Transition metal ions are critical to life as we know it. 30% of all proteins contain a metal ion cofactor and these proteins play essential roles in fundamental processes such as respiration, oxygen transport and storage, cell division and migration, and gene transcription. Paradoxically, these essential metals are also toxic and therefore cells must tightly regulate metal accumulation, transport, distribution and export. Not surprisingly, metal imbalance has profound implications at both the cellular and organismal level and is correlated with a host of pathological conditions such as Alzheimer's disease, neurodegeneration, diabetes, prostate cancer, and Wilson's and Menkes disease. The long term goals of our research are to identify the mechanisms by which cells balance metal ions, to define conditions under which cells use metals as signaling agents, and to elucidate how metal imbalance leads to disease and degeneration. The current proposal focuses on Zn2+ as there is emerging evidence that transient Zn2+ signals can be generated within the cell, representing an exciting new paradigm in how metal ions influence cellular function. Moreover, Zn2+ is unique among transition metal ions as it is concentrated into secretory vesicles in a sub-set of cells where it plays a specialized, but poorly defined role in cellular function. Disruption of Zn2+ in these cells has devastating consequences, highlighting the need for a deeper understanding of the physiological role of Zn2+ as well as the means by which Zn2+ disrupts cellular processes. Our current understanding of cellular Zn2+ homeostasis is limited by the lack of appropriate tools to interrogate Zn2+ distribution with high spatial resolution. We propose to address this need by developing a comprehensive family of fluorescent Zn2+ sensors that can be genetically encoded, i.e. explicitly targeted to distinct organelles and sub-domains of the cell. These sensors will be localized to the ER, Golgi, and mitochondria to image Zn2+ distribution and translocation in living cells. We hypothesize that cells contain labile pools of zinc that can be mobilized in response to cellular signals and stresses, and that cellular organelles play a critical role in modulating these zinc signals. Our proposed work has 3 specific aims: (1) Development of genetically encodable fluorescent zinc sensors by systematic investigation of naturally occurring Zn2+ binding domains and microfluidic screening of sensor libraries;(2) Biophysical characterization and in situ validation of sensors;and (3) Identify sources of and sinks for Zn2+ upon mobilization by cellular signals such as nitric oxide, and cellular stresses such as redox destabilization.

Public Health Relevance

Quantitative imaging of metal ion localization and translocation in living cells would transform our current knowledge of metal homeostasis, providing insight into the fundamental workings of the cell, and shedding light on cellular processes that are perturbed when metal regulation goes awry. Quantitative imaging of transition metal ions in living cells will transform our understanding of how cells regulate metal ion availability, and conversely how metal ions influence cellular function. Because metal imbalance and dysregulation have been correlated with a wide variety of diseases, such as Alzheimers, 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)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Anderson, Vernon
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University of Colorado at Boulder
Schools of Arts and Sciences
United States
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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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