期刊
ACS CHEMICAL BIOLOGY
卷 10, 期 9, 页码 2126-2134出版社
AMER CHEMICAL SOC
DOI: 10.1021/acschembio.5b00211
关键词
-
资金
- ECHO grant from The Netherlands Organization of Scientific Research [700.59.013]
- ERC starting grant [ERC-2011-StG 280255]
- STSM Grant from COST Action (ZincNet) [TD1304]
- Wellcome Trust Senior Investigator Award [WT098424AIA]
- Royal Society Wolfson Research Merit Award
- Wellcome Trust University award [091991/Z/10/Z]
- Wellcome Trust [091991/Z/10/Z] Funding Source: Wellcome Trust
- MRC [MR/K001981/1] Funding Source: UKRI
- Medical Research Council [MR/K001981/1] Funding Source: researchfish
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 Cys(2)His(2) 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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据