Understanding of selenium utilization and redox homeostasis through imaging and high-throughput approaches

Imaging and high-throughput approaches provide opportunities to obtain systems level understanding of biological processes and identify their new components. This is also true for redox regulation and metal homeostasis, which are not easily amenable to such approaches. In this work, we addressed these questions in several ways. First, we analyzed metabolism of hydrogen peroxide in mammalian cells. We used a hydrogen peroxide sensor HyPer as a basis to develop probes targeted to various cellular compartments. Using these sensors, we observed HyPer in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix. In contrast, HyPer was mostly oxidized in the endoplasmic reticulum. Using these biosensors, we characterized timing and control of hydrogen peroxide in various cell systems. These data provide information on compartmentalization, dynamics and homeostatic control of hydrogen peroxide in mammalian cells. Second, we used X-ray fluorescence microscopy to map the distribution of trace elements in various tissues of mice and naked mole rats, focusing on selenium in the liver, kidney and testes of mice. In mouse testes, selenium was enriched in elongating spermatids. This selenium was provided by Selenoprotein P specifically for the mitochondrial form of glutathione peroxidase 4 and was then preserved in the mature sperm. In mouse kidney, selenium was present in two pools. Highly localized Se pool on the proximal basement membranes was not dependent on Selenoprotein P expression and was identified as component of glutathione peroxidase 3. Overall, XFM allowed visualization of selenium in mammalian tissues at submicron resolution and provided important information on the role and distribution of selenium in these organisms. Finally, we developed a sensitive high-throughput approach to profile trace elements in mammalian cells grown in 96-well plates. Twenty one thousand human gene knockdowns in HeLa cells were analyzed for changes in trace elements using inductively-coupled plasma mass spectrometry. This approach identified known genes and transporters, and provided a dataset of candidate genes involved in regulation of trace elements. These findings, approaches and tools should lead to many avenues of investigation to understand redox regulation and metabolism of trace elements.