Src-family kinases stabilize the neuromuscular synapse in vivo via protein interactions, phosphorylation, and cytoskeletal linkage of acetylcholine receptors.
Postnatal stabilization and maturation of the postsynaptic membrane are important for development and function of the neuromuscular junction (NMJ), but the underlying mechanisms remain poorly characterized. We examined the role of Src-family kinases (SFKs) in vivo. Electroporation of kinase-inactive Src constructs into soleus muscles of adult mice caused NMJ disassembly: acetylcholine receptor (AChR)-rich areas became fragmented; the topology of nerve terminal, AChRs, and synaptic nuclei was disturbed; and occasionally nerves started to sprout. Electroporation of kinase-overactive Src produced similar but milder effects. We studied the mechanism of SFK action using cultured src(-/-);fyn(-/-) myotubes, focusing on clustering of postsynaptic proteins, their interaction with AChRs, and AChR phosphorylation. Rapsyn and the utrophin-glycoprotein complex were recruited normally into AChR-containing clusters by agrin in src(-/-);fyn(-/-) myotubes. But after agrin withdrawal, clusters of these proteins disappeared rapidly in parallel with AChRs, revealing that SFKs are of general importance in postsynaptic stability. At the same time, AChR interaction with rapsyn and dystrobrevin and AChR phosphorylation decreased after agrin withdrawal from mutant myotubes. Unexpectedly, levels of rapsyn protein were increased in src(-/-);fyn(-/-) myotubes, whereas rapsyn-cytoskeleton interactions were unaffected. The overall cytoskeletal link of AChRs was weak but still strengthened by agrin in mutant cells, consistent with the normal formation but decreased stability of AChR clusters. These data show that correctly balanced activity of SFKs is critical in maintaining adult NMJs in vivo. SFKs hold the postsynaptic apparatus together through stabilization of AChR-rapsyn interaction and AChR phosphorylation. In addition, SFKs control rapsyn levels and AChR-cytoskeletal linkage.
Sadasivam
Gayathri
G
Department of Neurochemistry, Brain Research Institute, University of Zürich, CH-8057 Zürich, Switzerland.
Willmann
Raffaella
R
Lin
Shuo
S
Erb-Vögtli
Susanne
S
Kong
Xian Chu
XC
Rüegg
Markus A
MA
Fuhrer
Christian
C
D018171:Agrin / Q000494:pharmacology; D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D000074765:Dysbindin; D049030:Dystroglycans / Q000378:metabolism; D049029:Dystrophin-Associated Proteins / Q000378:metabolism; D018274:Electroporation / Q000379:methods; D005786:Gene Expression Regulation / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D021621:Imaging, Three-Dimensional / Q000379:methods; D015151:Immunoblotting / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D066298:In Vitro Techniques; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D008954:Models, Biological; D018485:Muscle Fibers, Skeletal / Q000502:physiology; D009124:Muscle Proteins / Q000378:metabolism; D016296:Mutagenesis / Q000502:physiology; D016900:Neurofilament Proteins / Q000378:metabolism; D009469:Neuromuscular Junction / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D051076:Proto-Oncogene Proteins c-fyn / Q000172:deficiency / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology; D011950:Receptors, Cholinergic / Q000378:metabolism*; D016708:Synaptophysin / Q000378:metabolism; D013997:Time Factors; D049411:Utrophin / Q000378:metabolism; D019061:src-Family Kinases / Q000172:deficiency / Q000502:physiology*
D018171:Agrin; D002038:Bungarotoxins; D002352:Carrier Proteins; C498992:Dtnbp1 protein, mouse; D000074765:Dysbindin; D049029:Dystrophin-Associated Proteins; D009124:Muscle Proteins; D016900:Neurofilament Proteins; D011950:Receptors, Cholinergic; D016708:Synaptophysin; D049411:Utrophin; C098885:dystrobrevin; C051609:peripheral membrane protein 43K; D049030:Dystroglycans; D049452:Green Fluorescent Proteins; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases