Frontiers in Cellular Neuroscience (Dec 2022)

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

  • Sven Dannhäuser,
  • Achmed Mrestani,
  • Achmed Mrestani,
  • Achmed Mrestani,
  • Florian Gundelach,
  • Florian Gundelach,
  • Martin Pauli,
  • Fabian Komma,
  • Philip Kollmannsberger,
  • Markus Sauer,
  • Manfred Heckmann,
  • Mila M. Paul,
  • Mila M. Paul

DOI
https://doi.org/10.3389/fncel.2022.1074304
Journal volume & issue
Vol. 16

Abstract

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IntroductionNeurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release.MethodsWe employ minos-mediated integration cassette (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.Results and discussionElectrophysiological 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 subclusters that move toward the AZ center during PHP with unaltered Unc-13GFSTF protein levels.

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