Unraveling the Mode of Action of a Neuron‐Specific Fluorescent Probe, NeuO: Intracellular Phosphorylation Through PAK6 Kinase

Abstract NeuO is an intriguing molecular probe capable of selectively labeling live neurons within a heterogeneous population of brain cells. The precise mechanism responsible for this neuronal labeling has remained a longstanding enigma. We investigated four potential mechanisms underlying NeuO's fluorescence, including its interaction with neuronal proteins, transportation into neurons, export from glial cells, and structural alterations within neurons. Our study found that NeuO undergoes structural changes via phosphorylation by the PAK6 kinase, resulting in high fluorescence in neurons, likely due to fluorogenic activation and intracellular retention within neurons. This discovery improves our understanding of the molecular mechanism of NeuO in neurons and facilitates the development of new chemical compounds that can be selectively modified by kinases in living cells.