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

Abstract

Nitric oxide (NO.) 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 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, hence, open a new era of NO. bioimaging. The combination of geNOps with a Ca2+ sensor allowed us to visualize NO. and Ca2+ signals simultaneously in single endothelial cells. Moreover, targeting of the NO. probes was used to detect NO. signals within mitochondria. The geNOps are useful new tools to further investigate and understand the complex patterns of NO. signalling on the single (sub)cellular level.