title: Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils.
abstract: BACKGROUND
  SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood.
  
  
  METHODS
  Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17.
  
  
  RESULTS
  STX17 was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-α) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-α-stimulated eosinophils.
  
  
  CONCLUSIONS
  The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos.
authors.affiliation: Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil.
authors.forename: Lívia A S
authors.initials: LAS
authors.lastname: Carmo
authors.affiliation: Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil.
authors.forename: Felipe F
authors.initials: FF
authors.lastname: Dias
authors.affiliation: Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil.
authors.forename: Kássia K
authors.initials: KK
authors.lastname: Malta
authors.affiliation: Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil.
authors.forename: Kátia B
authors.initials: KB
authors.lastname: Amaral
authors.affiliation: Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Revital
authors.initials: R
authors.lastname: Shamri
authors.affiliation: Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Weller
authors.affiliation: Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil.|Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Rossana C N
authors.initials: RCN
authors.lastname: Melo
mesh_terms: D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D004804:Eosinophils / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D016253:Microscopy, Immunoelectron; D050765:Qa-SNARE Proteins / Q000378:metabolism*; D022142:Secretory Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D013347:Subcellular Fractions
chemical_list: D016207:Cytokines; D050765:Qa-SNARE Proteins; C551267:STX17 protein, human
keywords: Eosinophil sombrero vesicles; Human eosinophils; Immunogold electron microscopy; SNARES; Secretory granules; Syntaxin17; Transmission electron microscopy; Vesicular trafficking