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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 cleaves ephrinB2 and regulates ephrinB2 protein levels(a) ADAM10 is responsible for shedding of the ephrinB2 ectodomain. Western analysis of embryos expressing ephrinB2-HA with ADAM10-V5 and/or the ADAM10 specific inhibitor GI254023X. The full-length ephrinB2-HA and the C-terminal fragments (CTFs) of ephrinB2-HA are indicated. ADAM10-V5 is indicated, as is the Erk2 loading control.. (b) EphrinB2 associates with ADAM10. ADAM10-HA immunoprecipitation and Western analysis from embryonic lysates co-expressing ephrinB2-Flag and ADAM10-HA or ADAM17-HA. Direct lysates are probed with anti-Flag or HA as indicated. (c) ADAM10 associates with ephrinB1 as well as ephrinB2. Western analysis of the indicated IPs or direct lysates from oocytes expressing ephrinB2-HA or ephrinB1-HA alone or with ADAM10-V5. (d) ADAM10 specifically targets ephrinB2. Western analysis with indicated antibodies of lysates from embryos exogenously expressing increasing amounts of ADAM10-V5 along with ephrinB1-HA or ephrinB2-HA. (e) Endogenous ADAM10 associates with ephrinB2. EphrinB2 was immunoprecipitated from HT-29 cells and Western analysis was performed using anti-ephrinB2 or ADAM10 antibodies. EphrinB2 and ADAM10 expression levels in HT-29 cell lysates are shown. (f) In the presence of MG132, F1aMO leads to increased association between exogenously expressed ephrinB2 and ADAM10. Western analysis of the HA (ephrinB2) IPs and direct lysates from embryos co-expressing ephrinB2-HA and an ADAM10 mutant with compromised protease activity (ADAM10 PD-V5) and injected with the indicated MOs. (g) Endogenous ephrinB2 is cleaved and degraded in the presence of F1aMO, but is partially rescued by an ADAM10 specific inhibitor. Western analysis of lysates from neural folds of embryos injected with the indicated MOs, and the ADAM10 inhibitor GI254023X (st. 9 injection into the blastocoel). Westerns were probed using C-18 (pan-ephrinB) and ERK2 antibodies. (h) ephrinB2 is degraded by both the proteosomal and dynamin-dependent degradation pathways in the absence of flotillin-1. Western analysis with the indicated antibodies of embryos injected with ephrinB2-HA RNA alone or with F1aMO, and treated with vehicle control DMSO (−), or MG132 (M), and/or dynamin inhibitor dynasore (D). Erk2 is used as loading control.
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Figure 5: ADAM10 cleaves ephrinB2 and regulates ephrinB2 protein levels(a) ADAM10 is responsible for shedding of the ephrinB2 ectodomain. Western analysis of embryos expressing ephrinB2-HA with ADAM10-V5 and/or the ADAM10 specific inhibitor GI254023X. The full-length ephrinB2-HA and the C-terminal fragments (CTFs) of ephrinB2-HA are indicated. ADAM10-V5 is indicated, as is the Erk2 loading control.. (b) EphrinB2 associates with ADAM10. ADAM10-HA immunoprecipitation and Western analysis from embryonic lysates co-expressing ephrinB2-Flag and ADAM10-HA or ADAM17-HA. Direct lysates are probed with anti-Flag or HA as indicated. (c) ADAM10 associates with ephrinB1 as well as ephrinB2. Western analysis of the indicated IPs or direct lysates from oocytes expressing ephrinB2-HA or ephrinB1-HA alone or with ADAM10-V5. (d) ADAM10 specifically targets ephrinB2. Western analysis with indicated antibodies of lysates from embryos exogenously expressing increasing amounts of ADAM10-V5 along with ephrinB1-HA or ephrinB2-HA. (e) Endogenous ADAM10 associates with ephrinB2. EphrinB2 was immunoprecipitated from HT-29 cells and Western analysis was performed using anti-ephrinB2 or ADAM10 antibodies. EphrinB2 and ADAM10 expression levels in HT-29 cell lysates are shown. (f) In the presence of MG132, F1aMO leads to increased association between exogenously expressed ephrinB2 and ADAM10. Western analysis of the HA (ephrinB2) IPs and direct lysates from embryos co-expressing ephrinB2-HA and an ADAM10 mutant with compromised protease activity (ADAM10 PD-V5) and injected with the indicated MOs. (g) Endogenous ephrinB2 is cleaved and degraded in the presence of F1aMO, but is partially rescued by an ADAM10 specific inhibitor. Western analysis of lysates from neural folds of embryos injected with the indicated MOs, and the ADAM10 inhibitor GI254023X (st. 9 injection into the blastocoel). Westerns were probed using C-18 (pan-ephrinB) and ERK2 antibodies. (h) ephrinB2 is degraded by both the proteosomal and dynamin-dependent degradation pathways in the absence of flotillin-1. Western analysis with the indicated antibodies of embryos injected with ephrinB2-HA RNA alone or with F1aMO, and treated with vehicle control DMSO (−), or MG132 (M), and/or dynamin inhibitor dynasore (D). Erk2 is used as loading control.

Mentions: ADAM10 cleavage of ephrinB2 should result in two fragments: a 25-kDa amino-terminal fragment, and a 15-kDa carboxyl-terminal fragment (CTF). Without any treatment, the approximately 40-kDa full length ephrinB2-HA protein and two ephrinB2 CTF bands of approximately 15 kDa were detected in embryonic lysates, suggesting that ephrinB2 is cleaved by endogenous ADAM10 and further processed (Fig. 5a). Since it is known that MMP-8 can cleave the ephrinB1 ectodomain and that subsequently γ-secretase cleaves the juxtamembrane region of the intracellular domain20, we speculated that γ-secretase may also cleave the ephrinB2 intracellular domain. This could produce the two observed CTF bands: an upper band generated by ADAM10 cleavage, and a lower band resulting from both ADAM10 and γ-secretase cleavage. When ADAM10 was over-expressed, full-length ephrinB2 was reduced and the upper CTF band was increased, indicating that ADAM10 indeed cleaves ephrinB2 and reduces its expression (Fig. 5a). Conversely, when embryos were treated with the ADAM10 specific inhibitor GI254023X, full-length ephrinB2 was slightly increased and the upper CTF band was reduced, with the greatest difference detected in the embryos over-expressing ADAM10 (Fig. 5a). In addition, we used co-expression and co-immunoprecipitation analysis in embryos to determine whether ADAM10 associates with ephrinB2. We found that ephrinB2 is robustly present in ADAM10 immune complexes, but found at relatively low levels in ADAM17 immmune-complexes, supporting the idea that ephrinB2 is specifically cleaved by the ADAM10 metalloprotease (Fig. 5b). Moreover, although ephrinB1 also associates with ADAM10 in co-IPs from Xenopus oocytes co-expressing both proteins (Fig 5c), only ephrinB2 is a target of ADAM10 cleavage in a dose-dependent manner in embryos (Fig 5d). In HT-29 cells, endogenous ADAM10 was co-immunoprecipitated in ephrinB2 immune complexes (Fig. 5e), confirming that ephrinB2 and ADAM10 associate either directly or indirectly in vivo.


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 cleaves ephrinB2 and regulates ephrinB2 protein levels(a) ADAM10 is responsible for shedding of the ephrinB2 ectodomain. Western analysis of embryos expressing ephrinB2-HA with ADAM10-V5 and/or the ADAM10 specific inhibitor GI254023X. The full-length ephrinB2-HA and the C-terminal fragments (CTFs) of ephrinB2-HA are indicated. ADAM10-V5 is indicated, as is the Erk2 loading control.. (b) EphrinB2 associates with ADAM10. ADAM10-HA immunoprecipitation and Western analysis from embryonic lysates co-expressing ephrinB2-Flag and ADAM10-HA or ADAM17-HA. Direct lysates are probed with anti-Flag or HA as indicated. (c) ADAM10 associates with ephrinB1 as well as ephrinB2. Western analysis of the indicated IPs or direct lysates from oocytes expressing ephrinB2-HA or ephrinB1-HA alone or with ADAM10-V5. (d) ADAM10 specifically targets ephrinB2. Western analysis with indicated antibodies of lysates from embryos exogenously expressing increasing amounts of ADAM10-V5 along with ephrinB1-HA or ephrinB2-HA. (e) Endogenous ADAM10 associates with ephrinB2. EphrinB2 was immunoprecipitated from HT-29 cells and Western analysis was performed using anti-ephrinB2 or ADAM10 antibodies. EphrinB2 and ADAM10 expression levels in HT-29 cell lysates are shown. (f) In the presence of MG132, F1aMO leads to increased association between exogenously expressed ephrinB2 and ADAM10. Western analysis of the HA (ephrinB2) IPs and direct lysates from embryos co-expressing ephrinB2-HA and an ADAM10 mutant with compromised protease activity (ADAM10 PD-V5) and injected with the indicated MOs. (g) Endogenous ephrinB2 is cleaved and degraded in the presence of F1aMO, but is partially rescued by an ADAM10 specific inhibitor. Western analysis of lysates from neural folds of embryos injected with the indicated MOs, and the ADAM10 inhibitor GI254023X (st. 9 injection into the blastocoel). Westerns were probed using C-18 (pan-ephrinB) and ERK2 antibodies. (h) ephrinB2 is degraded by both the proteosomal and dynamin-dependent degradation pathways in the absence of flotillin-1. Western analysis with the indicated antibodies of embryos injected with ephrinB2-HA RNA alone or with F1aMO, and treated with vehicle control DMSO (−), or MG132 (M), and/or dynamin inhibitor dynasore (D). Erk2 is used as loading control.
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Figure 5: ADAM10 cleaves ephrinB2 and regulates ephrinB2 protein levels(a) ADAM10 is responsible for shedding of the ephrinB2 ectodomain. Western analysis of embryos expressing ephrinB2-HA with ADAM10-V5 and/or the ADAM10 specific inhibitor GI254023X. The full-length ephrinB2-HA and the C-terminal fragments (CTFs) of ephrinB2-HA are indicated. ADAM10-V5 is indicated, as is the Erk2 loading control.. (b) EphrinB2 associates with ADAM10. ADAM10-HA immunoprecipitation and Western analysis from embryonic lysates co-expressing ephrinB2-Flag and ADAM10-HA or ADAM17-HA. Direct lysates are probed with anti-Flag or HA as indicated. (c) ADAM10 associates with ephrinB1 as well as ephrinB2. Western analysis of the indicated IPs or direct lysates from oocytes expressing ephrinB2-HA or ephrinB1-HA alone or with ADAM10-V5. (d) ADAM10 specifically targets ephrinB2. Western analysis with indicated antibodies of lysates from embryos exogenously expressing increasing amounts of ADAM10-V5 along with ephrinB1-HA or ephrinB2-HA. (e) Endogenous ADAM10 associates with ephrinB2. EphrinB2 was immunoprecipitated from HT-29 cells and Western analysis was performed using anti-ephrinB2 or ADAM10 antibodies. EphrinB2 and ADAM10 expression levels in HT-29 cell lysates are shown. (f) In the presence of MG132, F1aMO leads to increased association between exogenously expressed ephrinB2 and ADAM10. Western analysis of the HA (ephrinB2) IPs and direct lysates from embryos co-expressing ephrinB2-HA and an ADAM10 mutant with compromised protease activity (ADAM10 PD-V5) and injected with the indicated MOs. (g) Endogenous ephrinB2 is cleaved and degraded in the presence of F1aMO, but is partially rescued by an ADAM10 specific inhibitor. Western analysis of lysates from neural folds of embryos injected with the indicated MOs, and the ADAM10 inhibitor GI254023X (st. 9 injection into the blastocoel). Westerns were probed using C-18 (pan-ephrinB) and ERK2 antibodies. (h) ephrinB2 is degraded by both the proteosomal and dynamin-dependent degradation pathways in the absence of flotillin-1. Western analysis with the indicated antibodies of embryos injected with ephrinB2-HA RNA alone or with F1aMO, and treated with vehicle control DMSO (−), or MG132 (M), and/or dynamin inhibitor dynasore (D). Erk2 is used as loading control.
Mentions: ADAM10 cleavage of ephrinB2 should result in two fragments: a 25-kDa amino-terminal fragment, and a 15-kDa carboxyl-terminal fragment (CTF). Without any treatment, the approximately 40-kDa full length ephrinB2-HA protein and two ephrinB2 CTF bands of approximately 15 kDa were detected in embryonic lysates, suggesting that ephrinB2 is cleaved by endogenous ADAM10 and further processed (Fig. 5a). Since it is known that MMP-8 can cleave the ephrinB1 ectodomain and that subsequently γ-secretase cleaves the juxtamembrane region of the intracellular domain20, we speculated that γ-secretase may also cleave the ephrinB2 intracellular domain. This could produce the two observed CTF bands: an upper band generated by ADAM10 cleavage, and a lower band resulting from both ADAM10 and γ-secretase cleavage. When ADAM10 was over-expressed, full-length ephrinB2 was reduced and the upper CTF band was increased, indicating that ADAM10 indeed cleaves ephrinB2 and reduces its expression (Fig. 5a). Conversely, when embryos were treated with the ADAM10 specific inhibitor GI254023X, full-length ephrinB2 was slightly increased and the upper CTF band was reduced, with the greatest difference detected in the embryos over-expressing ADAM10 (Fig. 5a). In addition, we used co-expression and co-immunoprecipitation analysis in embryos to determine whether ADAM10 associates with ephrinB2. We found that ephrinB2 is robustly present in ADAM10 immune complexes, but found at relatively low levels in ADAM17 immmune-complexes, supporting the idea that ephrinB2 is specifically cleaved by the ADAM10 metalloprotease (Fig. 5b). Moreover, although ephrinB1 also associates with ADAM10 in co-IPs from Xenopus oocytes co-expressing both proteins (Fig 5c), only ephrinB2 is a target of ADAM10 cleavage in a dose-dependent manner in embryos (Fig 5d). In HT-29 cells, endogenous ADAM10 was co-immunoprecipitated in ephrinB2 immune complexes (Fig. 5e), confirming that ephrinB2 and ADAM10 associate either directly or indirectly in vivo.

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