title: 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.
authors.affiliation: Laboratory of Chemical Biology and Institute of Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology , Eindhoven, The Netherlands.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Hessels
authors.affiliation: Section of Cell Biology and Functional Genomics, Division of Medicine, Imperial College London , London, United Kingdom.
authors.forename: Pauline
authors.initials: P
authors.lastname: Chabosseau
authors.affiliation: Laboratory of Chemical Biology and Institute of Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology , Eindhoven, The Netherlands.
authors.forename: Maarten H
authors.initials: MH
authors.lastname: Bakker
authors.affiliation: Laboratory of Chemical Biology and Institute of Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology , Eindhoven, The Netherlands.
authors.forename: Wouter
authors.initials: W
authors.lastname: Engelen
authors.affiliation: Section of Cell Biology and Functional Genomics, Division of Medicine, Imperial College London , London, United Kingdom.
authors.forename: Guy A
authors.initials: GA
authors.lastname: Rutter
authors.affiliation: Breast Cancer Molecular Pharmacology Group, School of Pharmacy and Pharmaceutical Sciences, Cardiff University , Cardiff, United Kingdom.
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Taylor
authors.affiliation: Laboratory of Chemical Biology and Institute of Complex Molecular Systems (ICMS), Department of Biomedical Engineering, Eindhoven University of Technology , Eindhoven, The Netherlands.
authors.forename: Maarten
authors.initials: M
authors.lastname: Merkx
mesh_terms: D001665:Binding Sites; D002413:Cations, Divalent / Q000032:analysis / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D061848:Optical Imaging / Q000379:methods; D015032:Zinc / Q000032:analysis* / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D015032:Zinc