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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

Embryonic cell reaggregation assay in the presence of IgG of anti-Epith-2 monoclonal antibody in H. pulcherrimus. (A) Whole embryonic cells of swimming blastulae immediately after dissociation (0 h). (B–D) Re-aggregated embryonic cells at 5 h after dissociation. (B) In plain artificial seawater (ASW alone). (C) With 10 μg/ml IgG (10 μg/ml IgG). (D) With 50 μg/ml IgG (50 μg/ml IgG). Scale bars, 100 μm. (E) Average size of cell aggregates with no IgG (0), 10 μg/ml (10) and 50 μg/ml (50) IgG. Bars, SD (n = 80). *P = 0.0344, **P = 0.0001. Unpaired t test. (F) Proportion of three cell aggregate sizes in 0 μg/ml (0), 10 μg/ml (10), and 50 μg/ml (50) anti-Epith-2 mAb IgG. Bars, SD (n = 80). *P = 0.0055, **P = 0.0588. Unpaired t test.
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Figure 7: Embryonic cell reaggregation assay in the presence of IgG of anti-Epith-2 monoclonal antibody in H. pulcherrimus. (A) Whole embryonic cells of swimming blastulae immediately after dissociation (0 h). (B–D) Re-aggregated embryonic cells at 5 h after dissociation. (B) In plain artificial seawater (ASW alone). (C) With 10 μg/ml IgG (10 μg/ml IgG). (D) With 50 μg/ml IgG (50 μg/ml IgG). Scale bars, 100 μm. (E) Average size of cell aggregates with no IgG (0), 10 μg/ml (10) and 50 μg/ml (50) IgG. Bars, SD (n = 80). *P = 0.0344, **P = 0.0001. Unpaired t test. (F) Proportion of three cell aggregate sizes in 0 μg/ml (0), 10 μg/ml (10), and 50 μg/ml (50) anti-Epith-2 mAb IgG. Bars, SD (n = 80). *P = 0.0055, **P = 0.0588. Unpaired t test.

Mentions: The dissociated H. pulcherrimus swimming blastulae in CMDASW were washed once with fresh CMDASW and diluted at 1.6 × 106 cells/ml. The anti-Epith-2 mAb IgG was added to the cell suspension in 24-well plates at 10 or 50 μg/ml and was placed in an incubator at 15°C for 5 h. The cell suspensions were examined under a light field microscope and photographed. The major axis of randomly chosen 80 cell aggregates was measured manually using printouts of micrographs of each sample, and the statistical significance was examined between the averages of two subjects using the unpaired t test by an online GraphPad software, QuickCalcs1 as shown in Figures 7E,F.


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)

Embryonic cell reaggregation assay in the presence of IgG of anti-Epith-2 monoclonal antibody in H. pulcherrimus. (A) Whole embryonic cells of swimming blastulae immediately after dissociation (0 h). (B–D) Re-aggregated embryonic cells at 5 h after dissociation. (B) In plain artificial seawater (ASW alone). (C) With 10 μg/ml IgG (10 μg/ml IgG). (D) With 50 μg/ml IgG (50 μg/ml IgG). Scale bars, 100 μm. (E) Average size of cell aggregates with no IgG (0), 10 μg/ml (10) and 50 μg/ml (50) IgG. Bars, SD (n = 80). *P = 0.0344, **P = 0.0001. Unpaired t test. (F) Proportion of three cell aggregate sizes in 0 μg/ml (0), 10 μg/ml (10), and 50 μg/ml (50) anti-Epith-2 mAb IgG. Bars, SD (n = 80). *P = 0.0055, **P = 0.0588. Unpaired t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Embryonic cell reaggregation assay in the presence of IgG of anti-Epith-2 monoclonal antibody in H. pulcherrimus. (A) Whole embryonic cells of swimming blastulae immediately after dissociation (0 h). (B–D) Re-aggregated embryonic cells at 5 h after dissociation. (B) In plain artificial seawater (ASW alone). (C) With 10 μg/ml IgG (10 μg/ml IgG). (D) With 50 μg/ml IgG (50 μg/ml IgG). Scale bars, 100 μm. (E) Average size of cell aggregates with no IgG (0), 10 μg/ml (10) and 50 μg/ml (50) IgG. Bars, SD (n = 80). *P = 0.0344, **P = 0.0001. Unpaired t test. (F) Proportion of three cell aggregate sizes in 0 μg/ml (0), 10 μg/ml (10), and 50 μg/ml (50) anti-Epith-2 mAb IgG. Bars, SD (n = 80). *P = 0.0055, **P = 0.0588. Unpaired t test.
Mentions: The dissociated H. pulcherrimus swimming blastulae in CMDASW were washed once with fresh CMDASW and diluted at 1.6 × 106 cells/ml. The anti-Epith-2 mAb IgG was added to the cell suspension in 24-well plates at 10 or 50 μg/ml and was placed in an incubator at 15°C for 5 h. The cell suspensions were examined under a light field microscope and photographed. The major axis of randomly chosen 80 cell aggregates was measured manually using printouts of micrographs of each sample, and the statistical significance was examined between the averages of two subjects using the unpaired t test by an online GraphPad software, QuickCalcs1 as shown in Figures 7E,F.

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