Genetically Encoded Sensors to Elucidate Spatial Distribution of Cellular Zinc

Abstract

Transition metals are essential enzyme cofactors that are required for a wide range of cellular processes. Paradoxically, whereas metal ions are essential for numerous cellular processes, they are also toxic. Therefore cells must tightly regulate metal accumulation, transport, distribution, and export. Improved tools to interrogate metal ion availability and spatial distribution within living cells would greatly advance our understanding of cellular metal homeostasis. In this work, we present genetically encoded sensors for Zn2+ based on the principle of fluorescence resonance energy transfer. We also develop methodology to calibrate the probes within the cellular environment. To identify both sources of and sinks for Zn2+, these sensors are genetically targeted to specific locations within the cell, including cytosol, plasma membrane, and mitochondria. Localized probes reveal that mitochondria contain an elevated pool of Zn2+ under resting conditions that can be released into the cytosol upon glutamate stimulation of hippocampal neurons. We also observed that Zn2+ is taken up into mitochondria following glutamate/Zn2+ treatment and that there is heterogeneity in both the magnitude and kinetics of the response. Our results suggest that mitochondria serve as a source of and a sink for Zn2+ signals under different cellular conditions. Transition metals are essential enzyme cofactors that are required for a wide range of cellular processes. Paradoxically, whereas metal ions are essential for numerous cellular processes, they are also toxic. Therefore cells must tightly regulate metal accumulation, transport, distribution, and export. Improved tools to interrogate metal ion availability and spatial distribution within living cells would greatly advance our understanding of cellular metal homeostasis. In this work, we present genetically encoded sensors for Zn2+ based on the principle of fluorescence resonance energy transfer. We also develop methodology to calibrate the probes within the cellular environment. To identify both sources of and sinks for Zn2+, these sensors are genetically targeted to specific locations within the cell, including cytosol, plasma membrane, and mitochondria. Localized probes reveal that mitochondria contain an elevated pool of Zn2+ under resting conditions that can be released into the cytosol upon glutamate stimulation of hippocampal neurons. We also observed that Zn2+ is taken up into mitochondria following glutamate/Zn2+ treatment and that there is heterogeneity in both the magnitude and kinetics of the response. Our results suggest that mitochondria serve as a source of and a sink for Zn2+ signals under different cellular conditions. Although mammalian cells are known to concentrate transition metals, it is now well established that under resting conditions, “free” (e.g. unbound) metals are maintained at extremely low levels. Estimates of the total Zn2+ concentration in mammalian cells typically range from 100 to 500 μm (1Eide D.J. Biochim. Biophys. Acta. 2006; 1763: 711-722Crossref PubMed Scopus (677) Google Scholar); yet free Zn2+ concentrations are tightly buffered by proteins such as metallothionein to maintain cytosolic Zn2+ concentrations in the picomolar to nanomolar range (2Maret W. Krezel A. Mol. Med. 2007; 13: 371-375Crossref PubMed Scopus (164) Google Scholar, 3Thompson R.B. Curr. Opin. Chem. Biol. 2005; 9: 526-532Crossref PubMed Scopus (97) Google Scholar, 4Frederickson C.J. Koh J.Y. Bush A.I. Nat. Rev. Neurosci. 2005; 6: 449-462Crossref PubMed Scopus (1505) Google Scholar, 5Suhy D.A. Simon K.D. Linzer D.I. O'Halloran T.V. J. Biol. Chem. 1999; 274: 9183-9192Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). However, there is emerging evidence that this static picture is dramatically altered by different cellular conditions, such as redox perturbations caused by oxidative stress (6Maret W. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 237-241Crossref PubMed Scopus (317) Google Scholar, 7Maret W. Neurochem. Int. 1995; 27: 111-117Crossref PubMed Scopus (143) Google Scholar) and cellular signals such as nitric oxide (8Bossy-Wetzel E. Talantova M.V. Lee W.D. Scholzke M.N. Harrop A. Mathews E. Gotz T. Han J. Ellisman M.H. Perkins G.A. Lipton S.A. Neuron. 2004; 41: 351-365Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Consequently, there is a pool of labile Zn2+ that, if mobilized by cellular signals, would result in the generation of transient Zn2+ signals. Recent studies suggest that these Zn2+ signals influence critical biological processes, such as mitochondrial function (7Maret W. Neurochem. Int. 1995; 27: 111-117Crossref PubMed Scopus (143) Google Scholar, 9Sensi S.L. Ton-That D. Sullivan P.G. Jonas E.A. Gee K.R. Kaczmarek L.K. Weiss J.H. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 6157-6162Crossref PubMed Scopus (360) Google Scholar, 10Kroncke K.D. Fehsel K. Schmidt T. Zenke F.T. Dasting I. Wesener J.R. Bettermann H. Breunig K.D. Kolb-Bachofen V. Biochem. Biophys. Res. Commun. 1994; 200: 1105-1110Crossref PubMed Scopus (266) Google Scholar). Elucidation of the sources and dynamics of these Zn2+ signals would greatly advance our understanding of the interplay between metal regulation and cellular function. There has been a huge effort in the past few years to develop sensitive and selective fluorescent probes to monitor Zn2+ in biological systems. The majority of this work has focused on the generation of small molecule fluorescent indicators (reviewed by Que et al. (11Que E.L. Domaille D.W. Chang C.J. Chem. Rev. 2008; 108: 1517-1549Crossref PubMed Scopus (1790) Google Scholar)). Yet there are also examples of sensors based partially on Zn2+-binding proteins, such as carbonic anhydrase (12Bozym R.A. Thompson R.B. Stoddard A.K. Fierke C.A. ACS Chem. Biol. 2006; 1: 103-111Crossref PubMed Scopus (213) Google Scholar) and metallothionein (13Hong S.H. Maret W. Proc. Natl. Acad. Sci. 2003; 100: 2255-2260Crossref PubMed Scopus (51) Google Scholar), and peptide scaffolds (14Walkup G.K. Imperiali B. J. Am. Chem. Soc. 1996; 118: 3053-3054Crossref Google Scholar). Although many of these sensors have begun to provide insight into Zn2+ concentrations within cells, one limitation is that it is challenging to explicitly target them to subdomains within the cell. Localized probes are necessary to generate a complete picture of cellular Zn2+ homeostasis in mammalian cells. For this reason, sensors that are genetically encoded (i.e. generated by translation of a nucleic acid sequence) are attractive platforms for engineering metal-specific sensors. Encoded sensors provide additional benefits such as retention of the sensor over days to weeks permitting long term imaging and the ability to systematically vary the sensor concentration to evaluate the extent to which the sensor perturbs resting Zn2+ concentrations. Here we present genetically encoded sensors designed with a “Zn2+-sensing domain” sandwiched between two fluorescent proteins. The fluorescent proteins are chosen so that they are capable of undergoing fluorescence resonance energy transfer (FRET). 2The abbreviations used are: FRETfluorescence resonance energy transferCFPcyan fluorescent proteinYFPyellow fluorescent proteinMOPS4-morpholinepropanesulfonic acidTPENN,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine. Because the mechanism of FRET involves dipole-dipole coupling, it is exquisitely dependent on the distance and orientation of the fluorophores with respect to one another. Therefore, if the binding of Zn2+ induces a conformational change in the sensor, it will alter the energy transfer between the two fluorescent proteins. The advantage of using FRET as the optical readout is that the donor emission will decrease and the acceptor emission will increase upon Zn2+ binding. Hence, by taking the ratio of the acceptor to the donor emission, we can create a ratiometric sensor. These sensors are targeted to the cytosol, mitochondria, and plasma membrane by attachment of signal sequences and fusion to other proteins. These sensors reveal differences in the spatial distribution of Zn2+ and highlight the power and utility of localized probes. fluorescence resonance energy transfer cyan fluorescent protein yellow fluorescent protein 4-morpholinepropanesulfonic acid N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine. Details of sensor construction, protein purification, and buffered metal solutions are presented in the supplemental text. Purified sensor protein (0.5 μm) was buffer exchanged into 10 mm MOPS, 100 mm KCl (pH 6.8) and titrated with Zn2+ to obtain the apparent dissociation constant, K′d. We determined the K′d using two approaches. In one approach, the fluorescence intensity at 529 nm (emission maximum of YFP) was plotted as a function of Zn2+, and in the second approach, the change in FRET ratio (R − Rmin) was plotted as a function of Zn2+, where the FRET ratio (R) is defined as the emission maximum of YFP divided by the emission maximum of CFP upon excitation of CFP, and Rmin is the minimum FRET ratio observed under Zn2+-free conditions. These measurements yielded K′d values within experimental error of one another. It should be noted that we measure the apparent Kd because we are monitoring the conformational change that to FRET as to the Zn2+ binding metal buffer solutions G.K. J. Am. Chem. Soc. Scopus Google Scholar) used to buffer Zn2+ from nm to μm and μm The sensor was and maintained under conditions supplemental for Biochem. Biophys. PubMed Scopus Google Scholar) was used to the of The observed FRET ratio change (R − Rmin) for the sensor with the of between and of The of complete from our to maintain a environment. to the of protein yielded the range for the sensor of measurements on a fluorescence FRET (R) for by the YFP intensity 529 by CFP intensity upon CFP excitation at The emission was 10 The sensor was targeted to the of mammalian cells using The sensor was targeted to the mitochondrial to as by of the signal to the of the sensor T. S.A. V. D. Chem. Biol. 2006; 13: Full Text Full Text PDF PubMed Scopus Google Scholar). into cells using to the and The imaging on an fluorescence with a and with CFP YFP and YFP FRET by a and using on sensors at whereas mitochondrial at The cells in To the in K′d the cells with μm to obtain the minimum FRET ratio by a and the of concentrations of a well defined Zn2+ For sensors and the cells with μm to obtain the minimum FRET ratio by a and the of μm and a Zn2+ to the maximum FRET ratio The was used to the plasma membrane and Zn2+ into the cell. The treatment was to cells and was at the of an For mitochondrial the Zn2+ μm) was with to Zn2+ the mitochondrial Because for the sensor, the sensor concentration was in magnitude to the and the FRET ratio was with the following K.D. Mol. Biol. 2004; Google sensor sensor sensor where was taken to be sensor concentration was determined as was from to 10 μm in the with change in the For the sensor, the to a one binding YFP intensity was to sensor protein concentration using an established A. Proc. Natl. Acad. Sci. 1999; PubMed Scopus Google Scholar). a protein was generated by the of sensor protein of known concentrations in a using as the imaging The FRET ratio (R) was from FRET and CFP fluorescence and for if The FRET to Zn2+ concentrations using the following J. Biol. Chem. Full Text PDF PubMed Scopus Google Scholar) with and in K′d It should be noted that Rmin and are in and in cell. hippocampal from with K. Scholar). the and on with and and in with and The to by at with of and to days in The at days in as using in a The sensor of two fluorescent proteins and a domain” that a conformational change upon Zn2+ binding. sensor is presented in of Zn2+ to the the distance and orientation between the cyan fluorescent protein and the fluorescent protein to FRET from CFP to The is a from the mammalian The was because that these are in the of metal ion and into a upon Zn2+ binding 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). sensor a a and a to Zn2+ binding. To the sensors in the sensor protein was by and that Zn2+ binding to the sensor a decrease in CFP and an increase in YFP emission upon CFP excitation caused by an increase in The observed for the sensor the sensor is and the sensor is presented as the FRET ratio The range of the sensor is defined as and was for the sensor and for the sensor, these of the sensitive FRET sensors based on conformational For sensors for a ratio change T. S.A. V. D. Chem. Biol. 2006; 13: Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. A. Proc. Natl. Acad. Sci. U.S.A. 2004; PubMed Scopus Google Scholar), sensors typically a ratio change J. Mol. 2007; PubMed Scopus Google Scholar), and glutamate sensors a ratio change S.A. Proc. Natl. Acad. Sci. 2008; PubMed Scopus Google Scholar). We that the in the range between the and sensors results from in the and orientation of the fluorescent proteins with respect to one another. There is for this because a of sensors in the range for sensors with different between the two fluorescent proteins T. T. A. Proc. Natl. Acad. Sci. U.S.A. 2004; PubMed Scopus Google Scholar). To the apparent dissociation for Zn2+ the FRET intensity and FRET ratio for sensor as a function of Zn2+ These measurements yielded K′d The FRET ratio (R) binding are presented in and The in for the and sensors determined to be and The sensor a Zn2+ the μm 10 We that this is because attachment of the fluorescent proteins the apparent of to to a in Zn2+ to generate both a and low sensor. of the alter the K′d of sensor, that the in Zn2+ binding. of to the sensor that Zn2+ binding to the Zn2+ of the sensor is required for the observed FRET ratio change Although the cellular is cellular be the transient in redox The of the sensor are sensitive to To the of on the FRET we the in FRET of the and sensor. Rmin was by sensor the of the sensor was to the maximum FRET ratio with the of Zn2+ These results that in FRET would be generated by sensor that upon the sensor will be sensitive to in a For sensors to Zn2+ dynamics within the of the cell, it is that they be specific for Zn2+ over other ions and and transition metals and To the metal we other metals a FRET ratio change in the and sensors with the Zn2+ The majority of metals a FRET change and with the Zn2+ response. The two and caused a small of Zn2+ binding to both sensors at the concentration an FRET with Zn2+ binding to the sensor such that the maximum Zn2+ be at the concentration The μm is the cytosolic free concentration of Schmidt R.A. T.V. 1999; PubMed Scopus Google Scholar). to and a FRET change at the concentration that signals would be to FRET The metal results suggest that metal will be a in the cellular environment. However, that ions the for we Zn2+ over in cells Although genetically encoded sensors for Zn2+ have been they have been to function in cells J. Am. Chem. Soc. 2007; PubMed Scopus Google Scholar, J. Mol. Biol. 2007; PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar) used explicitly to measure cellular Zn2+ W. D.J. Proc. Natl. Acad. Sci. 2006; PubMed Scopus Google Scholar). Therefore we to our and sensors in mammalian cells and the in response. We to target the low sensor to the of the plasma membrane and the sensor to both the cytosol and the mitochondrial FRET ratio of cells the cytosolic sensor. The of a results in a decrease in the FRET whereas an of Zn2+ results in an and sensors targeted to mitochondria and the plasma membrane, the sensors To measure the apparent dissociation in the cells with to the minimum FRET ratio in Zn2+ by membrane with and the of known concentrations of a a Zn2+ and the binding The in Zn2+ of sensor was with that observed in μm in μm in for and 10 μm in μm in for the cytosolic sensor FRET ratio change upon of Zn2+ to cells. The range of both sensors was in cells as with in in for and for in the for Zn2+ was The range for was in the cytosol and mitochondria, that redox differences in these locations the sensor and Zn2+ We that the range in be to in the cellular the fluorophores of the sensor to be in the of Zn2+, in an is by our that Rmin is in cells in protein under the experimental conditions on the Although the range was in cells, the sensors yielded to in Zn2+ and of Zn2+ dynamics in of sensors in this and mitochondria, and plasma in a we determined cellular ions with Zn2+ measurements in cells. FRET by the of specific acid to a minimum FRET ratio upon of that the in resting specific to Zn2+ and by The targeted sensor a in over the small molecule supplemental localized within mitochondria upon membrane whereas into the cytosol under these conditions. The in cellular Zn2+ homeostasis is to the of Zn2+ in different locations within the cell. There has been over mitochondria contain a labile pool of Zn2+ that is elevated over the cytosol S.L. Ton-That D. Sullivan P.G. Jonas E.A. Gee K.R. Kaczmarek L.K. Weiss J.H. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 6157-6162Crossref PubMed Scopus (360) Google Scholar, J. Neurochem. 2005; PubMed Scopus Google Scholar). To we resting Zn2+ concentrations in the cytosol and mitochondria using our sensor. Because the of a sensor cellular Zn2+ of the “free” Zn2+ concentrations should be over a range of sensor to the “free” at in sensor concentrations is a of transient where the of can vary from to cell. of sensor will be with the of and the of and the concentration of Zn2+ as a function of sensor concentration in the cytosol and mitochondria, For the cytosolic sensor YFP intensity was to protein concentration using a protein A. Proc. Natl. Acad. Sci. 1999; PubMed Scopus Google Scholar) In mitochondria, the intensity was to a protein concentration because of the in mitochondrial We a between sensor protein and the Zn2+ values in the cytosol, where sensor concentrations to Zn2+ levels. the and a resting cytosolic Zn2+ concentration of nm to sensor For of the we cells with low μm) to of cellular Zn2+ levels. We a between intensity and Zn2+ within mitochondria. is because the sensor was low of fluorescence of and the vary The Zn2+ within mitochondria was to be from both cytosol and mitochondria and the elevated resting Zn2+ within mitochondria. These the that mitochondria contain a pool of Zn2+ that serve as a source of Zn2+ signals, as by et al. S.L. Ton-That D. Sullivan P.G. Jonas E.A. Gee K.R. Kaczmarek L.K. Weiss J.H. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 6157-6162Crossref PubMed Scopus (360) Google Scholar). of a sensor is the ability to in living cells. and Zn2+ in in hippocampal yet the function of this Zn2+ (11Que E.L. Domaille D.W. Chang C.J. Chem. Rev. 2008; 108: 1517-1549Crossref PubMed Scopus (1790) Google Scholar). et al. J. J. Am. Chem. Soc. 2006; PubMed Scopus Google Scholar) that glutamate induces the of Zn2+ into the cytosol of hippocampal neurons. of this work is to the of Zn2+ Because numerous studies have that mitochondria Zn2+ (8Bossy-Wetzel E. Talantova M.V. Lee W.D. Scholzke M.N. Harrop A. Mathews E. Gotz T. Han J. Ellisman M.H. Perkins G.A. Lipton S.A. Neuron. 2004; 41: 351-365Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, J. Neurochem. 2005; PubMed Scopus Google Scholar, S.L. Weiss J.H. J. Neurosci. PubMed Scopus Google Scholar), we transient Zn2+ in the cytosol are to mitochondria in hippocampal neurons. treatment with glutamate Zn2+, we observed an increase of Zn2+ within mitochondria for the sensor the sensor of cells from different Although there was in the magnitude of the of ratio of the cells an increase in mitochondrial Zn2+ upon treatment with glutamate the transient mitochondrial Zn2+ signals are dependent on the of because stimulation with glutamate and Zn2+ in to increase in mitochondrial The mechanism of Zn2+ is the has been to Zn2+ J. Neurochem. 2005; PubMed Scopus Google Scholar). We are the mechanism of using of different in and supplemental we heterogeneity in the magnitude and kinetics of Zn2+ the FRET ratio as a function of for of whereas the The two cells at different with the at and the at there is in the within a cell. we observed transient by the for one two These to mitochondria small of mitochondria. Although we are the cellular of this the the of by of Zn2+ signals. with Zn2+ change to the FRET ratio in the mitochondria of hippocampal neurons. However, treatment of hippocampal with glutamate in a transient decrease in mitochondrial Zn2+ The small molecule Zn2+ that this mitochondrial Zn2+ is released into the the decrease in mitochondrial Zn2+ was observed with the low sensor. Zn2+ was by because glutamate treatment in buffer altered the kinetics the of the response. was in cells from two where the of ratio change was and of Zn2+ upon treatment with glutamate stimulation of to by of the that fluorescent proteins, the can be we it was to evaluate the of the mitochondrial targeted sensor and the extent of in mitochondria. in the sensor was by between and the FRET ratio at The decrease in FRET ratio is caused by of YFP fluorescence as the of is D.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To the within the mitochondrial upon glutamate stimulation in the and of Zn2+, we targeted D.A. PubMed Scopus Google Scholar) to mitochondria. glutamate treatment in the and of Zn2+, the sensor was by with of known in glutamate stimulation to a in the from to There is observed on the sensor in this Transition metals such as Zn2+ are both essential and toxic. The of metal homeostasis is that mammalian cells concentrate metal ions from the majority of these metals are to proteins cellular is emerging in which metals be mobilized from labile such as metallothionein The of transient metal is to cellular of metal availability and influence on cellular function. To obtain a picture of it is necessary to of labile Zn2+ as well as the of mobilized In this work, we present genetically targeted Zn2+ sensors and utility in monitoring the spatial distribution of Our targeted sensor an elevated resting Zn2+ in mitochondria to the treatment of hippocampal with glutamate results in transient of this into the cytosol, that mitochondria can serve as a source of Zn2+ signals. is with the by et al. S.L. Ton-That D. Sullivan P.G. Jonas E.A. Gee K.R. Kaczmarek L.K. Weiss J.H. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 6157-6162Crossref PubMed Scopus (360) Google Scholar) that mitochondrial Zn2+ be mobilized of cytosolic We also the work of et al. J. J. Am. Chem. Soc. 2006; PubMed Scopus Google Scholar) and that upon treatment with glutamate Zn2+, Zn2+ is taken up into and is into mitochondria, a transient Zn2+ signal in mitochondria. Our results that the of mitochondria in Zn2+ signals is glutamate treatment to Zn2+ whereas treatment with glutamate Zn2+ to Zn2+ have biological because hippocampal contain glutamate in whereas contain both glutamate and Zn2+ C.J. Koh J.Y. Bush A.I. Nat. Rev. Neurosci. 2005; 6: 449-462Crossref PubMed Scopus (1505) Google Scholar). Our results suggest that different will different cellular it is to that the cellular of these Zn2+ signals are There is a in cellular Zn2+ concentrations. In this work we that cellular Zn2+ are by of the low μm) sensor. is that our sensor concentration is also in the low and is to the Zn2+ as a function of we to to an of nm Zn2+ in the is using other probes in which Zn2+ are to be in the picomolar to nanomolar range (2Maret W. Krezel A. Mol. Med. 2007; 13: 371-375Crossref PubMed Scopus (164) Google Scholar, 3Thompson R.B. Curr. Opin. Chem. Biol. 2005; 9: 526-532Crossref PubMed Scopus (97) Google Scholar, 4Frederickson C.J. Koh J.Y. Bush A.I. Nat. Rev. Neurosci. 2005; 6: 449-462Crossref PubMed Scopus (1505) Google Scholar, 5Suhy D.A. Simon K.D. Linzer D.I. O'Halloran T.V. J. Biol. Chem. 1999; 274: 9183-9192Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). We that of these including our that our measurements must be at a range of sensor concentrations. is for which into cells at concentrations result in a in which the is the is to to of the cellular free Zn2+ levels. Although we for this in our we our measurements Zn2+ because our sensor is at the of we the of cytosolic and mitochondrial Zn2+ are that the sensor is used to monitor Zn2+ in these different To the and measurements of cellular Zn2+, we as a will a of probes with different K′d where the is the We are to our sensors under of an so that concentrations can be tightly and over a range for advantage of our sensor is that the fluorescent can be by the fluorescent proteins. We are different fluorescent protein as well as the of the proteins so that sensors can be into cells at in an effort to the of cellular In this genetically encoded probes an advantage over small molecule indicators concentration is to because cellular is a of and Biol. 1994; PubMed Scopus Google Scholar). the concentration of small molecule probes intensity is by Zn2+ is Our localized many of the of as a mitochondrial Zn2+ sensor. For it is from mitochondria upon membrane and the is We to to mitochondrial Zn2+ dynamics in the was so that we obtain the mitochondrial presented a advance in our ability to monitor mitochondrial However, the sensors presented can be in they are at low and which would of Zn2+ in The sensor on to Zn2+, and is to we to signals, the sensors would from an In we have genetically targeted sensors for Zn2+ and used these to that mitochondria contain a labile and pool of Zn2+ under resting conditions. In mitochondria can serve as a source of Zn2+ signals by Zn2+ into the cytosol, as well as a sink by elevated cytosolic The of a mitochondrial pool of Zn2+ the of other Zn2+ availability by as sources sinks for We for with up the protein with