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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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ADAM10 is responsible for the loss of ephrinB2 expression in the absence of flotillin-1(a) Metalloproteases are responsible for the loss of ephrinB2 expression in the absence of flotillin-1. Western analysis of ephrinB2-HA expression in the presence of CoMO or F1aMO and increasing amounts of the broad-spectrum metalloprotease inhibitors BB-94 and GM6001, as indicated. (b) ADAM10 reduces ephrinB2 expression, but is inhibited by the presence of endogenous flotillin-1. Western analysis of ephrinB2-HA in embryos injected with ADAM10MO and/or F1aMO, and ADAM10-V5 RNA as indicated. Erk2 is used as a loading control. (c) Specific knockdown of ADAM10 rescues ephrinB2 loss in the presence of F1aMO. Western analysis of ephrinB2-HA in the presence of F1aMO alone or with the indicated ADAM MO. Erk2 is used as a loading control. (d) ADAM10 overexpression reduces ephrinB2 expression in a dose-dependent manner. Western analysis of embryos expressing ephrinB2-HA with increasing amounts of ADAM10-V5 or ADAM17-V5. (e) EphrinB2 amino acid sequence. Black line indicates the globular region of the ephrinB2 ectodomain that is known to bind Eph receptors. The black boxes indicate amino acids 168–177, 182–194, and 197–218 in the juxtamembrane region of the ephrinB2 ectodomain, the grey box denotes the transmembrane domain, and the six asterisks indicate the six tyrosine residues in the intracellular domain of ephrinB2. (f) Amino acids 182–214 of ephrinB2 are important for the decrease in ephrinB2 mediated by F1MO. Western analysis of ephrinB2 mutants lacking the indicated amino acids or juxtamembrane domain in the presence or absence of F1aMO. (g) Amino acids 182–214 of ephrinB2 are important for the decrease in ephrinB2 mediated by ADAM10. Western analysis of ephrinB2 mutants lacking the indicated amino acids or juxtamembrane domain in the presence or absence of ADAM10.
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Figure 4: ADAM10 is responsible for the loss of ephrinB2 expression in the absence of flotillin-1(a) Metalloproteases are responsible for the loss of ephrinB2 expression in the absence of flotillin-1. Western analysis of ephrinB2-HA expression in the presence of CoMO or F1aMO and increasing amounts of the broad-spectrum metalloprotease inhibitors BB-94 and GM6001, as indicated. (b) ADAM10 reduces ephrinB2 expression, but is inhibited by the presence of endogenous flotillin-1. Western analysis of ephrinB2-HA in embryos injected with ADAM10MO and/or F1aMO, and ADAM10-V5 RNA as indicated. Erk2 is used as a loading control. (c) Specific knockdown of ADAM10 rescues ephrinB2 loss in the presence of F1aMO. Western analysis of ephrinB2-HA in the presence of F1aMO alone or with the indicated ADAM MO. Erk2 is used as a loading control. (d) ADAM10 overexpression reduces ephrinB2 expression in a dose-dependent manner. Western analysis of embryos expressing ephrinB2-HA with increasing amounts of ADAM10-V5 or ADAM17-V5. (e) EphrinB2 amino acid sequence. Black line indicates the globular region of the ephrinB2 ectodomain that is known to bind Eph receptors. The black boxes indicate amino acids 168–177, 182–194, and 197–218 in the juxtamembrane region of the ephrinB2 ectodomain, the grey box denotes the transmembrane domain, and the six asterisks indicate the six tyrosine residues in the intracellular domain of ephrinB2. (f) Amino acids 182–214 of ephrinB2 are important for the decrease in ephrinB2 mediated by F1MO. Western analysis of ephrinB2 mutants lacking the indicated amino acids or juxtamembrane domain in the presence or absence of F1aMO. (g) Amino acids 182–214 of ephrinB2 are important for the decrease in ephrinB2 mediated by ADAM10. Western analysis of ephrinB2 mutants lacking the indicated amino acids or juxtamembrane domain in the presence or absence of ADAM10.

Mentions: It has previously been shown that the ectodomain of ephrins can be cleaved by metalloproteases. For example, ephrinB1 is cleaved by matrix metalloproteinase-8 (MMP-8), and ephrin-A2 and ephrin-A5 are cleaved by ADAM1020–21. Thus, we tested the possibility that a metalloprotease cleaves ephrinB2 and causes its decreased expression in the absence of flotillin-1. We found that two different broad-spectrum metalloprotease inhibitors, BB-94 and GM6001, both stabilized ephrinB2 expression in F1aMO-injected embryos (Fig. 4a). Recently, it was shown that Xenopus ephrinB1 and ephrinB2 are cleaved by ADAM1311. Among identified Xenopus orthologues of ADAM family proteins22, we selected ADAM10, ADAM13, and ADAM17 to test whether these ectodomain sheddases22–25 may target ephrinB2 for cleavage and lead to the loss of its expression in the absence of flotillin-1. Surprisingly, knockdown of ADAM10 prevented a considerable portion of the loss of ephrinB2 protein that results from the introduction of the F1aMO, while knockdown of ADAM13 or ADAM17 had no such effect (Fig. 4b, c). Reintroduction of ADAM10 in the presence of the ADAM10 MO led to reduced ephrinB2 levels, supporting that ADAM10 specifically reduces ephrinB2 protein levels (Fig. 4b). Together, these data suggest that flotillin-1 inhibits ADAM10-mediated ephrinB2 loss. To confirm that ADAM10 is the specific metalloprotease that reduces ephrinB2 protein expression, ADAM10 and ADAM17 were over-expressed with ephrinB2 (Fig. 4d). Indeed, ADAM10 over-expression significantly lowers ephrinB2 protein levels in a dose-dependent manner, while ADAM17 has more subtle effects (Fig. 4d). To control for non-specific competitive inhibition of ephrinB2 translation, GFP was over-expressed using the same RNA concentrations, and this had no effect on ephrinB2 protein levels (Supplementary Fig. 2).


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)

ADAM10 is responsible for the loss of ephrinB2 expression in the absence of flotillin-1(a) Metalloproteases are responsible for the loss of ephrinB2 expression in the absence of flotillin-1. Western analysis of ephrinB2-HA expression in the presence of CoMO or F1aMO and increasing amounts of the broad-spectrum metalloprotease inhibitors BB-94 and GM6001, as indicated. (b) ADAM10 reduces ephrinB2 expression, but is inhibited by the presence of endogenous flotillin-1. Western analysis of ephrinB2-HA in embryos injected with ADAM10MO and/or F1aMO, and ADAM10-V5 RNA as indicated. Erk2 is used as a loading control. (c) Specific knockdown of ADAM10 rescues ephrinB2 loss in the presence of F1aMO. Western analysis of ephrinB2-HA in the presence of F1aMO alone or with the indicated ADAM MO. Erk2 is used as a loading control. (d) ADAM10 overexpression reduces ephrinB2 expression in a dose-dependent manner. Western analysis of embryos expressing ephrinB2-HA with increasing amounts of ADAM10-V5 or ADAM17-V5. (e) EphrinB2 amino acid sequence. Black line indicates the globular region of the ephrinB2 ectodomain that is known to bind Eph receptors. The black boxes indicate amino acids 168–177, 182–194, and 197–218 in the juxtamembrane region of the ephrinB2 ectodomain, the grey box denotes the transmembrane domain, and the six asterisks indicate the six tyrosine residues in the intracellular domain of ephrinB2. (f) Amino acids 182–214 of ephrinB2 are important for the decrease in ephrinB2 mediated by F1MO. Western analysis of ephrinB2 mutants lacking the indicated amino acids or juxtamembrane domain in the presence or absence of F1aMO. (g) Amino acids 182–214 of ephrinB2 are important for the decrease in ephrinB2 mediated by ADAM10. Western analysis of ephrinB2 mutants lacking the indicated amino acids or juxtamembrane domain in the presence or absence of ADAM10.
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Figure 4: ADAM10 is responsible for the loss of ephrinB2 expression in the absence of flotillin-1(a) Metalloproteases are responsible for the loss of ephrinB2 expression in the absence of flotillin-1. Western analysis of ephrinB2-HA expression in the presence of CoMO or F1aMO and increasing amounts of the broad-spectrum metalloprotease inhibitors BB-94 and GM6001, as indicated. (b) ADAM10 reduces ephrinB2 expression, but is inhibited by the presence of endogenous flotillin-1. Western analysis of ephrinB2-HA in embryos injected with ADAM10MO and/or F1aMO, and ADAM10-V5 RNA as indicated. Erk2 is used as a loading control. (c) Specific knockdown of ADAM10 rescues ephrinB2 loss in the presence of F1aMO. Western analysis of ephrinB2-HA in the presence of F1aMO alone or with the indicated ADAM MO. Erk2 is used as a loading control. (d) ADAM10 overexpression reduces ephrinB2 expression in a dose-dependent manner. Western analysis of embryos expressing ephrinB2-HA with increasing amounts of ADAM10-V5 or ADAM17-V5. (e) EphrinB2 amino acid sequence. Black line indicates the globular region of the ephrinB2 ectodomain that is known to bind Eph receptors. The black boxes indicate amino acids 168–177, 182–194, and 197–218 in the juxtamembrane region of the ephrinB2 ectodomain, the grey box denotes the transmembrane domain, and the six asterisks indicate the six tyrosine residues in the intracellular domain of ephrinB2. (f) Amino acids 182–214 of ephrinB2 are important for the decrease in ephrinB2 mediated by F1MO. Western analysis of ephrinB2 mutants lacking the indicated amino acids or juxtamembrane domain in the presence or absence of F1aMO. (g) Amino acids 182–214 of ephrinB2 are important for the decrease in ephrinB2 mediated by ADAM10. Western analysis of ephrinB2 mutants lacking the indicated amino acids or juxtamembrane domain in the presence or absence of ADAM10.
Mentions: It has previously been shown that the ectodomain of ephrins can be cleaved by metalloproteases. For example, ephrinB1 is cleaved by matrix metalloproteinase-8 (MMP-8), and ephrin-A2 and ephrin-A5 are cleaved by ADAM1020–21. Thus, we tested the possibility that a metalloprotease cleaves ephrinB2 and causes its decreased expression in the absence of flotillin-1. We found that two different broad-spectrum metalloprotease inhibitors, BB-94 and GM6001, both stabilized ephrinB2 expression in F1aMO-injected embryos (Fig. 4a). Recently, it was shown that Xenopus ephrinB1 and ephrinB2 are cleaved by ADAM1311. Among identified Xenopus orthologues of ADAM family proteins22, we selected ADAM10, ADAM13, and ADAM17 to test whether these ectodomain sheddases22–25 may target ephrinB2 for cleavage and lead to the loss of its expression in the absence of flotillin-1. Surprisingly, knockdown of ADAM10 prevented a considerable portion of the loss of ephrinB2 protein that results from the introduction of the F1aMO, while knockdown of ADAM13 or ADAM17 had no such effect (Fig. 4b, c). Reintroduction of ADAM10 in the presence of the ADAM10 MO led to reduced ephrinB2 levels, supporting that ADAM10 specifically reduces ephrinB2 protein levels (Fig. 4b). Together, these data suggest that flotillin-1 inhibits ADAM10-mediated ephrinB2 loss. To confirm that ADAM10 is the specific metalloprotease that reduces ephrinB2 protein expression, ADAM10 and ADAM17 were over-expressed with ephrinB2 (Fig. 4d). Indeed, ADAM10 over-expression significantly lowers ephrinB2 protein levels in a dose-dependent manner, while ADAM17 has more subtle effects (Fig. 4d). To control for non-specific competitive inhibition of ephrinB2 translation, GFP was over-expressed using the same RNA concentrations, and this had no effect on ephrinB2 protein levels (Supplementary Fig. 2).

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