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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 antigen of anti-Epith-1 mAb and -2 mAb in T. hardwicki using isoelectric points and molecular mass in Daltons separation (ISO-DALT) 2D immunoblotting and immunocrossreactivity of these two mAbs among seven species of sea urchins. (A) ISO-DALT 2D immunoblotting with anti-Epith-1 mAb shows an immunopositive spot at 160 kDa region and pH 4.98 (blue). The spot was artificially colored. (B) ISO-DALT 2D immunoblotting with anti-Epith-2 mAb shows an immunopositive spot (red) at the same region as (A). The spot was artificially colored. (C) Merged image between (A,B). (D) Immunocrossreactivity of anti-Epith-1 mAb (lanes 1–7) and anti-Epith-2 mAb (lanes 8–14). Lanes 1, 8; T. hardwicki (Th) swimming blastulae, lanes 2, 9; H. pulcherrimus (Hp) swimming blastulae, lanes 3, 10; C. japonicus (Cj) swimming blastulae, lanes 4, 11; M. globules (Mg) swimming blastulae, lanes 5, 12; L. pictus (Lp) swimming blastulae, lanes 6, 13; P. depressus (Pd) gastrulae, lanes 7, 14; S. intermedius mesenchyme blastulae (Si). Arrows, 160 kDa region. Arrowhead-1, 143 kDa region. Arrowhead-2, 137 kDa region.
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Figure 1: Immunochemical property of antigen of anti-Epith-1 mAb and -2 mAb in T. hardwicki using isoelectric points and molecular mass in Daltons separation (ISO-DALT) 2D immunoblotting and immunocrossreactivity of these two mAbs among seven species of sea urchins. (A) ISO-DALT 2D immunoblotting with anti-Epith-1 mAb shows an immunopositive spot at 160 kDa region and pH 4.98 (blue). The spot was artificially colored. (B) ISO-DALT 2D immunoblotting with anti-Epith-2 mAb shows an immunopositive spot (red) at the same region as (A). The spot was artificially colored. (C) Merged image between (A,B). (D) Immunocrossreactivity of anti-Epith-1 mAb (lanes 1–7) and anti-Epith-2 mAb (lanes 8–14). Lanes 1, 8; T. hardwicki (Th) swimming blastulae, lanes 2, 9; H. pulcherrimus (Hp) swimming blastulae, lanes 3, 10; C. japonicus (Cj) swimming blastulae, lanes 4, 11; M. globules (Mg) swimming blastulae, lanes 5, 12; L. pictus (Lp) swimming blastulae, lanes 6, 13; P. depressus (Pd) gastrulae, lanes 7, 14; S. intermedius mesenchyme blastulae (Si). Arrows, 160 kDa region. Arrowhead-1, 143 kDa region. Arrowhead-2, 137 kDa region.

Mentions: Prior to onset of immunocrossreactivity assay of anti-Epith-1 mAb and -2 mAb in several species of the sea urchins as will be described below, immunochemical property of antigens of these mAbs was examined by ISO-DALT immunoblotting using T. hardwicki. The ISO-DALT immunoblotting showed both mAbs bound to a spot at 160 kDa and pH 4.98 region (Figures 1A–C), which is, regarding these two mAbs were raised as sister mAbs, predictable result.


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 antigen of anti-Epith-1 mAb and -2 mAb in T. hardwicki using isoelectric points and molecular mass in Daltons separation (ISO-DALT) 2D immunoblotting and immunocrossreactivity of these two mAbs among seven species of sea urchins. (A) ISO-DALT 2D immunoblotting with anti-Epith-1 mAb shows an immunopositive spot at 160 kDa region and pH 4.98 (blue). The spot was artificially colored. (B) ISO-DALT 2D immunoblotting with anti-Epith-2 mAb shows an immunopositive spot (red) at the same region as (A). The spot was artificially colored. (C) Merged image between (A,B). (D) Immunocrossreactivity of anti-Epith-1 mAb (lanes 1–7) and anti-Epith-2 mAb (lanes 8–14). Lanes 1, 8; T. hardwicki (Th) swimming blastulae, lanes 2, 9; H. pulcherrimus (Hp) swimming blastulae, lanes 3, 10; C. japonicus (Cj) swimming blastulae, lanes 4, 11; M. globules (Mg) swimming blastulae, lanes 5, 12; L. pictus (Lp) swimming blastulae, lanes 6, 13; P. depressus (Pd) gastrulae, lanes 7, 14; S. intermedius mesenchyme blastulae (Si). Arrows, 160 kDa region. Arrowhead-1, 143 kDa region. Arrowhead-2, 137 kDa region.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Immunochemical property of antigen of anti-Epith-1 mAb and -2 mAb in T. hardwicki using isoelectric points and molecular mass in Daltons separation (ISO-DALT) 2D immunoblotting and immunocrossreactivity of these two mAbs among seven species of sea urchins. (A) ISO-DALT 2D immunoblotting with anti-Epith-1 mAb shows an immunopositive spot at 160 kDa region and pH 4.98 (blue). The spot was artificially colored. (B) ISO-DALT 2D immunoblotting with anti-Epith-2 mAb shows an immunopositive spot (red) at the same region as (A). The spot was artificially colored. (C) Merged image between (A,B). (D) Immunocrossreactivity of anti-Epith-1 mAb (lanes 1–7) and anti-Epith-2 mAb (lanes 8–14). Lanes 1, 8; T. hardwicki (Th) swimming blastulae, lanes 2, 9; H. pulcherrimus (Hp) swimming blastulae, lanes 3, 10; C. japonicus (Cj) swimming blastulae, lanes 4, 11; M. globules (Mg) swimming blastulae, lanes 5, 12; L. pictus (Lp) swimming blastulae, lanes 6, 13; P. depressus (Pd) gastrulae, lanes 7, 14; S. intermedius mesenchyme blastulae (Si). Arrows, 160 kDa region. Arrowhead-1, 143 kDa region. Arrowhead-2, 137 kDa region.
Mentions: Prior to onset of immunocrossreactivity assay of anti-Epith-1 mAb and -2 mAb in several species of the sea urchins as will be described below, immunochemical property of antigens of these mAbs was examined by ISO-DALT immunoblotting using T. hardwicki. The ISO-DALT immunoblotting showed both mAbs bound to a spot at 160 kDa and pH 4.98 region (Figures 1A–C), which is, regarding these two mAbs were raised as sister mAbs, predictable result.

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