Cargando…

Fluorescence Lifetime Imaging Microscopy reveals rerouting of SNARE trafficking driving dendritic cell activation

SNARE proteins play a crucial role in intracellular trafficking by catalyzing membrane fusion, but assigning SNAREs to specific intracellular transport routes is challenging with current techniques. We developed a novel Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET...

Descripción completa

Detalles Bibliográficos
Autores principales: Verboogen, Daniëlle Rianne José, González Mancha, Natalia, ter Beest, Martin, van den Bogaart, Geert
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473687/
https://www.ncbi.nlm.nih.gov/pubmed/28524818
http://dx.doi.org/10.7554/eLife.23525
_version_ 1783244327005192192
author Verboogen, Daniëlle Rianne José
González Mancha, Natalia
ter Beest, Martin
van den Bogaart, Geert
author_facet Verboogen, Daniëlle Rianne José
González Mancha, Natalia
ter Beest, Martin
van den Bogaart, Geert
author_sort Verboogen, Daniëlle Rianne José
collection PubMed
description SNARE proteins play a crucial role in intracellular trafficking by catalyzing membrane fusion, but assigning SNAREs to specific intracellular transport routes is challenging with current techniques. We developed a novel Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM)-based technique allowing visualization of real-time local interactions of fluorescently tagged SNARE proteins in live cells. We used FRET-FLIM to delineate the trafficking steps underlying the release of the inflammatory cytokine interleukin-6 (IL-6) from human blood-derived dendritic cells. We found that activation of dendritic cells by bacterial lipopolysaccharide leads to increased FRET of fluorescently labeled syntaxin 4 with VAMP3 specifically at the plasma membrane, indicating increased SNARE complex formation, whereas FRET with other tested SNAREs was unaltered. Our results revealed that SNARE complexing is a key regulatory step for cytokine production by immune cells and prove the applicability of FRET-FLIM for visualizing SNARE complexes in live cells with subcellular spatial resolution. DOI: http://dx.doi.org/10.7554/eLife.23525.001
format Online
Article
Text
id pubmed-5473687
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher eLife Sciences Publications, Ltd
record_format MEDLINE/PubMed
spelling pubmed-54736872017-06-19 Fluorescence Lifetime Imaging Microscopy reveals rerouting of SNARE trafficking driving dendritic cell activation Verboogen, Daniëlle Rianne José González Mancha, Natalia ter Beest, Martin van den Bogaart, Geert eLife Biophysics and Structural Biology SNARE proteins play a crucial role in intracellular trafficking by catalyzing membrane fusion, but assigning SNAREs to specific intracellular transport routes is challenging with current techniques. We developed a novel Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM)-based technique allowing visualization of real-time local interactions of fluorescently tagged SNARE proteins in live cells. We used FRET-FLIM to delineate the trafficking steps underlying the release of the inflammatory cytokine interleukin-6 (IL-6) from human blood-derived dendritic cells. We found that activation of dendritic cells by bacterial lipopolysaccharide leads to increased FRET of fluorescently labeled syntaxin 4 with VAMP3 specifically at the plasma membrane, indicating increased SNARE complex formation, whereas FRET with other tested SNAREs was unaltered. Our results revealed that SNARE complexing is a key regulatory step for cytokine production by immune cells and prove the applicability of FRET-FLIM for visualizing SNARE complexes in live cells with subcellular spatial resolution. DOI: http://dx.doi.org/10.7554/eLife.23525.001 eLife Sciences Publications, Ltd 2017-05-19 /pmc/articles/PMC5473687/ /pubmed/28524818 http://dx.doi.org/10.7554/eLife.23525 Text en © 2017, Verboogen et al http://creativecommons.org/licenses/by/4.0/ This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Biophysics and Structural Biology
Verboogen, Daniëlle Rianne José
González Mancha, Natalia
ter Beest, Martin
van den Bogaart, Geert
Fluorescence Lifetime Imaging Microscopy reveals rerouting of SNARE trafficking driving dendritic cell activation
title Fluorescence Lifetime Imaging Microscopy reveals rerouting of SNARE trafficking driving dendritic cell activation
title_full Fluorescence Lifetime Imaging Microscopy reveals rerouting of SNARE trafficking driving dendritic cell activation
title_fullStr Fluorescence Lifetime Imaging Microscopy reveals rerouting of SNARE trafficking driving dendritic cell activation
title_full_unstemmed Fluorescence Lifetime Imaging Microscopy reveals rerouting of SNARE trafficking driving dendritic cell activation
title_short Fluorescence Lifetime Imaging Microscopy reveals rerouting of SNARE trafficking driving dendritic cell activation
title_sort fluorescence lifetime imaging microscopy reveals rerouting of snare trafficking driving dendritic cell activation
topic Biophysics and Structural Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473687/
https://www.ncbi.nlm.nih.gov/pubmed/28524818
http://dx.doi.org/10.7554/eLife.23525
work_keys_str_mv AT verboogendanielleriannejose fluorescencelifetimeimagingmicroscopyrevealsreroutingofsnaretraffickingdrivingdendriticcellactivation
AT gonzalezmanchanatalia fluorescencelifetimeimagingmicroscopyrevealsreroutingofsnaretraffickingdrivingdendriticcellactivation
AT terbeestmartin fluorescencelifetimeimagingmicroscopyrevealsreroutingofsnaretraffickingdrivingdendriticcellactivation
AT vandenbogaartgeert fluorescencelifetimeimagingmicroscopyrevealsreroutingofsnaretraffickingdrivingdendriticcellactivation