Quantitative imaging of mitochondrial and cytosolic free zinc levels in an in vitro model of ischemia/reperfusion

Abstract

The role of zinc ion in cytotoxicity following ischemic stroke, prolonged status epilepticus, and traumatic brain injury remains controversial, but likely is the result of mitochondrial dysfunction. We describe an excitation ratiometric fluorescence biosensor based on human carbonic anhydrase II variants expressed in the mitochondrial matrix, permitting free zinc levels to be quantitatively imaged therein. We observed an average mitochondrial matrix free zinc concentration of 0.2 pM in the PC12 rat pheochromacytoma cell culture line. Cytoplasmic and mitochondrial free zinc levels were imaged in a cellular oxygen glucose deprivation (OGD) model of ischemia/reperfusion. We observed a significant increase in mitochondrial zinc 1 hr following 3 hr OGD, at a time point when cytosolic zinc levels were depressed. Following the increase, mitochondrial zinc levels returned to physiological levels, while cytosolic zinc increased gradually over a 24 hr time period in viable cells. The increase in intramitochondrial zinc observed during reoxygenation after OGD may contribute to bioenergetic dysfunction and cell death that occurs with both in vitro and in vivo models of reperfusion.

Zinc and diabetes

“…Two main families of FRET-based Zn 2+ sensors have been developed, namely the Zap-sensors by the Palmer group [51], and the eCALWYsensors by the Merkx group and ourselves [52]. Free cytosolic zinc concentrations measured using these FRET-sensors were approximately in the same range as those measured with chemical probes or hybrid sensors, and it is generally agreed now that free zinc is around a few hundreds of picomolar [53][54][55]. Both FRET sensor families have been targeted to intracellular organelles such as the ER, the nucleus, the mitochondria or the Golgi apparatus by in-frame fusion with suitable targeting motifs [51,[56][57][58].…”

“…Both FRET sensor families have been targeted to intracellular organelles such as the ER, the nucleus, the mitochondria or the Golgi apparatus by in-frame fusion with suitable targeting motifs [51,[56][57][58]. Of note, concentrations measured in the ER and in the mitochondria varied greatly depending on the FRET or hybrid sensors used, discrepancies that still have to be explained [51,[55][56][57].…”

The coming of age of the mitochondria–ER contact: a matter of thickness

“…The diffusion laws and the principles discussed above are poised to guide the movement of any type of ion at the MERCs; for example, Zn 2+ , 41,42 which is uptaken by mitochondria to modulate ATP production. 43,44 Another ion that accumulates in the mitochondrial matrix is copper.…”

Reaction-based fluorescent sensor for investigating mobile Zn 2+ in mitochondria of healthy versus cancerous prostate cells

“…The capricious localization of fluorescein-based probes contrasts with protein-based zinc sensors, which offer programmable probe localization (17,18). Although protein-based (19) and peptide-based (20) targeting strategies have recently been used to direct the localization of fluorescein-based zinc sensors, controlling the subcellular accumulation of such probes remains a significant challenge.…”