Abstract
The zinc ion (Zn(2+)) is emerging as an important signaling molecule. Here, we engineered an improved Zn(2+) 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(2+) concentrations. We further developed a set of GZnP2 derived imaging tools to determine the labile Zn(2+) 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(2+) concentration in the matrix was less than 1 pM, while the labile Zn(2+) concentration in the IMS was comparable to the cytosol (∼100 pM). With these sensors, we showed that upon exposure to high Zn(2+), only the cytosol and the IMS were overloaded with Zn(2+), while the mitochondrial matrix was unable to sequester excess labile Zn(2+) in depolarized INS-1 cells. This work highlighted the importance of distinguishing the labile Zn(2+) concentrations and dynamics between the mitochondrial matrix and IMS.