Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Eroglu, E.
Shining a Light on Cellular Nitric Oxide Signals
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Graz Medical University; 2016. pp.
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- Autor*innen der Med Uni Graz:
- EROGLU Emrah
- Betreuer*innen:
- Malli Roland
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- Abstract:
- Nitric oxide (NO) is a small radical which mediates multiple important cellular functions in mammals, bacteria, and plants. Despite the existence of a huge number of methods for de- tecting NO in vivo and in vitro, it turned out that real-time monitoring of NO at the single-cell level is very challenging. Recently, we expanded the palette of NO indicators by introducing fluorescent protein-based NO-probes, the geNOps that directly respond to cellular NO fluc- tuations and hence, open a new era of NO bioimaging. The aim of this thesis was to further develop, characterize, and apply geNOps in order to grow our current knowledge about the complex formation, diffusion, and degradation of one of nature´s most versatile signaling molecules. For this purpose a cell line stably expressing the green G-geNOp was developed and successfully used to test the NO releasing capability of different NO donors. Moreover, targeting of the geNOps into the mitochondrial lumen enabled the visualization of local NO signals within this organelle. Performing multichannel imaging experiments unveiled that NO rises simultaneously within the mitochondria and the cytosol upon Ca 2+ mobilization in en- dothelial cells. Furthermore, the geNOps technology was applied in HEK cells stably express- ing nNOS, eNOS or iNOS to examine the enzymatic generation of NO by different NOS isoforms on the single cell level. Our data clearly demonstrate that the Ca 2+ -dependent NO signals in eNOS and nNOS expressing HEK cells significantly differ in terms of their amplitude and kinetics. Interestingly, nNOS-derived NO signals occurred within seconds upon cell treatment with a Ca 2+ mobilizing agonist, while NO formation in eNOS expressing cells was much slower. To visualize NO signals in iNOS expressing HEK cells a novel double fluorescent protein-based ratiometric geNOp, referred to as CR-geNOp, was developed. The CR-geNOp was suitable to detect high basal NO levels in iNOS expressing HEK cells and track the strict dependency of NO generation from arginine. These experiments demonstrate that geNOps are proper tools to visualize the generation and degradation of single cell NO signals in real- time in diverse cellular model systems and experimental setups.