Fluorescent strategies to study the labile copper pool

Abstract

Copper plays a critical role in the structure and function of essential proteins. These proteins must draw copper from a labile and rapidly exchanging copper pool, which is able to change dynamically in response to cellular stresses or stimuli. When mismanaged, this pool can compromise the health and correct functioning of the cell. There is evidence that misregulation of the bioavailable copper pool is linked to neurodegenerative diseases and cancer, and recent studies have uncovered potential roles for copper as a signalling agent. This work has involved the strategic development of diverse sensing systems to image bioavailable copper in cells. First, the development of a ratiometric fluorescent copper sensor InCCu1 has enabled the selective imaging of mitochondrial copper pools. InCCu1 takes advantage of a dual fluorophore system, where one fluorophore is electronically conjugated to the receptor and therefore responsive to the concentration of copper, while the other provides a constant emission peak to internally standardise the probe's response. This probe was utilised in investigations of the relationship between cisplatin and copper homeostasis, as well as in multicellular organisms. The majority of fluorescent probes use fluorescence emission intensity or wavelength as the monitored parameter. Fluorescence lifetime offers another parameter that can be studied, but there are no reported lifetime-based Cu(I) probes. Here, CB-NS4, the first novel sensor for Cu(I) that exhibits changes in fluorescence lifetime, is reported. Interestingly, the probe displayed dual behaviour; its fluorescence lifetime was highly sensitive to the presence of Cu(I) but not Cu(II), while the reverse was observed in the emission parameter. Changes in copper levels in cells could be monitored using CB-NS4 by fluorescence lifetime imaging microscopy (FLIM). Finally, the development of reaction-based sensors for Cu(I) was explored. Reaction-based probes provide complementary information to previously reported probes, and may give insight about short-term fluctuations in metal concentration. To this end, ACCu1 was developed and its kinetics characterised. ACCu1 contains a trigger group masking a coumarin fluorophore, rendering it nearly non-fluorescent. Upon selective reaction with Cu(I), the trigger group is cleaved, releasing a highly fluorescent form of coumarin. While developing fluorescent sensors for species such as copper is challenging, this work demonstrates the utility of a number of distinct approaches, each of which can give rise to diverse probes with complementary properties and advantages. Importantly, this work has led to probes that respond in different parameters of detection, and novel sensors have been successfully applied to biological studies. This work highlights the importance of fluorescent sensors to enriching our understanding of the roles of copper in biology.