Fluorescence lifetime imaging microscopy reveals rerouting of SNARE trafficking driving dendritic cell activation
Abstract
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.
Article and author information
Author details
Funding
Seventh Framework Programme (336479)
- Geert van den Bogaart
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO-ALW VIDI 864.14.001)
- Geert van den Bogaart
Human Frontier Science Program (CDA-00022/2014)
- Geert van den Bogaart
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Human subjects: Monocytes were isolated from blood of healthy individuals (informed consent and consent to publish obtained, approved by Sanquin ethical committee and according to Radboudumc institutional guidelines).
Reviewing Editor
- Christian Rosenmund, Charité-Universitätsmedizin Berlin, Germany
Publication history
- Received: November 22, 2016
- Accepted: May 18, 2017
- Accepted Manuscript published: May 19, 2017 (version 1)
- Version of Record published: June 16, 2017 (version 2)
Copyright
© 2017, Verboogen et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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Topoisomerase V is a unique topoisomerase that combines DNA repair and topoisomerase activities. The enzyme has an unusual arrangement, with a small topoisomerase domain followed by 12 tandem (HhH)2 domains, which include three AP lyase repair domains. The uncommon architecture of this enzyme bears no resemblance to any other known topoisomerase. Here we present structures of topoisomerase V in complex with DNA. The structures show that the (HhH)2 domains wrap around the DNA and in this manner appear to act as a processivity factor. There is a conformational change in the protein to expose the topoisomerase active site. The DNA bends sharply to enter the active site, which melts the DNA and probably facilitates relaxation. The structures show a DNA binding mode not observed before and provide information on the way this atypical topoisomerase relaxes DNA. In common with type IB enzymes, topoisomerase V relaxes DNA using a controlled rotation mechanism, but the structures show that topoisomerase V accomplishes this in different manner. Overall, the structures firmly establish that type IC topoisomerases form a distinct type of topoisomerases, with no similarities to other types at the sequence, structural, or mechanistic level. They represent a completely different solution to DNA relaxation.
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