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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 regulates vesicular internalization of the ephrin-A5–EphA4 complexes.(A–L) Ephrin-A5-Fc fusion proteins bound to EphA4-HA transformants equally regardless of Meltrin β expression at a temperature that inhibits endocytosis (12°C; A–D). In contrast, when the cells were put at a temperature that promotes endocytosis (37°C), Meltrin β substantially inhibited endocytosis of ephrin–Eph complexes (E–H, arrowheads: intracellular vesicles containing ephrin-Eph complexes). Control human Fc fragments do not promote endocytosis (I–L). Asterisk: Meltrin β positive cells. Blue signals in panels C, G, and K are DAPI staining of nuclei. Bar: 10 µm. (M–P) The same experiments were performed for Meltrin β EQ mutant–expressing cells. EQ mutant proteins also inhibit internalization of ephrin–Eph complexes. (Q) The number of the cells that did not have intracellular vesicles containing ephrin–Eph complexes was counted. The number of cells without endosomes containing ephrin–Eph complexes was approximately 7 times or 5 times higher in the Meltrin β WT–positive cells or in the Meltrin β EQ mutant–positive cells respectively than in the control cells (P<0.01, Student's t-test).
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pone-0003322-g006: Meltrin β regulates vesicular internalization of the ephrin-A5–EphA4 complexes.(A–L) Ephrin-A5-Fc fusion proteins bound to EphA4-HA transformants equally regardless of Meltrin β expression at a temperature that inhibits endocytosis (12°C; A–D). In contrast, when the cells were put at a temperature that promotes endocytosis (37°C), Meltrin β substantially inhibited endocytosis of ephrin–Eph complexes (E–H, arrowheads: intracellular vesicles containing ephrin-Eph complexes). Control human Fc fragments do not promote endocytosis (I–L). Asterisk: Meltrin β positive cells. Blue signals in panels C, G, and K are DAPI staining of nuclei. Bar: 10 µm. (M–P) The same experiments were performed for Meltrin β EQ mutant–expressing cells. EQ mutant proteins also inhibit internalization of ephrin–Eph complexes. (Q) The number of the cells that did not have intracellular vesicles containing ephrin–Eph complexes was counted. The number of cells without endosomes containing ephrin–Eph complexes was approximately 7 times or 5 times higher in the Meltrin β WT–positive cells or in the Meltrin β EQ mutant–positive cells respectively than in the control cells (P<0.01, Student's t-test).

Mentions: To test whether Meltrin β regulates the internalization of the ephrin-A5-EphA4 complex, we added ephrin-A5-Fc or human Fc fragment (as a negative control) to EphA4-HA transformants, and analyzed the complex with anti-Fc fragment antibodies by using a confocal microscope (Fig. 6). When the cells were incubated at 12°C—a process that inhibits endocytosis—ephrin-A5-Fc bound to the cell surface uniformly whether or not the cells were exogenously expressing Meltrin β (Fig. 6A–D), suggesting that Meltrin β does not affect the affinity between ephrin-A5-Fc and EphA4. When cells not transfected with Meltrin β were incubated at 37°C—a process that allows endocytotic activity—ephrin-A5-Fc was internalized into the cells after it interacted with EphA4, and numerous intracellular particles containing ephrin-Eph complexes were observed (Fig. 6E, arrowheads). In contrast, the internalization of the ephrin-Eph complexes was inhibited in cells transfected with Meltrin β, although ephrin-A5-Fc bound to the cell surface (Fig. 6E, asterisk). Human Fc fragments did not bind to or was not endocytosed significantly in this assay (Fig. 6I–L), indicating that internalization of the complexes was dependent on the association of ephrin-A5 with EphA4. As quantitative results, the number of cells without endosomes containing the ephrin-Eph complexes (that is, in which endocytosis was inhibited) was approximately 7 times higher for cells expressing Meltrin β than for control cells (Fig. 6Q). Meltrin β EQ mutant proteins also inhibited the endocytotic activity as much as Meltrin β WT did (Fig. 6M–P, Q), indicating that the protease activity of Meltrin β is not needed for this regulation of endocytosis.


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 regulates vesicular internalization of the ephrin-A5–EphA4 complexes.(A–L) Ephrin-A5-Fc fusion proteins bound to EphA4-HA transformants equally regardless of Meltrin β expression at a temperature that inhibits endocytosis (12°C; A–D). In contrast, when the cells were put at a temperature that promotes endocytosis (37°C), Meltrin β substantially inhibited endocytosis of ephrin–Eph complexes (E–H, arrowheads: intracellular vesicles containing ephrin-Eph complexes). Control human Fc fragments do not promote endocytosis (I–L). Asterisk: Meltrin β positive cells. Blue signals in panels C, G, and K are DAPI staining of nuclei. Bar: 10 µm. (M–P) The same experiments were performed for Meltrin β EQ mutant–expressing cells. EQ mutant proteins also inhibit internalization of ephrin–Eph complexes. (Q) The number of the cells that did not have intracellular vesicles containing ephrin–Eph complexes was counted. The number of cells without endosomes containing ephrin–Eph complexes was approximately 7 times or 5 times higher in the Meltrin β WT–positive cells or in the Meltrin β EQ mutant–positive cells respectively than in the control cells (P<0.01, Student's t-test).
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Related In: Results  -  Collection

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pone-0003322-g006: Meltrin β regulates vesicular internalization of the ephrin-A5–EphA4 complexes.(A–L) Ephrin-A5-Fc fusion proteins bound to EphA4-HA transformants equally regardless of Meltrin β expression at a temperature that inhibits endocytosis (12°C; A–D). In contrast, when the cells were put at a temperature that promotes endocytosis (37°C), Meltrin β substantially inhibited endocytosis of ephrin–Eph complexes (E–H, arrowheads: intracellular vesicles containing ephrin-Eph complexes). Control human Fc fragments do not promote endocytosis (I–L). Asterisk: Meltrin β positive cells. Blue signals in panels C, G, and K are DAPI staining of nuclei. Bar: 10 µm. (M–P) The same experiments were performed for Meltrin β EQ mutant–expressing cells. EQ mutant proteins also inhibit internalization of ephrin–Eph complexes. (Q) The number of the cells that did not have intracellular vesicles containing ephrin–Eph complexes was counted. The number of cells without endosomes containing ephrin–Eph complexes was approximately 7 times or 5 times higher in the Meltrin β WT–positive cells or in the Meltrin β EQ mutant–positive cells respectively than in the control cells (P<0.01, Student's t-test).
Mentions: To test whether Meltrin β regulates the internalization of the ephrin-A5-EphA4 complex, we added ephrin-A5-Fc or human Fc fragment (as a negative control) to EphA4-HA transformants, and analyzed the complex with anti-Fc fragment antibodies by using a confocal microscope (Fig. 6). When the cells were incubated at 12°C—a process that inhibits endocytosis—ephrin-A5-Fc bound to the cell surface uniformly whether or not the cells were exogenously expressing Meltrin β (Fig. 6A–D), suggesting that Meltrin β does not affect the affinity between ephrin-A5-Fc and EphA4. When cells not transfected with Meltrin β were incubated at 37°C—a process that allows endocytotic activity—ephrin-A5-Fc was internalized into the cells after it interacted with EphA4, and numerous intracellular particles containing ephrin-Eph complexes were observed (Fig. 6E, arrowheads). In contrast, the internalization of the ephrin-Eph complexes was inhibited in cells transfected with Meltrin β, although ephrin-A5-Fc bound to the cell surface (Fig. 6E, asterisk). Human Fc fragments did not bind to or was not endocytosed significantly in this assay (Fig. 6I–L), indicating that internalization of the complexes was dependent on the association of ephrin-A5 with EphA4. As quantitative results, the number of cells without endosomes containing the ephrin-Eph complexes (that is, in which endocytosis was inhibited) was approximately 7 times higher for cells expressing Meltrin β than for control cells (Fig. 6Q). Meltrin β EQ mutant proteins also inhibited the endocytotic activity as much as Meltrin β WT did (Fig. 6M–P, Q), indicating that the protease activity of Meltrin β is not needed for this regulation of endocytosis.

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