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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). (CâE) Eighteen-hour-old spinal neurons were fixed and coimmunolabeled with anti-p120ctn and antiâsynapsin-I antibodies. A diffuse distribution of p120ctn was observed in axons with no specific concentration at SV puncta. (FâH) No signal was detected if the primary antibody was replaced with antiâhemagglutinin tag antibody (G). SV clusters labeled by antiâsynapsin-I antibody often existed at the base of filopodia or at varicosities (C, E and F, H).
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FIGURE 2:. The involvement of p120ctn in the formation of axonal filopodia and SV clusters. (AâC) Control axons expressing GFP exhibited filopodia and SV clusters (arrows in C) along their length. (DâF) Overexpression of GFP-linked, wild-type p120ctn (WTp120) in neurons enhanced axonal filopodial formation (arrowheads in D) and SV clustering (arrows in F). (GâI) Expression of mutant p120ctn with a deletion of the Rho-regulatory domain (Îp120, 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 Îp120-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 Îp120-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.
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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 antiâsynapsin-I labeling (B), respectively. WTp120-neurons (C, D) grew more filopodia and showed more SV clusters than did control neurons, whereas Îp120-neurons (E, F) exhibited fewer filopodia and a reduced density of SV clusters. (GâL) 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 Îp120 had few filopodia and displayed correspondingly diminished F-actin labeling, except along the axonal shaft.
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FIGURE 4:. The effect of RhoA inhibitor C3 transferase on filopodial assembly and SV clustering. (AâC) Control neurons expressing GFP had filopodia and SV clusters along axons, and after treatment with C3 toxin (Aâ²âCâ²) they showed an increase in axonal filopodia and SV clustering. (DâF) WTp120 expression enhanced both filopodial assembly and SV clustering, and addition of C3 toxin to these neurons (Dâ²âFâ²) did not further elevate the effect. (GâI) Îp120 expression suppressed the formation of filopodia and SV clusters, and, significantly, this inhibitory effect of Îp120 was reversed in the presence of C3 toxin (Gâ²âIâ²). 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.
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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. (AâC) GFP-neurons developed filopodia and SV clusters along axons, and treatment with Y27632 (Aâ²âCâ²) increased axonal filopodia and SV clustering in these neurons. (DâF) WTp120 expression by itself increased filopodial formation and SV clustering in neurons, and this was not further elevated by treatment with the inhibitor (Dâ²âFâ²). (GâI) Îp120 expression inhibited filopodial growth and SV clustering, and the suppressive effect of Îp120 was partially overcome in the presence of Y27632 (Gâ²âIâ²). 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.
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FIGURE 6:. The effect of p120ctn on bFGF induction of filopodia and SV clusters in neurons. (AâC) Filopodial and SV cluster formation in control GFP-expressing neurons. (Aâ²âCâ²) Treatment with bFGF elevated the density of both filopodia and SV clusters. (DâF) Overexpression of WTp120 on its own increased these two axonal specializations, and treatment with bFGF (Dâ²âFâ²) slightly enhanced filopodial formation (see J for quantification). (GâI) Îp120 expression suppressed both specializations and also inhibited the effect of bFGF (Gâ²âIâ²) 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.
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FIGURE 7:. Influence of bFGF on p120ctn's tyrosine phosphorylation and cadherin association. NIH3T3-L1 fibroblasts were serum starved for 16â18 h and then stimulated for 30 min without (â) 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 â¼30% after bFGF treatment (quantified on the right); the control anti-hemagglutinin antibody captured neither p120ctn nor cadherin. Ratios of band densities presented in AâC 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.
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FIGURE 8:. The function of neuronal p120ctn in NMJ formation. Spinal neurons expressing GFP (AâC), WTp120 (DâF), or Rho-mutant Îp120 (GâI) were cocultured with 3-d-old muscle cells cultured from uninjected embryos, and NMJ formation along nerveâmuscle 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, Îp120ctn-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.
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