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bioRxiv · 10.1101/2022.08.31.506141

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

Dannhäuser, S.·Mrestani, A.·Gundelach, F.·Pauli, M.·Komma, F.·Kollmannsberger, P.·Sauer, M.·Heckmann, M.·Paul, M.
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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-13GFSTF 3rd 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-13GFSTF, Bruchpilot and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13GFSTF nanoclusters that move towards the AZ center during PHP with unaltered Unc-13GFSTF protein levels.

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Dannhäuser, S., Mrestani, A., Gundelach, F., Pauli, M., Komma, F., Kollmannsberger, P., Sauer, M., Heckmann, M., Paul, M.. 2022-09-03. Endogenous tagging of Unc-13 reveals nanocluster reorganization at active zones during presynaptic homeostatic potentiation. https://doi.org/10.1101/2022.08.31.506141

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