Journal: PLoS ONE

Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

doi: 10.1371/journal.pone.0008478

Figure Lengend Snippet: Cultured embryonic Xenopus muscle cells labeled with rhodamine-α-bungarotoxin (R-BTX) were stimulated overnight with polystyrene beads coated with heparan-binding growth-associated molecule (HB-GAM) (A, D; asterisks) to induce AChR clusters (C, F). Cells were then fixed and labeled with affinity-purified polyclonal antibodies against the Arp2/3 complex proteins Arp2 (B) and p34arc (E) followed by FITC-linked anti-rabbit secondary antibodies. Separately, bead-stimulated muscle cells (G) were labeled with anti-p34arc polyclonal (H) and anti-cortactin monoclonal (I; mAb4F11) antibodies and then FITC-conjugated anti-rabbit and rhodamine-conjugated anti-mouse secondary antibodies. AChRs, Arp2 and p34arc were clustered at bead-muscle contacts (A-F; arrows) where cortactin localized and overlapped in distribution with p34arc (H and I; arrows). In primary muscle cultures non-muscle cells were occasionally found (J) and in these cells p34arc (K) and cortactin (L) localized along the cell periphery (arrowheads) but were not clustered at bead-cell contacts (“b” in K and L corresponds to bead indicated by asterisk in J).

Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

Techniques: Cell Culture, Labeling, Binding Assay, Affinity Purification

Journal: PLoS ONE

Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

doi: 10.1371/journal.pone.0008478

Figure Lengend Snippet: Xenopus muscle cells were labeled with R-BTX and after fixation with an antibody that specifically recognizes Y482-phospho-cortactin (plus FITC-linked anti-rabbit antibodies) (A-F). In some cases muscle cells were first co-cultured for 1 d with spinal neurons and then labeled with R-BTX and anti-phospho-cortactin and secondary antibodies (G-I). In pure muscle cultures (A, D) large “pre-patterned” AChR clusters were present (B, E; arrows) and at these sites staining by anti-phospho-cortactin was significantly stronger than elsewhere in muscle cells (C, F; arrows). Labeling for phospho-cortactin was detected at almost all pre-patterned clusters examined (see ), although within the clusters certain regions at times appeared to be more enriched in phospho-cortactin than others (as in F; arrow versus arrowhead). The anti-phospho-cortactin antibody also labeled muscle cell edges (C, F) where cortactin is known to be localized. In nerve-muscle co-cultures (G) AChRs were selectively concentrated at synaptic contacts (H; arrows) and these nerve-induced AChR clusters were also labeled by the anti-phospho-cortactin antibody (I; arrows).

Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

Techniques: Labeling, Cell Culture, Staining

Journal: PLoS ONE

Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

doi: 10.1371/journal.pone.0008478

Figure Lengend Snippet: R-BTX-labeled Xenopus muscle cells were stimulated overnight with HB-GAM-beads (A-C) or neural agrin (D-F). In cells exposed to beads (A; asterisks) AChRs aggregated at bead-muscle contacts (B; arrows) and strong labeling was detected at these bead-induced AChR clusters for Y482-phospho-cortactin (C; arrows). Treatment of muscle cells with agrin (D) generated numerous small (∼0.5-3 µm) AChR clusters (D; arrows) and antibody labeling showed that phospho-cortactin was enriched at these clusters (E; arrows) and also along myopodia that formed near the AChR clusters (F; arrows and arrowheads).

Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

Techniques: Labeling, Generated, Antibody Labeling

Journal: PLoS ONE

Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

doi: 10.1371/journal.pone.0008478

Figure Lengend Snippet: Cultured C2 mouse myotubes were exposed to medium without (-) or with added agrin (+) before preparing extracts for immuno-precipitation (A) with a monoclonal antibody against cortactin (IP: cort) or an unrelated protein (IP: ctl). When these samples were immuno-blotted for cortactin (IB: cort) and total phosphotyrosine (IB: PY; mAb4G10), cortactin was found to be captured only by the anti-cortactin antibody (upper lanes), and anti-phosphotyrosine staining showed that cortactin from extracts of agrin-treated cells was tyrosine phosphorylated significantly more than that captured from control extracts (lower lanes). This increase in cortactin phosphorylation was quantified from four experiments (A, graph) by measuring band densities, normalizing for cortactin loading (see ), and calculating the phosphotyrosine level change relative to control. B. To test whether the src-target sites in cortactin were phosphorylated in response to agrin-treatment, myotube extracts were blotted with antibodies against total cortactin and cortactin phosphorylated on Y421. Agrin-treatment did not alter the amount of cortactin present in extracts (upper lanes) but the staining of cortactin by the anti-Y421-phospho-cortactin antibody (IB: pCort) was enhanced by agrin-treatment more than two-fold, as shown by quantification from three experiments (B, graph). Positions of pre-stained MW markers (Bio-Rad) are indicated on the right side of blots, and in the graphs * represents P<0.02 in t-tests.

Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

Techniques: Cell Culture, Immunoprecipitation, Staining, Control, Phospho-proteomics

Journal: PLoS ONE

Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

doi: 10.1371/journal.pone.0008478

Figure Lengend Snippet: To examine the effect of exogenous cortactin proteins on AChR clustering, C2 myotubes were transfected with mRNAs encoding GFP (Ctl) or GFP-tagged phospho-mutant (3YF) cortactin or wild-type (WT) cortactin. After treating myotubes with agrin overnight, cells expressing exogenous proteins (A, D, G; asterisks) were identified by green fluorescence (B, E, H) and the AChR clusters present on the surface of these cells were examined by R-BTX-labeling (C, F, I; arrows). Forced expression of the phospho-mutant, but not wild-type, cortactin reduced the number and lengths of agrin-induced AChR clusters in myotubes. J. To biochemically confirm the expression of exogenous cortactin proteins in myotubes, extracts prepared from myotubes transfected with mRNAs encoding GFP, GFP-tagged WT and 3YF cortactin were immuno-blotted with anti-cortactin monoclonal antibody mAb4F11. Myotubes transfected with GFP mRNA (G) contained full-length endogenous cortactin (arrow on left), but those transfected with WT- and 3YF-cortactin mRNAs contained endogenous cortactin plus a protein (∼25 kD larger) corresponding to exogenous, GFP-tagged cortactin (asterisk). MW marker positions are indicated on the right. K-L. Myotubes transfected with GFP or GFP-tagged cortactin proteins were selected randomly and the numbers and lengths of the AChR clusters present on their surface were determined; data from five separate transfection experiments were pooled and normalized relative to values obtained from GFP-tranfected cells. Fewer (K) and smaller (L) AChR clusters were present in myotubes expressing phospho-mutant cortactin than in cells expressing GFP alone or WT-cortactin-GFP. Mean and SEM values are shown, *P<0.05.

Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

Techniques: Transfection, Mutagenesis, Expressing, Fluorescence, Labeling, Marker

Journal: PLoS ONE

Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

doi: 10.1371/journal.pone.0008478

Figure Lengend Snippet: C2 myotubes generated from myoblasts transfected with control siRNAs (A-C) or a pool of siRNAs directed against mouse cortactin (D-F) (both mixed with a cDNA encoding GFP) were incubated overnight in differentiation medium containing agrin before labeling with R-BTX. Transfected myotubes (A, D; asterisks) were identified by green fluorescence (B, E), and the AChR clusters present on their surface (C, F; arrows) were counted and the lengths of these clusters were measured. G. To demonstrate that siRNAs against cortactin knocked down cortactin expression, in each experiment extracts were prepared from myotubes generated from myoblasts transfected in parallel and maintained under conditions identical to those used for examining agrin-induced AChR clustering. Extracts of cells transfected with GFP cDNA plus control (p120ctn) siRNA (Ctl; left lane), cortactin siRNA (middle lane) or GFP cDNA alone (right lane) were immuno-blotted with antibodies against cortactin (upper blot) or tubulin (lower blot). The cortactin siRNA suppressed the expression of cortactin without affecting unrelated proteins (such as tubulin, which is also shown here to demonstrate equal protein loading), and cortactin's expression was not affected by control siRNAs or by transfection procedures (where only GFP cDNA was used). From four transfection experiments AChR cluster data from control (Ctl) and mouse cortactin (msCort) siRNA-transfected myotubes were pooled and normalized relative to those obtained from cells transfected with the control siRNA. These results showed that agrin-induced AChR cluster numbers (H) and lengths (I) were significantly lower in myotubes expressing reduced levels of endogenous cortactin compared to those expressing normal levels of cortactin. Mean and SEM values are shown, *P<0.05.

Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

Techniques: Generated, Transfection, Control, Incubation, Labeling, Fluorescence, Expressing

Journal: PLoS ONE

Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

doi: 10.1371/journal.pone.0008478

Figure Lengend Snippet: Xenopus embryonic muscle cells expressing GFP (A-D) and GFP-tagged wild-type cortactin (WT-cort; E-H) and phospho-mutant cortactin (3YF-cort; I-N) were co-cultured with spinals neurons for 1 d and then labeled with R-BTX to visualize AChR clusters. Cells expressing the exogenous proteins fluoresced green and AChR clusters appeared red, as shown in this figure with 2-3 representative examples of nerves contacting muscle cells with GFP or GFP-tagged cortactin proteins. In the GFP and WT-cort muscle cells (A-H), AChRs were tightly clustered (arrows) along nerve-contacts identified (traced in white in colored panels) but this was not the case in 3YF-cort cells where nerves often induced no AChR clustering (J) or induced few clusters that were loosely organized (L; arrowheads). We found cases where the same neurites moved across normal muscle cells and 3YF-cells (M-N) and in such cases synaptic AChR clustering was robust in the normal cells (arrows) but not mutant cells (arrowheads). O. The percentages of nerve-contacts with AChR clusters were determined by examining several co-cultures with muscle cells expressing GFP or the GFP-tagged cortactin proteins (see ) and these values were normalized relative to numbers obtained from examining nerve-contacts on GFP-cells. In muscle cells expressing phospho-mutant cortactin, synaptic AChR clustering was almost halved. Nerve-muscle contacts examined: GFP cells, 168; WT-cort cells, 214; 3YF-cort cells, 225; mean and SEM shown, *P<0.0001.

Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

Techniques: Expressing, Mutagenesis, Cell Culture, Labeling

Journal: PLoS ONE

Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

doi: 10.1371/journal.pone.0008478

Figure Lengend Snippet: Activation of MuSK by agrin induces AChR clustering in an actin polymerization-dependent manner. This model depicts a possible way in which cortactin signaling might promote the AChR clustering process. Initiation of intracellular signaling by the activated MuSK complex could enhance cortactin's tyrosine phosphorylation through src family tyrosine kinases (SFKs) (and possibly other kinases such as abl), and cortactin, in turn, could increase actin polymerization. Alternatively, cortactin might trigger actin polymerization by activating the Arp2/3 complex, either on its own or in concert with WASP-related proteins (N-WASP, WIP, etc.) to which it could be linked by the adapter Nck. In parallel, via other signaling intermediates, MuSK could stimulate Rho-family GTPases and, through them, F-actin assembly. Such enhanced and dynamic actin polymerization at synaptic sites could generate a scaffold which “traps” AChRs through rapsyn.

Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

Techniques: Activation Assay, Phospho-proteomics