TY  - JOUR
A1  - Dannhäuser, Sven
A1  - Mrestani, Achmed
A1  - Gundelach, Florian
A1  - Pauli, Martin
A1  - Komma, Fabian
A1  - Kollmannsberger, Philip
A1  - Sauer, Markus
A1  - Heckmann, Manfred
A1  - Paul, Mila M.
T1  - Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation
JF  - Frontiers in Cellular Neuroscience
N2  - 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.
KW  - active zone
KW  - Unc-13
KW  - MiMIC
KW  - presynaptic homeostasis
KW  - nanoarchitecture
KW  - localization microscopy
KW  - STORM
KW  - HDBSCAN
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-299440
SN  - 1662-5102
VL  - 16
ER  -