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EphrinB2 affects apical constriction in Xenopus embryos and is regulated by ADAM10 and flotillin-1.

Ji YJ, Hwang YS, Mood K, Cho HJ, Lee HS, Winterbottom E, Cousin H, Daar IO - Nat Commun (2014)

Bottom Line: The Eph/ephrin signalling pathways have a critical function in cell adhesion and repulsion, and thus play key roles in various morphogenetic events during development.Here we show that a decrease in ephrinB2 protein causes neural tube closure defects during Xenopus laevis embryogenesis.This dramatic decline in ephrinB2 protein levels on the absence of flotillin-1 expression is specific, and is partly the result of an increased susceptibility to cleavage by the metalloprotease ADAM10.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell and Developmental Signaling, National Cancer Institute-Frederick, Frederick, Maryland 21702, USA.

ABSTRACT
The Eph/ephrin signalling pathways have a critical function in cell adhesion and repulsion, and thus play key roles in various morphogenetic events during development. Here we show that a decrease in ephrinB2 protein causes neural tube closure defects during Xenopus laevis embryogenesis. Such a decrease in ephrinB2 protein levels is observed on the loss of flotillin-1 scaffold protein, a newly identified ephrinB2-binding partner. This dramatic decline in ephrinB2 protein levels on the absence of flotillin-1 expression is specific, and is partly the result of an increased susceptibility to cleavage by the metalloprotease ADAM10. These findings indicate that flotillin-1 regulates ephrinB2 protein levels through ADAM10, and is required for appropriate neural tube morphogenesis in the Xenopus embryo.

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ephrinB2 protein half-life is decreased in the absence of flotillin-1 and partially rescued by the ADAM10 inhibitor(a) Two-cell stage embryos were injected with carefully titrated ephrinB2-HA RNA along with control MO or F1aMO to yield roughly equivalent ephrinB2 protein levels when embryos reached the early neurula stages. A group of the injected embryos was subjected to a secondary injection into the blastocoel at stage 9 with ADAM10 inhibitor (10 nl of 1 mM), and the archenteron cavity (stage 14) with cycloheximide (10 nl of 75 ug/ul), and externally incubated in cycloheximide (7.5 ug/ul) to block further protein synthesis for the indicated times. Western blot analysis was performed on the embryonic lysates using HA antibodies, or Erk2 antibodies (as a loading control). (b) A graph of the mean band intensities as measured by Image J software shows the approximate half-lives in the presence of cycloheximide and the indicated MOs and ADAM10 inhibitor. The ephrinB2 C-terminal fragments (short arrow) and the full length protein (long arrow) are indicated. These data are the result of three independent experiments and +/− represents sd. (c) Endogenous surface ephrinB2 levels are reduced by knockdown of flotillin-1, but prominently rescued by the ADAM10 inhibitor. Embryos were injected with the indicated MOs and inhibitors. Neural folds were excised and left non-biotinylated (lane 1) or biotinylated. Lysates were prepared and cell surface proteins immunoprecipitated with streptavidin conjugated sepharose. Western analysis of biotin labelled cell surface proteins was performed using anti ephrinB antibody. Direct Western analysis of neural fold lysates were probed for ephrinB. Erk2 expression is shown as a loading control.
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Figure 6: ephrinB2 protein half-life is decreased in the absence of flotillin-1 and partially rescued by the ADAM10 inhibitor(a) Two-cell stage embryos were injected with carefully titrated ephrinB2-HA RNA along with control MO or F1aMO to yield roughly equivalent ephrinB2 protein levels when embryos reached the early neurula stages. A group of the injected embryos was subjected to a secondary injection into the blastocoel at stage 9 with ADAM10 inhibitor (10 nl of 1 mM), and the archenteron cavity (stage 14) with cycloheximide (10 nl of 75 ug/ul), and externally incubated in cycloheximide (7.5 ug/ul) to block further protein synthesis for the indicated times. Western blot analysis was performed on the embryonic lysates using HA antibodies, or Erk2 antibodies (as a loading control). (b) A graph of the mean band intensities as measured by Image J software shows the approximate half-lives in the presence of cycloheximide and the indicated MOs and ADAM10 inhibitor. The ephrinB2 C-terminal fragments (short arrow) and the full length protein (long arrow) are indicated. These data are the result of three independent experiments and +/− represents sd. (c) Endogenous surface ephrinB2 levels are reduced by knockdown of flotillin-1, but prominently rescued by the ADAM10 inhibitor. Embryos were injected with the indicated MOs and inhibitors. Neural folds were excised and left non-biotinylated (lane 1) or biotinylated. Lysates were prepared and cell surface proteins immunoprecipitated with streptavidin conjugated sepharose. Western analysis of biotin labelled cell surface proteins was performed using anti ephrinB antibody. Direct Western analysis of neural fold lysates were probed for ephrinB. Erk2 expression is shown as a loading control.

Mentions: We also examined whether the observed F1aMO-mediated reduction in ephrinB2 protein was due to a decrease in the half-life of ephrinB2 (as would be expected if the protein is being degraded), and whether cleavage by ADAM10 promotes this activity. For this purpose, we carefully titrated the amount of injected ephrinB2 RNA along with control MO (CoMO) or F1aMO to yield roughly equivalent ephrinB2 protein levels when embryos reached the neurula stage. The embryos were then injected with cycloheximide or with both cycloheximide and ADAM10 inhibitor, and cultured in cycloheximide to block further protein synthesis for 5 h (Fig. 6a, b). Western blot analysis shows that ephrinB2 levels are reduced by half within 153 min (~2.5 h) after the addition of cycloheximide to control MO-containing embryos. In the F1aMO-containing embryos, the half-life of ephrinB2 is reduced to 57 min (~1 h) (Fig. 6a, b). In contrast, the half-life of ephrinB2 is actually extended to 102 min (~1.7h) in the F1aMO plus ADAM10 inhibitor injected embryos (Fig. 6a, b). These data are consistent with flotillin-1 hindering ephrinB2 degradation, in part, by inhibiting its cleavage by ADAM10.


EphrinB2 affects apical constriction in Xenopus embryos and is regulated by ADAM10 and flotillin-1.

Ji YJ, Hwang YS, Mood K, Cho HJ, Lee HS, Winterbottom E, Cousin H, Daar IO - Nat Commun (2014)

ephrinB2 protein half-life is decreased in the absence of flotillin-1 and partially rescued by the ADAM10 inhibitor(a) Two-cell stage embryos were injected with carefully titrated ephrinB2-HA RNA along with control MO or F1aMO to yield roughly equivalent ephrinB2 protein levels when embryos reached the early neurula stages. A group of the injected embryos was subjected to a secondary injection into the blastocoel at stage 9 with ADAM10 inhibitor (10 nl of 1 mM), and the archenteron cavity (stage 14) with cycloheximide (10 nl of 75 ug/ul), and externally incubated in cycloheximide (7.5 ug/ul) to block further protein synthesis for the indicated times. Western blot analysis was performed on the embryonic lysates using HA antibodies, or Erk2 antibodies (as a loading control). (b) A graph of the mean band intensities as measured by Image J software shows the approximate half-lives in the presence of cycloheximide and the indicated MOs and ADAM10 inhibitor. The ephrinB2 C-terminal fragments (short arrow) and the full length protein (long arrow) are indicated. These data are the result of three independent experiments and +/− represents sd. (c) Endogenous surface ephrinB2 levels are reduced by knockdown of flotillin-1, but prominently rescued by the ADAM10 inhibitor. Embryos were injected with the indicated MOs and inhibitors. Neural folds were excised and left non-biotinylated (lane 1) or biotinylated. Lysates were prepared and cell surface proteins immunoprecipitated with streptavidin conjugated sepharose. Western analysis of biotin labelled cell surface proteins was performed using anti ephrinB antibody. Direct Western analysis of neural fold lysates were probed for ephrinB. Erk2 expression is shown as a loading control.
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Figure 6: ephrinB2 protein half-life is decreased in the absence of flotillin-1 and partially rescued by the ADAM10 inhibitor(a) Two-cell stage embryos were injected with carefully titrated ephrinB2-HA RNA along with control MO or F1aMO to yield roughly equivalent ephrinB2 protein levels when embryos reached the early neurula stages. A group of the injected embryos was subjected to a secondary injection into the blastocoel at stage 9 with ADAM10 inhibitor (10 nl of 1 mM), and the archenteron cavity (stage 14) with cycloheximide (10 nl of 75 ug/ul), and externally incubated in cycloheximide (7.5 ug/ul) to block further protein synthesis for the indicated times. Western blot analysis was performed on the embryonic lysates using HA antibodies, or Erk2 antibodies (as a loading control). (b) A graph of the mean band intensities as measured by Image J software shows the approximate half-lives in the presence of cycloheximide and the indicated MOs and ADAM10 inhibitor. The ephrinB2 C-terminal fragments (short arrow) and the full length protein (long arrow) are indicated. These data are the result of three independent experiments and +/− represents sd. (c) Endogenous surface ephrinB2 levels are reduced by knockdown of flotillin-1, but prominently rescued by the ADAM10 inhibitor. Embryos were injected with the indicated MOs and inhibitors. Neural folds were excised and left non-biotinylated (lane 1) or biotinylated. Lysates were prepared and cell surface proteins immunoprecipitated with streptavidin conjugated sepharose. Western analysis of biotin labelled cell surface proteins was performed using anti ephrinB antibody. Direct Western analysis of neural fold lysates were probed for ephrinB. Erk2 expression is shown as a loading control.
Mentions: We also examined whether the observed F1aMO-mediated reduction in ephrinB2 protein was due to a decrease in the half-life of ephrinB2 (as would be expected if the protein is being degraded), and whether cleavage by ADAM10 promotes this activity. For this purpose, we carefully titrated the amount of injected ephrinB2 RNA along with control MO (CoMO) or F1aMO to yield roughly equivalent ephrinB2 protein levels when embryos reached the neurula stage. The embryos were then injected with cycloheximide or with both cycloheximide and ADAM10 inhibitor, and cultured in cycloheximide to block further protein synthesis for 5 h (Fig. 6a, b). Western blot analysis shows that ephrinB2 levels are reduced by half within 153 min (~2.5 h) after the addition of cycloheximide to control MO-containing embryos. In the F1aMO-containing embryos, the half-life of ephrinB2 is reduced to 57 min (~1 h) (Fig. 6a, b). In contrast, the half-life of ephrinB2 is actually extended to 102 min (~1.7h) in the F1aMO plus ADAM10 inhibitor injected embryos (Fig. 6a, b). These data are consistent with flotillin-1 hindering ephrinB2 degradation, in part, by inhibiting its cleavage by ADAM10.

Bottom Line: The Eph/ephrin signalling pathways have a critical function in cell adhesion and repulsion, and thus play key roles in various morphogenetic events during development.Here we show that a decrease in ephrinB2 protein causes neural tube closure defects during Xenopus laevis embryogenesis.This dramatic decline in ephrinB2 protein levels on the absence of flotillin-1 expression is specific, and is partly the result of an increased susceptibility to cleavage by the metalloprotease ADAM10.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell and Developmental Signaling, National Cancer Institute-Frederick, Frederick, Maryland 21702, USA.

ABSTRACT
The Eph/ephrin signalling pathways have a critical function in cell adhesion and repulsion, and thus play key roles in various morphogenetic events during development. Here we show that a decrease in ephrinB2 protein causes neural tube closure defects during Xenopus laevis embryogenesis. Such a decrease in ephrinB2 protein levels is observed on the loss of flotillin-1 scaffold protein, a newly identified ephrinB2-binding partner. This dramatic decline in ephrinB2 protein levels on the absence of flotillin-1 expression is specific, and is partly the result of an increased susceptibility to cleavage by the metalloprotease ADAM10. These findings indicate that flotillin-1 regulates ephrinB2 protein levels through ADAM10, and is required for appropriate neural tube morphogenesis in the Xenopus embryo.

Show MeSH
Related in: MedlinePlus