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.DOI: http://dx.doi.org/10.7554/eLife.23525.001

Secretory vesicles of immune cells contain only a limited number of interleukin 6 molecules

“…The lower estimate is assuming completely nonpolarized secretion, as suggested by a macrophage study where there was no evidence found for polarized secretion of IL‐6 in macrophages, whereas TNFα was predominantly secreted at the nascent cup of phagosomes . The higher estimate is for the case that IL‐6 is exclusively released at the ventral membrane, which we consider unlikely given that we recently showed the presence of VAMP3 in postfusion cis ‐SNARE complexes (i.e., the SNARE for IL‐6 release ) at the dorsal membrane. In the case where release of IL‐6 would be higher at the dorsal than the ventral membranes, we underestimate the rate of exocytic events and the average number of IL‐6 molecules per vesicle will be even lower.…”

“…C). This rate increased on average about twofold to ~ 0.0027 bursts·s −1 ·μm −2 upon overnight stimulation with LPS, likely due to the increased expression of trafficking proteins and the rerouting of intracellular trafficking . These are coarse estimates, with a 10‐fold variation among cells (Fig.…”

“…Given that the molecular weight of the main form of IL‐6 is 23.7 kDa , this equals about 1300 IL‐6 molecules·cell −1 ·h −1 . We then stimulated the cells with LPS for 24 h. During this period, cells secrete more IL‐6 which accumulates in medium in an almost linear fashion for over 24 h . Overnight stimulation of the DCs with LPS increased the IL‐6 secretion on average roughly 40‐fold to ~ 46 000 molecules·cell −1 ·h −1 (Fig.…”

“…Cellular activation, for instance, during infection or tissue damage, triggers the secretion of pro‐ or anti‐inflammatory cytokines to orders of magnitude higher than at resting conditions . One of the most prominent examples is the secretion of the soluble cytokine interleukin 6 (IL‐6) within hours after stimulation of macrophages and dendritic cells by the bacterial cell membrane component lipopolysaccharide (LPS) . Here, LPS binds to the pattern recognition receptor Toll‐like receptor 4 on the plasma membrane of these immune cells and this triggers activation of NF‐κB and other transcription factors, which in turn result in the transcription and translation of IL‐6.…”

“…Cellular activation, for instance, during infection or tissue damage, triggers the secretion of proor anti-inflammatory cytokines to orders of magnitude higher than at resting conditions [1,2]. One of the most prominent examples is the secretion of the soluble cytokine interleukin 6 (IL-6) within hours after stimulation of macrophages and dendritic cells by the bacterial cell membrane component lipopolysaccharide (LPS) [3][4][5].…”