eZinCh-2: a versatile, genetically-encoded FRET sensor for cytosolic and intra-organelle Zn2+ imaging

A.M. Hessels, P. Chabosseau, M.H. Bakker, W. Engelen, G.A. Rutter, K.M. Taylor, M. Merkx

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Abstract

Zn2+ plays essential and diverse roles in numerous cellular processes. To get a better understanding of intracellular Zn2+ homeostasis and the putative signaling role of Zn2+, various fluorescent sensors have been developed that allow monitoring of Zn2+ concentrations in single living cells in real time. Thus far, two families of genetically encoded FRET-based Zn2+ 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 Zn2+, but distinctly different concentrations have been reported when using these sensors to measure Zn2+ 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 Zn2+ 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 Zn2+ in the cytosol but was also successfully used for measuring Zn2+ 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 Zn2+ simultaneously in the cytosol and the ER or mitochondria.
Original languageEnglish
Pages (from-to)2126-2134
JournalACS Chemical Biology
Volume10
Issue number9
DOIs
Publication statusPublished - 2015

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    Hessels, A. M., Chabosseau, P., Bakker, M. H., Engelen, W., Rutter, G. A., Taylor, K. M., & Merkx, M. (2015). eZinCh-2: a versatile, genetically-encoded FRET sensor for cytosolic and intra-organelle Zn2+ imaging. ACS Chemical Biology, 10(9), 2126-2134. https://doi.org/10.1021/acschembio.5b00211