Hessels, Anne M., Chabosseau, Pauline, Bakker, Maarten H., Engelen, Wouter, Rutter, Guy A., Taylor, Kathryn M.  ORCID: https://orcid.org/0000-0002-9576-9490 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: | Schools > Pharmacy |
| Subjects: | R Medicine > RM Therapeutics. Pharmacology |
| Additional Information: | This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. |
| Publisher: | American Chemical Society |
| ISSN: | 1554-8929 |
| Funders: | Wellcome Trust |
| Date of First Compliant Deposit: | 30 March 2016 |
| Date of Acceptance: | 7 July 2015 |
| Last Modified: | 04 May 2023 11:15 |
| URI: | https://orca.cardiff.ac.uk/id/eprint/75158 |
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