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Characterization and Endocytic Internalization of Epith-2 Cell Surface Glycoprotein during the Epithelial-to-Mesenchymal Transition in Sea Urchin Embryos.

Wakayama N, Katow T, Katow H - Front Endocrinol (Lausanne) (2013)

Bottom Line: In suramin-treated embryos, dissociated mesenchyme cells migrated partially by retaining their epithelial morphology.In HA-treated embryos, no mesenchyme cells migrated.Thus, the EMT occurs in relation to internalization of Epith-2 from presumptive PMC and SMC.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Marine Biology, Tohoku University, Aomori , Aomori , Japan.

ABSTRACT
The epithelial cells of the sea urchin Hemicentrotus pulcherrimus embryo express an Epith-2, uncharacterized glycoprotein, on the lateral surface. Here, we describe internalization of Epith-2 during mesenchyme formation through the epithelial-to-mesenchymal transition (EMT). Epith-2 was first expressed on the entire egg surface soon after fertilization and on the blastomeres until the 4-cell stage, but was localized to the lateral surface of epithelial cells at and after the 16-cell stage throughout the later developmental period. However, primary mesenchyme cells (PMC) and secondary mesenchyme cells (SMC) that ingress by EMT lost Epith-2 from their cell surface by endocytosis during dissociation from the epithelium, which was associated with the appearance of cytoplasmic Epith-2 dots. The cytoplasmic Epith-2 retained a similar relative molecular mass to that of the cell surface immediately after ingression through the early period of the spreading to single cells. Then, Epith-2 was completely lost from the cytoplasm. Tyrosine residues of Epith-2 were phosphorylated. The endocytic retraction of Epith-2 was inhibited by herbimycin A (HA), a protein tyrosine kinase (PTK) inhibitor, and suramin, a growth factor receptor (GFR) inhibitor, suggesting the involvement of the GFR/PTK (GP) signaling pathway. These two GP inhibitors also inhibited PMC and SMC spreading to individual cells after ingression, but the dissociation of PMC and SMC from the epithelium was not inhibited. In suramin-treated embryos, dissociated mesenchyme cells migrated partially by retaining their epithelial morphology. In HA-treated embryos, no mesenchyme cells migrated. Thus, the EMT occurs in relation to internalization of Epith-2 from presumptive PMC and SMC.

No MeSH data available.


Related in: MedlinePlus

Immunochemical property of Epith-2 of H. pulcherrimus (A) and T. hardwicki (B) analyzed by immunoblotting. (A) Immunoblotting of Epith-2 extracted in water (lane 1) and in non-ionic detergent 1% Triton X-100 (lane 2) of swimming blastulae. Whole embryo lysate stained with concanavalin A (lane 3). (B)Mr of Epith-2 glycoprotein under non-reducing conditions (lane 1; 160 kDa region) and reducing conditions (lane 2; 174 kDa region).
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Figure 3: Immunochemical property of Epith-2 of H. pulcherrimus (A) and T. hardwicki (B) analyzed by immunoblotting. (A) Immunoblotting of Epith-2 extracted in water (lane 1) and in non-ionic detergent 1% Triton X-100 (lane 2) of swimming blastulae. Whole embryo lysate stained with concanavalin A (lane 3). (B)Mr of Epith-2 glycoprotein under non-reducing conditions (lane 1; 160 kDa region) and reducing conditions (lane 2; 174 kDa region).

Mentions: The subcellular localization of Epith-2 was examined using lyophilized swimming blastulae of H. pulcherrimus. According to the immunoblotting, Epith-2 was not detected in the water-soluble fraction (Figure 3A, lane 1) but was detected in the non-ionic detergent-extract fraction at the 143 kDa region (Figure 3A, lane 2), which was consistent with previous Epith-1 analysis in T. hardwicki (10). The result suggested that Epith-2 is embedded in the plasma membrane or has a transmembrane domain (38). The Con-A staining of separated gels did not detect a 143 kDa band region (Figure 3A, lane 3), suggesting that unlike in the blastocoelar extracellular matrix in sea urchin embryos (26), Epith-2 has few or lacks mannose moieties.


Characterization and Endocytic Internalization of Epith-2 Cell Surface Glycoprotein during the Epithelial-to-Mesenchymal Transition in Sea Urchin Embryos.

Wakayama N, Katow T, Katow H - Front Endocrinol (Lausanne) (2013)

Immunochemical property of Epith-2 of H. pulcherrimus (A) and T. hardwicki (B) analyzed by immunoblotting. (A) Immunoblotting of Epith-2 extracted in water (lane 1) and in non-ionic detergent 1% Triton X-100 (lane 2) of swimming blastulae. Whole embryo lysate stained with concanavalin A (lane 3). (B)Mr of Epith-2 glycoprotein under non-reducing conditions (lane 1; 160 kDa region) and reducing conditions (lane 2; 174 kDa region).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757445&req=5

Figure 3: Immunochemical property of Epith-2 of H. pulcherrimus (A) and T. hardwicki (B) analyzed by immunoblotting. (A) Immunoblotting of Epith-2 extracted in water (lane 1) and in non-ionic detergent 1% Triton X-100 (lane 2) of swimming blastulae. Whole embryo lysate stained with concanavalin A (lane 3). (B)Mr of Epith-2 glycoprotein under non-reducing conditions (lane 1; 160 kDa region) and reducing conditions (lane 2; 174 kDa region).
Mentions: The subcellular localization of Epith-2 was examined using lyophilized swimming blastulae of H. pulcherrimus. According to the immunoblotting, Epith-2 was not detected in the water-soluble fraction (Figure 3A, lane 1) but was detected in the non-ionic detergent-extract fraction at the 143 kDa region (Figure 3A, lane 2), which was consistent with previous Epith-1 analysis in T. hardwicki (10). The result suggested that Epith-2 is embedded in the plasma membrane or has a transmembrane domain (38). The Con-A staining of separated gels did not detect a 143 kDa band region (Figure 3A, lane 3), suggesting that unlike in the blastocoelar extracellular matrix in sea urchin embryos (26), Epith-2 has few or lacks mannose moieties.

Bottom Line: In suramin-treated embryos, dissociated mesenchyme cells migrated partially by retaining their epithelial morphology.In HA-treated embryos, no mesenchyme cells migrated.Thus, the EMT occurs in relation to internalization of Epith-2 from presumptive PMC and SMC.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Marine Biology, Tohoku University, Aomori , Aomori , Japan.

ABSTRACT
The epithelial cells of the sea urchin Hemicentrotus pulcherrimus embryo express an Epith-2, uncharacterized glycoprotein, on the lateral surface. Here, we describe internalization of Epith-2 during mesenchyme formation through the epithelial-to-mesenchymal transition (EMT). Epith-2 was first expressed on the entire egg surface soon after fertilization and on the blastomeres until the 4-cell stage, but was localized to the lateral surface of epithelial cells at and after the 16-cell stage throughout the later developmental period. However, primary mesenchyme cells (PMC) and secondary mesenchyme cells (SMC) that ingress by EMT lost Epith-2 from their cell surface by endocytosis during dissociation from the epithelium, which was associated with the appearance of cytoplasmic Epith-2 dots. The cytoplasmic Epith-2 retained a similar relative molecular mass to that of the cell surface immediately after ingression through the early period of the spreading to single cells. Then, Epith-2 was completely lost from the cytoplasm. Tyrosine residues of Epith-2 were phosphorylated. The endocytic retraction of Epith-2 was inhibited by herbimycin A (HA), a protein tyrosine kinase (PTK) inhibitor, and suramin, a growth factor receptor (GFR) inhibitor, suggesting the involvement of the GFR/PTK (GP) signaling pathway. These two GP inhibitors also inhibited PMC and SMC spreading to individual cells after ingression, but the dissociation of PMC and SMC from the epithelium was not inhibited. In suramin-treated embryos, dissociated mesenchyme cells migrated partially by retaining their epithelial morphology. In HA-treated embryos, no mesenchyme cells migrated. Thus, the EMT occurs in relation to internalization of Epith-2 from presumptive PMC and SMC.

No MeSH data available.


Related in: MedlinePlus