De Cicco, T. | bioRxiv Science

bioRxiv2024

SH3BP2 regulates the organization of the neuromuscular synapses through protein-driven phase separation

The molecular mechanisms underlying the development and maintenance of the neuromuscular junction are poorly understood, even though the malfunction of these specialized synapses is associated with severe genetic and autoimmune disorders. The identity of factors controlling the maintenance mechanisms of postsynaptic acetylcholine receptors (AChR) in high-density has been elusive and is of great interest to the pharma industry, searching for possible new targets for disease interventions. Here, we report the identification of a scaffold protein SH3BP2, which exhibits polyvalent interaction with the dystrophin-glycoprotein complex (DGC) and AChR pentamers, promoting AChR clustering through phase separation. Muscle-specific SH3BP2 deletion in mice leads to impaired organization of the neuromuscular synapses, defects in synaptic transmission, and reduced muscle strength. Our studies identified a novel regulator of the postsynaptic machinery involved in clustering AChR and linking it to the DGC.

neuroscience↗

bioRxiv2023

Cortactin interacts with αDystrobrevin-1 and regulates neuromuscular junction morphology

BackgroundNeuromuscular 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. Dystrobrevin-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 Dystrobrevin-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 Dystrobrevin-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 animals ability to run on voluntary wheels and a treadmill. ResultsOur proteomic screen identified a number of putative Dystrobrevin-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 Dystrobrevin-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.

neuroscience↗