Development and Applications of New Chemical Tools for Studying the Cell Biology of Metals

Cells have evolved intricate mechanisms that coordinate the activity of transporters, chaperones, and small-molecule ligands to control the spatial and temporal positioning of metal ions. Alkali and alkaline earth metal ions are known to play essential roles in cellular signaling and charge balance whereas transition metals are typically found in the active sites of enzymes to carry out organic transformations. In order to carry out these functions the cell maintains static and labile metal ion pools. These pools can be characterized by bulk molecular biology and bioinorganic techniques on fixed samples in combination with fluorescent indicators that afford the ability to track metal ions in real-time through molecular imaging. In this regard, cellular calcium has been well characterized in variety of biological contexts, but methods to monitor biologically relevant transition metal ions in real-time have not been well established. This dissertation describes the synthesis, characterization, and applications of new fluorescent sensors for Ni(II) and Cu(I), as well as approaches to discover new roles of biological copper in cell signaling. Nickelsensor-1 is a new water-soluble, turn-on fluorescent sensor that is capable of selectively responding to Ni(II) in aqueous solution and in living cells. Coppersensor-3 and X-ray fluorescence microscopy reveal that neuronal cells mobilize significant pools of copper from their cell bodies to peripheral processes in a calcium dependent fashion upon depolarization with KCl. Mitochondrial Coppersensor-1 (Mito-CS1) is a bifunctional reporter that combines a Cu(I)-responsive fluorescent platform with a mitochondrial-targeting triphenylphosphonium moiety for the reversible detection of endogenous, exchangeable mitochondrial Cu(I) pools. Mito-CS1 in conjunction with ICP metal analyses, show that both the exchangeable Cu(I) and total mitochondrial copper pools are only mildly perturbed in fibroblasts with mutant SCO1 and SCO2 mitochondrial copper chaperones. Molecular imaging of neural calcium transients reveals that that endogenous copper is used to tune inhibitory and excitatory inputs during neural circuit development through the dynamic functions of CTR1. Finally, methods for high-throughput RNAi screening and immunofluorescence have been optimized to understand how the kinome regulates the copper mediated mobilization of ATP7A in mammalian cells