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

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

doi:10.3389/fncel.2022.1074304

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

Front Cell Neurosci. 2022 Dec 14:16:1074304. doi: 10.3389/fncel.2022.1074304. eCollection 2022.

Authors

Sven Dannhäuser¹, Achmed Mrestani^{1

2

3}, Florian Gundelach^{1

4}, Martin Pauli¹, Fabian Komma¹, Philip Kollmannsberger⁵, Markus Sauer⁶, Manfred Heckmann¹, Mila M Paul^{1

7}

Affiliations

¹ Department of Neurophysiology, Institute of Physiology, University of Würzburg, Würzburg, Germany.
² Department of Neurology, Leipzig University Medical Center, Leipzig, Germany.
³ Division of General Biochemistry, Rudolf Schönheimer Institute of Biochemistry, Medical Faculty, Leipzig University, Leipzig, Germany.
⁴ Center of Mental Health, Department of Psychiatry, Psychotherapy, and Psychosomatics, University Hospital of Würzburg, Würzburg, Germany.
⁵ Center for Computational and Theoretical Biology, Julius-Maximilians-Universität Würzburg, Würzburg, Germany.
⁶ Department of Biotechnology and Biophysics, Julius-Maximilians-Universität Würzburg, Würzburg, Germany.
⁷ Department of Orthopedic Trauma, Hand, Plastic and Reconstructive Surgery, University Hospital of Würzburg, Würzburg, Germany.

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-13 ^GFSTF 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^GFSTF, Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13^GFSTF subclusters that move toward the AZ center during PHP with unaltered Unc-13^GFSTF protein levels.

Keywords: HDBSCAN; MiMIC; Unc-13; active zone; dSTORM; localization microscopy; nanoarchitecture; presynaptic homeostasis.