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eZinCh-2: a versatile, genetically encoded FRET sensor for cytosolic and intraorganelle Zn2+Imaging

Hessels, Anne M., Chabosseau, Pauline, Bakker, Maarten H., Engelen, Wouter, Rutter, Guy A., Taylor, Kathryn Mary and Merkx, Maarten 2015. eZinCh-2: a versatile, genetically encoded FRET sensor for cytosolic and intraorganelle Zn2+Imaging. ACS Chemical Biology 10 (9) , pp. 2126-2134. 10.1021/acschembio.5b00211

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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.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Pharmacy
Subjects: R Medicine > RM Therapeutics. Pharmacology
Publisher: American Chemical Society
ISSN: 1554-8929
Funders: Wellcome Trust
Date of First Compliant Deposit: 30 March 2016
Date of Acceptance: 24 June 2015
Last Modified: 04 Jun 2017 08:16
URI: http://orca.cf.ac.uk/id/eprint/75158

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