Transition metal ions are critical to life as we know it and play essential roles in a wide swath of fundamental processes. Paradoxically, these essential metals are also toxic and therefore cells must tightly regulate metal accumulation, distribution and export. Not surprisingly, metal imbalance has profound implications for human health and is correlated with a host of pathophysiologies, including neurodegeneration, diabetes, cancer, and immune dysfunction. The long term goals of our research are to identify the mechanisms by which cells balance metal ions, define conditions under which cells use metals as signaling agents, and elucidate how metal imbalance leads to disease and degeneration. The current proposal focuses on zinc (Zn2+) as there is emerging evidence that transient Zn2+ signals can be generated within the cell, representing an exciting new paradigm for how metal ions influence cellular function. Zn2+ is an essential micronutrient required for human life. Its deficiency leads to impaired cognition, immune dysfunction, diarrhea, and death. Close to 3,000 genes in the human genome contain Zn2+ finger motifs, indicating that Zn2+ binding proteins are essential cell constituents. This is a truly staggering number, and represents close to 10% of the proteins encoded by the human genome. Our overall hypothesis is that Zn2+ serves as an important regulator of cell function, coordinating the activity of numerous cellular pathways, such that changes in Zn2+ status with disease alter downstream signaling targets, profoundly influencing cellular physiology. The basic premise of this hypothesis is that Zn2+ is dynamically regulated, and that changes in free Zn2+ influence canonical signaling pathways such as Ca2+, as well as alter the metal ion occupancy of the proteome, fine tuning the activity of hundreds, if not thousands of Zn2+-dependent proteins. Historically, our understanding of cellular Zn2+ homeostasis has been limited by the lack of tools to visualize and quantify free Zn2+ in specific locations (i.e. intracellular organelles) in living cells with high spatial and temporal resolution. In the last grant cycle, we addressed this need by developing a suite of fluorescent Zn2+ sensors genetically targeted to the cytosol, nucleus, ER, Golgi, and mitochondria. With these sensors we made remarkable discoveries about Zn2+ dynamics, interplay between Zn2+ and Ca2+, and provided the first glimpse of how the distribution of free Zn2+ may be altered in disease. In the next cycle, we will build on these discoveries and extend them to expand the repertoire of organelle-targeted sensors, thoroughly profile free Zn2+ distribution in normal versus diseased cells, define the mechanism(s) by which Zn2+ is altered, and identify the consequences of Zn2+ dysregulation for cellular function. Our proposed work has 3 specific aims: (1) Create new Zn2+ sensors that quantitatively report on free Zn2+ in organelles to enable comprehensive quantitative mapping of free Zn2+;(2) Define the changes in free Zn2+ in prostate cancer and identify the mechanism of dysregulation;and (3) Identify whether Zn2+ dysregulation plays a causative role in influencing downstream targets.

Public Health Relevance

Quantitative imaging of transition metal ions in living cells will transform our understanding of how cells regulate these essential micronutrients, and how the distribution of metal ions influences cellular function. Because metal imbalance and dysregulation have been correlated with a wide variety of diseases, such as Alzheimers, immune dysregulation, cancer, and diabetes, metal homeostasis has profound implications for human health. Understanding the detailed mechanisms by which organisms control metal ions will highlight potential avenues for intervention, and could ultimately lead to targeted therapies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Special Emphasis Panel (ZRG1-BCMB-R (02))
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Anderson, Vernon
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University of Colorado at Boulder
Schools of Arts and Sciences
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Carter, Kyle P; Young, Alexandra M; Palmer, Amy E (2014) Fluorescent sensors for measuring metal ions in living systems. Chem Rev 114:4564-601
Park, J Genevieve; Palmer, Amy E (2014) Quantitative measurement of Ca2+ and Zn2+ in mammalian cells using genetically encoded fluorescent biosensors. Methods Mol Biol 1071:29-47
Qin, Yan; Miranda, Jose G; Stoddard, Caitlin I et al. (2013) Direct comparison of a genetically encoded sensor and small molecule indicator: implications for quantification of cytosolic Zn(2+). ACS Chem Biol 8:2366-71
Dean, Kevin M; Qin, Yan; Palmer, Amy E (2012) Visualizing metal ions in cells: an overview of analytical techniques, approaches, and probes. Biochim Biophys Acta 1823:1406-15
Ma, Hairong; Gibson, Emily A; Dittmer, Philip J et al. (2012) High-throughput examination of fluorescence resonance energy transfer-detected metal-ion response in mammalian cells. J Am Chem Soc 134:2488-91
Qin, Yan; Dittmer, Philip J; Park, J Genevieve et al. (2011) Measuring steady-state and dynamic endoplasmic reticulum and Golgi Zn2+ with genetically encoded sensors. Proc Natl Acad Sci U S A 108:7351-6
Palmer, Amy E; Qin, Yan; Park, Jungwon Genevieve et al. (2011) Design and application of genetically encoded biosensors. Trends Biotechnol 29:144-52
Ravier, M A; Cheng-Xue, R; Palmer, A E et al. (2010) Subplasmalemmal Ca(2+) measurements in mouse pancreatic beta cells support the existence of an amplifying effect of glucose on insulin secretion. Diabetologia 53:1947-57
Perocchi, Fabiana; Gohil, Vishal M; Girgis, Hany S et al. (2010) MICU1 encodes a mitochondrial EF hand protein required for Ca(2+) uptake. Nature 467:291-6
Palmer, Amy E; Dittmer, Philip J (2010) SNAP-shots of hydrogen peroxide in cells. Chem Biol 17:318-9

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