Protein proximity ligation probe with wide sensing range resolving dynamics of subcellular ph by super-resolution imaging

Abstract

Subcellular pH plays a key role to modulate protein structures or specific active sites to impact diverse biological processes. The heterogeneity and high dynamics of intracellular pH require probes that can detect pH in the vicinity of the protein and have a wide pH response range. Here, we reported a protein proximity ligation fluorescent probe for super-resolution imaging of subcellular pH dynamics. Relying on SNAP-tag protein labeling technology, the pH-sensitive fluorophore BG-NDM based on photo-induced electron transfer mechanism was localized to the adjacent positions of the target protein in different subcellular organelles. The equilibrium between the non-fluorescent aggregate and the fluorogenic monomer enabled BG-NDM to have the ability of wash-free super-resolution imaging in live cells. And the wide pH response ranges from pH 6 to pH 12 made the probe have the ability of recognizing pH dynamics. The pH changes around functional proteins during mitophagy, cellular alkalosis and acidosis were revealed. Through long-term super-resolution imaging, we could further record the pH dynamics around mitochondrial proteins COX8A during mitochondrial contacts, fusion and fission at the resolution of a single mitochondria.