Cortactin interacts with αDystrobrevin-1 and regulates neuromuscular junction morphology

Abstract

AbstractBackgroundNeuromuscular junctions allow for transmitting signals from the nervous system to skeletal muscles, triggering their contraction, and their proper organization is essential for breathing and voluntary movements. αDystrobrevin-1 is a cytoplasmic component of the dystrophin-glycoprotein complex and has pivotal functions in regulating the integrity of muscle fibres and neuromuscular junctions. Previous studies identified that αDystrobrevin-1 functions in the organization of the neuromuscular junction and that its phosphorylation in the C-terminus is required in this process.MethodsWe used synthetic peptides corresponding to the phosphorylated tyrosine Y730 at the C-terminal part of αDystrobrevin-1 to precipitate interacting proteins from homogenate of differentiated muscle cells. Isolated proteins were identified by mass spectrometry, and co-immunoprecipitation and bimolecular fluorescence complementation experiments in skeletal muscles were used to validate interactions. We used immunohistochemical analysis and muscle electroporation to study cortactin localization in skeletal muscles. To study the function of cortactin in the neuromuscular system, we used cortactin KO mice. Synaptic morphology was studied using unbiased automatic image analysis, and muscle strength was assessed in grip-strength experiments and an animal’s ability to run on voluntary wheels and a treadmill.ResultsOur proteomic screen identified a number of putative αDystrobrevin-1 interactors recruited to the Y730 site in both its phosphorylated and unphosphorylated state. Amongst various actin regulators, we identified the Arp2/3 complex regulator cortactin. We showed that similarly to αDystrobrevin-1, cortactin is strongly enriched at the neuromuscular postsynaptic machinery and obtained results suggesting that these two proteins interact in cell homogenates and at the neuromuscular junctions. Analysis of synaptic morphology cortactin knockout mice showed abnormalities in the slow-twitching soleus muscle and not in the fast-twitching tibialis. However, muscle strength examination did not reveal apparent deficits in knockout animals.ConclusionsOur studies identified cortactin as a novel interactor of the dystrophin-glycoprotein complex, pivotal in maintaining muscle and neuromuscular junction integrity. We have shown that cortactin is a novel postsynaptic machinery component that can be essential in organizing the neuromuscular junctions.