Sadasivam Gayathri, Willmann Raffaella, Lin Shuo, Erb-Vögtli Susanne, Kong Xian Chu, Rüegg Markus A, Fuhrer Christian
Department of Neurochemistry, Brain Research Institute, University of Zürich, CH-8057 Zürich, Switzerland.
J Neurosci. 2005 Nov 9;25(45):10479-93. doi: 10.1523/JNEUROSCI.2103-05.2005.
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.
突触后膜的产后稳定和成熟对于神经肌肉接头（NMJ）的发育和功能至关重要，但其潜在机制仍不清楚。我们在体内研究了Src家族激酶（SFK）的作用。将激酶失活的Src构建体电穿孔到成年小鼠的比目鱼肌中会导致NMJ解体：富含乙酰胆碱受体（AChR）的区域变得碎片化；神经末梢、AChR和突触核的拓扑结构受到干扰；偶尔神经开始发芽。激酶过度活跃的Src电穿孔产生类似但较轻的影响。我们使用培养的src（-/-）；fyn（-/-）肌管研究了SFK作用的机制，重点是突触后蛋白的聚集、它们与AChR的相互作用以及AChR磷酸化。在src（-/-）；fyn（-/-）肌管中，Rapsyn和抗肌萎缩蛋白-糖蛋白复合物通过聚集素正常地被招募到含AChR的簇中。但在聚集素撤除后，这些蛋白质的簇与AChR同时迅速消失，表明SFK在突触后稳定性中具有普遍重要性。与此同时，从突变肌管中撤除聚集素后，AChR与Rapsyn和肌萎缩素结合蛋白的相互作用以及AChR磷酸化减少。出乎意料的是，src（-/-）；fyn（-/-）肌管中Rapsyn蛋白水平增加，而Rapsyn与细胞骨架的相互作用未受影响。AChR的整体细胞骨架连接较弱，但在突变细胞中仍通过聚集素得到加强，这与AChR簇的正常形成但稳定性降低一致。这些数据表明，SFK的正确平衡活性对于维持体内成年NMJ至关重要。SFK通过稳定AChR-Rapsyn相互作用和AChR磷酸化将突触后装置聚集在一起。此外，SFK控制Rapsyn水平和AChR-细胞骨架连接。
