Fluorescence lifetime imaging of physiological free Cu(ii) levels in live cells with a Cu(ii)-selective carbonic anhydrase-based biosensor

Abstract

Copper is a required trace element that plays key roles in a number of human enzymes, such that copper deficiency or genetic defects in copper transport lead to serious or fatal disease. Rae, et al., had famously predicted that free copper ion levels in the cell cytoplasm were extremely low, typically too low to be observable. We recently developed a variant of human apocarbonic anhydrase II for sensing metal ions that exhibits 25-fold better selectivity for Cu(II) over Zn(II) than the wild type protein, enabling us to accurately measure Cu(II) in the presence of ordinary cellular (picomolar) concentrations of free zinc. We inserted a fluorescent labeled Cu(II)-specific variant of human apocarbonic anhydrase into PC-12 cells and found that the levels are indeed extremely low (in the femtomolar range). We imaged the free Cu(II) levels in living cells by means of frequency-domain fluorescence lifetime microscopy. Implications of this finding are discussed.

Fluorescence lifetime imaging (FLIM): Basic concepts and some recent developments

“…An Oregon Green-labelled apocarbonic anhydrase II Cu 2+ sensor has also recently been reported, where the Oregon Green fluorescence is quenched by FRET upon binding of Cu 2+ to the apocarbonic anhydrase II (McCranor et al, 2014). Furthermore, FRET-based K + and Na + probes are important for hypertension measurements in blood, and fluorescence lifetime measurements for this purpose have been reported (Szmacinski and Lakowicz, 1999), albeit without imaging.…”

The role of copper ions in pathophysiology and fluorescent sensors for the detection thereof

“…134 Using the interconnection between the copper and Ca 2+ signalling pathways described before, 126 the transient calcium concentration (using a calcium selective fluorescent probe) was monitored upon the addition of the copper chelator, revealing an increase in correlated neuronal activity in the cultured hippocampal neurons and an increase in excitability in developing retinal tissues. 136 Whereas hCa II normally is a metalloenzyme incorporating a Zn 2+ ion, genetic modification of the metal binding site resulted in a Cu 2+ specific ligand, with a K d (the concentration of Cu 2+ necessary to saturate half of the binding sites) as low as 0.1 pM. 135 As such, this might reveal one of the mechanisms underlying cognitive impairment in MD patients.…”

Sensing of Intracellular Environments by Fluorescence Lifetime Imaging of Exogenous Fluorophores

“…41 Oregon Green-labeled human carbonic anhydrase II (hCA II) was used as a Cu 2+ indicator at subpicomolar levels. 42 FRET between Oregon Green and hCA II occurs when Cu 2+ binds to hCA II, resulting in a decrease in the fluorescence lifetime of the Oregon Green moiety. 42 Excited state proton transfer (ESPT) of xanthene dyes, such as fluorescein, significantly occurs when ionic species suitable for the proton donor and acceptor exist in a medium with a relatively high concentration, which results in a decrease in the fluorescence lifetime with increasing concentration of the ionic species.…”

“…42 FRET between Oregon Green and hCA II occurs when Cu 2+ binds to hCA II, resulting in a decrease in the fluorescence lifetime of the Oregon Green moiety. 42 Excited state proton transfer (ESPT) of xanthene dyes, such as fluorescein, significantly occurs when ionic species suitable for the proton donor and acceptor exist in a medium with a relatively high concentration, which results in a decrease in the fluorescence lifetime with increasing concentration of the ionic species. 43,44 The decrease in the fluorescence lifetime of the xanthene derivative with the occurrence of ESPT was shown to be useful to measure the concentration of phosphate ions of more than 10 mM in cells.…”