Madhavan R et al. (2006), Involvement of p120 catenin in myopodial assemb...

XB-ART-35118

J Neurobiol 2006 Nov 01;6613:1511-27. doi: 10.1002/neu.20320.

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Involvement of p120 catenin in myopodial assembly and nerve-muscle synapse formation.

Madhavan R , Zhao XT , Reynolds AB , Peng HB .

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At developing neuromuscular junctions (NMJs), muscles initially contact motor axons by microprocesses, or myopodia, which are induced by nerves and nerve-secreted agrin, but it is unclear how myopodia are assembled and how they influence synaptic differentiation at the NMJ. Here, we report that treatment of cultured muscle cells with agrin transiently depleted p120 catenin (p120ctn) from cadherin junctions in situ, and increased the tyrosine phosphorylation and decreased the cadherin-association of p120ctn in cell extracts. Whereas ectopic expression of wild-type p120ctn in muscle generated myopodia in the absence of agrin, expression of a specific dominant-negative mutant form of p120ctn, which blocks filopodial assembly in nonmuscle cells, suppressed nerve- and agrin-induction of myopodia. Significantly, approaching neurites triggered reduced acetylcholine receptor (AChR) clustering along the edges of muscle cells expressing mutant p120ctn than of control cells, although the ability of the mutant cells to cluster AChRs was itself normal. Our results indicate a novel role of p120ctn in agrin-induced myopodial assembly and suggest that myopodia increase muscle-nerve contacts and muscle's access to neural agrin to promote NMJ formation.

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Species referenced: Xenopus
Genes referenced: ctnnd1 dag1 isyna1 ppp3ca ptpn11

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	Figure 1 Myopodia in Xenopus nerve–muscle cocultures. Cocultures of spinal neurons and myotomal muscle cells derived from Xenopus embryos were examined for myopodia by phase-contrast microscopy (A–C). Myopodia (arrows) were detected along the edges of muscle cells (‘‘m’’) close to nerves (‘‘n’’) or neuritic processes. Clustering of muscle AChRs was visualized using rhodamineconjugated -bungarotoxin (R-BTX) (D).
	Figure 2 Agrin induction of myopodia and AChR microclusters. Xenopus muscle cells were treated without (A) or with (B) agrin for 2 h and stained with rhodamine-conjugated con A and a monoclonal antibody against phosphotyrosine (mAb4G10), followed by FITC-conjugated antimouse secondary antibody. In quiescent Xenopus muscle cells, myopodia were seldom detected along the edges (A), but they were observed in agrin-treated cells (B). In the agrin-treated cells, AChR microclusters (C, F) were found preferentially localized near myopodia, visualized by antiphosphotyrosine antibody labeling (D, G; thin arrows), and regions of cells lacking AChR clusters were mostly devoid of myopodia (thick arrows). Both AChR microclusters and myopodia were strongly labeled by the antiphosphotyrosine antibody (merged images; E, H). (I) This graph shows the correlation between agrin-induced myopodial formation and AChR clustering. 40 10 m2 regions were randomly selected along the edges of agrin-treated muscle cells (at least 2 per cell) and all myopodia and AChR microclusters within them were counted. A total of 66 regions were examined which contained 0–14 myopodia (n ¼ 313) and 0–27 AChR microclusters (n ¼ 464); linear regression, r2 ¼ 0.92.
	Figure 3 VVAB4 labeling of myopodial and muscle junctions. R-BTX-labeled Xenopus muscle cells were treated with agrin (2 h) and stained with FITC-conjugated VVAB4 (A–C). Agrin induced AChR microclusters and myopodia and, in this example myopodia from the muscle cell on the left contacted the cell on the right. Contacts between the myopodia and the neighboring muscle were strongly labeled by VVAB4 (C, arrows) but the general muscle membrane and regions of myopodia not touching the neighboring cell were not. VVAB4 stained all cell–cell junctions between quiescent muscle cells (D, F), and these sites were strongly labeled by an antibody against EP-cadherin (E, arrows) but not -dystroglycan (G, arrowheads).
	Figure 4 Localization and agrin-induced redistribution of p120ctn in situ. Labeling of Xenopus muscle (A–D) by anti-p120ctn and anti-EP-cadherin antibodies was examined in the presence or absence of agrin. In untreated cells, p120ctn was concentrated at cell–cell contacts (A; arrowheads), but it was depleted from these sites in cells exposed to agrin for 1 h (B; arrows). EP-cadherin was concentrated at muscle–muscle junctions (arrowheads) after 1 h agrintreatment (C), and so was p120ctn in muscle cells exposed to agrin for 4 h (D). Agrin-treatment did not affect the junctional localization of p120ctn in nonmuscle cells (E, F; arrowheads).
	Figure 5 Effect of agrin on the tyrosine phosphorylation and cadherin-association of p120ctn in cultured mouse myotubes. (A) C2 mouse myotube cultures were treated without (‘‘þP’’; all panels) or with agrin (‘‘þAP’’; all panels) for 1 h, and Triton X-100 extracts prepared from them were examined by immunoblotting (‘‘IBs’’; all panels), using antibodies against p120ctn, Shp2, and cadherin; agrin-treatment did not affect the total levels of these proteins in muscle extracts. (B) Immunoprecipitations (‘‘IPs’’; all panels) were carried out using monoclonal antibodies against Shp2 (‘‘shp’’) or p120ctn (‘‘p120’’) from extracts of myotubes treated with or without agrin (1 h). IP samples were immunoblotted with anti-Shp2 and anti-p120ctn antibodies (top) or with antiphosphotyrosine antibody (bottom). Anti-Shp2 and anti-p120ctn antibodies specifically captured their target proteins and the anti-phosphotyrosine antibody more strongly stained p120ctn captured from agrin-treated cells compared with that from untreated cells (lane 4 vs. 2). In this and following panels, positions of MW markers are indicated on the left; asterisks on the right denote a nonspecific mouse secondary antibody-stained band. (C) Mouse myotubes were treated with agrin for different times (0.5–4 h) and p120ctn was immunoprecipitated from their extracts. After loading equal amounts of p120ctn (anti-p120ctn staining; top blot), blots were stained with antibodies against phosphotyrosine and cadherin. Agrin-treatment increased the tyrosine phosphorylation of p120ctn up to 1 h, which then decreased (middle blot). Cadherin coprecipitated with p120ctn (bottom blot), and the association between cadherin and p120ctn was diminished by 1 h agrin-treatment (bottom blot and relative band intensity graph). (D) Agrin-treatment reduced cadherin association with p120ctn. Immunoblotting with the anticadherin antibody was carried out on equally loaded p120ctn IP samples from untreated and agrin-treated (1 h) myotube extracts. The cadherin band staining intensities were measured and normalized relative to that obtained in IP samples from untreated myotube extracts. The coprecipitation of cadherin with p120ctn was significantly reduced by agrin-treatment (4 experiments; p < 0.002). (E) Cadherin coprecipitated with -catenin (top) and -catenin (bottom) from myotube extracts, and this was not significantly affected by agrin-treatment. As controls in the IPs, for the anti--catenin antibody (top) a monoclonal anti-Shp2 antibody was used, whereas for the anti-cadherin antibody (bottom) a rabbit polyclonal antibody against calcineurin was used.
	Figure 6 Ectopic p120ctn expression and myopodial formation in Xenopus muscle cells. Muscle cells were cultured from Xenopus embryos injected with mRNAs encoding a p120ctn-GFP fusion protein (p120GFP) (A–E) or GFP only (F). In p120ctn-GFP-expressing cells, elongated, ‘‘dendritic’’ structures (A) and myopodia (B) were present, and exogenous p120ctn-GFP was recruited to muscle–muscle (arrows) and myopodia-muscle (arrowheads) contacts (C). As shown by merged images in panels (D) and (C), respectively, in these cells, AChR hot spots and agrin-induced AChR clusters appeared normal. GFP expressed in muscle cells was not recruited to cell contacts, and it did not induce dendritic structures or myopodia (F). Scale bar ¼ 20 m in panels (A, D–F), 6.67 m in panel (B), and 10 m in panel (C).
	Figure 7 Ectopic expression of wild-type and mutant p120ctn proteins and agrin-induced myopodial assembly. To directly address the role of p120ctn in agrin-induced formation of myopodia, muscle cells cultured from Xenopus embryos injected with mRNAs encoding GFP or GFPtagged wild-type and mutant p120ctn proteins were examined without (A, C, E, G) or with (B, D, F, H) agrin-treatment (2 h). Muscle cells and myopodia (arrows) were visualized by GFP fluorescence, and percentages of fluorescent cells with myopodia were determined (I). Agrin induced myopodia in GFP expressing cells (A, B) to a level that could be achieved by wild-type p120ctn (WT p120) expression alone (C); agrin-treatment increased the fraction of p120ctn-expressing cells with myopodia (D). Expression of the p120ctn deletion mutant protein (D p120) did not induce myopodia (E) and suppressed agrin’s ability to generate myopodia (F). Myopodia were generated by overexpression of a p120ctn mutant protein lacking src phosphorylation sites (8YF p120) (G), but here agrin-treatment did not increase myopodial formation (H). Data were pooled from experiments carried out on 4–6 separate culture preparations, using mRNA-injected embryos (I); number of cells examined for GFP ¼ 347; WT p120 ¼ 526; D p120 ¼ 408; and 8YF p120 ¼ 301. Asterisks indicate t test p values below 0.005.
	Figure 8 Ectopic expression of wild-type and mutant p120ctn proteins and nerve-induction of myopodia and AChR clusters. Xenopus muscle cells expressing exogenous wild-type p120ctn (left) or the deletion-mutant p120ctn (right) were cocultured for one day with spinal nerves. In live cultures, edges of muscle cells close to neuritic processes, classified as partial contacts, were examined for myopodia. At such sites, myopodia were readily detected in cells expressing wild-type p120ctn (A, B) (arrows) but few were present in cells expressing mutant p120ctn (C, D). Next, by R-BTX labeling, numerous AChR microclusters (arrows) were found in wild-type p120ctn-expressing cells (E–G; K–M) but fewer clusters were detected in the mutant cells (H–J; N–P). However, where neurites directly contacted muscle expressing wild-type (Q–S) or mutant p120ctn (T–V), AChR clusters were induced to similar levels. Myopodial and AChR microcluster densities at partial neuritemuscle contacts are presented in Table 1.