eZinCh-2: A Versatile, Genetically Encoded FRET Sensor for Cytosolic and Intraorganelle Zn Imaging

Zn plays essential and diverse roles in numerous cellular processes. To get a better understanding of intracellular Zn homeostasis and the putative signaling role of Zn, various fluorescent sensors have been developed that allow monitoring of Zn concentrations in single living cells in real time. Thus far, two families of genetically encoded FRET-based Zn sensors have been most widely applied, the eCALWY sensors developed by our group and the ZapCY sensors developed by Palmer and co-workers. Both have been successfully used to measure cytosolic free Zn, but distinctly different concentrations have been reported when using these sensors to measure Zn concentrations in the ER and mitochondria. Here, we report the development of a versatile alternative FRET sensor containing a de novo Cys2His2 binding pocket that was created on the surface of the donor and acceptor fluorescent domains. This eZinCh-2 sensor binds Zn with a high affinity that is similar to that of eCALWY-4 (Kd = 1 nM at pH 7.1), while displaying a substantially larger change in emission ratio. eZinCh-2 not only provides an attractive alternative for measuring Zn in the cytosol but was also successfully used for measuring Zn in the ER, mitochondria, and secretory vesicles. Moreover, organelle-targeted eZinCh-2 can also be used in combination with the previously reported redCALWY sensors to allow multicolor imaging of intracellular Zn simultaneously in the cytosol and the ER or mitochondria. A lthough zinc is sometimes still referred to as a “trace metal ion”, zinc ions play a range of essential roles in numerous cellular processes. In addition to serving as a cofactor in enzyme catalysis and protein stabilization, Zn ions have been postulated to be involved in a variety of signaling processes, ranging from a relatively well-established role in neuromodulation, insulin secretion, and fertilization to its proposed role as a secondary messenger in intracellular signaling. The high intrinsic affinity of Zn for the amino acid side chains of cysteines, histidines, as well as carboxylic acids makes the free Zn ion a potent inhibitor of enzymes and a potential modulator of protein−protein interactions. The level of free Zn in the cytosol is therefore believed to be tightly controlled between 100 pM and 1 nM, which is sufficient for Zn to bind to native Zn-binding proteins but low enough not to interfere with normal metabolic and signaling processes. The concentration of free Zn can be very different in other parts of the cell, however, as millimolar concentrations of total Zn have been reported for secretory vesicles in pancreatic β cells, oocytes, neuronal cells, and mast cells. Triggered release of Zn from organelles has been implicated in transient increases in cytosolic free Zn, potentially regulating the activity of regulatory enzymes such as protein phosphatases and caspases. To get a better understanding of intracellular Zn homeostasis and the putative signaling role of Zn, a variety of fluorescent sensors have been developed that allow monitoring of Zn concentrations in single living cells in real time. Although small molecule sensors are still the most commonly used imaging probes, it has proven challenging to control their subcellular localization and concentration. In contrast, genetically encoded, protein-based sensors can be conveniently targeted to specific subcellular locations and, at least in the cytosol, were found to not perturb intracellular free Zn levels. Thus far, two families of genetically encoded FRET-based Zn sensors have been most widely applied: the eCALWY sensors developed by our group and the ZapCY Received: March 24, 2015 Accepted: July 7, 2015 Articles pubs.acs.org/acschemicalbiology © XXXX American Chemical Society A DOI: 10.1021/acschembio.5b00211 ACS Chem. Biol. XXXX, XXX, XXX−XXX This is an open access art icle published under an ACS AuthorChoice License, which permits copying and redist ribut ion of the art icle or any adaptat ions for non-commercial purposes.