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

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

JOURNAL=Frontiers in Cellular Neuroscience

VOLUME=16

YEAR=2022

URL=https://www.frontiersin.org/journals/cellular-neuroscience/articles/10.3389/fncel.2022.1074304

DOI=10.3389/fncel.2022.1074304

ISSN=1662-5102

ABSTRACT=<sec><title>Introduction</title><p>Neurotransmitter 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 <italic>Drosophila melanogaster</italic> is essential for docking of synaptic vesicles and transmitter release.</p></sec><sec><title>Methods</title><p>We employ minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated <italic>Drosophila</italic> Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system.</p></sec><sec><title>Results and discussion</title><p>Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in <italic>unc-13<italic><sup>GFSTF</sup></italic></italic> 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-13<sup>GFSTF</sup>, Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13<sup>GFSTF</sup> subclusters that move toward the AZ center during PHP with unaltered Unc-13<sup>GFSTF</sup> protein levels.</p></sec>