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

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.

Nitric Oxide Sensors for Biological Applications

“…Ronald Malli and colleagues developed a system in which the NO responsive transcription factor of Escherichia coli (E. coli) is conjugated to fluorescent protein variants that are then taken up by cells [110][111][112]. When these fluorescent probes were tested with HeLa cells they were shown to be taken up and expressed at similar rates compared to other genetically encoded probes [110]. The NO probes were shown to respond to NO immediately with a reversible signal, allowing for long term NO dynamics investigations within cells in vitro [110].…”

“…When these fluorescent probes were tested with HeLa cells they were shown to be taken up and expressed at similar rates compared to other genetically encoded probes [110]. The NO probes were shown to respond to NO immediately with a reversible signal, allowing for long term NO dynamics investigations within cells in vitro [110]. Ongoing research efforts are looking at the possibility of utilizing this system to develop an in vivo model [110].…”

“…An interesting new way to detect NO within a single cell is through the addition of genetic materials that will code for a visual signal response in the presence of NO [110]. Ronald Malli and colleagues developed a system in which the NO responsive transcription factor of Escherichia coli (E. coli) is conjugated to fluorescent protein variants that are then taken up by cells [110][111][112].…”

“…The NO probes were shown to respond to NO immediately with a reversible signal, allowing for long term NO dynamics investigations within cells in vitro [110]. Ongoing research efforts are looking at the possibility of utilizing this system to develop an in vivo model [110]. One major obstacle that still prevents the utilization of the probes in vivo is that the fluorescence emission range that is currently being utilized is difficult to visualize at any depth within tissue.…”

Filling a GAP—An Optimized Probe for ER Ca 2+ Imaging In Vivo

“…However, a direct interaction of the analyte with amino acids forming the chromophore might be accomplished by positioning a respective binding domain that facilitates communication with the FP barrel ( Figure 1B). A similar principle has been recently suggested for a novel class of genetically encoded nitric oxide (NO) probes (Eroglu et al, 2016) that only consist of a bacteria-derived NO binding domain fused to certain FP variants. Binding of NO to this chimeric constructs induces a significant quenching of the FP fluorescence which allows NO imaging in real time.…”