Abstract
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 Ca2+ sensor allowed us to visualize and Ca2+ 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.
Highlights
Nitric oxide (NO) is a free radical with a wide range of biological effects, but practically impossible to visualize in single cells
We assumed that specific NO binding close to fluorescent protein (FP) in such constructs considerably influences the fluorescence signal by affecting the electron density within certain amino acids forming the chromophore. We demonstrate that such fluorescent chimeras, referred to as genetically encoded NO probes (geNOps), represent a completely novel class of NO indicators that allow direct imaging ofcellular NO dynamics in real time
These results proved that fusion of the bacterial NO-binding GAF domain to FP variants results in cyan geNOp (C-geNOp), M-geNOp, green geNOp (G-geNOp), Y-geNOp and O-geNOp (Fig. 1b), allowing imaging of cellular NO dynamics in real time and in a multichromatic manner
Summary
Nitric oxide (NO) is a free radical with a wide range of biological effects, but practically impossible to visualize in single cells. We report the development of novel multicoloured fluorescent quenching-based NO probes by fusing a bacteria-derived NO-binding domain close to distinct fluorescent protein variants. These genetically encoded NO probes, referred to as geNOps, provide a selective, specific and real-time read-out of cellular NO dynamics and, open a new era of NO bioimaging. The combination of geNOps with a Ca2 þ sensor allowed us to visualize NO and Ca2 þ signals simultaneously in single endothelial cells. We demonstrate that such fluorescent chimeras, referred to as geNOps, represent a completely novel class of NO indicators that allow direct imaging of (sub)cellular NO dynamics in real time
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