities at 527 and 475 nm, respectively. For both eCALWY-1 and eCALWY-4, the copper titrations were performed by addition of 0.1-3.0 |M Cu+.

Cell culture. INS-1(832/13) cells were grown in RPMI-1640 medium containing 10% (vol/vol) FBS, 10 mM Hepes, 2 mM glutamine, 1 mM sodium-pyruvate, 50 |M P-mercaptoethanol, 100 units ml-1 penicillin and 100 |g ml-1 streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. HEK293 cells were grown in DMEM (Sigma) containing 10% (vol/vol) FBS (Life Technologies), 3 mM glucose, 2 mM glutamine, 100 units ml-1 penicillin and 100 |g ml-1 streptomycin at the same temperature and CO2 levels. Cells were plated on poly(L-lysine)-coated glass coverslips and transiently transfected with 0.5-1.0 |g of plas-mid DNA by using Lipofectamine 2000 (Invitrogen) according to the manufacturers' instructions. Cells were imaged 2 d after transfection.

Intracellular FRET imaging. Brightfield imaging was performed on an Olympus IX-70 microscope fitted with a monochromator (Polychrome IV; TILL Photonics) and an Imago charge-coupled device (CCD) camera (TILL Photonics) controlled by Tillvision software (TILL Photonics). For FRET measurements, a 455DRLP dichroic mirror (Chroma Technology) and two emission filters (Chroma; D465/30 for Cerulean and D535/30 for Citrine) alternated by a filter changer (Lambda 10-2; Sutter Instruments) were used. Images were acquired at 1 Hz using a 100-ms exposure time and a 433 nm excitation wavelength. Confocal images were made using a Zeiss Axiovert 200 M confocal microscope fitted with a PlanApo x63 oil-immersion objective and a X1.5 optivar. Samples were illuminated at 440 and 561 nm for Cerulean and

mCherry, respectively, and data acquisition was controlled with an Improvision/Nokigawa spinning disc system running Volocity (Improvision) software. Chroma filtersets of ET480/40, ET535/30 and ET620/60 were used to detect Cerulean, Citrine and mCherry emission respectively. Images were captured using a Hamamatsu electron-multiplying CCD digital camera model C9100-13. Live-cell images were acquired at 0.05 Hz using a 100-ms exposure time.

Cells were imaged in modified Krebs-Hepes-bicarbonate buffer (KHB), consisting of 140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, 2 mM NaHCO3 and 3 mM glucose, which was pre-equilibrated with 95:5 O2:CO2 (pH 7.4). TPEN and pyrithione were prepared fresh on the day of use in 25 mM and 1 mM stock solutions in DMSO, respectively. During KCl stimuli, testing of culture conditions and monensin treatment, KHB plus (KHBP) buffer was used, consisting of 140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, 25 mM NaHCO3 and 3 mM glucose (pH 7.4). In KHBP, 25 mM NaHCO3 was used instead of 2 mM to prevent possible cytosolic pH changes from affecting Citrine fluorescence. Buffers were added using perifusion (2 ml min-1) with KHB or KHBP plus additions as stated (37 °C). Where indicated, cells were permeabilized by adding 20 |l of 250 |g ml-1 a-toxin dissolved in intracellular buffer (IB) to 100 |l of INS-1(832/13) cells in IB. IB comprised 140 mM KCl, 5 mM KH2PO4, 100 |M ATP, 2 mM Na+ succinate, 20 mM Hepes and 5.5 mM glucose, which was pre-equilibrated with 95:5 O2:CO2 (pH 7.05). After 30 s of incubation with a-toxin, perifusion was used to incubate the cells in fresh IB (2 ml min-1). Next, cells were exposed to IB containing different amounts of Ca2+, Mg2+, Zn2+ that were buffered using combinations of EGTA and EDTA or HEDTA (Supplementary Table 5).

KCl stimulation. INS-1(832/13) cells were grown and transfected as described above. One day before the fluorescence microscopy imaging, cells were starved overnight in RPMI-1640 medium containing 3 mM instead of 10 mM glucose. Cells were imaged in KHBP pre-equilibrated with 95:5 O2:CO2. TPEN and pyrithione were prepared fresh on the day of use in 25 mM and 1 mM stock solutions in DMSO respectively. Calcium imaging experiments were performed by incubating nontransfected INS-1(832-13) cells with 5 |M of Fluo-3-AM for 15 min, followed by KCl stimulation according to the protocol described above. For Ca2+ imaging experiments, microscope settings were identical to those used for Zn2+ imaging, except for using an excitation wavelength of 488 nm instead and a standard GFP filter block.

Effect of growth conditions. INS-1(832/13) cells were grown for 16-20 h in normal growth medium to which either 100 |M EDTA or a buffered solution of 5 |M free zinc was added to create growth conditions deprived of zinc or rich in zinc, respectively. The latter was created via addition of 1 mM EGTA, 0.5 mM CaCl2 and 0.5 mM ZnCl2. During imaging, cells were perifused with KHBP, to which 100 |M EDTA, 50 |M TPEN, 5 |M pyrithione and 100 |M ZnCl2 or nothing was added. At the start of the experiment (t = 0 until t = 180 s), KHBP contained additives to mimic the zinc levels during growth: 100 |M EDTA for the zinc-deprived cells, and to create a buffered Zn2+ solution with 5 |M of free zinc, 1 mM of EGTA was added together with 0.8 mM CaCl2 and 0.2 mM ZnCl2.

NATURE METHODS

doi:10.1038/nmeth.1368

Imaging concentrations are different from culturing concentrations as KHBP contains 1.5 mM instead of 0.42 mM Ca2+.

Localization studies. Cells were transfected as described above. Two days after transfection cells were fixated using 4% paraformaldehyde (PFA) in PBS (pH 7.4), followed by washing with PBS three times and mounting using Prolong antifade gold (Invitrogen). Colocalization studies on fixed cells were carried out on the confocal setup described above. z-dimension stacks

were recorded with 0.15-|m steps after manually determining the top and bottom of the cell. Exposure time per image was 0.2 s and 0.1 s for Cerulean and mCherry emission, respectively.

16. Evers, T.H., Dongen, E.M.W.M., van Faesen, A.C., Meijer, E.W. & Merkx, M. Biochemistry 45, 13183-13192 (2006).

17. Silen, L.S. Stability Constants of Metal-ion Complexes, 2nd edn. (The Chemical Society, London, 1964).

18. Nguyen, A.W. & Daugherty, P.S. Nat. Biotechnol. 23, 355-360 (2005).

doi:10.1038/nmeth.1368

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