The challenges of using a copper fluorescent sensor (CS1) to track intracellular distributions of copper in neuronal and glial cells† Katherine A. Price, a James L. Hickey, bc Zhiguang Xiao, bc Anthony G. Wedd, bc Simon A. James, d Jeffrey R. Liddell, a Peter J. Crouch, a Anthony R. White‡ * a and Paul S. Donnelly‡ * bc Received 31st March 2012, Accepted 10th June 2012 DOI: 10.1039/c2sc20397a Copper is an essential biometal involved in critical cell functions including respiration. However, the mechanisms controlling its sub-cellular localization during health and disease remain poorly understood. This is partially due to the difficulty of detecting a metal ion that is bound tightly to metallo-chaperone and detoxification molecules in the cell. A BODIPY-based Cu fluorescent probe CS1 (Cu sensor 1) has been applied in innovative attempts to visualize monovalent Cu pools within cells (Zeng et al., J. Am. Chem. Soc., 2006, 128, 10–11). Inspired by this work, we sought to use CS1 to identify sub-cellular localization of Cu delivered to M17 neuronal or U87MG glial cells by a cell- permeable bis(thiosemicarbazonato)Cu(II) complex, Cu II (gtsm). This complex increases cellular Cu concentrations by factors of 10–100 when compared to treatment with equivalent concentrations of CuCl 2 (Donnelly et al., J. Biol. Chem., 2008, 283, 4568–4577). However, we were unable to identify any specific increase in CS1 fluorescence in neurons or glia treated with CuCl 2 or with Cu II (gtsm), despite controls revealing a large increase in total cellular Cu with the latter treatment. Further in vitro characterization of CS1 suggests that, consistent with its relatively weak affinity for Cu I (K D z 10 11 M), it is unlikely to compete with endogenous proteins with sub-picomolar affinities, nor with glutathione, the endogenous redox buffer essential for functional maintenance of many proteins, including those that bind Cu I . Moreover, we show that CS1 is localized predominantly to lysosomes and that the observed background fluorescence may be attributed to increased concentrations of apo- CS1 in this organelle or to the probe gaining access to Cu I made available via recycling of nutrient Cu in the acidic lysosome. It was possible to observe a consistent increase in CS1 fluorescence in neuronal cells exposed to stress. For example, treatment with buthionine sulfoximine decreased cellular glutathione levels and led to enhanced CS1 fluorescence, but the total cellular Cu levels did not correlate with the increased fluorescence. In addition, cells treated with reagents that are known to alter cellular pH homeostasis provided an enhanced fluorescence. Our findings demonstrate that the source of enhanced CS1 fluorescence in Cu-supplemented cells must be interpreted with caution. It may be a consequence of altered cell pH, compromised vesicle maturation, increased CS1 uptake and/or trapping of CS1 in the lysosomal compartment. Introduction Copper is an essential element in biological systems, being a critical component of many enzymes and, in particular, of mitochondrial cytochrome c oxidase. Its Cu II /Cu I redox couple drives its role in cellular metabolism, but this reactivity also means that, when uncontrolled, Cu ions can generate damaging reactive oxygen species. 1 Cu II/I also has the potential to bind adventitiously to protein surfaces and to displace other biometals from their active sites. 2 In mitigation, cells have developed elaborate systems to manage this essential nutrient. 3 Upon uptake by the Cu transporter Ctr1 at the mammalian plasma membrane, Cu I is trafficked to a number of cellular locations including the mitochondria, endoplasmic reticulum, a Department of Pathology, The University of Melbourne, 3010, and Mental Health Research Institute, Parkville, 3052, Victoria, Australia. E-mail: arwhite@unimelb.edu.au; Fax: +61 (3) 8344 4004; Tel: +61 (3) 8344 1805 b Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, 3052, Australia. E-mail: pauld@unimelb. edu.au; Tel: +61 (3) 8344 2399 c School of Chemistry, The University of Melbourne, Victoria, 3010, Australia d CSIRO Materials Science and Engineering, Clayton, Victoria, Australia and the CSIRO Preventive Health Flagship † Electronic supplementary information (ESI) available: Evaluation of CS1 as a Cu I probe in cell-free experiments. See DOI: 10.1039/c2sc20397a ‡ These authors contributed equally. 2748 | Chem. Sci., 2012, 3, 2748–2759 This journal is ª The Royal Society of Chemistry 2012 Dynamic Article Links C < Chemical Science Cite this: Chem. Sci., 2012, 3, 2748 www.rsc.org/chemicalscience EDGE ARTICLE