application-genetically-encoded-fluorescent-nitric-oxide-no-probes

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Medical University of Graz

Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store.

Generation of Red-Shifted Cameleons for Imaging Ca²⁺ Dynamics of the Endoplasmic Reticulum.

Nitric oxide () is a free radical with a wide range of biological effects, but practically impossible to visualize in single cells. Here we report the development of novel multicoloured fluorescent quenching-based probes by fusing a bacteria-derived -binding domain close to distinct fluorescent protein variants. These genetically encoded probes, referred to as geNOps, provide a selective, specific and real-time read-out of cellular dynamics and, hence, open a new era of bioimaging. The combination of geNOps with a Ca(2+) sensor allowed us to visualize and Ca(2+) signals simultaneously in single endothelial cells. Moreover, targeting of the probes was used to detect signals within mitochondria. The geNOps are useful new tools to further investigate and understand the complex patterns of signalling on the single (sub)cellular level.

Development of novel FP-based probes for live-cell imaging of nitric oxide dynamics.

Mitochondrial Ca(2+) uptake regulates diverse endothelial cell functions and has also been related to nitric oxide (NO(•)) production. However, it is not entirely clear if the organelles support or counteract NO(•) biosynthesis by taking up Ca(2+). The objective of this study was to verify whether or not mitochondrial Ca(2+) uptake influences Ca(2+)-triggered NO(•) generation by endothelial NO(•) synthase (eNOS) in an immortalized endothelial cell line (EA.hy926), respective primary human umbilical vein endothelial cells (HUVECs) and eNOS-RFP (red fluorescent protein) expressing human embryonic kidney (HEK293) cells. We used novel genetically encoded fluorescent NO(•) probes, the geNOps, and Ca(2+) sensors to monitor single cell NO(•) and Ca(2+) dynamics upon cell treatment with ATP, an inositol 1,4,5-trisphosphate (IP3)-generating agonist. Mitochondrial Ca(2+) uptake was specifically manipulated by siRNA-mediated knock-down of recently identified key components of the mitochondrial Ca(2+) uniporter machinery. In endothelial cells and the eNOS-RFP expressing HEK293 cells we show that reduced mitochondrial Ca(2+) uptake upon the knock-down of the mitochondrial calcium uniporter (MCU) protein and the essential MCU regulator (EMRE) yield considerable attenuation of the Ca(2+)-triggered NO(•) increase independently of global cytosolic Ca(2+) signals. The knock-down of mitochondrial calcium uptake 1 (MICU1), a gatekeeper of the MCU, increased both mitochondrial Ca(2+) sequestration and Ca(2+)-induced NO(•) signals. The positive correlation between mitochondrial Ca(2+) elevation and NO(•) production was independent of eNOS phosphorylation at serine(1177). Our findings emphasize that manipulating mitochondrial Ca(2+) uptake may represent a novel strategy to control eNOS-mediated NO(•) production.

Article	Year
Generation of Red-Shifted Cameleons for Imaging Ca²⁺ Dynamics of the Endoplasmic Reticulum. Sensors (Basel, Switzerland)\| PubMed ID: 26053751	2015
Development of novel FP-based probes for live-cell imaging of nitric oxide dynamics. Nature communications\| PubMed ID: 26842907	2016
Intact mitochondrial Ca(2+) uniport is essential for agonist-induced activation of endothelial nitric oxide synthase (eNOS). Free radical biology & medicine\| PubMed ID: 27923677	2017

Helmut Bischof

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