PUPIL enables mapping and stamping of transient electrical connectivity in developing nervous systems

• Published in: Cell reports (Cambridge) 2021-10, Vol.37 (3), p.109853-109853, Article 109853
• Main Authors: Xie, Shu, Li, Haixiang, Yao, Fenyong, Huang, Jiechang, Yang, Xiaomei, Chen, Xin, Liu, Qiang, Zhuang, Min, He, Shuijin
• Format: Article
• Language: English
• Subjects: Age Factors; Alkaline Phosphatase - genetics; Alkaline Phosphatase - metabolism; Animals; Animals, Newborn; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Cerebral Cortex - growth & development; Cerebral Cortex - metabolism; Cerebral Cortex - ultrastructure; Connexin 26 - genetics; Connexin 26 - metabolism; Connexins - genetics; Connexins - metabolism; Electric Conductivity; Electrical Synapses - metabolism; Electrical Synapses - physiology; Electrical Synapses - ultrastructure; Female; Gap Junction delta-2 Protein; Gap Junctions - metabolism; Gap Junctions - physiology; Gap Junctions - ultrastructure; Gestational Age; HEK293 Cells; HeLa Cells; Humans; Mice; Mice, Inbred ICR; Mice, Knockout; Microscopy, Confocal; Neurons - metabolism; Neurons - physiology; Neurons - ultrastructure; Optogenetics; PafA; Pregnancy; PUPIL; pupylation; stamping; Synaptic Potentials; transient electrical synapses
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2021.109853

Currently, many genetic methods are available for mapping chemical connectivity, but analogous methods for electrical synapses are lacking. Here, we present pupylation-based interaction labeling (PUPIL), a genetically encoded system for noninvasively mapping and stamping transient electrical synapses in the mouse brain. Upon fusion of connexin 26 (CX26) with the ligase PafA, pupylation yields tag puncta following conjugation of its substrate, a biotin- or fluorescent-protein-tagged PupE, to the neighboring proteins of electrical synapses containing CX26-PafA. Tag puncta are validated to correlate well with functional electrical synapses in immature neurons. Furthermore, puncta are retained in mature neurons when electrical synapses mostly disappear—suggesting successful stamping. We use PUPIL to uncover spatial subcellular localizations of electrical synapses and approach their physiological functions during development. Thus, PUPIL is a powerful tool for probing electrical connectivity patterns in complex nervous systems and has great potential for transient receptors and ion channels as well. [Display omitted] •Application of a genetically encoded pupylation system for proximity labeling•PUPIL reveals subcellular localization of CX26-containing gap junctions•Proximity labeling stamps neonatal CX26-containing gap junctions in adult neurons•PUPIL is a useful tool for imaging of gap junctions in living nervous tissues Xie et al. apply a bacterial pupylation system to proximity label electrical and chemical synapses in the mouse cortex by fusing the ligase PafA with CX26/36 and with gephyrin, respectively. Proximity labeling enables stamping of transient electrical synapses, demonstrating utility for decoding physiological functions in the developing nervous system.