TY  - JOUR
A1  - Lichter, Katharina
A1  - Paul, Mila Marie
A1  - Pauli, Martin
A1  - Schoch, Susanne
A1  - Kollmannsberger, Philip
A1  - Stigloher, Christian
A1  - Heckmann, Manfred
A1  - Sirén, Anna-Leena
T1  - Ultrastructural analysis of wild-type and RIM1α knockout active zones in a large cortical synapse
JF  - Cell Reports
N2  - Rab3A-interacting molecule (RIM) is crucial for fast Ca\(^{2+}\)-triggered synaptic vesicle (SV) release in presynaptic active zones (AZs). We investigated hippocampal giant mossy fiber bouton (MFB) AZ architecture in 3D using electron tomography of rapid cryo-immobilized acute brain slices in RIM1α\(^{−/−}\) and wild-type mice. In RIM1α\(^{−/−}\), AZs are larger with increased synaptic cleft widths and a 3-fold reduced number of tightly docked SVs (0–2 nm). The distance of tightly docked SVs to the AZ center is increased from 110 to 195 nm, and the width of their electron-dense material between outer SV membrane and AZ membrane is reduced. Furthermore, the SV pool in RIM1α\(^{−/−}\) is more heterogeneous. Thus, RIM1α, besides its role in tight SV docking, is crucial for synaptic architecture and vesicle pool organization in MFBs.
KW  - active zone
KW  - acute brain slices
KW  - CA3
KW  - electron tomography
KW  - high-pressure freezing
KW  - hippocampal mossy fiber bouton
KW  - RIM1α
KW  - SV pool
KW  - synaptic ultrastructure
KW  - presynaptic
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300913
VL  - 40
IS  - 12
ER  - 
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  -