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

Abstract

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 (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 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.

Engineering BRET-Sensor Proteins

“…11) (Aper et al, 2016). The Zn 2+ levels obtained using BRET detection were similar to those previously determined using the eZinCh-2 FRET-sensor (Hessels et al, 2015;Hessels, Taylor, & Merkx, 2016), but BRET-based detection resulted in lower cell-to-cell variation in emission ratio because of the absence of background fluorescence. The BLZinCh sensors, thus, not only allow straightforward monitoring of intracellular Zn 2+ levels using a plate reader, but should also be very useful for all those imaging applications where laser excitation is undesirable, such as prolonged imaging, imaging in optogenetics, imaging in light-sensitive cells, and in vivo imaging.…”

“…In contrast to the eCALWY sensors, eZinCh-2 does not contain distinct Zn 2+ -binding domains, but has a Zn 2+ -binding histidine and cysteine residue located on the surface of both Cerulean and Citrine, which are fused via a long, flexible linker (Fig. 9A) (Hessels et al, 2015). The coordination of one Zn 2+ ion in between the two histidines and two cysteines brings the two fluorescent domains close together, inducing a $5-fold increase of FRET.…”

Dual Readout BRET/FRET Sensors for Measuring Intracellular Zinc

“…The eZinCh-2 FRET sensor does not contain distinct Zn 2+ binding domains but displays a Zn 2+ coordinating histidine and cysteine residue on the surface of both Cerulean and Citrine. 15 Together, these four residues allow the formation of a stable tetrahedral Zn 2+ complex, bringing both fluorescent domains closer together and inducing an increase in FRET. To minimize the perturbation of the FRET sensor properties as much as possible, we introduced the NLuc domain at the N-terminus of the Cerulean domain in both sensors.…”

“…5,15,17,22,23 In addition, red-shifted variants have been developed that allow multiparameter imaging together with the original CFP-YFP-based sensors, allowing simultaneous Zn 2+ imaging in different cellular compartments in the same cell. 5,15,24,25 …”

Zinc and diabetes

“…Although raising controversies that remain to be resolved, this new information suggests that modulation of ZnT8 activity may provide an interesting new target for diabetes therapies which can be used in a personalized manner to treat the aetiological deficiency in carriers of risk alleles. [56]) and Vamp2-eZinCh2 (yellow, [61]). Modified from [27].…”