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.

In this issue of Cell Chemical Biology, Navas-Navarro et al. (2016) demonstrate that fusion of engineered derivatives of the long-known jellyfish proteins green fluorescent protein (GFP) and aequorin yield optimized genetically encoded fluorescent probes for detecting Ca(2+) signals within the endoplasmic reticulum (ER) of living animals.

Filling a GAP-An Optimized Probe for ER Ca(2+) Imaging In Vivo.

Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol.

Resveratrol Specifically Kills Cancer Cells by a Devastating Increase in the Ca2+ Coupling Between the Greatly Tethered Endoplasmic Reticulum and Mitochondria.

Recent studies revealed that mitochondrial Ca(2+) channels, which control energy flow, cell signalling and death, are macromolecular complexes that basically consist of the pore-forming mitochondrial Ca(2+) uniporter (MCU) protein, the essential MCU regulator (EMRE), and the mitochondrial Ca(2+) uptake 1 (MICU1). MICU1 is a regulatory subunit that shields mitochondria from Ca(2+) overload. Before the identification of these core elements, the novel uncoupling proteins 2 and 3 (UCP2/3) have been shown to be fundamental for mitochondrial Ca(2+) uptake. Here we clarify the molecular mechanism that determines the UCP2/3 dependency of mitochondrial Ca(2+) uptake. Our data demonstrate that mitochondrial Ca(2+) uptake is controlled by protein arginine methyl transferase 1 (PRMT1) that asymmetrically methylates MICU1, resulting in decreased Ca(2+) sensitivity. UCP2/3 normalize Ca(2+) sensitivity of methylated MICU1 and, thus, re-establish mitochondrial Ca(2+) uptake activity. These data provide novel insights in the complex regulation of the mitochondrial Ca(2+) uniporter by PRMT1 and UCP2/3.

PRMT1-mediated methylation of MICU1 determines the UCP2/3 dependency of mitochondrial Ca(2+) uptake in immortalized cells.

Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN), resulting in activation of soluble guanylate cyclase (sGC) and cGMP-mediated vasodilation. We have previously shown that a minor reaction of ALDH2-catalyzed GTN bioconversion, accounting for about 5% of the main clearance-based turnover yielding inorganic nitrite, results in direct NO formation and concluded that this minor pathway could provide the link between vascular GTN metabolism and activation of sGC. However, lack of detectable NO at therapeutically relevant GTN concentrations (≤1 μm) in vascular tissue called into question the biological significance of NO formation by purified ALDH2. We addressed this issue and used a novel, highly sensitive genetically encoded fluorescent NO probe (geNOp) to visualize intracellular NO formation at low GTN concentrations (≤1 μm) in cultured vascular smooth muscle cells (VSMC) expressing an ALDH2 mutant that reduces GTN to NO but lacks clearance-based GTN denitration activity. NO formation was compared with GTN-induced activation of sGC. The addition of 1 μm GTN to VSMC expressing either wild-type or C301S/C303S ALDH2 resulted in pronounced intracellular NO elevation, with maximal concentrations of 7 and 17 nm, respectively. Formation of GTN-derived NO correlated well with activation of purified sGC in VSMC lysates and cGMP accumulation in intact porcine aortic endothelial cells infected with wild-type or mutant ALDH2. Formation of NO and cGMP accumulation were inhibited by ALDH inhibitors chloral hydrate and daidzin. The present study demonstrates that ALDH2-catalyzed NO formation is necessary and sufficient for GTN bioactivation in VSMC.

Formation of Nitric Oxide by Aldehyde Dehydrogenase-2 Is Necessary and Sufficient for Vascular Bioactivation of Nitroglycerin.

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.

Emrah Eroglu

Institute of Molecular Biology and Biochemistry

Medical University of Graz

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