Abstract
BackgroundA 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.ResultsUsing 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 SIRPα1 (signal regulatory protein α1) also enhanced the disassembly of AChR clusters, whereas the expression of a truncated SIRPα1 mutant that suppresses Shp2 signaling inhibited cluster disassembly.ConclusionOur results suggest that Shp2 activation by synaptogenic stimuli, through signaling intermediates such as SIRPα1, promotes the dispersal of pre-patterned AChR clusters to facilitate the selective accumulation of AChRs at developing NMJs.
Highlights
A crucial event in the development of the vertebrate neuromuscular junction (NMJ) is the postsynaptic enrichment of muscle acetylcholine (ACh) receptors (AChRs)
We began by staining the myotomal muscle cell cultures with antibodies against twelve different tyrosine phosphatases and found that an anti-Shp2 antibody, unlike other antibodies tested, labeled the cells strongly and uniformly, which led us to determine if Shp2 was present in embryonic Xenopus muscle in vivo
We divided the number of pre-patterned AChR clusters in bead-stimulated cells by the number of pre-patterned clusters in cells not exposed to beads; here, this compensated for the reduced formation of pre-patterned AChR clusters in cells over-expressing Shp2. These ratios were further normalized using those calculated for cells expressing green fluorescent protein (GFP) alone. These results indicated that the presence of excess wild-type or constitutively active Shp2 proteins in muscle cells is sufficient for promoting AChR cluster dispersal (Figure 7J)
Summary
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. In innervated muscle, AChRs become concentrated at incipient synapses where MuSK is locally stimulated by the nerve-derived factor agrin [4,5,6] This synaptic accumulation of AChRs is coupled with the disassembly of the pre-patterned AChR clusters [7,8,9,10], and these two distinct processes together help generate a 1000-fold higher density of AChRs at the NMJ compared to extra-synaptic muscle [11]
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