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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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
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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. |
format | Online Article Text |
id | pubmed-9797049 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
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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