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

Daniëlle Rianne José Verboogen,Martin Ter Beest,Geert Van Den Bogaart,Natalia González Mancha

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.

Highlights

One of the central paradigms in cell biology is that all intracellular membrane fusion, except for mitochondrial fusion, is catalyzed by soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor (SNARE) proteins (Hong, 2005; Jahn and Scheller, 2006)
Our results showed an increase in Forster resonance energy transfer (FRET) of fluorescently labeled syntaxin 4 (Stx4), but not of syntaxin 3 (Stx3), with vesicle-associated membrane protein 3 (VAMP3) upon cellular activation with the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS), indicating that LPS promotes the complex formation of Stx4 with VAMP3
Based on the high structural homology of SNARE proteins, we expected that mCitrine and mCherry attached to the C-termini of VAMP3 or VAMP8 and

Summary

Introduction

One of the central paradigms in cell biology is that all intracellular membrane fusion, except for mitochondrial fusion, is catalyzed by soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor (SNARE) proteins (Hong, 2005; Jahn and Scheller, 2006). The interaction of one R motif and three Q motifs (termed Qa, Qb and Qc) of cognate SNARE proteins located in opposing membranes leads to the formation of a 4-helix coiled-coil bundle. This trans-SNARE complex formation brings the membranes in close proximity and catalyzes their fusion. From studies in yeast and many mammalian cell types, including neurons, neuroendocrine cells, adipocytes and epithelial cells, it is clear that different intracellular trafficking steps are catalyzed by specific sets of SNARE proteins (Pelham, 2001; Hong, 2005; Jahn and Scheller, 2006).

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