Intracellular Metal Detectors Amy M. Barrios* Department of Chemistry, University of Southern California, Los Angeles, California 90089
Z
inc is the second most abundant “trace” element in the body with an estimated 2–3 g distributed among over 1000 different proteins (1, 2 ). Zinc plays important roles in numerous biological processes including gene expression, apoptosis, enzyme regulation, immune system modulation and metabolism, to name a few (3). Although the total cellular concentration of zinc is approximately 200 µM (2 ), most is tightly bound to biomolecules and is not readily accessible to chelation by small molecules (3 ). However, it has been established that in specialized cells in the brain, pancreas, and prostate, significant pools of readily exchangeable zinc are accumulated (3 ). Because of the well-established roles of protein-bound and exchangeable zinc in both healthy and diseased tissue, the question of how much zinc is available in a cell has emerged at the forefront of chemical biology. On page 103 of this issue of ACS Chemical Biology, Bozym et al. report the first use of a fluorescence resonance energy transfer (FRET) based ratiometric zinc sensor to directly image and quantify the concentration of zinc in resting eukaryotic cells (4 ). Interest in the development of reagents to sense intracellular and extracellular zinc levels has skyrocketed over the past decade, resulting in a multitude of probes based on molecules, peptides, or proteins that exhibit zinc-dependent fluorescence (5 ). These probes exhibit various dynamic ranges, excitation and emission profiles, and cellular permeabilities and localize differently in cells. Biological metal ion sensing continues to be the subject of www.acschemicalbiolog y.o rg
intense research in part because it is clear that no one probe will be ideal for all applications. However, there are certain features that are desirable in zinc probes for a biological toolkit. First, the probes must exhibit excellent selectivity for zinc over metal ions such as calcium and magnesium, which are present in cells at a much higher concentration. Second, probes should be useful over a broad concentration range, for use in imaging the very low resting concentrations of zinc present in most cells as well as the high resting zinc concentrations in certain specialized cell types. Third, probes may also need fast zinc association and dissociation rates in order to facilitate dynamic imaging of changes in zinc concentrations over time and in response to various stimuli. Fourth, the probes should ideally localize in the organelle of interest, distribute evenly throughout the cell, or remain in the extracellular environment, depending on the experiment. Finally, the probes should not be cytotoxic or perturb the resting state of the cells. Despite recent advances in intracellular zinc sensing, it remains difficult to quantify the concentration of exchangeable zinc in resting eukaryotic cells that do not accumulate large amounts of zinc. The zinc biosensor reported by Bozym et al. (4 ) is based on FRET from a zinc-bound aryl sulfonamide to a fluorophore covalently attached to carbonic anhydrase (CA), an enzyme with exquisite selectivity and sensitivity to zinc (Figure 1) (4 ). By labeling CA with a TAT cell-penetrating peptide, the authors are able to introduce the zinc sensor into both PC-12 (a rat pheochromo-
A B S T R A C T Metal ions play numerous crucial roles in biology, and there is great interest in obtaining an accurate measurement of the intracellular concentrations of both tightly bound and exchangeable metal ions. Measuring the concentration of readily exchangeable transition metal ions in a cell has been particularly difficult because of the extremely small concentrations involved, interference from other metal ions and biomolecules, and the challenge of introducing probes into the cell with minimal perturbations. Recent work has made quantification of the intracellular exchangeable zinc pool possible for the first time using a cell-permeable, ratio metric, fluorescence resonance energy transfer based zinc biosensor.
*To whom correspondence should be addressed. E-mail:
[email protected].
Published online March 17, 2006 10.1021/cb600080a CCC: $33.50 © 2006 by American Chemical Society
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With the right toolkit, the tasks of quantifying metal ion concentrations and imaging changes in metal concentrations in response to external stimuli, intracellular signaling events, physiological and pathological processes will become routine.
Figure 1. Ratiometric zinc determination using Alexa Fluor 594‑modified carbonic anhydrase and dapoxyl sulfonamide. The emission intensity at 617 nm following excitation at 365 nm is normalized to the emission intensity at 617nm following excitation at 543 nm, leading to a quantitative measure of zinc concentration. On the right is shown a PC‑12 cell false-colored to indicate exchangeable cytoplasmic zinc concentrations with a concentration calibration bar at the far right. Cell image from Bozym et al. (ACS Chem. Biol. 1, 103–111)
cytoma cell line) and CHO (Chinese hamster ovary) cells with no apparent ill effects, circumventing the need for microinjection, electroporation, or other, less “gentle” methods of introducing sensors into cells, one of the major difficulties in this field. The zinc concentration inside the cells is quantitatively measured from the ratio of fluorescence intensities at two different excitation wavelengths (6 ). The advantages of ratiometric imaging methods include minimal background from variations in excitation intensity, specimen thickness, and fluorophore concentration (7 ). With a CA-based zinc sensor, the intracellular concentration of available zinc is measured at approximately 5 pM throughout both types of cells (Figure 1, inset). This concentration, while quite low, is significantly higher than the femtomolar levels proposed for prokaryotic cells (8 ). In these experiments, the zinc seems to be evenly distributed throughout the cytoplasm. One unexpected result of this research is the rapid equilibration between apoprotein and metal-bound protein in the cells. In vitro, the zinc-binding kinetics of apo-carbonic anhydrase are quite slow, and equilibration of the apoprotein with picomolar levels of intracellular exchangeable zinc would be expected to require several hours. In the imaging experiments, however, the equilibrium seemed to occur in minutes. This 68
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suggests that catalysis of zinc insertion into the CA may be occurring. This work represents a critical step forward in sensing intracellular metal ion concentrations in that it provides the first example of a quantitative measurement of zinc levels in eukaryotic cells in a resting state. As is often the case, the results raise even more questions and highlight the prospects for future research in this field. For example, is 5 pM a standard level of exchangeable zinc in many types of differentiated eukaryotic cells? Is this zinc really distributed evenly throughout the cytoplasm, and how do exchangeable zinc levels differ in various subcellular compartments? What effects, if any, do the probes have on the cells? We can expect many more exciting breakthroughs in sensing biological metal ion concentrations over the next several years, building upon recent advances in the field. Genetically encoded biosensors, “reagentless” sensing systems, and cellpermeable small molecule sensors are all promising directions to pursue. Chemists are charged with designing novel probes with high selectivity, optimal binding affinities, and on/off rates that can be selectively delivered to a desired subcellular compartment. Techniques that allow improved spatial resolution as well as reliable quantification of analyte concentrations will
Free [Zn] 50 nM
500 pM
5 pM
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be of paramount importance. Biologists are presented with the challenge of developing “gentle” methods for delivering sensors into cells, elucidating the biological effects of the added sensors, and interpreting the results of metal ion concentration measurements. With the right toolkit, the tasks of quantifying metal ion concentrations and imaging changes in metal concentrations in response to external stimuli, intracellular signaling events, physiological and pathological processes will become routine. REFERENCES 1. Berg, J. M., and Shi, Y. (1996) The galvanization of biology: a growing appreciation for the roles of zinc, Science 271, 1081–1085. 2. Maret, W. (2001) Zinc biochemistry, physiology, and homeostasis — recent insights and current trends, BioMetals 14, 187–190. 3. Vallee, B. L., and Falchuk, K. H. (1993) The biochemical basis of zinc physiology, Physiol. Rev. 73, 79–118. 4. Bozym, R., Thompson, R., Stoddard, A., and Fierke, C. (2006) Measuring picomolar intracellular exchangeable zinc in PC-12 cells using a ratiometric fluorescence biosensor, ACS Chem. Biol. 1, 103–111. 5. Kikuchi, K., Komatsu, K., and Nagano, T. (2004) Zinc sensing for cellular application, Curr. Opin. Chem. Biol. 8, 182–191. 6. Fierke, C. A., and Thompson, R. B. (2001) Fluorescence-based biosensing of zinc using carbonic anhydrase, BioMetals 14, 205–222. 7. Tsien, R. Y. (1989) Fluorescent Probes of Cell Signaling in Annual review of neuroscience, pp 227–253, Annual Review, Inc., Palo Alto, CA. 8. Finney, L. A., and O’Halloran, T. V. (2003) Transition metal speciation in the cell: insights from the chemistry of metal ion receptors, Science 300, 931–936.
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