eZinCh-2: A Versatile, Genetically Encoded FRET Sensor for Cytosolic and Intraorganelle Zn2+ Imaging

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• 作者：Anne M. Hessels ; Pauline Chabosseau ; Maarten H. Bakker ; Wouter Engelen ; Guy A. Rutter ; Kathryn M. Taylor ; Maarten Merkx
• 刊名：ACS Chemical Biology
• 出版年：2015
• 出版时间：September 18, 2015
• 年：2015
• 卷：10
• 期：9
• 页码：2126-2134
• 全文大小：565K
• ISSN：1554-8937

文摘

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 (K_d = 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.