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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

Isoelectric points and molecular mass in Daltons separation (ISO-DALT) analysis of Epith-2 of H. pulcherrimus (A–C) and proteinase sensitivity of Epith-2 T. hardwicki (D). (A) ISO-DALT 2D immunoblotting shows an immunopositive spot at the 143 kDa and pH 4.7 (red) region. The spot is artificially colored. (B) ISO-DALT 2D pattern stained with silver. (C) Merged image between (A,B). (D) Protease digestion of Epith-2 of swimming blastulae. Cont, control; the numbers, dilution ratios of protease, Tryp, trypsin digested. Chytrp, chymotrypsin digested.
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Figure 2: Isoelectric points and molecular mass in Daltons separation (ISO-DALT) analysis of Epith-2 of H. pulcherrimus (A–C) and proteinase sensitivity of Epith-2 T. hardwicki (D). (A) ISO-DALT 2D immunoblotting shows an immunopositive spot at the 143 kDa and pH 4.7 (red) region. The spot is artificially colored. (B) ISO-DALT 2D pattern stained with silver. (C) Merged image between (A,B). (D) Protease digestion of Epith-2 of swimming blastulae. Cont, control; the numbers, dilution ratios of protease, Tryp, trypsin digested. Chytrp, chymotrypsin digested.

Mentions: To examine the similarities between the epitopes of anti-Epith-2 mAb of H. pulcherrimus and T. hardwicki, further proteomic analysis was conducted using ISO-DALT 2D immunoblotting using swimming blastulae. The immunoblotting localized an anti-Epith-2 mAb-binding spot at the acidic region [isoelectric point (pI) = 4.7; Figure 2A], which is similar to the previous report regarding T. hardwicki [pI = 4.98, Ref. (10)]. However, according to silver-stained ISO-DALT separation gel analysis, no positive spot was observed at the equivalent spot to the anti-Epith-2 mAb-binding spot region (Figure 2B, arrow), which was confirmed by a merged image (Figure 2C). In T. hardwicki, however, the 160 kDa region was weakly silver-stained using ISO-DALT 2D gel analysis (10). This weak stain may not be due to the small proportion of peptide but may be due to the highly acid property of Epith-2 (36).


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)

Isoelectric points and molecular mass in Daltons separation (ISO-DALT) analysis of Epith-2 of H. pulcherrimus (A–C) and proteinase sensitivity of Epith-2 T. hardwicki (D). (A) ISO-DALT 2D immunoblotting shows an immunopositive spot at the 143 kDa and pH 4.7 (red) region. The spot is artificially colored. (B) ISO-DALT 2D pattern stained with silver. (C) Merged image between (A,B). (D) Protease digestion of Epith-2 of swimming blastulae. Cont, control; the numbers, dilution ratios of protease, Tryp, trypsin digested. Chytrp, chymotrypsin digested.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Isoelectric points and molecular mass in Daltons separation (ISO-DALT) analysis of Epith-2 of H. pulcherrimus (A–C) and proteinase sensitivity of Epith-2 T. hardwicki (D). (A) ISO-DALT 2D immunoblotting shows an immunopositive spot at the 143 kDa and pH 4.7 (red) region. The spot is artificially colored. (B) ISO-DALT 2D pattern stained with silver. (C) Merged image between (A,B). (D) Protease digestion of Epith-2 of swimming blastulae. Cont, control; the numbers, dilution ratios of protease, Tryp, trypsin digested. Chytrp, chymotrypsin digested.
Mentions: To examine the similarities between the epitopes of anti-Epith-2 mAb of H. pulcherrimus and T. hardwicki, further proteomic analysis was conducted using ISO-DALT 2D immunoblotting using swimming blastulae. The immunoblotting localized an anti-Epith-2 mAb-binding spot at the acidic region [isoelectric point (pI) = 4.7; Figure 2A], which is similar to the previous report regarding T. hardwicki [pI = 4.98, Ref. (10)]. However, according to silver-stained ISO-DALT separation gel analysis, no positive spot was observed at the equivalent spot to the anti-Epith-2 mAb-binding spot region (Figure 2B, arrow), which was confirmed by a merged image (Figure 2C). In T. hardwicki, however, the 160 kDa region was weakly silver-stained using ISO-DALT 2D gel analysis (10). This weak stain may not be due to the small proportion of peptide but may be due to the highly acid property of Epith-2 (36).

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