GFP nanobodies reveal recently-exocytosed pHluorin molecules

Neurotransmitter release requires vesicle recycling, which consists of exocytosis, endocytosis and the reformation of new fusion-competent vesicles. One poorly understood aspect in this cycle is the fate of the vesicle proteins after exocytosis, when they are left on the plasma membrane. Such proteins are often visualized by coupling to pH-sensitive GFP moieties (pHluorins). However, pHluorin imaging is typically limited by diffraction to spots several-fold larger than the vesicles. Here we show that pHuorin-tagged vesicle proteins can be easily detected using single-domain antibodies (nanobodies) raised against GFP. By coupling the nanobodies to chemical fluorophores that were optimal for super-resolution imaging, we could analyze the size and intensity of the groups of pHluorin-tagged proteins under a variety of conditions, in a fashion that would have been impossible based solely on the pHluorin fluorescence. We conclude that nanobody-based pHluorin detection is a promising tool for investigating post-exocytosis events in neurons.