Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
A crucial event in the development of the vertebrate neuromuscular junction (NMJ) is the postsynaptic enrichment of muscle acetylcholine (ACh) receptors (AChRs). This process involves two distinct steps: the local clustering of AChRs at synapses, which depends on the activation of the muscle-specific receptor tyrosine kinase MuSK by neural agrin, and the global dispersal of aneural or "pre-patterned" AChR aggregates, which is triggered by ACh or by synaptogenic stimuli. We and others have previously shown that tyrosine phosphatases, such as the SH2 domain-containing phosphatase Shp2, regulate AChR cluster formation in muscle cells, and that tyrosine phosphatases also mediate the dispersal of pre-patterned AChR clusters by synaptogenic stimuli, although the specific phosphatases involved in this latter step remain unknown. Using an assay system that allows AChR cluster assembly and disassembly to be studied separately and quantitatively, we describe a previously unrecognized role of the tyrosine phosphatase Shp2 in AChR cluster disassembly. Shp2 was robustly expressed in embryonic Xenopus muscle in vivo and in cultured myotomal muscle cells, and treatment of the muscle cultures with an inhibitor of Shp2 (NSC-87877) blocked the dispersal of pre-patterned AChR clusters by synaptogenic stimuli. In contrast, over-expression in muscle cells of either wild-type or constitutively active Shp2 accelerated cluster dispersal. Significantly, forced expression in muscle of the Shp2-activator SIRPalpha1 (signal regulatory protein alpha1) also enhanced the disassembly of AChR clusters, whereas the expression of a truncated SIRPalpha1 mutant that suppresses Shp2 signaling inhibited cluster disassembly. Our results suggest that Shp2 activation by synaptogenic stimuli, through signaling intermediates such as SIRPalpha1, promotes the dispersal of pre-patterned AChR clusters to facilitate the selective accumulation of AChRs at developing NMJs.
Figure 1. Shp2 expression in embryonic Xenopus muscle. In situ hybridization assays for Shp2 mRNA were carried out with anti-sense (A) and control sense (B) probes using stage 22 (A and B, upper) and 28 (lower) Xenopus embryos. Specific labeling was observed with Shp2 mRNA anti-sense probe in myotomal muscle and brain. C. TX-100 total protein extracts of Xenopus tadpoletailmuscle ("X") and cultured C2 myotubes ("C") were immunoblotted with an anti-Shp2 monoclonal antibody. A band of about 70 kD was observed in both samples. Positions of molecular weight markers are indicated on the left.
Figure 2. Effect of Shp2 inhibitor NSC-87877 on AChR micro-cluster formation. Cultured Xenopus muscle cells, used here and in all experiments described below, were labeled with R-BTX and then incubated overnight in control culture medium (A, B) or in medium containing 1 μM NSC-87877 (NSC) (C, D). Large pre-patterned AChR clusters, often >10 μm in width, were detected in both control and NSC-treated cells (B, D; large arrows), but in cells exposed to NSC, AChR micro-clusters were also frequently observed (D; small arrows). E. Quantification of pooled data from multiple experiments revealed that AChR micro-clusters developed in nearly twice the number of muscle cells incubated in 1 μM NSC-87877 as those maintained in control medium. Mean and SEM values are shown here and in all following figures; control cells, n = 103; NSC-treated cells, n = 109; t-test *p < 0.005.
Figure 3. Effect of NSC-87877 on agrin-induced dispersal of pre-patterned AChR clusters. Muscle cells labeled with R-BTX were incubated overnight in culture medium containing agrin (ctl; A, B), agrin plus 2 μM NSC-87877 (NSC; C, D), or agrin plus 20 μM pervanadate (PV; E, F). In agrin-treated muscle cells AChR micro-clusters (~0.5–2 mm in diameter) were found but larger clusters were not generally detected (B). However, in cells incubated with agrin and NSC (D) or agrin and PV (F), both micro-clusters and large pre-patterned clusters were present (arrows).
Figure 4. Inhibition of HB-GAM bead-induced dispersal of pre-patterned AChR clusters by NSC-87877. R-BTX-labeled muscle cells were stimulated overnight with HB-GAM beads in the absence (A, B) or presence of 1 μM NSC-87877 (C, D). In control cells only bead-induced AChR clusters were present (B; asterisks) but in the NSC-treated cells both bead-induced (D; asterisks) and pre-patterned AChR clusters (arrows) were detected. The number of pre-patterned AChR clusters in bead-stimulated cells and the fraction of beads that induced new AChR clusters were quantified from multiple experiments using 0.1 or 1 μM NSC and the results were normalized relative to cells maintained in control medium (E, F). NSC-treatment inhibited the dispersal of pre-patterned AChR clusters but did not significantly affect the induction of new AChR clusters by HB-GAM beads. Control, n = 95; 0.1 μM NSC, n = 115; 1 μM NSC, n = 129; *p < 0.05, 1 μM NSC relative to control.
Figure 5. Blockage of tyrosine dephosphorylation of pre-patterned AChR clusters by NSC-treatment. R-BTX-labeled muscle cells were exposed to HB-GAM beads for 4 h in the absence (A-C) or presence of 1 μM NSC-87877 (D-F), fixed and then stained with anti-phosphotyrosine (mAb4G10) and FITC-conjugated secondary antibodies. In the example of the control muscle cell shown here, phosphotyrosine is detected at the bead-induced cluster (B, C; asterisks) but not at the pre-patterned cluster (B, C; arrow), but in the NSC-treated muscle cell, both the bead-induced cluster (E, F: asterisks) and the pre-patterned cluster (E, F: arrows) contain phosphotyrosine. G. Pooled data from multiple experiments were quantified after sorting the pre-patterned AChR clusters present on bead-stimulated cells as being fully tyrosine phosphorylated or partially or fully dephosphorylated. Control, n = 127; 0.1 μM NSC, n = 129; 1 μM NSC, n = 94; *p < 0.05 NSC relative to control.
Figure 6. Effects of over-expression of wild-type or constitutively active Shp2 on pre-patterned AChR cluster formation. Wild-type and mutant Shp2 proteins were over-expressed in Xenopus muscle cells by mRNA injection. Muscle cells expressing green fluorescent protein alone (GFP; A, B) or GFP together with wild-type Shp2 (Shp2 WT; C, D), constitutively active Shp2 (Shp2 E76A; E, F) or dominant-negative Shp2 (Shp2 deltaP; images not shown) were stained with R-BTX (B, D, F) to visualize pre-patterned AChR clusters. In muscle cells expressing GFP, normal pre-patterned AChR clusters were found (B; arrow), but in cells with wild-type or constitutively active Shp2 fewer clusters were detected (D, F). G. Data from multiple micro-injection experiments were quantified and the numbers of pre-patterned clusters in cells expressing wild-type and active Shp2 were normalized relative to the number obtained from GFP-expressing cells. GFP-cells, n = 356; Shp2 WT, n = 98; Shp2 E76A, n = 327; Shp2 deltaP, n = 356; *p < 0.05; **p < 0.001 relative to GFP-cells.
Figure 7. Acceleration in the dispersal of pre-patterned AChR clusters resulting from exogenous wild-type or constitutively active Shp2 expression. R-BTX labeled muscle cells expressing GFP only (A-C), or GFP plus Shp2 WT (D-F), Shp2 E76A (E-G) or Shp2 deltaP (not shown) were stimulated overnight with HB-GAM beads. In a fraction of bead-stimulated cells expressing GFP alone, pre-patterned AChR clusters were still present, but the fraction of such cells expressing wild-type or active Shp2 that retained pre-patterned clusters was significantly lower. To quantify these results, the number of pre-patterned clusters counted in bead-stimulated cells was divided by the number obtained from muscle cells that had not been exposed to beads (to offset differences in pre-patterned cluster formation; see text). Results normalized relative to GFP-cells are shown in panel J. GFP-cells, n = 212; Shp2 WT, n = 60; Shp2 E76A, n = 138; Shp2 deltaP, n = 127; *p < 0.01; **p < 0.05. Panel K shows that the expression of Shp2 proteins did not affect bead-induced AChR clustering.
Figure 8. SIRPα1's regulation of pre-patterned AChR cluster dispersal. R-BTX labeled muscle cells expressing GFP alone (A-C) or GFP and wild-type SIRPα1 (SIRP-FL; D-F) or dominant-negative SIRPα1 (SIRP-TR, G-I) were exposed to HB-GAM beads overnight. In the muscle cell expressing dominant-negative SIRPα1, a large pre-patterned AChR cluster was detected (I; arrow) in addition to the bead-induced clusters, which were present in control and Shp2-expressing cells (C, F, I; asterisks). Results, quantified as described in Fig. 7, revealed that the expression of wild-type SIRPα1 promoted the dispersal of pre-patterned AChR clusters whereas expression of dominant-negative SIRPα1 inhibited dispersal (panel J). GFP-cells, n = 198; SIRP-FL, n = 156; SIRP-TR, n = 169; *p < 0.05 relative to GFP-cells.
Figure 9. A model for Shp2-dependent regulation of AChR redistribution. Synaptogenic stimuli initiate two SIRPα1/Shp2-involving signaling cascades. Agrin/MuSK stimulates src family tyrosine kinases that, in addition to phosphorylating MuSK, phosphorylate SIRPα1 to activate Shp2, which then dephosphorylates (on tyr527) and stimulates more src, and thus more SIRPα1/Shp2, etc., to propagate the dispersal signal that disassembles pre-patterned AChR clusters at extrasynaptic sites. Locally at postsynaptic sites, SIRPα1/Shp2 signaling triggered by MuSK/src acts through a feedback loop to restrain the MuSK-initiated AChR clustering signal, which helps generate new AChR clusters selectively at postsynaptic sites. This form of regenerative signaling through src, SIRPα1 and Shp2 also has built-in self-regulation because Shp2 can dephosphorylate SIRPα1 to control its own activation state.
ptpn11b (protein tyrosine phosphatase, non-receptor type 11, b) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22 and 28, lateral view, anteriorleft, dorsal up.
Anderson,
Nerve-induced and spontaneous redistribution of acetylcholine receptors on cultured muscle cells.
1977, Pubmed,
Xenbase
Anderson,
Nerve-induced and spontaneous redistribution of acetylcholine receptors on cultured muscle cells.
1977,
Pubmed
,
Xenbase
Anderson,
Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells.
1977,
Pubmed
,
Xenbase
Balice-Gordon,
Long-term synapse loss induced by focal blockade of postsynaptic receptors.
1994,
Pubmed
Banks,
The postsynaptic submembrane machinery at the neuromuscular junction: requirement for rapsyn and the utrophin/dystrophin-associated complex.
2003,
Pubmed
Bloch,
Loss of acetylcholine receptor clusters induced by treatment of cultured rat myotubes with carbachol.
1986,
Pubmed
Borges,
Agrin-induced phosphorylation of the acetylcholine receptor regulates cytoskeletal anchoring and clustering.
2001,
Pubmed
Brandon,
Aberrant patterning of neuromuscular synapses in choline acetyltransferase-deficient mice.
2003,
Pubmed
Bruneau,
The dynamics of recycled acetylcholine receptors at the neuromuscular junction in vivo.
2006,
Pubmed
Camilleri,
Tyrosine phosphatases such as SHP-2 act in a balance with Src-family kinases in stabilization of postsynaptic clusters of acetylcholine receptors.
2007,
Pubmed
Chen,
Rapsyn interaction with calpain stabilizes AChR clusters at the neuromuscular junction.
2007,
Pubmed
Chen,
Discovery of a novel shp2 protein tyrosine phosphatase inhibitor.
2006,
Pubmed
Daggett,
Full-length agrin isoform activities and binding site distributions on cultured Xenopus muscle cells.
1996,
Pubmed
,
Xenbase
Dai,
A role of tyrosine phosphatase in acetylcholine receptor cluster dispersal and formation.
1998,
Pubmed
,
Xenbase
Dai,
The actin-driven movement and formation of acetylcholine receptor clusters.
2000,
Pubmed
,
Xenbase
Dong,
Shp2 is dispensable in the formation and maintenance of the neuromuscular junction.
,
Pubmed
Duclert,
Acetylcholine receptor gene expression at the developing neuromuscular junction.
1995,
Pubmed
Fertuck,
Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after 125I-alpha-bungarotoxin binding at mouse neuromuscular junctions.
1976,
Pubmed
Finn,
Postsynaptic requirement for Abl kinases in assembly of the neuromuscular junction.
2003,
Pubmed
Fu,
Aberrant motor axon projection, acetylcholine receptor clustering, and neurotransmission in cyclin-dependent kinase 5 null mice.
2005,
Pubmed
Fuhrer,
Roles of rapsyn and agrin in interaction of postsynaptic proteins with acetylcholine receptors.
1999,
Pubmed
Fuhrer,
Functional interaction of Src family kinases with the acetylcholine receptor in C2 myotubes.
1996,
Pubmed
Gautam,
Failure of postsynaptic specialization to develop at neuromuscular junctions of rapsyn-deficient mice.
1995,
Pubmed
Gautam,
Defective neuromuscular synaptogenesis in agrin-deficient mutant mice.
1996,
Pubmed
Glass,
Agrin acts via a MuSK receptor complex.
1996,
Pubmed
Harland,
In situ hybridization: an improved whole-mount method for Xenopus embryos.
1991,
Pubmed
,
Xenbase
Kidokoro,
Redistribution of acetylcholine receptors during neuromuscular junction formation in Xenopus cultures.
1985,
Pubmed
,
Xenbase
Kummer,
Assembly of the postsynaptic membrane at the neuromuscular junction: paradigm lost.
2006,
Pubmed
Kuromi,
Nerve disperses preexisting acetylcholine receptor clusters prior to induction of receptor accumulation in Xenopus muscle cultures.
1984,
Pubmed
,
Xenbase
Lin,
Neurotransmitter acetylcholine negatively regulates neuromuscular synapse formation by a Cdk5-dependent mechanism.
2005,
Pubmed
Lin,
Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse.
2001,
Pubmed
Luo,
Regulation of AChR clustering by Dishevelled interacting with MuSK and PAK1.
2002,
Pubmed
,
Xenbase
Luo,
Signaling complexes for postsynaptic differentiation.
2003,
Pubmed
Madhavan,
Tyrosine phosphatase regulation of MuSK-dependent acetylcholine receptor clustering.
2005,
Pubmed
,
Xenbase
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
Madhavan,
A synaptic balancing act: local and global signaling in the clustering of ACh receptors at vertebrate neuromuscular junctions.
2003,
Pubmed
Madhavan,
The involvement of calcineurin in acetylcholine receptor redistribution in muscle.
2003,
Pubmed
,
Xenbase
Maile,
Regulation of insulin-like growth factor I receptor dephosphorylation by SHPS-1 and the tyrosine phosphatase SHP-2.
2002,
Pubmed
McMahan,
The agrin hypothesis.
1990,
Pubmed
Megeath,
Calcium-dependent maintenance of agrin-induced postsynaptic specializations.
2003,
Pubmed
Mei,
RNA splicing regulates the activity of a SH2 domain-containing protein tyrosine phosphatase.
1994,
Pubmed
Milholland,
L-type calcium channels mediate acetylcholine receptor aggregation on cultured muscle.
2007,
Pubmed
Milholland,
A role for acetylcholine receptors in their own aggregation on muscle cells.
2007,
Pubmed
Misgeld,
Roles of neurotransmitter in synapse formation: development of neuromuscular junctions lacking choline acetyltransferase.
2002,
Pubmed
Misgeld,
Agrin promotes synaptic differentiation by counteracting an inhibitory effect of neurotransmitter.
2005,
Pubmed
Mittaud,
A single pulse of agrin triggers a pathway that acts to cluster acetylcholine receptors.
2004,
Pubmed
Moody-Corbett,
Influence of nerve on the formation and survival of acetylcholine receptor and cholinesterase patches on embryonic Xenopus muscle cells in culture.
1982,
Pubmed
,
Xenbase
Neel,
The 'Shp'ing news: SH2 domain-containing tyrosine phosphatases in cell signaling.
2003,
Pubmed
Nizhynska,
Phosphoinositide 3-kinase acts through RAC and Cdc42 during agrin-induced acetylcholine receptor clustering.
2007,
Pubmed
Oh,
Regulation of early events in integrin signaling by protein tyrosine phosphatase SHP-2.
1999,
Pubmed
Okada,
The muscle protein Dok-7 is essential for neuromuscular synaptogenesis.
2006,
Pubmed
O'Reilly,
Activated mutants of SHP-2 preferentially induce elongation of Xenopus animal caps.
2000,
Pubmed
,
Xenbase
Panzer,
In vivo imaging of preferential motor axon outgrowth to and synaptogenesis at prepatterned acetylcholine receptor clusters in embryonic zebrafish skeletal muscle.
2006,
Pubmed
Peng,
Elimination of preexistent acetylcholine receptor clusters induced by the formation of new clusters in the absence of nerve.
1986,
Pubmed
,
Xenbase
Peng,
Tissue culture of Xenopus neurons and muscle cells as a model for studying synaptic induction.
1991,
Pubmed
,
Xenbase
Peng,
The role of heparin-binding growth-associated molecule (HB-GAM) in the postsynaptic induction in cultured muscle cells.
1995,
Pubmed
,
Xenbase
Roskoski,
Src kinase regulation by phosphorylation and dephosphorylation.
2005,
Pubmed
Ross,
Induction of phosphorylation and cell surface redistribution of acetylcholine receptors by phorbol ester and carbamylcholine in cultured chick muscle cells.
1988,
Pubmed
Sadasivam,
Src-family kinases stabilize the neuromuscular synapse in vivo via protein interactions, phosphorylation, and cytoskeletal linkage of acetylcholine receptors.
2005,
Pubmed
Sanes,
Development of the vertebrate neuromuscular junction.
1999,
Pubmed
Schaeffer,
Targeting transcription to the neuromuscular synapse.
2001,
Pubmed
Smith,
Src, Fyn, and Yes are not required for neuromuscular synapse formation but are necessary for stabilization of agrin-induced clusters of acetylcholine receptors.
2001,
Pubmed
Tang,
The SH2-containing protein-tyrosine phosphatase SH-PTP2 is required upstream of MAP kinase for early Xenopus development.
1995,
Pubmed
,
Xenbase
Tanowitz,
Regulation of neuregulin-mediated acetylcholine receptor synthesis by protein tyrosine phosphatase SHP2.
1999,
Pubmed
Wallace,
Regulation of the interaction of nicotinic acetylcholine receptors with the cytoskeleton by agrin-activated protein tyrosine kinase.
1995,
Pubmed
Weatherbee,
LDL-receptor-related protein 4 is crucial for formation of the neuromuscular junction.
2006,
Pubmed
Weston,
Agrin and laminin induce acetylcholine receptor clustering by convergent, Rho GTPase-dependent signaling pathways.
2007,
Pubmed
Yang,
Patterning of muscle acetylcholine receptor gene expression in the absence of motor innervation.
2001,
Pubmed
Zhao,
Regulation of ACh receptor clustering by the tyrosine phosphatase Shp2.
2007,
Pubmed
