Madhavan R et al. (2009), The function of cortactin in the clustering of ...

XB-ART-41310

PLoS One 2009 Dec 29;412:e8478. doi: 10.1371/journal.pone.0008478.

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The function of cortactin in the clustering of acetylcholine receptors at the vertebrate neuromuscular junction.

Madhavan R , Gong ZL , Ma JJ , Chan AW , Peng HB .

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BACKGROUND: Postsynaptic enrichment of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction (NMJ) depends on the activation of the muscle receptor tyrosine MuSK by neural agrin. Agrin-stimulation of MuSK is known to initiate an intracellular signaling cascade that leads to the clustering of AChRs in an actin polymerization-dependent manner, but the molecular steps which link MuSK activation to AChR aggregation remain incompletely defined. METHODOLOGY/PRINCIPAL FINDINGS: In this study we used biochemical, cell biological and molecular assays to investigate a possible role in AChR clustering of cortactin, a protein which is a tyrosine kinase substrate and a regulator of F-actin assembly and which has also been previously localized at AChR clustering sites. We report that cortactin was co-enriched at AChR clusters in situ with its target the Arp2/3 complex, which is a key stimulator of actin polymerization in cells. Cortactin was further preferentially tyrosine phosphorylated at AChR clustering sites and treatment of myotubes with agrin significantly enhanced the tyrosine phosphorylation of cortactin. Importantly, forced expression in myotubes of a tyrosine phosphorylation-defective cortactin mutant (but not wild-type cortactin) suppressed agrin-dependent AChR clustering, as did the reduction of endogenous cortactin levels using RNA interference, and introduction of the mutant cortactin into muscle cells potently inhibited synaptic AChR aggregation in response to innervation. CONCLUSION: Our results suggest a novel function of phosphorylation-dependent cortactin signaling downstream from agrin/MuSK in facilitating AChR clustering at the developing NMJ.

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Species referenced: Xenopus
Genes referenced: abl1 actl6a aicda ctnnd1 cttn musk nck1 rapsn rho rho.2 rrad was wasl wipf2

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	Figure 1. Localization of Arp2/3 complex proteins and cortactin at AChR clustering sites.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).
	Figure 2. Tyrosine phosphorylation of cortactin at pre-patterned and nerve-induced AChR clusters.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 Table S1), 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).
	Figure 3. Cortactin phosphorylation at AChR clusters induced by growth factor-coated beads and agrin.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).
	Figure 4. Agrin-dependent enhancement of cortactin tyrosine phosphorylation.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 Methods), 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.
	Figure 5. Inhibition of agrin-induced AChR clustering by forced expression of phospho-mutant cortactin in myotubes.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.
	Figure 6. Inhibition of agrin-induced AChR clustering by down-regulation of cortactin expression in myotubes.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.
	Figure 7. Suppression of synaptic AChR aggregation by phospho-mutant cortactin expressed selectively in muscle cells.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 Methods) 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.
	Figure 8. Cortactin signaling in agrin-dependent AChR clustering: a model.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.

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