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Electron tomography and immunonanogold electron microscopy for investigating intracellular trafficking and secretion in human eosinophils.

Melo RC, Dvorak AM, Weller PF - J. Cell. Mol. Med. (2008)

Bottom Line: Electron tomography (ET) has increasingly been used to understand the complexity of membrane systems and protein-trafficking events.By ET and immunonanogold electron microscopy, we recently defined a route for vesicular transport and release of granule-stored products from within activated human eosinophils, cells specialized in the secretion of numerous cytokines and other proteins during inflammatory responses.Here, we highlight these techniques as important tools to unveil a distinct eosinophil vesicular system and secretory pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Biology, Department of Biology Federal University of Juiz de Fora, UFJF, Juiz de Fora, Minas Gerais, Brazil. rossana.melo@ufjf.edu.br

ABSTRACT
Electron tomography (ET) has increasingly been used to understand the complexity of membrane systems and protein-trafficking events. By ET and immunonanogold electron microscopy, we recently defined a route for vesicular transport and release of granule-stored products from within activated human eosinophils, cells specialized in the secretion of numerous cytokines and other proteins during inflammatory responses. Here, we highlight these techniques as important tools to unveil a distinct eosinophil vesicular system and secretory pathway.

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Ultrastructural image of a human activated eosinophil. (Ai) Eosinophil sombrero vesicles (EoSVs) (lumens highlighted in pink) with typical morphology are observed in the cytoplasm by transmission electron microscopy. These vesicles show a ‘Mexican hat’ (sombrero) appearance or a ‘C’-shaped morphology in conventional cross-thin sections (∼80 nm of thickness) of the eosinophils. Secretory granules (Gr), seen in progressive stages of emptying, indicate occurrence of piecemeal degranulation. (Aii) is the boxed area of (Ai) and shows in higher magnification several EoSVs profiles in close apposition to a mobilized granule. Eosinophils, isolated from the blood by negative selection [8], were stimulated with recombinant stem cell factor and processed as described in Reference [4]. N, nucleus. Bars: 630 nm (Ai), 300 nm (Aii).
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fig01: Ultrastructural image of a human activated eosinophil. (Ai) Eosinophil sombrero vesicles (EoSVs) (lumens highlighted in pink) with typical morphology are observed in the cytoplasm by transmission electron microscopy. These vesicles show a ‘Mexican hat’ (sombrero) appearance or a ‘C’-shaped morphology in conventional cross-thin sections (∼80 nm of thickness) of the eosinophils. Secretory granules (Gr), seen in progressive stages of emptying, indicate occurrence of piecemeal degranulation. (Aii) is the boxed area of (Ai) and shows in higher magnification several EoSVs profiles in close apposition to a mobilized granule. Eosinophils, isolated from the blood by negative selection [8], were stimulated with recombinant stem cell factor and processed as described in Reference [4]. N, nucleus. Bars: 630 nm (Ai), 300 nm (Aii).

Mentions: Recent studies based on fully automated electron tomography (ET) and refined immunonanogold electron microscopy (EM) revealed that, during eosinophil secretion, a distinct population of large, tubular transport vesicles, termed eosinophil sombrero vesicles (EoSVs) (Fig. 1), bud from the cytoplasmic secretory granules (also referred to as specific granules), and in conjunction with small, round vesicles, transport granule-stored products to the plasma membrane for extracellular release [4]. This vesicle-mediated process of cell secretion (piecemeal degranulation) [5], also frequently identified in other cells [6, 7], was until recently believed to be accomplished only by small, round vesicles. However, recent data have provided conclusive evidence for both the active formation of large vesiculotubular carriers (EoSVs) in response to cell activation (Figs. 1 and 2A) and their participation in granule-to-plasma membrane trafficking [4]. Combining pre-embedding immunonanogold EM for precise epitope preservation, highly specific monoclonal antibodies and subcellular localization associated with very small gold particles (1.4 nm) as a probe, EoSVs were positively immunola-belled for typical granule products such as major basic protein (MBP) (Fig. 2B and C) and interleukin 4 (IL-4) [4, 8]. MBP is one of the most abundant cationic proteins stored within and recognized as a marker of the eosinophil specific granules, while IL-4, also stored in the specific granules, is a hallmark eosinophil cytokine, typical of allergic and anti-helminthic parasite immune responses [9].


Electron tomography and immunonanogold electron microscopy for investigating intracellular trafficking and secretion in human eosinophils.

Melo RC, Dvorak AM, Weller PF - J. Cell. Mol. Med. (2008)

Ultrastructural image of a human activated eosinophil. (Ai) Eosinophil sombrero vesicles (EoSVs) (lumens highlighted in pink) with typical morphology are observed in the cytoplasm by transmission electron microscopy. These vesicles show a ‘Mexican hat’ (sombrero) appearance or a ‘C’-shaped morphology in conventional cross-thin sections (∼80 nm of thickness) of the eosinophils. Secretory granules (Gr), seen in progressive stages of emptying, indicate occurrence of piecemeal degranulation. (Aii) is the boxed area of (Ai) and shows in higher magnification several EoSVs profiles in close apposition to a mobilized granule. Eosinophils, isolated from the blood by negative selection [8], were stimulated with recombinant stem cell factor and processed as described in Reference [4]. N, nucleus. Bars: 630 nm (Ai), 300 nm (Aii).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562554&req=5

fig01: Ultrastructural image of a human activated eosinophil. (Ai) Eosinophil sombrero vesicles (EoSVs) (lumens highlighted in pink) with typical morphology are observed in the cytoplasm by transmission electron microscopy. These vesicles show a ‘Mexican hat’ (sombrero) appearance or a ‘C’-shaped morphology in conventional cross-thin sections (∼80 nm of thickness) of the eosinophils. Secretory granules (Gr), seen in progressive stages of emptying, indicate occurrence of piecemeal degranulation. (Aii) is the boxed area of (Ai) and shows in higher magnification several EoSVs profiles in close apposition to a mobilized granule. Eosinophils, isolated from the blood by negative selection [8], were stimulated with recombinant stem cell factor and processed as described in Reference [4]. N, nucleus. Bars: 630 nm (Ai), 300 nm (Aii).
Mentions: Recent studies based on fully automated electron tomography (ET) and refined immunonanogold electron microscopy (EM) revealed that, during eosinophil secretion, a distinct population of large, tubular transport vesicles, termed eosinophil sombrero vesicles (EoSVs) (Fig. 1), bud from the cytoplasmic secretory granules (also referred to as specific granules), and in conjunction with small, round vesicles, transport granule-stored products to the plasma membrane for extracellular release [4]. This vesicle-mediated process of cell secretion (piecemeal degranulation) [5], also frequently identified in other cells [6, 7], was until recently believed to be accomplished only by small, round vesicles. However, recent data have provided conclusive evidence for both the active formation of large vesiculotubular carriers (EoSVs) in response to cell activation (Figs. 1 and 2A) and their participation in granule-to-plasma membrane trafficking [4]. Combining pre-embedding immunonanogold EM for precise epitope preservation, highly specific monoclonal antibodies and subcellular localization associated with very small gold particles (1.4 nm) as a probe, EoSVs were positively immunola-belled for typical granule products such as major basic protein (MBP) (Fig. 2B and C) and interleukin 4 (IL-4) [4, 8]. MBP is one of the most abundant cationic proteins stored within and recognized as a marker of the eosinophil specific granules, while IL-4, also stored in the specific granules, is a hallmark eosinophil cytokine, typical of allergic and anti-helminthic parasite immune responses [9].

Bottom Line: Electron tomography (ET) has increasingly been used to understand the complexity of membrane systems and protein-trafficking events.By ET and immunonanogold electron microscopy, we recently defined a route for vesicular transport and release of granule-stored products from within activated human eosinophils, cells specialized in the secretion of numerous cytokines and other proteins during inflammatory responses.Here, we highlight these techniques as important tools to unveil a distinct eosinophil vesicular system and secretory pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Biology, Department of Biology Federal University of Juiz de Fora, UFJF, Juiz de Fora, Minas Gerais, Brazil. rossana.melo@ufjf.edu.br

ABSTRACT
Electron tomography (ET) has increasingly been used to understand the complexity of membrane systems and protein-trafficking events. By ET and immunonanogold electron microscopy, we recently defined a route for vesicular transport and release of granule-stored products from within activated human eosinophils, cells specialized in the secretion of numerous cytokines and other proteins during inflammatory responses. Here, we highlight these techniques as important tools to unveil a distinct eosinophil vesicular system and secretory pathway.

Show MeSH
Related in: MedlinePlus