Issue Date: September 20, 2010
Monitoring Cellular Metals
Certain transition metals—such as zinc, copper, and iron, three of the most abundant metals in biological systems—are essential to life. Those same metals, however, can also be toxic at excessive concentrations. And although researchers have made progress in identifying individual metal-dependent enzymes and some of the mechanisms involved in essential metal uptake and regulation, there is still a lot to be learned about the location, speciation, and flux of metals within cells. At the American Chemical Society national meeting in Boston last month, several researchers spoke about new probes and techniques they’re developing to monitor and image metals to better understand cellular metal homeostasis and trafficking.
One way for researchers to visualize metal ions is to use a fluorescent probe attached to a metal-specific chelator. Without the metal, photoinduced electron transfer quenches the probe’s fluorescence emission. When certain metals bind to the probe, however, changes in molecular orbital energy prevent electron transfer, allowing the probe to fluoresce.
A popular target for such probes is zinc, which is found in mobile pools in cells as well as bound within proteins to serve as a structural or catalytic center. Mobile zinc has signaling and other roles in the brain; pancreas; intestine; and in the salivary, pituitary, and prostate glands, said Stephen J. Lippard, a chemistry professor at Massachusetts Institute of Technology. Excess zinc, however, is toxic and can suppress absorption of other metals. Lippard and colleagues have been working with fluorescein-based compounds that fluoresce upon binding to Zn2+ ions.
Lippard described experiments involving a chemical sensor called ZPP1, which has two zinc-binding pockets involving pyridine and pyrazine rings. Lippard and colleagues used ZPP1 to evaluate zinc concentrations in prostate cells. Those cells normally incorporate large amounts of zinc, which, together with citrate, bind to semenogelin proteins in ejaculate to assist sperm motility. But if cancer develops in the prostate, zinc import proteins are silenced, Lippard said. In one study, he and coworkers used the ZPP1 probe to watch Zn2+ concentrations fall as tumors developed in a mouse model of prostate cancer, demonstrating the probe’s potential for disease diagnosis (Cancer Res. 2010, 70, 6119).
Lippard and collaborators are also using zinc probes to understand the role of mobile zinc in the hippocampus, the center for learning and memory in the brain. The researchers are working on ways to attach the probes to cellular targets to elucidate the function of zinc in synaptic firing.
Copper is another essential metal for biological function. But although copper is necessary for the function of many enzymes, excess copper in cells can produce radical ions that could damage cell components. Developing fluorescent probes for copper and iron can be particularly challenging, noted Christoph J. Fahrni, a chemistry professor at Georgia Institute of Technology, because these metal ions often continue to quench fluorophores rather than enable fluorescence, as does Zn2+.
Fahrni and colleagues have been working with a 16-membered thiazacrown ligand to selectively recognize and bind Cu+. They tethered the ligand to a 1,3,5-triarylpyrazoline fluorophore. For this system, the researchers found that lack of fluorescence occurred through formation of ternary complexes with the solvent, rather than through electron-transfer quenching (J. Am. Chem. Soc. 2010, 132, 737).
The researchers have now redesigned the ligand system to prevent such complex formation. Preliminary results indicate that the new ligand system greatly improves fluorescence of the copper-bound probe in both methanol and aqueous solutions, Fahrni said. He and his team are now using the probes to get qualitative information about the location and mobility of copper pools in cells. He also plans to use the probes to characterize the metal-binding properties of copper-transporting proteins involved in cellular homeostasis.
Another approach to copper imaging is to couple a chelator to magnetic resonance imaging contrast agents. Emily L. Que, now a postdoctoral researcher at Northwestern University, described her graduate work in Christopher J. Chang’s laboratory at the University of California, Berkeley. She coupled a clinically used MRI agent, gadolinium-DO3A, to a copper-binding moiety containing acetate or thioether groups and found that the constructs could complex with Cu2+ or Cu+, respectively. The result was an increase in the solution MRI signal of up to 360% compared with that of the nonchelated agent.
Additional modifications to the Gd-binding domain of the construct increased the hydrophilicity, steric bulk, and charge of the complex, reducing its sensitivity to cellular anions such as bicarbonate, phosphate, citrate, and lactate and further increasing the MRI signal (Dalton Trans. 2010, 39, 469). Researchers in Chang’s group are now working to get cells to take up the agents so that they can observe copper pools in cells. They’d like to use the copper probes to get MRI maps of copper in living organisms and also to investigate models of neurodegenerative diseases linked to elevated copper levels, Que said.
Yi Lu, a chemistry professor at the University of Illinois, Urbana-Champaign, noted that most sensor design involves a time-consuming trial-and-error process. His group has developed a combinatorial approach that involves screening a pool of 1014 to 1015 DNA sequences to find DNAzymes—catalytic DNAs that will selectively bind to a particular metal ion to cleave a substrate DNA strand to which the catalytic strand is hybridized. A fluorescent tag and quencher molecule can be tethered to the catalytic and substrate DNA strands such that, prior to metal binding, the quencher suppresses fluorescence. When the construct finds the appropriate metal ion, however, the resulting cleavage of the substrate strand releases the tag, which then fluoresces.
The screening system can be optimized for specific metal ions. The researchers can also tune the sensors to detect metal ions in a particular concentration range. Lu’s lab is also now experimenting with tethering to the DNA biotin and MRI contrast agents, akin to the quencher and fluorescent tag: The MRI signal would change depending on whether the DNA substrate strand was hybridized to the catalytic DNA or cleaved and released.
Other researchers are working on visualizing metals directly, without the need for illuminating tags. Paul A. Lindahl, a chemistry professor at Texas A&M University, is using Mössbauer and other spectroscopic techniques to quantify the amount and species of iron within cells. Iron is critical for cell metabolism, but in excess quantities it can create reactive oxygen species and is linked to neurodegeneration. The two big hubs for iron trafficking within a cell are the mitochondria and vacuoles. “The driving need for iron in yeast cells is for respiratory complexes,” Lindahl said, and the vacuoles might provide something of an iron storage pool.
Lindahl and coworkers used Mössbauer spectroscopy to compare the iron located in the mitochondria of respiring cells, which use glucose and oxygen to generate adenosine triphosphate (ATP), with fermenting cells, which generate ATP by converting glucose to ethanol. The researchers found that respiring cells have most of their mitochondrial iron bound within heme groups or in iron-sulfur clusters, such as [Fe2S2]+ or [Fe4S4]2+, that are typical of enzymes involved in respiration. In the mitochondria of fermenting cells, however, the amount of iron localized to respiratory complexes is about two-thirds lower (Biochemistry 2010, 49, 5436). Instead, the researchers see mononuclear Fe2+ and Fe3+ complexes as well as ferric phosphate nanoparticles. Lindahl suspects that yeast mitochondria maintain a pool of iron in their mononuclear or nanoparticulate form during fermentation; that pool then gets drawn down to enable respiration when cells turn on that pathway.
Lindahl and colleagues are expanding their studies beyond mitochondria to try to pin down the location and species of iron in whole yeast cells. The researchers are also investigating the effects of having excess or limited iron in cell media as well as turning on and off specific iron transport genes and regulatory pathways.
Thomas V. O’Halloran, a chemistry professor at Northwestern University, described the use of synchrotron-based X-ray fluorescence microscopy (XFM) to analyze zinc concentrations in mouse oocytes, or immature egg cells. As part of a collaboration with Northwestern obstetrics and gynecology professor Teresa K. Woodruff to understand how to preserve cancer patients’ fertility, graduate student Alison M. Kim used XFM to map the total zinc content of mouse oocytes as they progressed through meiosis and matured into eggs capable of fertilization.
Kim found that zinc concentrations went up more than 50% as the oocytes matured (Nat. Chem. Biol. 2010, 6, 674). “The cell acquires 2 × 1010 zinc atoms in the last 12 hours of the maturation process,” probably fed by nurse cells in the ovary, O’Halloran said. Copper and iron concentrations, in contrast, stayed relatively stable. After fertilization, zinc concentrations decreased again. Feeding the cells a zinc chelator to sequester the metal indicated that the meiotic spindle, which separates chromosomes during cell division, is affected by insufficient zinc. Lack of zinc also affected oscillations in calcium concentrations that normally occur when an egg is fertilized. Although zinc-insufficient eggs could be fertilized, they did not generate productive embryos. O’Halloran and colleagues are now trying to pin down the enzyme and signaling pathways involved in the zinc flux.
The new probes and techniques represent an innovative approach to studying metal ions in cells, Fahrni noted. Historically, researchers added or subtracted metals from growth media, observed which genes were turned on or off, and then tracked the genes to the proteins involved. By looking at metal ions more directly, scientists should be able to reach a deeper understanding of the role of inorganic chemistry in biology.
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