Research Projects

Recent articles demonstrated the presence and important biological roles of labile copper pool in organelles of living mammalian cells. Solier and coworkers established that mitochondrial labile copper ions in inflammatory macrophages catalyze NAD(H) redox cycling by activating hydrogen peroxide. In another report, Cui and coworkers showed that copper chelators when targeted to mitochondria inhibited growth of triple negative breast cancer cells and increased the survival rates in mice. These reports indicate mapping mitochondrial copper concentrations and studying its transporters are crucial and will facilitate the development of new therapeutic avenues. Though many qualitative fluorescent probes for copper exist, reversible ratiometric fluorescent probes are extremely rare. Ratiometric probes provide absolute concentrations of analytes which is independent of the probe concentration and therefore corrects for any inhomogeneous probe distribution. This makes them more reliable than qualitative probes. Also, most of these existing probes are not targeted specifically to any particular organelle and provide copper concentrations of the entire cell. In this proposal, I will develop organelle-targeted fluorescent ratiometric probes for copper which will provide quantitative visual readout in living cells in real time. My probes will be based on small molecule scaffolds as they offer more versatility in chemical design and modularity than genetically encoded sensors. Ratiometric probes will be developed by chemically conjugating existing or new qualitative copper probes with a standard fluorophore (the latter being used for normalization). The normalizing dye will be robust, bright, non-toxic and unresponsive to any biological ions or analytes. I will introduce organelle targeting groups that will enhance accumulation of the probe in desired organelles. Next, I will map organellar copper concentrations in living cells using confocal microscopy. Finally, I will investigate the biological roles of organellar copper in healthy and patient-derived cells. In short, I will map the absolute copper concentrations in organelles of living cells at single organelle resolution for the first time. The immediate results of this proposal will be utilized to understand new biological pathways and regulation of intracellular copper. Identification of organellar copper regulators and development of high throughput screening platforms for diagnosis of copper-related diseases will be possible. In the long term, the inventions of this proposal will be commercialized.