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Meltrin beta/ADAM19 interacting with EphA4 in developing neural cells participates in formation of the neuromuscular junction.

Yumoto N, Wakatsuki S, Kurisaki T, Hara Y, Osumi N, Frisén J, Sehara-Fujisawa A - PLoS ONE (2008)

Bottom Line: Meltrin beta plays a regulatory role in formation of the NMJ.The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph.We propose that Meltrin beta stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Shogo-in, Kyoto, Japan.

ABSTRACT

Background: Development of the neuromuscular junction (NMJ) is initiated by the formation of postsynaptic specializations in the central zones of muscles, followed by the arrival of motor nerve terminals opposite the postsynaptic regions. The post- and presynaptic components are then stabilized and modified to form mature synapses. Roles of ADAM (A Disintegrin And Metalloprotease) family proteins in the formation of the NMJ have not been reported previously.

Principal findings: We report here that Meltrin beta, ADAM19, participates in the formation of the NMJ. The zone of acetylcholine receptor alpha mRNA distribution was broader and excess sprouting of motor nerve terminals was more prominent in meltrin beta-deficient than in wild-type embryonic diaphragms. A microarray analysis revealed that the preferential distribution of ephrin-A5 mRNA in the synaptic region of muscles was aberrant in the meltrin beta-deficient muscles. Excess sprouting of motor nerve terminals was also found in ephrin-A5 knockout mice, which lead us to investigate a possible link between Meltrin beta and ephrin-A5-Eph signaling in the development of the NMJ. Meltrin beta and EphA4 interacted with each other in developing motor neurons, and both of these proteins localized in the NMJ. Coexpression of Meltrin beta and EphA4 strongly blocked vesicular internalization of ephrin-A5-EphA4 complexes without requiring the protease activity of Meltrin beta, suggesting a regulatory role of Meltrin beta in ephrin-A5-Eph signaling.

Conclusion: Meltrin beta plays a regulatory role in formation of the NMJ. The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph. We propose that Meltrin beta stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development.

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Meltrin β is expressed at the NMJ and participates in the formation of the NMJ.(A–C) Adult tibialis anterior (T.A.) muscles were stained with an anti-Meltrin β antibody (A) and Alexa-594–conjugated α-bungarotoxin (BTX) (B). Meltrin β was expressed at NMJs (C, arrowheads). Meltrin β was also expressed in a part of muscle fibers. Bar: 10 µm. (D–I) The diaphragms of wild-type (D–F) and meltrin β−/− (G–I) mice at E18.5 were stained with an anti-synaptophysin antibody (D and G) to label axon terminals and with BTX (E and H) to label acetylcholine receptors (AChRs). AChR clusters were distributed more broadly (H and I, also compare the bars in E and H) and the nerve terminals sprouted excessively (G and I, arrowheads) in the meltrin β−/− muscle compared with in the wild-type muscle (F). Bar: 100 µm. (J and K) In situ hybridization with probes for AChR α-subunit mRNA in E18.5 diaphragms of wild-type (J) and meltrin β−/− (K) mice. The central zone expressing the AChR α-subunit mRNA is broader in meltrin β−/− (K) than in the wild-type diaphragm (J) (compare the bars in J and K). (L) Neonatal (P0) diaphragms were stained with BTX. AChR clusters were distributed more broadly in meltrin β−/− than in wild-type diaphragms (left panel). The average size of the individual AChR clusters was smaller in meltrin β−/− muscles relative to that in wild-type muscles (right panel). Bar: 100 µm.
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pone-0003322-g001: Meltrin β is expressed at the NMJ and participates in the formation of the NMJ.(A–C) Adult tibialis anterior (T.A.) muscles were stained with an anti-Meltrin β antibody (A) and Alexa-594–conjugated α-bungarotoxin (BTX) (B). Meltrin β was expressed at NMJs (C, arrowheads). Meltrin β was also expressed in a part of muscle fibers. Bar: 10 µm. (D–I) The diaphragms of wild-type (D–F) and meltrin β−/− (G–I) mice at E18.5 were stained with an anti-synaptophysin antibody (D and G) to label axon terminals and with BTX (E and H) to label acetylcholine receptors (AChRs). AChR clusters were distributed more broadly (H and I, also compare the bars in E and H) and the nerve terminals sprouted excessively (G and I, arrowheads) in the meltrin β−/− muscle compared with in the wild-type muscle (F). Bar: 100 µm. (J and K) In situ hybridization with probes for AChR α-subunit mRNA in E18.5 diaphragms of wild-type (J) and meltrin β−/− (K) mice. The central zone expressing the AChR α-subunit mRNA is broader in meltrin β−/− (K) than in the wild-type diaphragm (J) (compare the bars in J and K). (L) Neonatal (P0) diaphragms were stained with BTX. AChR clusters were distributed more broadly in meltrin β−/− than in wild-type diaphragms (left panel). The average size of the individual AChR clusters was smaller in meltrin β−/− muscles relative to that in wild-type muscles (right panel). Bar: 100 µm.

Mentions: To examine the role of Meltrin β in the development of the NMJ, we first investigated the expression of Meltrin β at the NMJ in muscles (Fig. 1A–C). Anti-Meltrin β antibody recognizes the C-terminus of Meltrin β, and show little affinity to other proteins in meltrin β−/− mice ([22], also see Figure S1S). Acetylcholine receptors (AChRs) were visualized with Alexa-594–α-bungarotoxin (BTX). In the adult, Meltrin β was expressed at NMJs (Fig. 1C, arrowheads). During development, expression of Meltrin β gradually clustered at the NMJ during the late stage of embryogenesis (Figure S1).


Meltrin beta/ADAM19 interacting with EphA4 in developing neural cells participates in formation of the neuromuscular junction.

Yumoto N, Wakatsuki S, Kurisaki T, Hara Y, Osumi N, Frisén J, Sehara-Fujisawa A - PLoS ONE (2008)

Meltrin β is expressed at the NMJ and participates in the formation of the NMJ.(A–C) Adult tibialis anterior (T.A.) muscles were stained with an anti-Meltrin β antibody (A) and Alexa-594–conjugated α-bungarotoxin (BTX) (B). Meltrin β was expressed at NMJs (C, arrowheads). Meltrin β was also expressed in a part of muscle fibers. Bar: 10 µm. (D–I) The diaphragms of wild-type (D–F) and meltrin β−/− (G–I) mice at E18.5 were stained with an anti-synaptophysin antibody (D and G) to label axon terminals and with BTX (E and H) to label acetylcholine receptors (AChRs). AChR clusters were distributed more broadly (H and I, also compare the bars in E and H) and the nerve terminals sprouted excessively (G and I, arrowheads) in the meltrin β−/− muscle compared with in the wild-type muscle (F). Bar: 100 µm. (J and K) In situ hybridization with probes for AChR α-subunit mRNA in E18.5 diaphragms of wild-type (J) and meltrin β−/− (K) mice. The central zone expressing the AChR α-subunit mRNA is broader in meltrin β−/− (K) than in the wild-type diaphragm (J) (compare the bars in J and K). (L) Neonatal (P0) diaphragms were stained with BTX. AChR clusters were distributed more broadly in meltrin β−/− than in wild-type diaphragms (left panel). The average size of the individual AChR clusters was smaller in meltrin β−/− muscles relative to that in wild-type muscles (right panel). Bar: 100 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2552171&req=5

pone-0003322-g001: Meltrin β is expressed at the NMJ and participates in the formation of the NMJ.(A–C) Adult tibialis anterior (T.A.) muscles were stained with an anti-Meltrin β antibody (A) and Alexa-594–conjugated α-bungarotoxin (BTX) (B). Meltrin β was expressed at NMJs (C, arrowheads). Meltrin β was also expressed in a part of muscle fibers. Bar: 10 µm. (D–I) The diaphragms of wild-type (D–F) and meltrin β−/− (G–I) mice at E18.5 were stained with an anti-synaptophysin antibody (D and G) to label axon terminals and with BTX (E and H) to label acetylcholine receptors (AChRs). AChR clusters were distributed more broadly (H and I, also compare the bars in E and H) and the nerve terminals sprouted excessively (G and I, arrowheads) in the meltrin β−/− muscle compared with in the wild-type muscle (F). Bar: 100 µm. (J and K) In situ hybridization with probes for AChR α-subunit mRNA in E18.5 diaphragms of wild-type (J) and meltrin β−/− (K) mice. The central zone expressing the AChR α-subunit mRNA is broader in meltrin β−/− (K) than in the wild-type diaphragm (J) (compare the bars in J and K). (L) Neonatal (P0) diaphragms were stained with BTX. AChR clusters were distributed more broadly in meltrin β−/− than in wild-type diaphragms (left panel). The average size of the individual AChR clusters was smaller in meltrin β−/− muscles relative to that in wild-type muscles (right panel). Bar: 100 µm.
Mentions: To examine the role of Meltrin β in the development of the NMJ, we first investigated the expression of Meltrin β at the NMJ in muscles (Fig. 1A–C). Anti-Meltrin β antibody recognizes the C-terminus of Meltrin β, and show little affinity to other proteins in meltrin β−/− mice ([22], also see Figure S1S). Acetylcholine receptors (AChRs) were visualized with Alexa-594–α-bungarotoxin (BTX). In the adult, Meltrin β was expressed at NMJs (Fig. 1C, arrowheads). During development, expression of Meltrin β gradually clustered at the NMJ during the late stage of embryogenesis (Figure S1).

Bottom Line: Meltrin beta plays a regulatory role in formation of the NMJ.The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph.We propose that Meltrin beta stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development.

View Article: PubMed Central - PubMed

Affiliation: Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, Shogo-in, Kyoto, Japan.

ABSTRACT

Background: Development of the neuromuscular junction (NMJ) is initiated by the formation of postsynaptic specializations in the central zones of muscles, followed by the arrival of motor nerve terminals opposite the postsynaptic regions. The post- and presynaptic components are then stabilized and modified to form mature synapses. Roles of ADAM (A Disintegrin And Metalloprotease) family proteins in the formation of the NMJ have not been reported previously.

Principal findings: We report here that Meltrin beta, ADAM19, participates in the formation of the NMJ. The zone of acetylcholine receptor alpha mRNA distribution was broader and excess sprouting of motor nerve terminals was more prominent in meltrin beta-deficient than in wild-type embryonic diaphragms. A microarray analysis revealed that the preferential distribution of ephrin-A5 mRNA in the synaptic region of muscles was aberrant in the meltrin beta-deficient muscles. Excess sprouting of motor nerve terminals was also found in ephrin-A5 knockout mice, which lead us to investigate a possible link between Meltrin beta and ephrin-A5-Eph signaling in the development of the NMJ. Meltrin beta and EphA4 interacted with each other in developing motor neurons, and both of these proteins localized in the NMJ. Coexpression of Meltrin beta and EphA4 strongly blocked vesicular internalization of ephrin-A5-EphA4 complexes without requiring the protease activity of Meltrin beta, suggesting a regulatory role of Meltrin beta in ephrin-A5-Eph signaling.

Conclusion: Meltrin beta plays a regulatory role in formation of the NMJ. The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph. We propose that Meltrin beta stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development.

Show MeSH
Related in: MedlinePlus