Fluorescence lifetime imaging microscopy reveals rerouting of SNARE trafficking driving dendritic cell activation

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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.

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Daniëlle Rianne José Verboogen

Department of Tumor Immunology, Radboud University Medical Center, Nijmegen, Netherlands

Competing interests
The authors declare that no competing interests exist.
Natalia González Mancha

Department of Tumor Immunology, Radboud University Medical Center, Nijmegen, Netherlands

Competing interests
The authors declare that no competing interests exist.
Martin ter Beest

Department of Tumor Immunology, Radboud University Medical Center, Nijmegen, Netherlands

Competing interests
The authors declare that no competing interests exist.
Geert van den Bogaart

Department of Tumor Immunology, Radboud University Medical Center, Nijmegen, Netherlands

For correspondence
geert.vandenbogaart@radboudumc.nl

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-2180-6735

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).

Copyright

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.