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EZinCh-2: A Versatile, Genetically Encoded FRET Sensor for Cytosolic and Intraorganelle Zn(2+) Imaging.

ABSTRACT: Zn(2+) plays essential and diverse roles in numerous cellular processes. To get a better understanding of intracellular Zn(2+) homeostasis and the putative signaling role of Zn(2+), various fluorescent sensors have been developed that allow monitoring of Zn(2+) concentrations in single living cells in real time. Thus far, two families of genetically encoded FRET-based Zn(2+) 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(2+), but distinctly different concentrations have been reported when using these sensors to measure Zn(2+) 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(2+) 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(2+) in the cytosol but was also successfully used for measuring Zn(2+) 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(2+) simultaneously in the cytosol and the ER or mitochondria.

SUBMITTER: Hessels AM

PROVIDER: S-EPMC4577962 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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

Hessels Anne M AM Chabosseau Pauline P Bakker Maarten H MH Engelen Wouter W Rutter Guy A GA Taylor Kathryn M KM Merkx Maarten M

ACS chemical biology 20150720 9

Zn(2+) plays essential and diverse roles in numerous cellular processes. To get a better understanding of intracellular Zn(2+) homeostasis and the putative signaling role of Zn(2+), various fluorescent sensors have been developed that allow monitoring of Zn(2+) concentrations in single living cells in real time. Thus far, two families of genetically encoded FRET-based Zn(2+) sensors have been most widely applied, the eCALWY sensors developed by our group and the ZapCY sensors developed by Palmer ...[more]

PMID: 26151333

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Dataset Information

EZinCh-2: A Versatile, Genetically Encoded FRET Sensor for Cytosolic and Intraorganelle Zn(2+) Imaging.

Publications

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

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