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

Dannhäuser, Sven and Mrestani, Achmed and Gundelach, Florian and Pauli, Martin and Komma, Fabian and Kollmannsberger, Philip and Sauer, Markus and Heckmann, Manfred and Paul, Mila M. (2022) Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation. Frontiers in Cellular Neuroscience, 16. ISSN 1662-5102

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Abstract

Introduction: 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 Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release.

Methods: We 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 discussion: 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 subclusters that move toward the AZ center during PHP with unaltered Unc-13GFSTF protein levels.

Item Type: Article
Subjects: West Bengal Archive > Medical Science
Depositing User: Unnamed user with email support@westbengalarchive.com
Date Deposited: 23 Mar 2023 07:14
Last Modified: 04 Sep 2024 04:15
URI: http://article.stmacademicwriting.com/id/eprint/341

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