Evan W Miller

Publications of Evan W Miller

• Calcium-dependent copper redistributions in neuronal cells revealed by a fluorescent copper sensor and X-ray fluorescence microscopy.

  Authors: Sheel C Dodani, Dylan W Domaille, Christine I Nam, Evan W Miller, Lydia A Finney, Stefan Vogt, Christopher J Chang

  Proceedings of the National Academy of Sciences of the United States of America.

  Dynamic fluxes of s-block metals like potassium, sodium, and calcium are of broad importance in cell signaling. In contrast, the concept of mobile transition metals triggered by cell activationDynamic fluxes of s-block metals like potassium, sodium, and calcium are of broad importance in cell signaling. In contrast, the concept of mobile transition metals triggered by cell activation remains insufficiently explored, in large part because metals like copper and iron are typically studied as static cellular nutrients and there are a lack of direct, selective methods for monitoring their distributions in living cells. To help meet this need, we now report Coppersensor-3 (CS3), a bright small-molecule fluorescent probe that offers the unique capability to image labile copper pools in living cells at endogenous, basal levels. We use this chemical tool in conjunction with synchotron-based microprobe X-ray fluorescence microscopy (XRFM) to discover that neuronal cells move significant pools of copper from their cell bodies to peripheral processes upon their activation. Moreover, further CS3 and XRFM imaging experiments show that these dynamic copper redistributions are dependent on calcium release, establishing a link between mobile copper and major cell signaling pathways. By providing a small-molecule fluorophore that is selective and sensitive enough to image labile copper pools in living cells under basal conditions, CS3 opens opportunities for discovering and elucidating functions of copper in living systems.

• Light-Activated Regulation of Cofilin Dynamics Using a Photocaged Hydrogen Peroxide Generator.

  Authors: Evan W Miller, Nicolas Taulet, Carl S Onak, Elizabeth J New, Julie K Lanselle, Gillian S Smelick, Christopher J Chang

  Journal of the American Chemical Society.

  Hydrogen peroxide (H(2)O(2)) can exert diverse signaling and stress responses within living systems depending on its spatial and temporal dynamics. Here we report a new small-molecule probe forHydrogen peroxide (H(2)O(2)) can exert diverse signaling and stress responses within living systems depending on its spatial and temporal dynamics. Here we report a new small-molecule probe for producing H(2)O(2) on demand upon photoactivation and its application for optical regulation of cofilin-actin rod formation in living cells. This chemical method offers many potential opportunities for dissecting biological roles for H(2)O(2) as well as remote control of cell behavior via H(2)O(2)-mediated pathways.

• Aquaporin-3 mediates hydrogen peroxide uptake to regulate downstream intracellular signaling.

  Authors: Evan W Miller, Bryan C Dickinson, Christopher J Chang

  Proceedings of the National Academy of Sciences of the United States of America.

  Hydrogen peroxide (H(2)O(2)) produced by cell-surface NADPH Oxidase (Nox) enzymes is emerging as an important signaling molecule for growth, differentiation, and migration processes. However, howHydrogen peroxide (H(2)O(2)) produced by cell-surface NADPH Oxidase (Nox) enzymes is emerging as an important signaling molecule for growth, differentiation, and migration processes. However, how cells spatially regulate H(2)O(2) to achieve physiological redox signaling over nonspecific oxidative stress pathways is insufficiently understood. Here we report that the water channel Aquaporin-3 (AQP3) can facilitate the uptake of H(2)O(2) into mammalian cells and mediate downstream intracellular signaling. Molecular imaging with Peroxy Yellow 1 Methyl-Ester (PY1-ME), a new chemoselective fluorescent indicator for H(2)O(2), directly demonstrates that aquaporin isoforms AQP3 and AQP8, but not AQP1, can promote uptake of H(2)O(2) specifically through membranes in mammalian cells. Moreover, we show that intracellular H(2)O(2) accumulation can be modulated up or down based on endogenous AQP3 expression, which in turn can influence downstream cell signaling cascades. Finally, we establish that AQP3 is required for Nox-derived H(2)O(2) signaling upon growth factor stimulation. Taken together, our findings demonstrate that the downstream intracellular effects of H(2)O(2) can be regulated across biological barriers, a discovery that has broad implications for the controlled use of this potentially toxic small molecule for beneficial physiological functions.

• An Integrated Imaging Approach To The Study Of Oxidative Stress Generation By Mitochondrial Dysfunction In Living Cells.

  Authors: Wan-Yun Cheng, Haiyan Tong, Evan W Miller, Christopher J Chang, James Remington, Robert M Zucker, Philip A Bromberg, James M Samet, Thomas P J Hofer

  Environmental health perspectives.

  Background: The mechanisms of action of many environmental agents commonly involve oxidative stress resulting from mitochondrial dysfunction. Zinc is a common environmental metallic contaminant thatBackground: The mechanisms of action of many environmental agents commonly involve oxidative stress resulting from mitochondrial dysfunction. Zinc is a common environmental metallic contaminant that has been implicated in a variety of oxidant-dependent toxicological responses, although Zn2+ does not directly generate ROS (unlike transition metals such as Fe, Cu, V, etc). Objectives and methods: In order to characterize the role of oxidative stress in zinc-induced toxicity, we have used an integrated imaging approach that employs the H2O2-specific fluorophore PG1, the mitochondrial potential sensor JC-1, and the mitochondrial redox potential sensor MTroGFP1 in living cells. Results: Zinc treatment in the presence of the Zn2+ ionophore pyrithione of A431 skin carcinoma cells pre-loaded with the H2O2-specific indicator PG1 resulted in a significant increase in H2O2 production that could be significantly inhibited with the mitochondrial inhibitor CCCP. Mitochondria were further implicated as the source of zinc-induced H2O2 formation by the observation that exposure to zinc caused a loss of mitochondrial membrane potential. Using the genetically encoded fluorescent sensor MTroGFP1 we show that zinc exposure of A431 cells induces a rapid loss of reducing redox potential in mitochondria. We also demonstrated that zinc exposure results in rapid swelling of mitochondria isolated from mouse hearts. Conclusion: Taken together, these findings show a disruption of mitochondrial integrity, H2O2 formation and a shift towards positive redox potential in cells exposed to zinc. These data demonstrate the utility of a real time, live cell imaging to the study of the role of oxidative stress in toxicological responses.

• Mitochondria are the source of hydrogen peroxide for dynamic brain-cell signaling.

  Authors: Li Bao, Marat V Avshalumov, Jyoti C Patel, Christian R Lee, Evan W Miller, Christopher J Chang, Margaret E Rice

  The Journal of neuroscience : the official journal of the Society for Neuroscience. 29(28):9002-10.

  Hydrogen peroxide (H(2)O(2)) is emerging as a ubiquitous small-molecule messenger in biology, particularly in the brain, but underlying mechanisms of peroxide signaling remain an open frontier forHydrogen peroxide (H(2)O(2)) is emerging as a ubiquitous small-molecule messenger in biology, particularly in the brain, but underlying mechanisms of peroxide signaling remain an open frontier for study. For example, dynamic dopamine transmission in dorsolateral striatum is regulated on a subsecond timescale by glutamate via H(2)O(2) signaling, which activates ATP-sensitive potassium (K(ATP)) channels to inhibit dopamine release. However, the origin of this modulatory H(2)O(2) has been elusive. Here we addressed three possible sources of H(2)O(2) produced for rapid neuronal signaling in striatum: mitochondrial respiration, monoamine oxidase (MAO), and NADPH oxidase (Nox). Evoked dopamine release in guinea-pig striatal slices was monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry. Using direct fluorescence imaging of H(2)O(2) and tissue analysis of ATP, we found that coapplication of rotenone (50 nM), a mitochondrial complex I inhibitor, and succinate (5 mM), a complex II substrate, limited H(2)O(2) production, but maintained tissue ATP content. Strikingly, coapplication of rotenone and succinate also prevented glutamate-dependent regulation of dopamine release, implicating mitochondrial H(2)O(2) in release modulation. In contrast, inhibitors of MAO or Nox had no effect on dopamine release, suggesting a limited role for these metabolic enzymes in rapid H(2)O(2) production in the striatum. These data provide the first demonstration that respiring mitochondria are the primary source of H(2)O(2) generation for dynamic neuronal signaling.

• A red-emitting naphthofluorescein-based fluorescent probe for selective detection of hydrogen peroxide in living cells.

  Authors: Aaron E Albers, Bryan C Dickinson, Evan W Miller, Christopher J Chang

  Bioorganic & medicinal chemistry letters.

  We report the synthesis, properties, and cellular application of Naphtho-Peroxyfluor-1 (NPF1), a new fluorescent indicator for hydrogen peroxide based on a red-emitting naphthofluorescein platform.We report the synthesis, properties, and cellular application of Naphtho-Peroxyfluor-1 (NPF1), a new fluorescent indicator for hydrogen peroxide based on a red-emitting naphthofluorescein platform. Owing to its boronate cages, NPF1 features high selectivity for hydrogen peroxide over a panel of biologically relevant reactive oxygen species (ROS), including superoxide and nitric oxide, as well as excitation and emission profiles in the far-red region of the visible spectrum (>600nm). Flow cytometry experiments in RAW264.7 macrophages establish that NPF1 can report changes in peroxide levels in living cells.

• An ICT-based approach to ratiometric fluorescence imaging of hydrogen peroxide produced in living cells.

  Authors: Duangkhae Srikun, Evan W Miller, Dylan W Domaille, Christopher J Chang

  Journal of the American Chemical Society. 130(14):4596-7.

• Preparation and use of Leadfluor-1, a synthetic fluorophore for live-cell lead imaging.

  Authors: Evan W Miller, Qiwen He, Christopher J Chang

  Nature protocols. 3(5):777-83.

  Leadfluor-1 (LF1) is a small-molecule fluorescent sensor for detecting lead in biological and environmental samples, including live cells. This dye uses a xanthenone fluorescent scaffold coupled to aLeadfluor-1 (LF1) is a small-molecule fluorescent sensor for detecting lead in biological and environmental samples, including live cells. This dye uses a xanthenone fluorescent scaffold coupled to a dicarboxylate pseudocrown ether receptor to achieve selective detection of Pb(2+) in the presence of biologically relevant metal ions, including divalent calcium, magnesium and zinc. LF1 fluorescence increases by up to 18-fold on binding Pb(2+). In this protocol, we describe the synthesis and application of LF1 to imaging lead accumulation within live cells. The preparation of LF1 is anticipated to take 14-21 d, and the imaging assays can be performed in 1-2 d with cultured cells.

• Fluorescent probes for nitric oxide and hydrogen peroxide in cell signaling.

  Authors: Evan W Miller, Christopher J Chang

  Current opinion in chemical biology. 11(6):620-5.

  Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) have emerged as essential small molecules for cellular signal transduction owing largely to their ability to mediate oxidative posttranslationalNitric oxide (NO) and hydrogen peroxide (H(2)O(2)) have emerged as essential small molecules for cellular signal transduction owing largely to their ability to mediate oxidative posttranslational modifications (PTMs). Inventing new ways to track these small, diffusible, and reactive species with spatial and temporal resolution is a key challenge in elucidating their chemistry in living systems. Recent progress in the development of fluorescent probes that respond selectively to NO and H(2)O(2) produced at cell signaling levels offers a promising approach to interrogating their physiological production, accumulation, trafficking, and function.

• Molecular imaging of hydrogen peroxide produced for cell signaling.

  Authors: Evan W Miller, Orapim Tulyathan, Orapim Tulyanthan, Ehud Y Isacoff, Christopher J Chang

  Nature chemical biology. 3(5):263-7.

  Hydrogen peroxide (H2O2) is emerging as a newly recognized messenger in cellular signal transduction. However, a substantial challenge in elucidating its diverse roles in complex biologicalHydrogen peroxide (H2O2) is emerging as a newly recognized messenger in cellular signal transduction. However, a substantial challenge in elucidating its diverse roles in complex biological environments is the lack of methods for probing this reactive oxygen metabolite in living systems with molecular specificity. Here we report the synthesis and application of Peroxy Green 1 (PG1) and Peroxy Crimson 1 (PC1), two new fluorescent probes that show high selectivity for H2O2 and are capable of visualizing endogenous H2O2 produced in living cells by growth factor stimulation, including the first direct imaging of peroxide produced for brain cell signaling. The combined features of reactive oxygen species selectivity, sensitivity to signaling levels of H2O2, and live-cell compatibility presage many new opportunities for PG1, PC1 and related synthetic reagents for exploring the physiological roles of H2O2 in living systems with molecular imaging.

• A fluorescent sensor for imaging reversible redox cycles in living cells.

  Authors: Evan W Miller, Shelly X Bian, Christopher J Chang

  Journal of the American Chemical Society. 129(12):3458-9.

• A bright and specific fluorescent sensor for mercury in water, cells, and tissue.

  Authors: Sungho Yoon, Evan W Miller, Qiwen He, Patrick H Do, Christopher J Chang

  Angewandte Chemie (International ed. in English). 46(35):6658-61.

• A selective fluorescent sensor for detecting lead in living cells.

  Authors: Qiwen He, Evan W Miller, Audrey P Wong, Christopher J Chang

  Journal of the American Chemical Society. 128(29):9316-7.

  We present the synthesis, properties, and biological applications of Leadfluor-1 (LF1), a new water-soluble, turn-on fluorescent sensor that is capable of selectively imaging Pb2+ in aqueous solutionWe present the synthesis, properties, and biological applications of Leadfluor-1 (LF1), a new water-soluble, turn-on fluorescent sensor that is capable of selectively imaging Pb2+ in aqueous solution and in living cells. LF1 combines a fluorescein chromophore and a pseudocrown receptor to provide good selectivity for Pb2+ over a range of biologically and environmentally relevant metal ions in aqueous solution, with sensitivity to parts per billion EPA limits for allowable lead in drinking water. In addition to these attributes, imaging experiments further show that LF1 is the first small-molecule reagent that can be delivered into living cells and report changes in intracellular Pb2+ levels.

• Preparation and use of Coppersensor-1, a synthetic fluorophore for live-cell copper imaging.

  Authors: Evan W Miller, Li Zeng, Dylan W Domaille, Christopher J Chang

  Nature protocols. 1(2):824-7.

  Coppersensor-1 (CS1) is a small-molecule, membrane-permeable fluorescent dye for imaging labile copper pools in biological samples, including live cells. This probe, comprising a boron dipyrrometheneCoppersensor-1 (CS1) is a small-molecule, membrane-permeable fluorescent dye for imaging labile copper pools in biological samples, including live cells. This probe, comprising a boron dipyrromethene (BODIPY) chromophore coupled to a thioether-rich receptor, has a picomolar affinity for Cu+ with high selectivity over competing cellular metal ions. CS1 fluorescence increases up to 10-fold on binding to Cu+. In this protocol we describe the synthesis of CS1 and how to use this chemical tool to investigate intracellular levels of labile copper in cultured cells. The preparation of CS1 is anticipated to take 4-5 d, and imaging assays can be performed in 1-2 d with cultured cells.

• A selective turn-on fluorescent sensor for imaging copper in living cells.

  Authors: Li Zeng, Evan W Miller, Arnd Pralle, Ehud Y Isacoff, Christopher J Chang

  Journal of the American Chemical Society. 128(1):10-1.

  We present the synthesis, properties, and biological applications of Coppersensor-1 (CS1), a new water-soluble, turn-on fluorescent sensor for intracellular imaging of copper in living biologicalWe present the synthesis, properties, and biological applications of Coppersensor-1 (CS1), a new water-soluble, turn-on fluorescent sensor for intracellular imaging of copper in living biological samples. CS1 utilizes a BODIPY reporter and thioether-rich receptor to provide high selectivity and sensitivity for Cu+ over other biologically relevant metal ions, including Cu2+, in aqueous solution. This BODIPY-based probe is the first Cu+-responsive sensor with visible excitation and emission profiles and gives a 10-fold turn-on response for detecting this ion. Confocal microscopy experiments further establish that CS1 is membrane-permeable and can successfully monitor intracellular Cu+ levels within living cells.

• Boronate-based fluorescent probes for imaging cellular hydrogen peroxide.

  Authors: Evan W Miller, Aaron E Albers, Arnd Pralle, Ehud Y Isacoff, Christopher J Chang

  Journal of the American Chemical Society. 127(47):16652-9.

  The syntheses, properties, and biological applications of the Peroxysensor family, a new class of fluorescent probes for hydrogen peroxide, are presented. These reagents utilize a boronateThe syntheses, properties, and biological applications of the Peroxysensor family, a new class of fluorescent probes for hydrogen peroxide, are presented. These reagents utilize a boronate deprotection mechanism to provide high selectivity and optical dynamic range for detecting H2O2 in aqueous solution over similar reactive oxygen species (ROS) including superoxide, nitric oxide, tert-butyl hydroperoxide, hypochlorite, singlet oxygen, ozone, and hydroxyl radical. Peroxyresorufin-1 (PR1), Peroxyfluor-1 (PF1), and Peroxyxanthone-1 (PX1) are first-generation probes that respond to H2O2 by an increase in red, green, and blue fluorescence, respectively. The boronate dyes are cell-permeable and can detect micromolar changes in H2O2 concentrations in living cells, including hippocampal neurons, using confocal microscopy and two-photon microscopy. The unique combination of ROS selectivity, membrane permeability, and a range of available excitation/emission colors establishes the potential value of PR1, PF1, PX1, and related probes for interrogating the physiology and pathology of cellular H2O2.