Endogenous tagging of Unc-13 reveals nanocluster reorganization at active zones during presynaptic homeostatic potentiation

Abstract

Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein mesh- work the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release. We employ MiMIC-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV- 3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system. Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13 GFSTF 3 rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13 GFSTF , Bruchpilot and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13 GFSTF nanoclusters that move towards the AZ center during PHP with unaltered Unc-13 GFSTF protein levels.

Competing Interest Statement

The authors have declared no competing interest.