Optical Recording of Zn Dynamics in the Mitochondrial Matrix and Intermembrane Space with the GZnP2 Sensor.

The zinc ion (Zn) is emerging as an important signaling molecule. Here, we engineered an improved Zn probe GZnP2 based on a previously developed fluorescent sensor GZnP1 to provide a higher fluorescent readout (2-fold higher) that is proportional to cellular labile Zn concentrations. We further developed a set of GZnP2 derived imaging tools to determine the labile Zn concentrations in the mitochondrial matrix, mitochondrial intermembrane space (IMS), and cytosol in four different cell lines (HeLa, Cos-7, HEK293, and INS-1). The labile Zn concentration in the matrix was less than 1 pM, while the labile Zn concentration in the IMS was comparable to the cytosol (∼100 pM). With these sensors, we showed that upon exposure to high Zn, only the cytosol and the IMS were overloaded with Zn, while the mitochondrial matrix was unable to sequester excess labile Zn in depolarized INS-1 cells. This work highlighted the importance of distinguishing the labile Zn concentrations and dynamics between the mitochondrial matrix and IMS.