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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.
Figure 1 Myopodia in Xenopus nerveâmuscle cocultures.
Cocultures of spinal neurons and myotomal muscle
cells derived from Xenopus embryos were examined for
myopodia by phase-contrast microscopy (AâC). Myopodia
(arrows) were detected along the edges of muscle cells
(ââmââ) close to nerves (âânââ) 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 0â14 myopodia (n ¼ 313) and 0â27 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 (AâC). 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 cellâcell
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 (AâD) by
anti-p120ctn and anti-EP-cadherin antibodies was examined
in the presence or absence of agrin. In untreated cells,
p120ctn was concentrated at cellâcell 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 muscleâmuscle 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 (ââþPââ; all panels)
or with agrin (ââþAPââ; all panels) for 1 h, and Triton X-100 extracts prepared from them were
examined by immunoblotting (ââIBsââ; 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
(ââIPsââ; all panels) were carried out using monoclonal antibodies against Shp2
(ââshpââ) or p120ctn (ââp120ââ) 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 (0.5â4 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) (AâE) or GFP only (F). In p120ctn-GFP-expressing cells, elongated, ââdendriticââ
structures (A) and myopodia (B) were present, and exogenous p120ctn-GFP was recruited to
muscleâmuscle (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, DâF),
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 agrinâs 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 4â6 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
(EâG; KâM) but fewer clusters were detected in the mutant cells (HâJ; NâP). However, where neurites
directly contacted muscle expressing wild-type (QâS) or mutant p120ctn (TâV), AChR clusters
were induced to similar levels. Myopodial and AChR microcluster densities at partial neuritemuscle
contacts are presented in Table 1.
