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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 CCL11more » 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. - Highlights: • First demonstration of the Qa-SNARE syntaxin-17 (STX17) in human eosinophils. • High resolution immunogold EM shows STX17 in granules and tubular vesicles. • Unstimulated, TNF-α or CCL11-stimulated eosinophils express STX17. • Our findings suggest a role for STX17 in the transport of granule-derived cargos.« less

Authors:
; ; ;  [1]; ;  [2];  [1];  [3]
  1. Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG (Brazil)
  2. Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA (United States)
  3. (United States)
Publication Date:
OSTI Identifier:
22462331
Resource Type:
Journal Article
Journal Name:
Experimental Cell Research
Additional Journal Information:
Journal Volume: 337; Journal Issue: 2; Other Information: Copyright (c) 2015 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0014-4827
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ANTIBODIES; CARRIERS; EOSINOPHILS; GOLD; HUMAN POPULATIONS; INFLAMMATION; LABELLING; LIGANDS; MEMBRANES; PRESERVATION; PROTEINS; RADIOPROTECTIVE SUBSTANCES; RESOLUTION; STIMULI; TRANSMISSION ELECTRON MICROSCOPY

Citation Formats

Carmo, Lívia A.S., Dias, Felipe F., Malta, Kássia K., Amaral, Kátia B., Shamri, Revital, Weller, Peter F., Melo, Rossana C.N., E-mail: rossana.melo@ufjf.edu.br, and Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils. United States: N. p., 2015. Web. doi:10.1016/J.YEXCR.2015.07.003.
Carmo, Lívia A.S., Dias, Felipe F., Malta, Kássia K., Amaral, Kátia B., Shamri, Revital, Weller, Peter F., Melo, Rossana C.N., E-mail: rossana.melo@ufjf.edu.br, & Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils. United States. doi:10.1016/J.YEXCR.2015.07.003.
Carmo, Lívia A.S., Dias, Felipe F., Malta, Kássia K., Amaral, Kátia B., Shamri, Revital, Weller, Peter F., Melo, Rossana C.N., E-mail: rossana.melo@ufjf.edu.br, and Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. Thu . "Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils". United States. doi:10.1016/J.YEXCR.2015.07.003.
@article{osti_22462331,
title = {Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils},
author = {Carmo, Lívia A.S. and Dias, Felipe F. and Malta, Kássia K. and Amaral, Kátia B. and Shamri, Revital and Weller, Peter F. and Melo, Rossana C.N., E-mail: rossana.melo@ufjf.edu.br and Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA},
abstractNote = {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. - Highlights: • First demonstration of the Qa-SNARE syntaxin-17 (STX17) in human eosinophils. • High resolution immunogold EM shows STX17 in granules and tubular vesicles. • Unstimulated, TNF-α or CCL11-stimulated eosinophils express STX17. • Our findings suggest a role for STX17 in the transport of granule-derived cargos.},
doi = {10.1016/J.YEXCR.2015.07.003},
journal = {Experimental Cell Research},
issn = {0014-4827},
number = 2,
volume = 337,
place = {United States},
year = {2015},
month = {10}
}