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Participation of ezrin in bacterial uptake by trophoblast giant cells.

Watanabe K, Tachibana M, Kim S, Watarai M - Reprod. Biol. Endocrinol. (2009)

Bottom Line: Here we identified ezrin, a member of ezrin-radixin-moesin (ERM) protein family, as a molecule associated with Hsc70.The expression level of ezrin was higher in TG cells than in trophoblast stem (TS) cells, and ezrin knockdown TG cells showed a reduction in bacterial uptake ability.Ezrin associates with Hsc70 that locates on the membrane of TG cells and participates in the bacterial uptake by TG cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Public Health, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. kentaw@yamaguchi-u.ac.jp

ABSTRACT

Background: Trophoblast giant (TG) cells are involved in systematic removal of bacterial pathogens from the maternal-fetal interface of the placenta. In particular, TG cells have the ability to take up extracellular antigens by active phagocytosis induced by interferon-gamma (IFN-gamma). We previously reported that heat shock cognate protein 70 (Hsc70) present on the surface of TG cells mediated the uptake of Brucella abortus. However, the mechanism of bacterial uptake by TG cells is not completely understood. Here we identified ezrin, a member of ezrin-radixin-moesin (ERM) protein family, as a molecule associated with Hsc70.

Methods: Mouse TG cells were employed in all experiments, and B. abortus was used as the bacterial antigen. Confirmation of the binding capacity of ERM protein was assessed by pull-down assay and ELISA using recombinant Hsc70 and ERM proteins. Ezrin was depleted using siRNA and the depletion examined by immunoblotting or immunofluorescence staining.

Results: The expression level of ezrin was higher in TG cells than in trophoblast stem (TS) cells, and ezrin knockdown TG cells showed a reduction in bacterial uptake ability. Although tyrosine phosphorylation of ezrin was not related to bacterial uptake activity, localization of Hsc70 on the membrane was affected by the depletion of ezrin in TG cells.

Conclusion: Ezrin associates with Hsc70 that locates on the membrane of TG cells and participates in the bacterial uptake by TG cells.

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Distribution of tyrosine-phosphorylated ezrin in TG cells. (A) Immunofluorescence staining of F-actin and tyrosine-phosphorylated ezrin (p-Ezrin). Anti-phosphorylated tyrosine residues 146 (pTyr146) and 354 (pTyr354) of ezrin were used to detect p-Ezrin. Scale bar indicates 25 μm. (B) Immunofluorescence staining of p-Ezrin (red) and B. abortus (green). Cells were observed after 30 min of bacterial inoculation. Scale bar indicates 10 μm.
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Figure 5: Distribution of tyrosine-phosphorylated ezrin in TG cells. (A) Immunofluorescence staining of F-actin and tyrosine-phosphorylated ezrin (p-Ezrin). Anti-phosphorylated tyrosine residues 146 (pTyr146) and 354 (pTyr354) of ezrin were used to detect p-Ezrin. Scale bar indicates 25 μm. (B) Immunofluorescence staining of p-Ezrin (red) and B. abortus (green). Cells were observed after 30 min of bacterial inoculation. Scale bar indicates 10 μm.

Mentions: To evaluate whether ezrin phosphorylation is a requirement for bacterial uptake, we tried to detect tyrosine phosphorylation of ezrin at the site of bacterial uptake in TG cells. Although tyrosine-phosphorylated ezrin was strongly co-localized with the actin cytoskeleton (Fig. 5A), co-localization between phosphorylated ezrin and intracellular bacteria was not observed (Fig. 5B). The change in quantity of tyrosine phosphorylation of ezrin due to bacterial inoculation was not detected by immunoblotting (data not shown).


Participation of ezrin in bacterial uptake by trophoblast giant cells.

Watanabe K, Tachibana M, Kim S, Watarai M - Reprod. Biol. Endocrinol. (2009)

Distribution of tyrosine-phosphorylated ezrin in TG cells. (A) Immunofluorescence staining of F-actin and tyrosine-phosphorylated ezrin (p-Ezrin). Anti-phosphorylated tyrosine residues 146 (pTyr146) and 354 (pTyr354) of ezrin were used to detect p-Ezrin. Scale bar indicates 25 μm. (B) Immunofluorescence staining of p-Ezrin (red) and B. abortus (green). Cells were observed after 30 min of bacterial inoculation. Scale bar indicates 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2748081&req=5

Figure 5: Distribution of tyrosine-phosphorylated ezrin in TG cells. (A) Immunofluorescence staining of F-actin and tyrosine-phosphorylated ezrin (p-Ezrin). Anti-phosphorylated tyrosine residues 146 (pTyr146) and 354 (pTyr354) of ezrin were used to detect p-Ezrin. Scale bar indicates 25 μm. (B) Immunofluorescence staining of p-Ezrin (red) and B. abortus (green). Cells were observed after 30 min of bacterial inoculation. Scale bar indicates 10 μm.
Mentions: To evaluate whether ezrin phosphorylation is a requirement for bacterial uptake, we tried to detect tyrosine phosphorylation of ezrin at the site of bacterial uptake in TG cells. Although tyrosine-phosphorylated ezrin was strongly co-localized with the actin cytoskeleton (Fig. 5A), co-localization between phosphorylated ezrin and intracellular bacteria was not observed (Fig. 5B). The change in quantity of tyrosine phosphorylation of ezrin due to bacterial inoculation was not detected by immunoblotting (data not shown).

Bottom Line: Here we identified ezrin, a member of ezrin-radixin-moesin (ERM) protein family, as a molecule associated with Hsc70.The expression level of ezrin was higher in TG cells than in trophoblast stem (TS) cells, and ezrin knockdown TG cells showed a reduction in bacterial uptake ability.Ezrin associates with Hsc70 that locates on the membrane of TG cells and participates in the bacterial uptake by TG cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Public Health, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. kentaw@yamaguchi-u.ac.jp

ABSTRACT

Background: Trophoblast giant (TG) cells are involved in systematic removal of bacterial pathogens from the maternal-fetal interface of the placenta. In particular, TG cells have the ability to take up extracellular antigens by active phagocytosis induced by interferon-gamma (IFN-gamma). We previously reported that heat shock cognate protein 70 (Hsc70) present on the surface of TG cells mediated the uptake of Brucella abortus. However, the mechanism of bacterial uptake by TG cells is not completely understood. Here we identified ezrin, a member of ezrin-radixin-moesin (ERM) protein family, as a molecule associated with Hsc70.

Methods: Mouse TG cells were employed in all experiments, and B. abortus was used as the bacterial antigen. Confirmation of the binding capacity of ERM protein was assessed by pull-down assay and ELISA using recombinant Hsc70 and ERM proteins. Ezrin was depleted using siRNA and the depletion examined by immunoblotting or immunofluorescence staining.

Results: The expression level of ezrin was higher in TG cells than in trophoblast stem (TS) cells, and ezrin knockdown TG cells showed a reduction in bacterial uptake ability. Although tyrosine phosphorylation of ezrin was not related to bacterial uptake activity, localization of Hsc70 on the membrane was affected by the depletion of ezrin in TG cells.

Conclusion: Ezrin associates with Hsc70 that locates on the membrane of TG cells and participates in the bacterial uptake by TG cells.

Show MeSH
Related in: MedlinePlus