Chen C, Li PP, Madhavan R, Peng HB.

	FIGURE 1:. Filopodia and SV clusters in Xenopus spinal neurons. (A, B) Axonal filopodia contacted muscle cells in cocultures (arrowheads in A) and R-BTX labeling showed that AChR clusters formed at these contacts (arrows in B). (C–E) Eighteen-hour-old spinal neurons were fixed and coimmunolabeled with anti-p120ctn and anti–synapsin-I antibodies. A diffuse distribution of p120ctn was observed in axons with no specific concentration at SV puncta. (F–H) No signal was detected if the primary antibody was replaced with anti–hemagglutinin tag antibody (G). SV clusters labeled by anti–synapsin-I antibody often existed at the base of filopodia or at varicosities (C, E and F, H).
	FIGURE 2:. The involvement of p120ctn in the formation of axonal filopodia and SV clusters. (A–C) Control axons expressing GFP exhibited filopodia and SV clusters (arrows in C) along their length. (D–F) Overexpression of GFP-linked, wild-type p120ctn (WTp120) in neurons enhanced axonal filopodial formation (arrowheads in D) and SV clustering (arrows in F). (G–I) Expression of mutant p120ctn with a deletion of the Rho-regulatory domain (Δp120, also linked with GFP) suppressed filopodial and SV cluster formation along the axon. (J) Quantification based on data collected from cultures prepared from three separate batches of mRNA-injected embryos. The densities of filopodia (left y-axis) and SV puncta (right y-axis) per 10 μm of axonal length were calculated. (K) To examine filopodial dynamics in neurons expressing exogenous proteins, time-lapse recording was carried out. Neurons were imaged for 10 min at 1-min time intervals. and the rates of new filopodial growth were calculated for GFP-, WTp120-, and Δp120-expressing neurons; these values were normalized relative to that in GFP-expressing neurons. WTp120-expressing neurons showed a twofold increase in the rate of filopodial assembly compared with GFP-neurons, whereas in Δp120-expressing neurons the rate of filopodial formation was very low. Data are shown as mean and SEM. *p < 0.05, **p < 0.01, and ***,^^^p < 0.001 compared with control.
	FIGURE 3:. Induction of axonal filopodia and SV clusters by p120ctn and the association of F-actin with filopodia. In fixed cultures of control GFP-neurons, filopodia and SV clusters along axons could be detected by green fluorescence (A) and by anti–synapsin-I labeling (B), respectively. WTp120-neurons (C, D) grew more filopodia and showed more SV clusters than did control neurons, whereas Δp120-neurons (E, F) exhibited fewer filopodia and a reduced density of SV clusters. (G–L) Rhodamine-conjugated phalloidin was used to visualize F-actin enrichment in axons. In GFP axons (G, H) F-actin was enriched in filopodia, and in the WTp120-neurons (I, J), where more filopodia formed, F-actin labeling was also pronounced. (K, L) Axons expressing Δp120 had few filopodia and displayed correspondingly diminished F-actin labeling, except along the axonal shaft.
	FIGURE 4:. The effect of RhoA inhibitor C3 transferase on filopodial assembly and SV clustering. (A–C) Control neurons expressing GFP had filopodia and SV clusters along axons, and after treatment with C3 toxin (A′–C′) they showed an increase in axonal filopodia and SV clustering. (D–F) WTp120 expression enhanced both filopodial assembly and SV clustering, and addition of C3 toxin to these neurons (D′–F′) did not further elevate the effect. (G–I) Δp120 expression suppressed the formation of filopodia and SV clusters, and, significantly, this inhibitory effect of Δp120 was reversed in the presence of C3 toxin (G′–I′). These changes in the densities of filopodia and SV clusters are quantified in J and K. Mean and SEM are shown. *p < 0.05, ***p < 0.001, compared with control; ^p < 0.05, ^^p < 0.01, ^^^p < 0.001, compared with no treatment.
	FIGURE 5:. The effect of ROCK-1 inhibitor on filopodial assembly and SV clustering. The role of Rho GTPase signaling was further tested using Y27632, which inhibits the RhoA effector ROCK-1. (A–C) GFP-neurons developed filopodia and SV clusters along axons, and treatment with Y27632 (A′–C′) increased axonal filopodia and SV clustering in these neurons. (D–F) WTp120 expression by itself increased filopodial formation and SV clustering in neurons, and this was not further elevated by treatment with the inhibitor (D′–F′). (G–I) Δp120 expression inhibited filopodial growth and SV clustering, and the suppressive effect of Δp120 was partially overcome in the presence of Y27632 (G′–I′). Both J and K quantify these changes in filopodial formation and SV clustering. Mean and SEM are shown. *p < 0.05, **p < 0.01, compared with control; ^p < 0.05, ^^p < 0.01, compared with no treatment.
	FIGURE 6:. The effect of p120ctn on bFGF induction of filopodia and SV clusters in neurons. (A–C) Filopodial and SV cluster formation in control GFP-expressing neurons. (A′–C′) Treatment with bFGF elevated the density of both filopodia and SV clusters. (D–F) Overexpression of WTp120 on its own increased these two axonal specializations, and treatment with bFGF (D′–F′) slightly enhanced filopodial formation (see J for quantification). (G–I) Δp120 expression suppressed both specializations and also inhibited the effect of bFGF (G′–I′) on filopodial and SV cluster induction. The densities of bFGF-induced filopodia (J) and SV clusters (K) were calculated from data pooled from three separate mRNA injections and culture preparations. Mean and SEM shown; t test, **p < 0.01, compared with control; ^p < 0.05, ^^p < 0.01, compared with no treatment.
	FIGURE 7:. Influence of bFGF on p120ctn's tyrosine phosphorylation and cadherin association. NIH3T3-L1 fibroblasts were serum starved for 16–18 h and then stimulated for 30 min without (–) or with (+) bFGF (200 ng/ml) before preparing total protein extracts. (A) Extracts were immunoblotted with antibodies against p120ctn or p120ctn phosphorylated on three specific tyrosine residues (Y228, Y280, and Y291) and tubulin (loading control). Treatment with bFGF did not alter p120ctn levels in extracts, but it increased the phosphorylation of p120ctn at Y280 robustly and Y228 and Y291 slightly, as shown in the quantification on the right. (B) To confirm that bFGF stimulated the cells in these experiments, we also used a portion of the extracts for immunoprecipitating FRS2α, a downstream target of bFGF signaling. Staining with anti-FRS2α (top) and anti-phosphotyrosine mAb4G10 (pY; bottom) antibodies showed that bFGF stimulation significantly enhanced FRS2α's tyrosine phosphorylation (quantified on the right); a polyclonal GFP antibody used as a negative control did not capture FRS2α. (C) In parallel experiments when p120ctn was immunoprecipitated from extracts (top), under control conditions cadherin (bottom) coprecipitated with p120ctn, but the amount of cadherin associated with p120ctn was reduced by ∼30% after bFGF treatment (quantified on the right); the control anti-hemagglutinin antibody captured neither p120ctn nor cadherin. Ratios of band densities presented in A–C were quantified from three replicates for each set. The ratios of samples from bFGF-treated cells were normalized relative to those from control to obtain the fold change produced by bFGF. Mean and SEM are shown. *p < 0.05, **p < 0.01, ***p < 0.001, compared with control.
	FIGURE 8:. The function of neuronal p120ctn in NMJ formation. Spinal neurons expressing GFP (A–C), WTp120 (D–F), or Rho-mutant Δp120 (G–I) were cocultured with 3-d-old muscle cells cultured from uninjected embryos, and NMJ formation along nerve–muscle contacts was examined after 1 d. The expression of exogenous proteins in spinal neurons was confirmed by GFP fluorescence, and labeling with R-BTX identified AChR clusters in muscle. Neurons expressing GFP (B) and WTp120ctn (E) effectively induced AChR clusters where they contacted muscle cells (C, F), with clusters present at 66.3 ± 7.7 and 78.3 ± 15.3% of contacts between muscle cells and GFP- and WTp120ctn-neurons, respectively (J). In contrast, Δp120ctn-expressing neurons (H) induced AChR clustering (I) at only 37.5 ± 9.7% of contacts with muscle cells (J). Arrowheads point to innervating axons, and arrows mark AChR clusters. Mean and SEM are shown. *p < 0.05, compared with control.

Aktories, Clostridial Rho-inhibiting protein toxins. 2005, Pubmed

Aktories, Clostridial Rho-inhibiting protein toxins. 2005, Pubmed
Anastasiadis, Regulation of Rho GTPases by p120-catenin. 2001, Pubmed
Anastasiadis, Inhibition of RhoA by p120 catenin. 2000, Pubmed
Arikkath, Cadherins and catenins at synapses: roles in synaptogenesis and synaptic plasticity. 2008, Pubmed
Banks, The postsynaptic submembrane machinery at the neuromuscular junction: requirement for rapsyn and the utrophin/dystrophin-associated complex. 2003, Pubmed
Brigidi, Cadherin-catenin adhesion complexes at the synapse. 2011, Pubmed
Buchanan, Studies of nerve-muscle interactions in Xenopus cell culture: fine structure of early functional contacts. 1989, Pubmed , Xenbase
Ciesiolka, p120 catenin is required for morphogenetic movements involved in the formation of the eyes and the craniofacial skeleton in Xenopus. 2004, Pubmed , Xenbase
Coleman, Basic fibroblast growth factor sensitizes NIH 3T3 cells to apoptosis induced by cisplatin. 2000, Pubmed
Dai, Dynamics of synaptic vesicles in cultured spinal cord neurons in relationship to synaptogenesis. 1996, Pubmed , Xenbase
Dai, Presynaptic differentiation induced in cultured neurons by local application of basic fibroblast growth factor. 1995, Pubmed , Xenbase
Dent, Axon guidance by growth cones and branches: common cytoskeletal and signaling mechanisms. 2003, Pubmed
Downing, PDGF, CSF-1, and EGF induce tyrosine phosphorylation of p120, a pp60src transformation-associated substrate. 1991, Pubmed
Elia, p120 catenin regulates dendritic spine and synapse development through Rho-family GTPases and cadherins. 2006, Pubmed
Esser, Vascular endothelial growth factor induces VE-cadherin tyrosine phosphorylation in endothelial cells. 1998, Pubmed
Eswarakumar, Cellular signaling by fibroblast growth factor receptors. 2005, Pubmed
Fang, Vertebrate development requires ARVCF and p120 catenins and their interplay with RhoA and Rac. 2004, Pubmed , Xenbase
Fertuck, Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after 125I-alpha-bungarotoxin binding at mouse neuromuscular junctions. 1976, Pubmed
Fitzsimonds, Retrograde signaling in the development and modification of synapses. 1998, Pubmed
Fox, Seeking long-term relationship: axon and target communicate to organize synaptic differentiation. 2006, Pubmed
Fox, Distinct target-derived signals organize formation, maturation, and maintenance of motor nerve terminals. 2007, Pubmed
Gallo, Regulation of growth cone actin filaments by guidance cues. 2004, Pubmed
Glass, Agrin acts via a MuSK receptor complex. 1996, Pubmed
Glass, Sequential roles of agrin, MuSK and rapsyn during neuromuscular junction formation. 1997, Pubmed
Harris, Dendritic spines: cellular specializations imparting both stability and flexibility to synaptic function. 1994, Pubmed
Hotulainen, Defining mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis. 2009, Pubmed
Hughes, Molecular architecture of the neuromuscular junction. 2006, Pubmed
Ishizaki, Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. 2000, Pubmed
Johnson-Venkatesh, Secreted factors as synaptic organizers. 2010, Pubmed
Lee, The function of mitochondria in presynaptic development at the neuromuscular junction. 2008, Pubmed , Xenbase
Lee, Mitochondrial clustering at the vertebrate neuromuscular junction during presynaptic differentiation. 2006, Pubmed , Xenbase
Lee, Synapses are regulated by the cytoplasmic tyrosine kinase Fer in a pathway mediated by p120catenin, Fer, SHP-2, and beta-catenin. 2008, Pubmed
Li, Axonal filopodial asymmetry induced by synaptic target. 2011, Pubmed , Xenbase
Lin, Cytoskeletal remodeling during growth cone-target interactions. 1993, Pubmed
Loudon, RhoA-kinase and myosin II are required for the maintenance of growth cone polarity and guidance by nerve growth factor. 2006, Pubmed
Mackay, The Rho's progress: a potential role during neuritogenesis for the Rho family of GTPases. 1995, Pubmed
Madhavan, Molecular regulation of postsynaptic differentiation at the neuromuscular junction. 2005, Pubmed
Madhavan, Involvement of p120 catenin in myopodial assembly and nerve-muscle synapse formation. 2006, Pubmed , Xenbase
McCrea, The catenin family at a glance. 2010, Pubmed
Nakajima, The development of functional neuromuscular junctions in vitro: an ultrastructural and physiological study. 1980, Pubmed
Nobes, Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. 1995, Pubmed
Noren, p120 catenin regulates the actin cytoskeleton via Rho family GTPases. 2000, Pubmed
Olivo, Distinct involvement of cdc42 and RhoA GTPases in actin organization and cell shape in untransformed and Dbl oncogene transformed NIH3T3 cells. 2000, Pubmed
Paulson, Misexpression of the catenin p120(ctn)1A perturbs Xenopus gastrulation but does not elicit Wnt-directed axis specification. 1999, Pubmed , Xenbase
Peng, Tissue culture of Xenopus neurons and muscle cells as a model for studying synaptic induction. 1991, Pubmed , Xenbase
Reynolds, p120, a novel substrate of protein tyrosine kinase receptors and of p60v-src, is related to cadherin-binding factors beta-catenin, plakoglobin and armadillo. 1992, Pubmed , Xenbase
Reynolds, Transformation-specific tyrosine phosphorylation of a novel cellular protein in chicken cells expressing oncogenic variants of the avian cellular src gene. 1989, Pubmed
Reynolds, p120-catenin: Past and present. 2007, Pubmed
Reynolds, Exposing p120 catenin's most intimate affair. 2010, Pubmed
Reynolds, The novel catenin p120cas binds classical cadherins and induces an unusual morphological phenotype in NIH3T3 fibroblasts. 1996, Pubmed
Ritzenthaler, Myopodia (postsynaptic filopodia) participate in synaptic target recognition. 2003, Pubmed
Seidel, E- and N-cadherin differ with respect to their associated p120ctn isoforms and their ability to suppress invasive growth in pancreatic cancer cells. 2004, Pubmed
Siksou, Ultrastructural organization of presynaptic terminals. 2011, Pubmed
Terauchi, Distinct FGFs promote differentiation of excitatory and inhibitory synapses. 2010, Pubmed
Uhm, Synapse-forming axons and recombinant agrin induce microprocess formation on myotubes. 2001, Pubmed
Weldon, Development of synaptic ultrastructure at neuromuscular contacts in an amphibian cell culture system. 1979, Pubmed , Xenbase
Wildenberg, p120-catenin and p190RhoGAP regulate cell-cell adhesion by coordinating antagonism between Rac and Rho. 2006, Pubmed