The aggregation of muscle acetylcholine receptors (AChRs) into high-density clusters in the postsynaptic membrane is a hallmark of neuromuscular junction (NMJ) development. Before innervation, AChRs form pre-patterned clusters. After motor innervation, the nerve-derived factor agrin activates muscle-specific kinase MuSK to disperse pre-patterned AChR clusters and to induce new postsynaptic clusters. Previous studies suggest that tyrosine phosphatases are important for the dispersal of pre-patterned AChR clusters. Here, we investigated the role of tyrosine phosphatases in AChR cluster formation. Spinal neurons and beads coated with heparin-binding growth-associated molecules (HB-GAM) were used to induce AChR clustering. Both bead- and nerve-induced clusters conformed to the size of the c...[ Read more ]

The aggregation of muscle acetylcholine receptors (AChRs) into high-density clusters in the postsynaptic membrane is a hallmark of neuromuscular junction (NMJ) development. Before innervation, AChRs form pre-patterned clusters. After motor innervation, the nerve-derived factor agrin activates muscle-specific kinase MuSK to disperse pre-patterned AChR clusters and to induce new postsynaptic clusters. Previous studies suggest that tyrosine phosphatases are important for the dispersal of pre-patterned AChR clusters. Here, we investigated the role of tyrosine phosphatases in AChR cluster formation. Spinal neurons and beads coated with heparin-binding growth-associated molecules (HB-GAM) were used to induce AChR clustering. Both bead- and nerve-induced clusters conformed to the size of the contacts and showed discrete boundaries. However, in the presence of the phosphatase inhibitor bpV, induced clusters spread markedly. Furthermore, blocking the tyrosine kinase Shp2 produced AChR clustering akin to those seen with bpV treatment, indicating that Shp2 is critical for regulating AChR clustering. These results suggest that tyrosine phosphatases, especially Shp2, regulate AChR cluster assembly in response to synaptogenic stimuli by defining the zone of protein kinase activity to establish the boundary within which postsynaptic specializations develop. Furthermore, because Shp2-Src-SIRPα1 signaling was found to be critical for regulating NMJ formation and maintenance, we examined the function of Src kinase in live muscle cells using FRET probes that measure Src activation. Muscle cells were treated with neural agrin and HB-GAM-coated beads, and our results showed that Src was activated in muscle immediately after stimulation and became maximally activated rapidly, and that Src activity remained high even after AChR clusters were formed locally. Thus, using the Src probe, we have provided direct evidence of Src activation in live muscle cells during synaptogenic signaling, which is a critical step in the formation and maintenance of postsynaptic AChR clusters.