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

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 trans...

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Autores principales: Dannhäuser, Sven, Mrestani, Achmed, Gundelach, Florian, Pauli, Martin, Komma, Fabian, Kollmannsberger, Philip, Sauer, Markus, Heckmann, Manfred, Paul, Mila M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9797049/
https://www.ncbi.nlm.nih.gov/pubmed/36589286
http://dx.doi.org/10.3389/fncel.2022.1074304
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author Dannhäuser, Sven
Mrestani, Achmed
Gundelach, Florian
Pauli, Martin
Komma, Fabian
Kollmannsberger, Philip
Sauer, Markus
Heckmann, Manfred
Paul, Mila M.
author_facet Dannhäuser, Sven
Mrestani, Achmed
Gundelach, Florian
Pauli, Martin
Komma, Fabian
Kollmannsberger, Philip
Sauer, Markus
Heckmann, Manfred
Paul, Mila M.
author_sort Dannhäuser, Sven
collection PubMed
description 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.
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spelling pubmed-97970492022-12-29 Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation Dannhäuser, Sven Mrestani, Achmed Gundelach, Florian Pauli, Martin Komma, Fabian Kollmannsberger, Philip Sauer, Markus Heckmann, Manfred Paul, Mila M. Front Cell Neurosci Cellular Neuroscience 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. Frontiers Media S.A. 2022-12-14 /pmc/articles/PMC9797049/ /pubmed/36589286 http://dx.doi.org/10.3389/fncel.2022.1074304 Text en Copyright © 2022 Dannhäuser, Mrestani, Gundelach, Pauli, Komma, Kollmannsberger, Sauer, Heckmann and Paul. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cellular Neuroscience
Dannhäuser, Sven
Mrestani, Achmed
Gundelach, Florian
Pauli, Martin
Komma, Fabian
Kollmannsberger, Philip
Sauer, Markus
Heckmann, Manfred
Paul, Mila M.
Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation
title Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation
title_full Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation
title_fullStr Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation
title_full_unstemmed Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation
title_short Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation
title_sort endogenous tagging of unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation
topic Cellular Neuroscience
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9797049/
https://www.ncbi.nlm.nih.gov/pubmed/36589286
http://dx.doi.org/10.3389/fncel.2022.1074304
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