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eZinCh-2: A Versatile, Genetically Encoded FRET Sensor for Cytosolic and
Intraorganelle Zn(2+) Imaging
#MMPMID26151333
Hessels AM
; Chabosseau P
; Bakker MH
; Engelen W
; Rutter GA
; Taylor KM
; Merkx M
ACS Chem Biol
2015[Sep]; 10
(9
): 2126-34
PMID26151333
show ga
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.
|Binding Sites
[MESH]
|Cations, Divalent/analysis/metabolism
[MESH]
|Cytosol/chemistry/*metabolism
[MESH]
|Fluorescence Resonance Energy Transfer/*methods
[MESH]
free
free
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