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

Elife. 2017 May 19;6:e23525. doi: 10.7554/eLife.23525.

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.

Keywords: FRET-FLIM; SNARE proteins; biophysics; dendritic cells; human; immunology; membrane trafficking; structural biology.

MeSH terms

  • Dendritic Cells / physiology*
  • Fluorescence Resonance Energy Transfer
  • Humans
  • Interleukin-6 / metabolism
  • Microscopy, Fluorescence / methods*
  • Optical Imaging / methods*
  • Protein Transport
  • Qa-SNARE Proteins / metabolism*
  • Vesicle-Associated Membrane Protein 3 / metabolism*

Substances

  • IL6 protein, human
  • Interleukin-6
  • Qa-SNARE Proteins
  • VAMP3 protein, human
  • Vesicle-Associated Membrane Protein 3
  • syntaxin 4, human

Grant support

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.