Proteome analysis identified human neutrophil membrane tubulovesicular extensions (cytonemes, membrane tethers) as bactericide trafficking.

ABSTRACT

BACKGROUND: Following adhesion to fibronectin neutrophils can develop membrane tubulovesicular extensions (TVEs) that can be 200nm wide and several cell diameters long. TVEs attach neutrophils to the other cells, substrata or bacteria over distance. To understand the physiological significance of TVEs we performed proteome analysis of TVE content in neutrophils plated to fibronectin in the presence of compounds known to induce TVE formation (nitric oxide donor diethylamine NONOate, 4-bromophenacyl bromide, cytochalasin D). METHODS: Development of TVEs was confirmed by scanning electron microscopy. TVEs were disrupted following removal of inductors and biochemical, high-performance liquid chromatography and mass spectrometry investigations were employed to characterize the proteins within the incubation media. RESULTS: TVE disruption released (a) the granular bactericides lactoferrin, lipocalin, myeloperoxidase, cathepsin G and defensins; (b) energy metabolism enzymes; (c) actin cytoskeleton proteins; (d) S100 proteins; and (e) annexin 1. CONCLUSIONS: The data confirm that TVEs represent a means of secretory bactericide trafficking, where the protrusions fuse with the plasma membrane upon neutrophil adhesion or extend from the cell surface when fusion is impaired. It is proposed that proteins abundantly presented in TVE (energy metabolism enzymes, actin cytoskeleton and S100 proteins, annexin 1) play an important role in fusion of TVE with the plasma membrane. GENERAL SIGNIFICANCE: Our study confirms TVEs as neutrophil secretory protrusions that make direct contacts with cells and bacteria over distance. The membrane-packed content and outstanding length of TVEs might allow targeted neutrophil secretion of aggressive bactericides over a long distance without dilution or injury to surrounding tissues.