Applications of fluorescent probes to the study of small molecule cellular signaling and ion homeostasis in biological systems

Living organisms have evolved to harness energy from controlled chemical processes. For aerobic organisms, primary energy production requires molecular oxygen, which undergoes a series of transformations in the electron transport chain. The byproducts and reactive intermediates of these reactions, such as superoxide and hydrogen peroxide (H2O2), are potential sources of oxidative stress. The cell has therefore evolved a variety of mechanisms to manage the production of these reactive species, including enzymes to convert reactive oxygen species to more benign chemicals and the generation of reducing species to buffer against abrupt redox changes. This intricate balance of reducing and oxidizing species in biological systems is of great interest, as under- or overproduction of reactive species is observed in many disease states. Thus, tools to sense these chemicals in intact biological models would offer powerful new approaches to studying how these reactive small molecules affect cellular function./nHydrogen sulfide (H2S), a reactive sulfur species that has gained attention for its roles as an antioxidant and potential gasotransmitter, has previously been measured using methods such as gas chromatography, colorimetric assays, and microelectrodes. While these techniques have contributed greatly to our understanding of the effects of H2S in physiology and disease, the development of the first fluorescent probes that could detect H2S in intact, living cells offered new approaches to studying H2S biology. Following the first series of H2S probes, preparation of second generation probes with enhanced sensitivity and cellular retention has expanded the application scope for these indicator molecules. One of these probes, Sulfidefluor-7 acetoxymethyl ester (SF7-AM), was used to detect H2S production in human umbilical vein endothelial cells upon stimulation with vascular endothelial growth factor (VEGF). Furthermore, SF7-AM could be used in live cells to monitor H2S production in real-time, allowing for the H2S signaling pathway to be probed using a variety of inhibitors. Imaging results using SF7-AM confirmed that H2O2 plays a role in maintaining stimulation of the VEGF receptor upstream of H2S production. This first example of monitoring endogenously produced H2S provides a launching point for future investigations into the roles of this multifaceted reactive species./nWhile SF7-AM was tailored for applications in monolayer cultured cells, we sought to explore different molecular scaffolds with properties more suitable for tissue applications. Examining reactive species in tissue systems requires probes with superior permeability and the ability to penetrate multiple layers of cells. In collaboration with the Gong laboratory, we observed that mice lacking a specific isoform of NADPH oxidase, Nox2, displays mild punctate nuclear cataracts in their lenses. The interior of the lens cannot be examined readily using most analytical techniques without disruption of the delicate tissue structure, making fluorescent probes an appealing approach to studying reactive species in the lens. As cataracts are frequently associated with oxidative stress, we developed a highly permeable fluorescent H2O2 probe based on the hydroxymethyl diethylrhodol scaffold (HMDER) for use in the lens. This probe, Peroxy Yellow-1 Spiro Ether (PY1-SE), displays low background, excellent permeability in tissues, and a 10-fold turn-on response in the presence of 100 µM H2O2 in vitro. PY1-SE responded to 100 µM exogenous H2O2 in cultured lens epithelial cells, and could also be applied to whole, freshly isolated mouse lenses. Fluorescence imaging of live lenses showed that Nox2(-/-) mouse lenses had significantly lower amounts of H2O2 compared to wild type (WT) lenses. Further study is needed to determine the origins of the Nox2(-/-) nuclear cataracts, which present at an early age and do not increase in severity over time. These initial findings confirm that loss of Nox2 results in decreased levels of H2O2 in the lens. Thus, the absence of an ROS-producing enzyme interestingly gives rise in a mild cataract phenotype, suggesting that H2O2 or another ROS produced by Nox2 may play a role in early lens development./nThis dissertation concludes with preliminary work in a new direction for our laboratory, based on the development of fluorescent sodium sensors. Building off our previous investigation of H2O2 levels in the lens using fluorescent probes, we sought to apply new fluorescent sodium sensors to the mammalian lens. The fluid circulation model, which has been proposed as an important means of transport in the lens, is thought to be driven by intracellular sodium currents. The mechanisms by which the lens is able to sustain itself despite minimal metabolic activity in the majority of the tissue are not fully understood. A fluorescent sodium sensor based on the 1,7-diaza-15-crown-5 sodium-binding receptor with a Tokyo Green fluorophore was prepared. This sensor, Tokyo Green Sodium (TG-Sodium), features an asymmetric design which reduces the molecular size of the probe and allows for bath-loading of the dye into living cells without the use of detergents or cell permeabilization methods. Sodium titration experiments showed that TG-Sodium responds to changes in sodium concentration when loaded into HEK293T, a model cell line. While TG-Sodium was found to be unsuitable for live tissue applications, we developed a straightforward plate reader assay using this probe to quantify the bulk sodium content of lens tissue samples. Future advances in sodium sensor development will offer opportunities to measure the distribution of sodium in ex vivo lenses and elucidate the roles of sodium transport in the movement of other ions, nutrients, and small molecules in this complex tissue./nThis body of work presents the application of three different types of fluorescent sensing molecules to a number of biological systems, with an emphasis on methods optimization in mammalian cells and special attention to the tailoring of probe design for the application of interest. As the available selection of probes grows in both number and diversity, imaging methods and other fluorescence-based assays undergo continuous modification for improved robustness and generality. These findings provide deeper insight into the relationship between the molecular structures of these probes and their performance in different cellular systems, facilitating further advances in probe design as we continue to pursue complex questions in chemical biology.

Advisor: Chang, Christopher J